Novel plant growth promoting bradyrhizobium compositions

Abstract

The present invention relates to the field of sustainable agriculture. In particular, the present invention relates to an isolated Bradyrhizobium bacterium having enhanced characteristics, including but not limited to enhanced adaptation to the low and medium root zone temperature for example in North- West Europe.

Description

[0001] SoGo / BRADY-2 / 853

[0002] NOVEL PLANT GROWTH PROMOTING BRADYRHIZOBIUM COMPOSITIONS

[0003] Field of the invention

[0004] The present invention relates to the field of sustainable agriculture. Specifically, the invention provides microbial compositions and methods for improving the yield of nitrogen fixing plants. In particular, the present invention relates to a newly isolated Bradyrhizobium japonicum bacterium having enhanced characteristics, including but not limited to enhanced adaptation to low and / or medium soil temperatures, for example in North-West Europe.

[0005] Introduction to the invention

[0006] Soybean (Glycine max), the world's most extensively farmed legume, serves as a crucial source of plantbased protein for both human diets and animal feed1. Recently, there has been a push to extend the range of the crop to more northern regions. In Europe, these efforts align with the Farm to Fork strategy, part of the EU's Green Deal, and aim for three key objectives facilitating more sustainable agriculture: (1) reducing soy import from South American regions, where soy cultivation contributes to deforestation and biodiversity loss2; (2) encouraging a shift in the human diet from animal-based proteins to plantbased alternatives, in order to mitigate the environmental impact associated with a rising global demand for protein-rich foods3-4; and (3) decreasing the use of chemical fertilizers. Indeed, soybean can be grown without the need for nitrogen fertilization, thanks to a symbiotic relationship with diazotrophic rhizobial bacteria. Additionally, this capability allows the use of non-harvested parts of soybean plants as a natural nitrogen fertilizer in crop rotation systems5-7.

[0007] To make soybean production at northern latitudes profitable and competitive, a significant yield gap must be crossed8. One issue to address is that commercially available rhizobial inocula, such as BIODOZ® (B. diazoefficiens G49) and HiStick® (B. japonicum 532C), originate from warmer climates, and are thus not always compatible with local soybean varieties or not well adapted to the local climate9-11. The formation of actively nitrogen-fixing nodules, usually recognized by their red interior, requires compatibility between bacteria and host. Incompatibility translates to the formation of white, non-fixing nodules, or an inability to nodulate altogether12. A strategy to overcome this is to develop inocula from rhizobial strains isolated locally and to enhance our understanding of the interactions among bacterial strains, soybean varieties, and the environment10-11-13.

[0008] A straightforward way of obtaining endogenous rhizobia that are able to nodulate soybean is via so-called 'trapping'. Briefly, nodules of locally grown, non-inoculated soybean are harvested, and from these, bacteria are isolated and identified. This strategy has been applied successfully for isolation of local SoGo / BRADY-2 / 853 soybean-nodulating rhizobia in several African and South American countries, and recently also in Germany10-14-17.

[0009] SUMMARY

[0010] A trapping experiment was set up in diverse Belgian soils to capture native soybean nodulators. Therefore, the invention provides in a first aspect, an isolated Bradyrhizobium R-85129 strain, also designated herein as 521_C7_N1.3, having the deposit accession number LMG P-33693. Also enriched cultures and biologically pure cultures of said Bradyrhizobium strain are provided. In another aspect, a composition comprising said Bradyrhizobium strain or one of said cultures is provided. In one embodiment, the Bradyrhizobium strain is lyophilized, freeze-dried, dried in a form of a powder or present as an aqueous slurry. In another embodiment, said compositions further comprises growth medium appropriate for Bradyrhizobium species and / or a cryoprotectant. The composition may also further comprise an agriculturally compatible carrier. In another aspect a plant seed coated with one of the Bradyrhizobium strain of the invention or a plant seed coated with the Bradyrhizobium strain is provided. In one embodiment, the plant seed is a leguminous plant seed, more particularly a soybean.

[0011] Given that the Bradyrhizobium strain disclosed in current application is especially useful in the field of agriculture, methods are provided for enhancing growth, yield and / or nitrogen fixation of plants, more particularly of leguminous plants by administration of said Bradyrhizobium strain to said plants. This is equivalent as saying that the use of the Bradyrhizobium strain of the invention, the cultures or compositions comprising it is provided to enhance growth, yield and / or nitrogen fixation of a plant, more particularly a leguminous plant. In one embodiment, said yield refers to the protein content of the seeds, more particularly of soybeans. It is envisaged that the Bradyrhizobium strain of the invention can be administered by several ways, for example but not limited to by coating plant seeds, inoculating the soil or other plant growth supporting media, spraying or irrigating plants. Hence, in one embodiment, the methods comprise the steps of inoculating a plant growth medium with the Bradyrhizobium strain according to the invention or with one of the cultures or compositions comprising it, and growing the leguminous plant in said plant growth medium. In a particular embodiment, the Bradyrhizobium strain, cultures or compositions are applied to the plant growth medium as a powder, as a pellet, as a granule or as a liquid. In other embodiments, the methods comprise the steps of growing plants in an environment that supports plant growth and administering a sprayable formulation to said environment or to said plant, said formulation comprising the Bradyrhizobium strain of the invention or the cultures or compositions comprising it.

[0012] DEPOSIT OF BIOLOGICAL MATERIAL

[0013] Purified cultures of the microbial strains described in present application were deposited by VIB vzw (Suzanne Tassierstraat 1, 9052 Gent, Belgium) at the BCCM (Belgian Coordinated Collections of Micro- SoGo / BRADY-2 / 853

[0014] Organisms) consortium (BCCM represented by Laboratorium voor Microbiologie - Bacterienverzameling (LMG), Universiteit Gent, K.L. Ledeganckstraat 35, 9000 Gent, Belgium), recognized as an International Depositary Authority by the World Intellectual Property organization since March 1, 1992 and in accordance with the Budapest Treaty as specified in Rule 31(1) EPC for the purpose of patent procedure and the regulations thereunder.

[0015] The Bradyrhizobium sp. R-85129 strain of current application has been deposited with deposit number LMG P-33693 for which the original deposit has been done on 10 July 2024.

[0016] FIGURE LEGENDS

[0017] Figure 1: Selection and performance of indigenous rhizobial strains. (A) Presence of the core set of nif, nod, fix genes and components of the type 3 secretion system (T3SS) in the commercial strain G49 and five native Bradyrhizobium strains. Green, orange and red colour indicates respectively the presence of all, some or none of the symbiosis genes that are found in G49. (B-C) Phenotype (B) and number of red nodules (C) on soybean plants inoculated with the indicated strains and grown until four weeks past inoculation in sterile vermiculite. For each treatment, 12 plants were analysed, with each plant being represented by a single dot. (D) Nodule number and dry weight for soybean plants inoculated with the indicated strains and grown in a 1:1 mixture of fresh agricultural soil and sand for eight weeks. For each treatment and variety, nine plants, grouped in three pots, were analysed. Treatments were grouped in three separate experiments, each displayed in a separate graph. (E) Nodule and yield parameters for soybean plants inoculated with the indicated strains and in fields at two different locations ('Bottelare' and 'Merelbeke'). For each treatment, variety, and location, three separate plots were analysed. Nodule and yield parameters were determined per plot, except for nodule number, which was measured for respectively five plants per plot. (C-E) Seeds inoculated with G49 or non-inoculated (Control) seeds were used as a positive or negative control, respectively. Colours of dots and boxplots indicate the soybean variety used. The centre lines in the boxplots show the median, with the box limits representing the upper and lower quartiles, and the whiskers representing the maximum and minimum values. Dots drawn outside the whiskers mark outliers. Significant differences between treatments were determined using one-way ANOVA (C) or a linear mixed model (D,E) with Tukey multiple comparison correction. Letters indicate significance groups (P<0.05). SoGo / BRADY-2 / 853

[0018] Figure 2: Capacity for nodulation of native isolated strains. Soybean seeds of variety 'Arcadia' were inoculated with the indicated isolated strains and grown in sterile vermiculite. Seeds inoculated with G49 or non-inoculated (Control) seeds were used as a positive or negative control, respectively. At four weeks post-inoculation, nodules were counted, and nodule colour was noted. For each treatment, at least six plants were analysed, with each plant being represented by a single dot. Treatments were grouped in three separate experiments, each displayed in a separate graph. Each dot represents the number of nodules on a single plant, with colour indicating the colour of the most active nodule found on that plant (red > pink > white). Plants without nodules are presented in black. Colours of the boxplots represent the most prevalent colour of nodules across plants receiving the same treatment. The centre lines in the boxplots show the median, with the box limits representing the upper and lower quartiles, and the whiskers representing the maximum and minimum values. Dots drawn outside the whiskers mark outliers.

[0019] Figure 3: Nitrogen fixation efficiency of selected indigenous rhizobia strains. Soybean seeds of variety 'Arcadia' were inoculated with the indicated isolated strains and grown in a 1:1 mixture of fresh agricultural soil and sand. Seeds inoculated with G49 or 532C were used as a positive control. At four weeks post-inoculation (A) nitrogenase activity was determined by measuring the acetylene reduction activity (ARA) for each root system. ARA is represented as pmol of ethylene (C2H4) produced per hour. Per root system, nodule number (B), and nodule dry weight (C) were also assessed. For each treatment, at least nine plants were analysed, with each plant being represented by a single dot. The centre lines in the boxplots show the median, with the box limits representing the upper and lower quartiles, and the whiskers representing the maximum and minimum values. Dots drawn outside the whiskers mark outliers. Significant differences between treatments were determined using one-way ANOVA with Tukey multiple comparison correction. Letters indicate significance groups (P<0.05).

[0020] Figure 4: Complete results of the pot trials. Soybean seeds of 5 varieties were inoculated with the indicated isolated strains and grown in a 1:1 mixture of fresh agricultural soil and sand. Seeds inoculated with G49 or non-inoculated (Control) seeds were used as a positive or negative control, respectively. At eight weeks after sowing, nodule number (A), nodule dry weight (B), chlorophyll content (C) plant height (D), and above ground biomass (E) were determined. For each treatment and variety, nine plants, grouped in three pots, were analysed. Treatments were grouped in six separate experiments, each displayed in a separate graph. The centre lines in the boxplots show the median, with the box limits representing the upper and lower quartiles, and the whiskers representing the maximum and minimum values. Dots drawn outside the whiskers mark outliers. Box plot colour represents the soybean variety. Significant differences between treatments were determined using a linear mixed model with Tukey multiple comparison correction. Letters indicate significance groups (P<0.05). SoGo / BRADY-2 / 853

[0021] Figure 5: Complete results of field trials. Soybean seeds of variety 'Lenka' (yellow) and 'RGT Shouna' (blue) were inoculated with the indicated isolated strains and sown in fields at two different locations ('Bottelare' and 'Merelbeke'). Seeds inoculated with G49 or non-inoculated (Control) seeds were used as a positive or negative control, respectively. Nodule number (A), nodule dry weight (B), chlorophyll content (C) bean yield (D), bean protein content (E), and thousand kernel weight (F) were determined when plants reached growth stage R5 (A-C) or full maturity (D-F). For each treatment, variety, and location, three separate plots were analysed. Nodule and yield parameters were determined per plot, except for nodule number (A) and chlorophyll content (C), which were measured for respectively five plants and ten leaves per plot. The centre lines in the boxplots show the median, with the box limits representing the upper and lower quartiles, and the whiskers representing the maximum and minimum values. Dots drawn outside the whiskers mark outliers. Box plot colour represents the soybean variety. Significant differences between treatments were determined using a linear mixed model with Tukey multiple comparison correction. Letters indicate significance groups (P<0.05).

[0022] Figure 6: determination of the protein content of five different strains described herein when after inoculation of a soybean variety. The nulobject (first row in figure 6 is the soybean variety without inoculant) and the second row in figure 6 is the Bradyrhizobium strain 521_C7_N1.3 of the present invention.

[0023] Table 1: ANI values, representing the genetic similarity, of the isolated strains when compared to the respective type strains.

[0024] Isolate Type strain ANI , SoGo / BRADY-2 / 853

[0025] Isolate Type strain ANI

[0026] Harvest date 5 / 10 / 2022 23 / 09 / 2022

[0027] Preceding crop Fibre flax Fibre flax

[0028] Fertilization

[0029] Soil pH-KCI 5.8 6.0

[0030] NO3-N (kg / ha)17.1 6.0

[0031] NH4-N (kg / ha)16.3 7.4

[0032] 1Soil layer 0-30 cm SoGo / BRADY-2 / 853

[0033] DETAILED DESCRIPTION

[0034] In the current application a novel nitrogen fixing bacterial strain is disclosed, more particularly a Bradyrhizobium R-85129 strain. The strain has a high genetic homology to Bradyrhizobium japonicum USDA6 strain. The strain of the application is able to undergo a nitrogen fixing interaction with a leguminous plant, more particularly with soy. "Nitrogen-fixing interaction" as used herein refers to a plant-bacterial interaction in which atmospheric nitrogen is made available to the plant as a nutrient through the bacterium. Typically, this interaction results in the formation of root nodules containing the bacterium.

[0035] STRAIN

[0036] In one aspect of current application, a Bradyrhizobium sp. R-85129 strain is provided comprising a 16S rRNA sequence as depicted in SEQ. ID No. 1. In one embodiment, said Bradyrhizobium strain is a nitrogen fixing bacterial strain. In another embodiment, said Bradyrhizobium strain isa Bradyrhizobium japonicum strain, more particularly the Bradyrhizobium japonicum R-85129 strain. In another embodiment, said strain is an isolated strain. The term "isolated" means that the bacterial strain has been removed from its natural environment. "Isolated" thus implies a purification step. However, "isolated" does not necessarily reflect the extent to which the microorganism, more particularly the bacterium has been purified. A bacterial strain of current application is purified at least 2x, at least 5x, at least lOx, at least 50x or at least lOOx from the raw material from which it is isolated. As a non-limiting example, if a microorganism is isolated from soil as raw material, the microorganism can be isolated to an extent that its concentration in a given quantity of purified or partially purified material (e.g. soil) is at least 2x, at least 5x, at least lOx, at least 50x or at least lOOx that in the original raw material.

[0037] In yet another embodiment, said Bradyrhizobium strain improves the nitrogen status and / or nitrogen fixation of a soybean plant that was inoculated with said strain compared to other publicly available Bradyrhizobium strains, more particularly to the commercial Bradyrhizobium strain USDA6. With "nitrogen status" as used herein it is meant the nitrogen level or nitrogen concentration or nitrogen usage in the inoculated plant.

[0038] In another aspect of current application a bacterial strain is provided with deposit accession numbers LMG P-33693. In one embodiment, said bacterial strain is a nitrogen fixing bacterial strain. In another embodiment, said bacterial strain is the Bradyrhizobium sp. R-85129 strain. In another embodiment, said strain is an isolated strain.

[0039] From here on, the above bacterial strain will be referred to as the bacterial strain of current application. SoGo / BRADY-2 / 853

[0040] In another aspect of the application a culture of the bacterial strain of current application is provided. The term "culture" as used herein refers to a population of microorganisms that are propagated on or in media of various kinds. In one embodiment, said culture is an enriched culture of a bacterial strain of current application. This is equivalent as saying that a culture of microorganisms, more particularly a bacterial culture, is provided, wherein said culture is enriched with a bacterial strain of current application (i.e. Bradyrhizobium sp. R-85129 strain) and wherein "enriched" means that the total microbial (or more particularly the total bacterial) population of said culture contains more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% of the isolated bacterial strain of current application.

[0041] In another embodiment, a biologically pure culture of a bacterial strain of current application is provided. As used herein, "biologically pure" refers to a culture which contains substantially no other microorganisms than the desired strain and thus a culture wherein virtually all of the cells present are of the selected strain. In practice, a culture is defined biologically pure if the culture contains at least more than 96%, at least more than 97%, at least more than 98% or at least more than 99% of a bacterial strain of current application, more particularly of Bradyrhizobium sp. R-85129 with deposit number LMG P- 33693. When a biologically pure culture contains 100% of the desired microorganism a monoculture is reached. A monoculture thus only contains cells of the selected strain and is the most extreme form of a biologically pure culture.

[0042] In yet another embodiment, the culture of one of the Bradyrhizobium strains of the application comprises at least 1%, at least 5%, at least 10%, at least 25%, at least 50% or at least 75% living Bradyrhizobium R-85129 bacteria.

[0043] In a particular embodiment, the bacterial strains of current application may be lyophilized, freeze-dried or in a form of a dry powder. In another particular embodiment, an aqueous slurry of the bacterial strains of the current application or of any culture herein described comprising the strain is provided, the slurry being optionally dried to a powder at a temperature which does not adversely affect viability of the bacterial strain.

[0044] COMPOSITIONS

[0045] In another aspect, a composition is provided comprising a bacterial strain of current application. This is equivalent as saying that the composition comprises an inoculum of a bacterial strain of the application. As used herein, the term "inoculum" is intended to mean any form of bacterial cells, or spores, which is capable of propagating on or in the soil when the conditions of temperature, moisture, etc., are favourable for bacterial growth. A "spore" generally refers to a microorganism in its dormant, protected state. SoGo / BRADY-2 / 853

[0046] In a particular embodiment, the composition may be in the form of a liquid, a slurry, a wettable powder or a dry powder. In a particular embodiment, a bacterial strain of current application may be lyophilized, freeze-dried or in the form of a dry powder before it is used in the processing of the composition. In another particular embodiment, an aqueous slurry of a bacterial strain of the current application is provided, which is optionally dried to a powder at a temperature which does not adversely affect viability of the bacterial strain. The powder may then be mixed with an agriculturally compatible carrier. In other embodiments, a liquid suspension or slurry of a bacterial strain of the current application may be applied to an absorbent material, e.g. a granular mass, or may be used to coat plant seeds or other plant tissues. Also a powder comprising a bacterial strain of the application is suitable for coating seeds. When used to coat plant seeds, the composition may be applied to the seeds and allowed to dry. In embodiments wherein the composition is a powder (e.g. a wettable powder), a liquid, such as water, may need to be added to the powder before application to a seed.

[0047] In another embodiment, a composition is provided comprising a bacterial strain of current application further comprising a cryoprotectant and / or growth medium appropriate for Bradyrhizobium species. A "cryoprotectant" as used herein protects the bacteria by preventing the damaging effects of water crystals when cells are frozen, more particularly at -60°C, or -70°C or -80°C or in liquid nitrogen. Nonlimiting examples of a cryoprotectant is glycerol and trehalose. In another embodiment, a composition is provided comprising the bacterial strain herein disclosed wherein the bacterial strain is lyophilized, freeze dried or in the form of a dry powder. In one embodiment, the composition can further comprise a preservative.

[0048] In another particular embodiment, any of the compositions described herein further comprises an agriculturally compatible carrier. Said carrier can be inert (e.g. a detectable agent or label or liquid carrier) or active (e.g. a fertilizer), but should allow the bacterial strain of the application to remain efficacious and viable. An "agriculturally compatible carrier" may be a natural or synthetic, organic or inorganic material with which the active compounds (e.g. a bacterial strain of the current application) are combined to facilitate their application on the plant, a plant part, plant seed or to the plant growth medium. Said "agriculturally compatible carrier" which can be regarded as a vehicle, is generally inert and it must be acceptable in agriculture. Thus, the phrase "agriculturally compatible" denotes a substance that can be used routinely under field conditions without interfering with growers' planting equipment, and without adversely influencing crop development or the desired ecological balance in a cultivated area.

[0049] The agriculturally compatible carrier can be solid. Solid carriers can include but are not limited to clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers, a polymer, a granular mass, perlite, a SoGo / BRADY-2 / 853 perlite granule, peat, a peat pellet, soil, vermiculite, charcoal, sugar factory carbonation press mud, rice husk, carboxymethyl cellulose, fine sand, calcium carbonate, flour, alum, a starch, talc, polyvinyl pyrrolidone, or a combination thereof. The agriculturally compatible carrier can be a liquid. In one embodiment, the liquid carrier is water, sugar water, diluted or non-dilute growth medium to culture the bacterial strain of the application. Non-limiting examples of suitable growth media for said bacterial strain include yeast extract mannitol (YEM), yeast mannitol agar (YMA), yeast mannitol broth (YMB).

[0050] Other non-limited example of liquid carriers can include but are not limited to water, sugar wateralcohols, ketones, petroleum fractions, oils, aromatic or paraffinic hydrocarbons, chlorinated hydrocarbons, liquefied gases or a combination thereof. More particularly, the agriculturally compatible carrier can include a dispersant, a surfactant, an additive, a thickener, an anti-caking agent, residue breakdown, a composting formulation, a granular application, diatomaceous earth, a colouring agent, a stabilizer, a preservative, a polymer, a coating or a combination thereof.

[0051] The carrier can also be a slurry, optionally comprising a sticking agent capable of sticking the inoculum to the substrate of interest, for example to a plant seed. Non-limiting examples of sticking agents include alginate, mineral oil, syrup, gum arabic, honey, methyl cellulose, milk, wallpaper paste, and combinations thereof. One of the ordinary skills in the art can readily determine the appropriate carrier to be used taking into consideration factors such as a particular bacterial strain, plant to which the inoculum is to be applied, type of soil, climate conditions, whether the inoculum is in liquid, solid or powder form, and the like. The additive can comprise an oil, a gum, a resin, a clay, a polyoxyethylene glycol, a terpene, a viscid organic, a fatty acid ester, a sulfated alcohol, an alkyl sulfonate, a petroleum sulfonate, an alcohol sulfate, a sodium alkyl butane diamate, a polyester of sodium thiobutant dioate, a benzene acetonitrile derivative, a proteinaceous material, or a combination thereof. The proteinaceous material can include a milk product, wheat flour, soybean meal, blood, albumin, gelatine, or a combination thereof. The thickener can comprise a long chain alkylsulfonate of polyethylene glycol, polyoxyethylene oleate or a combination thereof. The surfactant can contain a heavy petroleum oil, a heavy petroleum distillate, a polyol fatty acid ester, a polyethoxylated fatty acid ester, an aryl alkyl polyoxyethylene glycol, an alkyl amine acetate, an alkyl aryl sulfonate, a polyhydric alcohol, an alkyl phosphate, or a combination thereof. The anti-caking agent can include a sodium salt such as a sodium sulfite, a sodium sulfate, a sodium salt of monomethyl naphthalene sulfonate, or a combination thereof, or a calcium salt such as calcium carbonate, diatomaceous earth, or a combination thereof. The agriculturally compatible carrier can also include a fertilizer, a micronutrient fertilizer material, an insecticide, a herbicide, a plant growth amendment, a fungicide, a molluscicide, an algicide, a bacterial inoculant, a fungal inoculant, or a combination thereof. Non-limiting examples are provided below. As way of example the bacterial strain of the current application may be mixed with an agriculturally compatible carrier. SoGo / BRADY-2 / 853

[0052] Non-limiting examples of the above provided composition in practice are soluble powders, wettable granules, dry flowables, aqueous flowables, wettable dispersible granules, emulsifiable concentrates, aqueous suspensions, a fertilizer granule, a sprayable formulation, an agrochemical formulation. Thus, in another embodiment, an agricultural composition comprising the bacterial strain of current application is provided. "Agricultural composition" as used herein refers to a composition for agricultural purposes. Given that the composition is of use to promote plant growth and development, more particularly to promote the quantitative and / or qualitative yield of a leguminous plant, even more particularly to promote nitrogen fixation of a leguminous plant, most particularly to increase the protein content of soybeans, also a plant growth promoting composition is provided. Plant growth promoting refers to a promoting effect on the growth and development of the cultured plant or crop. Said cultured plant or crop is the plant or crop of interest and does not include unwanted plants. As described above, the composition or "plant growth promoting composition" herein provided can include a herbicide, if said herbicide is used to remove unwanted plants or prevent germination of seeds of unwanted plants. The composition, agricultural composition or plant growth promoting composition can also comprise a fertilizer, a micronutrient fertilizer material, an insecticide, a plant growth amendment, a fungicide, a molluscicide, an algicide, a bacterial inoculant, a fungal inoculant, or a combination thereof. In some cases, the fertilizer is a liquid fertilizer. Liquid fertilizer can include without limitation, ammonium sulfate, ammonium nitrate, ammonium sulfate nitrate, ammonium chloride, ammonium bisulfate, ammonium polysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcitic limestone, calcium oxide, hampene (chelated iron), dolomitic limestone, hydrate lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, potassium nitrate, potassium bicarbonate, monopotassium phosphate, magnesium nitrate, magnesium sulfate, potassium sulfate, potassium chloride, sodium nitrates, magnesian limestone, magnesia, disodium dihydromolybdate, cobalt chlorid hexahydrate, nickel chloride hexahydrate, indole butyric acid, L- tryptophan, urea, urea-formaldehydes, urea ammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylidene diurea, K2SO4-2MgSO4, kainite, sylvinite, kieserite, Epsom salts, elemental sulfur, marl, ground oyster shells, fish meal, oil cakes, fish manure, blood meal, rock phosphate, super phosphates, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion or a combination thereof. The micronutrient fertilizer material can comprise boric acid, a borate, a boron frit, copper sulfate, a copper frit, a copper chelate, a sodium tetraborate decahydrate, an iron sulfate, an iron oxide, iron ammonium sulfate, an iron frit, an iron chelate, a manganese sulfate, a manganese oxide, a manganese chelate, a manganese chloride, a manganese frit, a sodium molybdate, molybdic acid, a zinc sulfate, a zinc oxide, a zinc SoGo / BRADY-2 / 853 carbonate, a zinc frit, zinc phosphate, a zinc chelate or a combination thereof. In a particular embodiment, said fertilizer or fertilizer material does not comprise insoluble selenium, selenium mineral, soluble selenium or salts thereof. The insecticide can include an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, a fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biologically-based insecticide, or a combination thereof. The herbicide can comprise a chlorophenoxy compound, a nitrophenolic compound, a nitrocresolic compound, a dipyridyl compound, an acetamide, an aliphatic acide, an anilide, a benzamide, a benzoic acid, a benzoic acid derivative, anisic acid, an anisic acid derivative, a benzonitrile, benzothiadiazinone dioxide, a thiocarbamate, a carmabate, carbanilate, chloropyridinyl, a cyclohexenone derivative, a dinitroaminobenzene derivative, a fluorodinitrotoluidine compound, isoxazolidinone, nicotinic acide, isopropylamine, an isopropulamine derivative, oxadiazolinone, a phosphate, a phthalate, a picolinic acid compound, a triazine, a triazole, a uracil, a urea derivative, endothall, sodium chlorate, or a combination thereof. The fungicide can comprise a substituted benzene, a thiocarbamate, an ethylene bis dithiocarbamate, a thiophthalidamide, a copper compound, an organomercury compound, an organotin compound, a cadmium compound, anilazine, benomyl, cyclohexamide, dodine, etridiazole, iprodione, metlaxyl, thiamimefon, triforine, or a combination thereof. The fungal inoculant can comprise a fungal noculant of the family Glomeraceae, a fungal inoculant of the family Claroidoglomeraceae, a fungal noculant of the family Acaulosporaceae, a fungal inoculant of the family Sacculospraceae, a fungal noculant of the family Entrophosporaceae, a fungal inoculant of the family Pacidsproraceae, a fungal noculant of the family Diversisporaceae, a fungal inoculant of the family Paraglomeraceae, a fungal noculant of the family Archaeosporaceae, a fungal inoculant of the family Geosiphonaceae, a fungal noculant of the family Ambisporacea, a fungal inoculant of the family Scutellosproaceae, a fungal noculant of the family Dentiscultataceae, a fungal inoculant of the family Racocetraceae, a fungal inoculant of the phylum Basidiomycota, a fungal inoculant of the phylum Ascomycota, a fungal inoculant of the phylum Zygomycota, a fungal inoculant of the genus Glomus or a combination thereof. The bacterial inoculant can include a bacterial inoculant of the genus Rhizobium, another bacterial inoculant of the genus Bradyrhizobium, bacterial inoculant of the genus Mesorhizobium, bacterial inoculant of the genus Azorhizobium, bacterial inoculant of the genus Allorhizobium, bacterial inoculant of the genus Burkholderia, bacterial inoculant of the genus Sinorhizobium, bacterial inoculant of the genus Kluyvera, bacterial inoculant of the genus Azotobacter, bacterial inoculant of the genus Pseudomonas, bacterial inoculant of the genus Azosprillium, bacterial inoculant of the genus Bacillus, bacterial inoculant of the genus Streptomyces, bacterial inoculant of the genus Paenibacillus, bacterial inoculant of the genus Paracoccus, bacterial inoculant of the genus Enterobacter, bacterial inoculant of the genus Alcaligenes, SoGo / BRADY-2 / 853 bacterial inoculant of the genus Mycobacterium, bacterial inoculant of the genus Trichoderma, bacterial inoculant of the genus Gliocladium, bacterial inoculant of the genus Klebsiella, or a combination thereof.

[0053] Also, the application provides a combination comprising a bacterial strain of current application and at least one microorganism selected from the list consisting of Bacillus subtilis strain 713, Bacillus amyloliquefaciens MBI 600, Bacillus pumillus QST2808, Pseudomonas fluorescens, Trichoderma vireus, Pseudomonas putida, Trichoderma harzianum Rifai strain T22, Penicillium bilaii, Mesorhizobium, Azospirillum, Azotobacter vinelandii and Clostridium pasteurianum.

[0054] In another embodiment, an agricultural or plant growth promoting composition comprising a bacterial strain of current application and an agriculturally compatible carrier is provided.

[0055] COATED SEEDS

[0056] In a next aspect, a plant seed or plant propagule coated with a microbial population comprising a bacterial strain of current application is provided. This is equivalent as saying that a plant seed or plant propagule is provided, wherein said plant seed or propagule having applied to the surface of said seed or of said propagule, a culture, an enriched culture or a biological pure culture of a bacterial strain of current application.

[0057] A "plant propagule" is any plant material for the purpose of plant propagation. Because of the totipotency of plants, any part of the plant may be used (e.g. a stem cutting, a leaf section, a portion of a root), though it is usually a highly meristematic part such as root and stem ends, buds, tubers, bulbs, rhizome, stolon or any plant part for vegetative reproduction. In sexual reproduction, a propagule is a seed or spore.

[0058] A "plant seed coated" or alternatively a "coated seed" as used in this application refers to a plant seed covered with a certain composition. This composition (i.e. the coating composition) can be a water composition or an oil composition or a polymer or any of the above described compositions comprising the bacterial strain of the application. "Coating" includes the simplest covering methods of dipping seeds or plant propagules in a microbial suspension or spraying seeds or propagules with a microbial suspension. In the latter case, the coating compositions are found to be film-forming, i.e. upon contacting with seeds or propagules they form a thin liquid film that adheres to the surface. "Coating" also includes rolling seeds / propagules in or dusting seeds / propagules with or brushing seeds / propagules with a powder comprising microorganisms, to more complex procedures as injecting plant seeds / propagules with a composition comprising microorganism or the use of complex coating layers including one or more adhesive, binder solvent and / or filler components. A person skilled in the art is familiar with a variety of conventional and more advanced methods to coat plants seeds (e.g. SoGo / BRADY-2 / 853

[0059] US5113619, EP0080999, WO1997036471, EP0010630, W02006131213, W02001045489, US4465017, EP2676536 which are here all incorporated as reference). The coating composition can include a number of ingredients, including but not limited to gelatin, a desiccant, water, tallow (e.g. to increase the release rate of any active ingredient in the composition), bulking agents (e.g. clay, vermiculite, perlite and / or bentonite to give more body to the liquid coating composition). Coating compositions which include bulking agents produce more rounded coated seeds. Such coated seeds are generally easier to plant when using mechanical planters. The concentration of the bulking agent can be up to about 50 % of the solids by volume. As way of example of a liquid coating procedure, seeds or propagules are fed into one or more tanks containing the liquid coating composition. The seeds or propagules are transported from the tanks into a drying zone where forced air dries and solidifies the coating applied to the seeds. The seeds or propagules are dipped at least once and preferably at least twice in the liquid coating composition of the present invention. The dried coated seeds or propagules can be sowed or planted using standard sowing or planting machinery or by hand. In the alternative, the coated seeds or propagules can be stored for later application. If the temperature and humidity are relatively high or if prolonged storage is contemplated, it is desirable to place on the surface of the coating an inert material, preferably a powder material, such as, chalk or talcum powder. Such inert material reduces the tendency for the seed to stick together or agglomerate. The coating should cover more than 50%, more than 60%, more than 70%, more than 80%, more than 90, more than 95% of the surface area of the seeds or propagules. In some embodiments, after the coating procedure, the seeds should comprise at least one living cell of an isolated bacterial strain of current application. The coating layer can also consist of one or more components. These components can be additional plant growth promoting microorganisms but can also be fertilizers, biocontrol agents, or pesticides including fungicides, insecticides and herbicides. Non-limiting examples of these components are provided above. The coating composition can also include protective colloids, adhesives, thickening agents, thixotropic agents, penetrating agents, stabilizing agents, sequestering agents, fertilizers, anti-freeze agents, repellents, color additives, corrosion inhibitors, water-repelling agents, siccatives, UV-stabilizers, pigments, dyes or polymers.

[0060] In another embodiment, when used as a seed treatment, the bacterial strain of current application is applied at a rate of about lxlO2to about lxlO11cfu / seed or at a rate of about lxlO3to about lxlO10cfu / seed or at a rate of at least lxlO2, at least lxlO3, at least lxlO4, at least lxlO5, at least 1x10s, at least lxlO7, at least 1x10s, at least lxlO9, at least lxlO10or at least lxlO11cfu / seed. In yet another embodiment, for coating purposes seeds are treated with a bacterial solution of at least lxlO5cfu of the bacterial strain of current application per ml, at least 1x10scfu of the bacterial strain of current application per ml, at least lxlO7cfu of the bacterial strain of current application per ml, at least 1x10s SoGo / BRADY-2 / 853 cfu of the bacterial strain of current application per ml, at least lxlO9cfu of the bacterial strain of current application per ml, at least lxlO10cfu of the bacterial strain of current application per ml or at least lxlO11cfu of the bacterial strain of current application per ml. After the coating procedure, the bacterial strain of current application is present on the seeds in a concentration of between lxlO4and lxlO7CFU, between lxlO5and 5x10sCFU per seed or at least lxlO5CFU, at least 1x10sCFU or at least lxlO7CFU per seed.

[0061] In a particular embodiment, a plant seed refers to a seed of a leguminous plant. A leguminous plant or alternatively phrased a legume is referred in current application as a plant from the family Fabaceae (or Leguminosae). When used as a dry grain, the seed is also called a pulse. Leguminous plants are grown agriculturally, primarily for human consumption, for livestock forage and silage, and as soil-enhancing green manure. Well-known leguminous plants include beans (Phaseolus), soybeans (Glycine max), broad beans (Vicia faba), peas (Pisum sativum), chickpeas (Cicer arietinum), bitter vetch (Vicia ervilia), peanuts (Arachis hypogaea), lentils (Lens culinaris), lupins (Lupinus), mesquite (Prosopis), carob (Ceratonia siligua), tamarind (Tamarindus indica), alfalfa (Medicagosativa), liquorice (Glycyrrhiza glabra) and clover (Trifolium sp.).

[0062] In another aspect, a method is provided of treating plant seeds, the method comprises the step of applying to said seeds an inoculum of a bacterial strain of the application. In one embodiment, said treating is coating. In another embodiment, said plant seeds are seeds from a leguminous plant, more particularly soybean.

[0063] APPLICATIONS

[0064] In a next aspect, the use of the Bradyrhizobium R-85129 strain of the application or of a microbial population comprising it or of any of the previously described cultures is provided to increase or improve plant yield, more particularly agricultural yield.

[0065] "Yield" as used herein, generally refers to a measurable product from a plant, and more particularly to the amount or quality of harvestable plant material or plant-derived product. "Yield" is normally defined as the measurable produce of economic value of a crop. For crop plants, "yield" also means the amount and / or quality of harvested material per hectare or unit of production. Yield may be defined in terms of quantity or quality. The harvested material may vary from crop to crop, for example, it may be seeds, above ground biomass, roots, fruits, fibres, any other part of the plant, or any plant-derived product which is of economic value. The term "yield" also encompasses yield potential, which is the maximum obtainable yield. Yield may be dependent on a number of yield components, which may be monitored SoGo / BRADY-2 / 853 by certain parameters. These parameters are well known to persons skilled in the art and vary from crop to crop. The yield can be determined using any convenient method, for example, kilograms of plant product produced per hectare of planting or bushels or pound of plant product produced per acre of planting. The term "yield" also encompasses harvest index, which is the ratio between the harvested biomass over the total amount of biomass. The harvest index is relatively stable under many environmental conditions, and so a robust correlation between plant size and yield is possible. Yield and yield increase (in comparison to a control plant) can be measured in a number of ways, and it is understood that a skilled person will be able to apply the correct meaning in view of the particular embodiments, the particular crop concerned and the specific purpose or application concerned. The terms "enhanced yield" or "improved yield" or "increased yield" can be used interchangeable. As used herein, the term "enhanced yield" means any statistically significant improvement of one or more yield parameters selected from the group consisting of biomass yield, dry biomass yield, aerial dry biomass yield, underground dry biomass yield, fresh-weight biomass yield, aerial fresh-weight biomass yield, underground fresh-weight biomass yield, enhanced yield of harvestable parts, either dry or fresh-weight or both, either aerial or underground or both, enhanced yield of seeds, either dry or fresh-weight or both, either aerial or underground or both, improved nutrient use efficiency, improved seed set and harvest, improved protein content per seed, increased stress tolerance., increased efficiency of nodulation and / or nitrogen fixation, increased efficiency of carbon assimilation, improvement of seedling vigour / early vigour and / or enhanced efficiency of germination (under stressed or non-stressed conditions).

[0066] For example, yield refers to biomass yield, e.g. to dry weight biomass yield and / or fresh-weight biomass yield. Biomass yield refers to the aerial or underground parts of a plant, depending on the specific circumstances (test conditions, specific crop of interest, application of interest, and the like). In one embodiment, biomass yield refers to the aerial and underground parts. Biomass yield may be calculated as fresh-weight, dry weight or a moisture adjusted basis. Biomass yield may be calculated on a per plant basis or in relation to a specific area (e.g. biomass yield per acre / square meter / or the like). "Yield" can also refer to seed yield which can be measured by one or more of the following parameters: number of seeds or number of filled seeds (per plant or per area (acre, square meter or the like); seed filling rate (ratio between number of filled seeds and total number of seeds); number of flowers per plant; seed biomass or total seeds weight (per plant or per area (acre, square meter or the like); thousand kernel weight (TKW; extrapolated from the number of filled seeds counted and their total weight; an increase in TKW may be caused by an increased seed size, an increased seed weight, an increased embryo size, and / or an increased endosperm) and protein content of the harvested seeds. Other parameters allowing to measure seed yield are also known in the art. Seed yield may be determined on a dry weight or on a SoGo / BRADY-2 / 853 fresh weight basis, or typically on a moisture adjusted basis, e.g. at 15.5 % moisture. For example, the term "increased yield" means that a plant, exhibits an increased growth rate, e.g. in the absence or presence of abiotic environmental stress, compared to the corresponding wild-type plant. An increased growth rate may be reflected inter alia by or confers an increased biomass production of the whole plant, or an increased biomass production of the aerial parts of a plant, or by an increased biomass production of the underground parts of a plant, or by an increased biomass production of parts of a plant, like stems, leaves, blossoms, fruits, and / or seeds. A prolonged growth comprises survival and / or continued growth of the plant, at the moment when the untreated control plant shows visual symptoms of deficiency and / or death.

[0067] In accordance with the invention, changes in different phenotypic traits may improve yield. For example, and without limitation, parameters such as floral organ development, seed number, seed weight, protein content of the seed, root initiation, root biomass, harvest index, leaf formation, phototropism, apical dominance, and fruit development, are suitable measurements of improved yield. Increased yield includes higher seed yields, higher protein content of the seed, higher fresh matter production, and / or higher dry matter production. Any increase in yield is an improved yield in accordance with the invention. For example, the improvement in yield can comprise a 0.1%, 0.2%, 0.5%, 0.8%, 1%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater increase in any measured parameter compared to a mock situation. For example, an increase in the seed / acre yield of soy treated with the bacterial strain of the invention, as compared with the seed / acre yield from untreated soy cultivated under the same conditions, is an improved yield in accordance with the invention. Also an increased protein content / seed of soy treated with the bacterial strain of the invention, as compared with the protein content / seed from untreated soy cultivated under the same conditions, is an improved yield in accordance with the invention.

[0068] The yield of a plant can depend on the specific plant or crop of interest as well as its intended application (such as food production, feed production, processed food production, biofuel, biogas or alcohol production, or the like) of interest in each particular case. In one embodiment, yield can be calculated as harvest index (see definition above), harvestable parts weight per area (acre, square meter, or the like); and the like. Measurements of plant size in early development, under standardized conditions in a growth chamber or greenhouse, are standard practices to measure potential yield advantages conferred by the presence of plant growth promoting or nitrogen fixing bacteria.

[0069] When the plant treated with a bacterial strain of the application is a leguminous plant, increased yield means, in one embodiment, increased seed yield. Increased seed yield refers to a statistically significant increase or an at least 0.1%, 0.2%, 0.5%, 0.8%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, SoGo / BRADY-2 / 853

[0070] 70%, 80%, 90% or more increase compared to an untreated but identical leguminous plant. In particular, increased yield for soy plants means increased seed yield, in particular for soy varieties used for feed or food. Non-limiting examples of soy varieties are Primus, Shouna, Bettina, Amarok, Lenka, Artemis and Hermes.

[0071] Increased seed yield of soy refers in one embodiment to an increased seed size or weight, an increased number of seeds per pod, an increased number of pods per plant, improvement of seed composition or an increased protein content of the seeds. In a particular embodiment, increased protein content means obtaining a protein content of between 35 and 48%, of between 39 and 45%, between 40 and 44%, between 41 and 43% or of least 42% or of least 43% or of least 44%. The protein content of seeds, more particularly of soybeans can be measured by several methods known by the skilled person. The classical method for whole seed protein determination is the complete extraction of whole seed protein followed by nitrogen determination using either the Kjeldahl method (ISO 5683-2) or the Bradford assay based on a protein / nitrogen ratio of 6.25 (e.g. Yu et al 2016 Food Chem 196; James and Aijun et al 2016 Food Chem 194). Hence, the protein content can be calculated according to the formula: protein content = nitrogen content x 6.25. Another non-limiting example of protein measurement is the combination of near infrared (NIR) spectroscopy and the Kjeldahl method. In a nutshell, dry seed material from a collection of samples is first analysed by NIR spectroscopy. Next, in order to calibrate the NIR spectroscopy data, protein content of a selection (e.g. 10%) of the samples covering the spectral variation of the complete set of samples is determined using the Kjeldahl method. Finally, based on the calibrated NIR spectroscopy data the protein content of all samples can be determined (Pannecoucque et al 2018 Eur J Agron 132; Pannecoucque et al 2018 J Agr Sci 156). In a particular embodiment of the invention, the protein content percentages herein disclosed are determined based on the Kjeldahl method (ISO 5683-2).

[0072] In another embodiment, increased seed yield of soy refers to an increased harvest of seeds per area, such as acre or hectare (ha). In a particular embodiment, increased seed yield means obtaining a seed yield of between 3.5 and 6 ton / ha, between 4 and 5.5 ton / ha or between 4.5 and 5 ton / ha or of at least 4 ton / ha, at least 4.5 ton / ha, at least 5 ton / h or of at least 5.5 ton / ha.

[0073] The above described increased or improved yield can be achieved in the absence or presence of stress conditions. The bacterial strain of the application is also provided to be of use to improve the adaptation of plants, more particular leguminous plants to cool growing conditions. More particularly to be of use to increase nitrogen fixation of the treated plant, more particularly leguminous plant in cool growing temperatures. How to measure nitrogen fixation in a plant is known by the person skilled in the art, e.g. SoGo / BRADY-2 / 853 as explained in Pannecoucque et al 2018 (Eur J Agron 132). In short, nitrogen (N) derived from the air is measured based on the different ratios of the stable nitrogen isotopes15N:14N in air and soil respectively. In yet another embodiment, the use of a bacterial strain of the application is provided to increase cold tolerance of a plant. This solution is of great agricultural importance as low temperatures often significantly affect plant growth and crop productivity with crop losses as result (Xin and Browse 2001 Plant Cell Environ 23:893-902). Cold tolerance in plants is a very complex trait, involving many different metabolic pathways and cell compartments. Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Plants differ in their tolerance to cold or chilling (0-17°C) and freezing (< 0°C) temperatures. Plants of tropical and subtropical origins (e.g. soy) are highly sensitive to cold or chilling stress and are injured or killed by non-freezing low temperatures or have a reduced nitrogen fixing capacity. They exhibit various symptoms of chilling injury such as chlorosis, necrosis, or growth retardation. In contrast, plants from temperate climatic regions can be cold or chilling tolerant with variable degree and can be able to grow at such non-freezing cold temperatures.

[0074] "Cold tolerance" or equivalently "chilling tolerance" or "low temperature tolerance" as used in current application is defined as the ability of a plant to tolerate low temperatures without or with limited injury, damage or yield drop, wherein said low temperatures are non-freezing temperatures. In one embodiment said low temperatures are temperatures between 5 and 20°C or between 8 and 18°C or between 10 and 15°C. In one embodiment, these temperatures are the temperatures of the soil or plant growth medium. Plants or plant roots are exposes to said low temperatures for at least 2h, at least 4h, at least 6h or at least 8h per day or said low temperatures are reached during at least a part of the day, for example during the night.

[0075] In particular embodiments, cold tolerance observed in plants that were treated with or were grown from seeds coated with the bacterial strain of current application leads to injury, damage or a drop in yield or nitrogen fixation due to low temperatures which is at least 10%, least 20%, least 30%, least 40%, least 50%, least 60%, least 70%, least 80%, least 90% or 100% less than the injury, damage or a drop in yield or nitrogen fixation observed in plants that were not treated with or were grown from seeds not coated with the bacterial strain of current application.

[0076] In particular embodiments, the use of a bacterial strain of the application is provided to increase tolerance to non-freezing low temperatures in plants, more particularly leguminous plants, wherein said low temperatures are between 5 and 20°C or between 8 and 18°C or between 10 and 15°C. More particularly, increased tolerance to said non-freezing low temperatures in leguminous plants means increased tolerance of nitrogen fixation to said non-freezing low temperatures in leguminous plants, more particularly in soybean. SoGo / BRADY-2 / 853

[0077] In a next aspect, a method is provided for enhancing growth, yield and / or cold tolerance of a plant comprising inoculating a plant growth medium with a microbial population, wherein said population comprises the bacterial strain of current application; and growing a plant in said plant growth medium; to enhance growth, yield and / or cold tolerance of said plant. In one embodiment, said yield is seed yield. In another embodiment, said enhancing yield is enhancing the protein content of seeds, more particularly of seeds of a leguminous plant. In one particular embodiment, said cold tolerance is cold tolerance of nitrogen fixation. Also provided is a method for enhancing nodulation or enhancing nitrogen fixation of a leguminous plant comprising inoculating a plant growth medium with a microbial population, wherein said population comprises the bacterial strain of current application and growing a leguminous plant in said plant growth medium to enhance nitrogen fixation of said plant. In one embodiment, said bacterial strains of current application are Bradyrhizobium sp. R-85129 with deposit number LMG P-33693. In another embodiment, said leguminous plant is soybean.

[0078] The term "enhancing nodulation" is defined herein as a statistically significant increase and / or an at least 5%, 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, 100% or more increase in the number of nodules per root system, more particular per cm root system or per gram fresh or dry weight of the root system.

[0079] The term "inoculating" as used herein refers to introducing at least one bacterium into a plant growth medium. By way of example and without the intention to be limiting, said introduction can be performed using a liquid, a powder, a granule, a pellet. "Plant growth medium" is defined as any environment wherein plants can grow. Non-limiting examples of a plant growth medium are soil, sand, gravel, a polysaccharide, mulch, compost, peat moss, straw, logs, clay, or a combination thereof. A plant growth medium can also include a hydroculture system or an in vitro culture system. Hydroculture is the growing of plants in a soilless medium or an aquatic based environment, while an in vitro culture system refers to the growing of plants or explants on or in a recipient with synthetic medium, in sterile conditions, in a controlled environment and in reduced space. Explants refer to parts of a plant, from all the aerial part to isolated cells, as parts of leaves, of roots, seeds, bulbs, tubers, buds. The inoculation of said plant growth medium with a microbial population can be done before, during and / or after sowing or before, during and / or after the start of the plant growth cycle in case of hydroculture or in vitro culture. The inoculation can be performed once or multiple times during the plant growth cycle. In one embodiment, the microbial population is applied to the plant growth medium as a powder, as a pellet, as a granule or as a liquid.

[0080] The term "plant" as used herein encompasses whole plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), bulbs, buds, flowers, and tissues and organs. The term "plant" SoGo / BRADY-2 / 853 also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores. Thus, in one embodiment, a method is provided for stimulating plant growth or yield comprising applying the microbial culture comprising the bacterial strain of current application to a plant, plant part, plant seed or to the plant growth medium. Unless otherwise specified, in the latter and further embodiments and aspects, "stimulating", "enhancing", "increasing" or "improving" refers to a statistically significant increase and / or an at least 5% increase or at least 6% increase or at least 7% increase or at least 8% increase or at least 9% increase or at least 10% increase or at least 12% increase or at least 15% increase or at least 20% increase or at least 25% increase or at least 30% increase or at least 50% increase or at least 75% increase or at least a 100% increase in the property being measured (e.g. plant growth, plant yield, nitrogen fixation) and compared to a mock or control situation.

[0081] Plants that are particularly useful in the methods of current application include in particular nitrogen fixing plants or leguminous plants. Non-limiting examples of leguminous plants are acacia (genus Acacia), alfalfa (Medicago sativa), almendro (Dipteryx oleifera), bean (genus Phaseolus), common bean (P. vulgaris), green bean (P. vulgaris), lima bean (P. lunatus), scarlet runner bean (P. coccineus), bird's-foot trefoil (Lotus corniculatus), bush clover (genus Lespedeza), broom (genus Cytisus), carob (Ceratonia siligua), chickpea (Cicer arietinum), clover (genus Trifolium), cowpea (Vigna unguiculata), crown vetch (Securigera varia), fenugreek (Trigonella foenum-graecum), honey locust (Gleditsia species), hyacinth bean (Lablab purpureus), indigo (genus Indigofera), jicama (Pachyrhizus erosus), kakabeak (genus Clianthus), Kentucky coffee tree (Gymnocladus dioica), kidney vetch (Anthyllis vulneraria), kudzu vine (Pueraria montana), laburnum (genus Laburnum), golden chain (L. anagyroides), genus Lathyrus, beach pea (L. japonicus), sweet pea (L. odoratus), lentil (Lens culinaris), licorice (Glycyrrhiza glabra), locoweed (Astragalus and Oxytropis species), locust (genus Robinia), logwood (Haematoxylum campechianum), lupine (genus Lupinus), Texas bluebonnet (L. texensis and L. subcarnosus), mesquite (genus Prosopis), mimosa (genus Mimosa), sensitive plant (M. pudica), narra (Pterocarpus species), pagoda tree (Styphnolobium japonicum), palo verde (genus Parkinsonia), pea (Pisum sativum), peanut (Arachis hypogaea), redbud (genus Cercis), rosary pea (Abrus precatorius), royal poinciana (Delonix regia), senna (genus Senna), silk tree (genus Albizia), smoke tree (Dalea spinosa), soybean (Glycine max), suicide tree (Tachigali versicolor), sunn hemp (Crotalaria juncea), tamarind (Tamarindus indica), vetch (genus Vicia), broad bean (V. faba), wisteria (genus Wisteria). In a particular embodiment, said leguminous plant is selected from the list consisting of alfalfa (Medicago sativa), bean (genus Phaseolus), common bean (P. vulgaris), green bean (P. vulgaris), lima bean (P. lunatus), scarlet runner bean (P. coccineus), chickpea (Cicer arietinum), clover (genus Trifolium), cowpea (Vigna unguiculata), fenugreek (Trigonella foenum- graecum), genus Lathyrus, beach pea (L. japonicus), sweet pea (L. odoratus), lentil (Lens culinaris), SoGo / BRADY-2 / 853 licorice (Glycyrrhiza glabra), pea (Pisum sativum), peanut (Arachis hypogaea), soybean (Glycine max), tamarind (Tamarindus indica), vetch (genus Vicia) and broad bean (V. faba). In a most particular embodiment, said leguminous plant is soybean (Glycine max), bean (Phaseolus sp.), lentils (Lens culinaris), chickpea (Cicer arietinum), clover (genus Trifolium), cowpea (Vigna unguiculata) or Lathyrus.

[0082] In another aspect, a method for enhancing growth, yield and / or cold tolerance of a plant is provided, wherein said method comprises growing coated seeds of a plant, wherein said seeds are coated with a microbial population comprising an isolated bacterial strain of current application, to obtain enhanced growth, yield and / or cold tolerance of said plant. In one embodiment, said yield is seed yield or amount of seeds (e.g. expressed in tons) per acre or hectares. In another embodiment, said enhancing yield is enhancing the protein content of seeds, more particularly of seeds of a leguminous plant. Also provided is a method for enhancing nodulation or enhancing nitrogen fixation of a leguminous plant, wherein said method comprises growing coated seeds of said plant, wherein said seeds are coated with a microbial population comprising an isolated bacterial strain of current application. In some embodiments, after the coating procedure, the seeds should comprise at least lxlO4, lxlO5or 1x10sliving cells or spores of a bacterial strain of current application.

[0083] An "effective amount" refers to an amount sufficient to effect beneficial or desired results. In a nonlimiting example, an "effective amount" leads to a statistically significant increase of plant growth and / or biomass and / or yield and / or cold tolerance and / or protein content of seed and / or nitrogen fixation as compared to the growth, biomass and / or yield and / or cold tolerance and / or protein content of seed and / or nitrogen fixation of the control plant. An effective amount can be administered in one or more administrations. A "control plant" as used in current application provides a reference point for measuring changes in phenotype of the subject plant and may be any suitable plant cell, seed, plant component, plant tissue, plant organ or whole plant. A control plant may comprise for example a plant or cell which is genetically identical to the subject plant or cell but which is not exposed to the same treatment (e.g. administration of the bacterial strain of current application) as the subject plant or cell.

[0084] In another embodiment, a method is provided for enhancing nutrient uptake and / or nutrient use efficiency of a plant, said method comprising growing coated seeds of a plant, wherein said seeds are coated with a microbial population comprising the bacterial strain of current application, to obtain enhanced nutrient uptake and / or nutrient use efficiency of said plant. In another embodiment, a method is provided for enhancing nodulation or enhancing the nitrogen fixating capacity of a plant, said method comprising growing coated seeds of a plant, wherein said seeds are coated with a microbial population SoGo / BRADY-2 / 853 comprising a bacterial strain of current application, to obtain enhanced nodulation or enhanced a nitrogen fixating capacity of said plant.

[0085] In yet another aspect, a method for enhancing growth, yield and / or cold tolerance of a plant is provided comprising: growing a plant in an environment that supports plant growth; and administering a sprayable formulation to said environment or to said plant, said formulation comprising a bacterial strain of current application; to obtain enhanced growth, yield and / or cold tolerance of said plant.

[0086] In one embodiment, said yield is seed yield. In another embodiment, said enhancing yield is enhancing the protein content of seeds of said plant, more particularly a leguminous plant. Also a method for enhancing nodulation or enhancing nitrogen fixation of a leguminous plant is provided, comprising the steps of growing said plant in an environment that supports plant growth and administering a sprayable formulation to said environment or to said plant, said formulation comprising a bacterial strain of current application.

[0087] A "sprayable formulation" as used herein is an agrochemical or a biological solution that can be sprinkled on a plant or soil. The formulation is composed in such a way that the active ingredients can be absorbed by the above-ground tissue of a plant or is available for the plant roots when administered to the soil. The above disclosed methods thus also includes irrigation with a liquid comprising the bacterial strain of current application. "Irrigating" or "irrigation" as used herein refers to the method in which water or other liquids are supplied to plants at regular intervals. Irrigation includes but is not limited to "localized irrigation" (i.e. a system where water is distributed under pressure through a piped network, in a predetermined pattern, and applied as a small discharge to each plant or adjacent to it. "Drip (or micro) irrigation", also known as "trickle irrigation" (i.e. a system where water falls drop by drop just at the position of roots or near the root zone of plants) and "sprinkler irrigation" (i.e. a system where water is distributed by overhead sprinklers) belong to this category of irrigation methods. In "sprinkler irrigation", sprinklers can also be mounted on moving platforms connected to the water source by a hose. Automatically moving wheeled systems known as traveling sprinklers may irrigate areas such as small farms, sports fields, parks and pastures unattended. Most of these utilize a length of polyethylene tubing wound on a steel drum. As the tubing is wound on the drum powered by the irrigation water or a small gas engine, the sprinkler is pulled across the field. When the sprinkler arrives back at the reel the system shuts off. This type of system is known to most people as a "waterreel" traveling irrigation sprinkler. SoGo / BRADY-2 / 853

[0088] Hence, in various embodiments, a method is provided for enhancing growth, yield and / or cold tolerance of a plant, said method comprising: growing said plant in an environment that supports plant growth; irrigating said environment using a liquid solution comprising a bacterial strain of current application; to obtain enhanced growth, yield and / or cold tolerance of said plant.

[0089] In one embodiment, said yield is seed yield. In another embodiment, enhancing yield is enhancing the protein content of seeds of said plant, more particularly the seeds of a leguminous plant. Also provided is a method to increase nodulation or the nitrogen fixation of a leguminous plant comprising the steps of growing said plant in an environment that supports plant growth and irrigating said environment using a liquid solution comprising a bacterial strain of current application.

[0090] In particular embodiments, when used as a soil treatment, a bacterial strain of current application can be applied as a soil surface drench, injected and / or applied in-furrow or by mixture with irrigation water. The rate of application for drench soil treatments, which may be applied at planting, during or after seeding, or after transplanting and at any stage of plant growth, is about lxlO11to about 8xl012cfu per acre. In some embodiments, the rate of application is about lxlO12to about 8xl012cfu per acre. The rate of application for in-furrow treatments, applied at planting, is about 2.5xlO10to about SxlO11cfu per 1000 row feet. In some embodiments, the rate of application is about 6xlO10to about 4xlOncfu per 1000 row feet. Those of skill in the art will understand how to adjust rates for broadcast treatments (where applications are at a lower rate but made more often) and other less common soil treatments.

[0091] In some embodiments, when a bacterial strain of current application is applied as microbial population or bacterial population or solution or culture or agricultural composition or sprayable formulation, the number of colony forming units (cfu) per milliliter (ml) of said bacterial strain of current application in the microbial populations or bacterial populations or solutions or cultures or agricultural compositions or sprayable formulations will be at least 1x10scfu / ml or at least lxlO7cfu / ml or at least 1x10scfu / ml or at least lxlO9cfu / ml or at least 2xl09cfu / ml or at least 3xl09cfu / ml or at least 4xl09cfu / ml or at least 5xl09cfu / ml or at least 6xl09cfu / ml or at least 7xl09cfu / ml or at least 8xl09cfu / ml or at least 9xl09cfu / ml or at least lxlO10cfu / ml or at least 2xl010cfu / ml or at least 3xlO10cfu / ml or at least 4xlO10cfu / ml or at least 5xlO10cfu / ml or at least 6xlO10cfu / ml or at least 7xlO10cfu / ml or at least 8xlO10cfu / ml or at least 9xlO10cfu / ml or at least lxlO11cfu / ml or at least 2xl0ncfu / ml or at least 3X1011cfu / ml or at least 4X1011cfu / ml or at least SxlO11cfu / ml or at least SxlO11cfu / ml or at least 7xlOncfu / ml or at least SxlO11cfu / ml or at least 9xlOncfu / ml or at least lxlO12cfu / ml or at least lxlO13cfu / ml or at least lxlO14cfu / ml. SoGo / BRADY-2 / 853

[0092] Additional to the above detailed description of the invention, terminology as used in describing the aspects of the invention is described in the following sections.

[0093] In all herein described aspects and embodiments - unless specified differently - "enhance" or "increase" or "improvement" refers to a statistically significant increase and / or an at least 1%, 2%, 3%, 4% or 5% increase or at least 6% increase or at least 7% increase or at least 8% increase or at least 9% increase or at least 10% increase or at least 15% increase or at least 20% increase or at least 25% increase or at least 30% increase or at least 50% increase or at least 75% increase or at least a 100% increase in the property being measured and compared to a control situation. Said control situation is a mock situation wherein the plant, plant seed or other plant part was not treated with the microbial population or bacterial strain herein disclosed. The skilled person is aware how a scientifically sound mock situation should be set up. "Treated" as used herein can be direct treatment (e.g. coating seeds or spraying plants) and / or indirect treatment (e.g. providing the substrate wherein the plant is growing with a bacterial population).

[0094] The term "statistically significant" or "statistically significantly" different is well known by the person skilled in the art. Statistical significance plays a pivotal role in statistical hypothesis testing. It is used to determine whether the null hypothesis should be rejected or retained. It states that the results are obtained because of chance and are not supporting a real change or difference between two data sets. The null hypothesis is the default assumption that what one is trying to prove did not happen. In contrast the alternative hypotheses states that the obtained results support the theory being investigated. For the null hypothesis to be rejected (and thus the alternative hypothesis to be accepted), an observed result has to be statistically significant, i.e. the observed p-value is less than the pre-specified significance level a. The p stands for probability and measures how likely it is that the null hypothesis is incorrectly rejected and thus that any observed difference between data sets is purely due to chance. In most cases the significance level a is set at 0.05.

[0095] "Microbial" as used herein refers to microorganisms, wherein said microorganisms can include bacteria, archaebacteria, fungi, yeasts, mycorrhiza, microscopic eukaryotes (e.g. protozoa and algae), viruses, viroids or a combination thereof. A "microbial population" as used herein can thus refer to a synthetic or artificial collection of different microorganisms with distinct geographical origins. In various more particular embodiments of this application, "microbial" refers to "bacterial".

[0096] For the purpose of current application, the term "bacterium" or "bacteria" includes any prokaryotic organism that does not have a distinct nucleus. While being both part of the group of microorganisms, SoGo / BRADY-2 / 853 bacteria and fungi are clearly distinct. The term "fungi" or "fungus" includes a wide variety of nucleated spore-bearing organisms that are devoid of chlorophyll.

[0097] In order to reconstruct the evolutionary relationships and sequence identity of one bacterial isolate to another, phylogenetic approaches are used standardly exploiting the 16S rRNA sequence or a portion of the 16S rRNA sequence of the bacteria, although any other sequence or the entire genome of the microorganisms to be analyzed can also be used. In microbiology, "16S rRNA sequence" refers to the sequence derived by characterizing the nucleotides that comprise the 16S ribosomal RNA gene(s). The bacterial 16S rRNA is approximately 1500 nucleotides in length.

[0098] In this application "sequence similarity", "sequence identity" and "sequence homology" are interchangeably used. The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. A gap, i.e., a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. Determining the percentage of sequence identity can be done manually, or by making use of computer programs that are available in the art. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al. J. Mol. Biol. 215: 403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0099] The term "plant growth promoting" as used herein, refers to a promoting effect on a wide range of growth and development properties of cultured plants or crops, including but not limited to increased root development, increased leaf area, increased plant yield, increased fresh or dry weight, increased seed yield, increased seed germination, increased photosynthesis, increase in accumulated biomass of the plant, increased nitrogen fixation or increased efficiency of nutrients such as nitrogen, phosphorus or potassium.

[0100] "CFU" or "cfu" as used herein refers to colony-forming unit. This unit is well-known by the person skilled in the art of microbiology (as well as the methodology how to determine the number of colony-forming units) and is used to estimate the number of viable bacteria or fungal cells in a sample. "Viable" is defined SoGo / BRADY-2 / 853 as the ability to multiply via binary fission under controlled conditions. Counting with colony-forming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all cells, living or dead.

[0101] The present invention is described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are nonlimiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0102] The terms or definitions provided herein are solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Michael R. Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0103] It is to be understood that although particular embodiments, specific configurations as well as materials and / or molecules, have been discussed herein for cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The Examples described below are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims. 1 SoGo / BRADY-2 / 853

[0104] EXAMPLES

[0105] 1.1200 gardens with diverse soil types delivered 267 nodules

[0106] The 'Soy in 1000 Gardens' project comprised a pipeline to trap bacteria that nodulate locally grown soybean combined with additional analyses to investigate the biotic and abiotic bulk soil characteristics that influence nodulation success19. At the start of the citizen science experiment, soil samples were collected to characterize the garden soils. Analysis of physicochemical soil characteristics (PSC), phospholipid fatty acid (PLFA) analysis, bacterial 16S rRNA gene sequencing, and fungal internal transcribed spacer (ITS) sequencing analyses were performed on soil samples from each garden to extract information on the soil texture and nutrient content, microbial biomass, and bacterial and fungal community structure. Soil textures ranged from sandy soil to loam and clay. Nitrate levels were generally low, while the phosphorus levels were higher. The lower nitrate levels confirmed our assumption that the citizen's gardens are N-poor and thus probably conducive to nodulation. The bulk soil 16S microbiome characterization showed that the relative abundance of the known soybean-nodulating Bradyrhizobium genus was generally low with a mean value of 0.019%.

[0107] After the initial sampling, citizen scientists reported on soybean growth and yield and, in total, returned 4436 plants from 1093 gardens back to the laboratory. Of these plants, 1004, coming out of 386 gardens, exhibited root nodules or nodule-like structures. We found 34 nodules with a red interior originating from 27 gardens, 133 white nodules coming from 80 gardens and 100 brown (necrotic) nodules coming from 52 gardens. The 737 remaining nodules were too small to determine the colour and were classified as nodule-like structures or nodules of 'unknown' colour.

[0108] 2.lsolation and identification of indigenous rhizobial bacteria from soybean nodules

[0109] In parallel to the nodule microbiome analysis, putative nitrogen-fixing bacteria were isolated from the nodules. Initially, 954 strains were retrieved, of which 392 isolates were unique after dereplication with MALDI-TOF MS. These strains originated from 186 nodules with distinct color phenotypes (32 red, 54 brown, 87 white and 13 of unknown color), obtained from 134 plants grown in 93 gardens. 60 genera were represented, of which the richest group was Bacillus sp., with 195 isolated strains, whereas in the microbiome this genus was present in low abundance. Meanwhile, genera that accounted for more than 50% of the rel. ab. in the nodule microbiome such as Bradyrhizobium, Rhizobium or Tardiphaga had less representatives during the isolation. Nonetheless, 17 Bradyrhizobium strains, belonging to ASV2 and ASV15, seven Rhizobium sp. (ASV4 and ASV109) and 26 isolates belonging to the genus Tardiphaga (ASV1) were obtained. Expectedly, most of these isolates originated from red, white, and brown nodules where there was a dominant Bradyrhizobium, Tardiphaga or Rhizobium ASV. SoGo / BRADY-2 / 853

[0110] 3.Nodulation capacity of selected strains

[0111] To identify the most promising nodulators among the isolated strains, we selected strains based on their ability to induce nodulation on a soybean host in a series of experiments presenting a consecutively more physiologically relevant environment.

[0112] First, we selected 39 native strains belonging to the genus Bradyrhizobium, Rhizobium or Tardiphaga and assessed their nodulation capacity in pots filled with sterile substrate (Fig. 1B,C and Fig. 2). While all tested strains could induce nodulation, most could only induce small white nodules (Extended Fig. 5). Only eight strains, belonging to the genera Bradyrhizobium or Rhizobium, consistently produced red nodules (Fig. 1 B and C and Fig. 2). For five Bradyrhizobium strains (521_C7_N1.3, 590_E9_N4.2, 1200_B8_Nl.l, 1200_D9_Nl.l and 1200_D9_N1.2) we demonstrated formation of red nodules in a quantity at least as high as the commercially used B. diazoefficiens strain G49, and these strains were taken along for further testing (Fig. 1C). A sixth strain, 604_D8_N2.3, is also tested here as a control for later analyses, as it displayed a significantly lower capacity to induce red nodules (Fig. 1C). Correspondingly, plants inoculated with strain 604_D8_N2.3 appeared shorter and less green, when compared to the other five strains (Fig. IB).

[0113] Also in pots filled with non-sterile agricultural soil and for each of five tested soybean varieties, all five tested Bradyrhizobium strains consistently induced red nodules, with nodule numbers and nodule dry weight reaching levels similar to plants inoculated with G49, or even higher as in the case of 1200_B8_N1.2 in terms of nodule dry weight (Fig. ID and Fig. 4). Nodule numbers and strain performance was not dependent on the soybean variety, and interestingly, tested strains that could induce small white nodules in sterile substrate were no longer able to do so in agricultural soil (Fig. 4). The five native strains all displayed similar nitrogen fixation efficiency compared to G49, as assessed in an acetylene reduction assay (ARA) (Fig. 3).

[0114] Finally, nodulation efficacy of two of the native strains (590_E5_N4.2 and 1200_B8_N1.2) was assessed in two field trials at different sites, Merelbeke and Bottelare, using two soybean varieties currently grown in Western-Europe, i.e. Glycine max cv. Lenka (Prograin, Canada) and RGT Shouna (RAGT, France) (Fig. IE and Fig. 5). In both field trials, no nodulation was observed on non-inoculated soybean. Moreover, the isolated strains induced significantly more nodules than G49, reflected in a significantly higher nodule dry weight. Again, the effects on nodulation were independent of the soybean variety.

[0115] In Merelbeke, the efficient nodulation appeared to correlate with a darker green and more extensive foliage, as well as an increased grain yield and protein content (Fig. IE and Fig. 5). Indeed, when compared to non-inoculated plants, inoculation with G49 resulted in an increased average grain yield SoGo / BRADY-2 / 853

[0116] (2.93 t / ha to 3.14 t / ha), though this was not statistically significant, and a significantly increased protein content (33.3% to 36.1%). Interestingly, inoculation with 590_E5_N4.2 (3.80 t / ha, 38.4%) or 1200_B8_N1.2 (3.45 t / ha, 37.5%) could increase both yield and protein content even more, though the difference with G49 was only statistically significant for 590_E5_N4.2. In Bottelare, a lower yield (2.17 t / ha) was obtained in non-inoculated plants, and inoculation with G49 or any of the native strains did not result in a significant increase in either yield or protein content.

[0117] We can conclude that despite the variable effects of nodulation on soybean yield in the two field trials, the two newly isolated strains that were tested performed at least as well as G49 in the field, and might even be able to outperform the commercial inoculant in the Merelbeke field trial in terms of both yield and protein content.

[0118] 4.Characterization of the phylogeny and presence of the symbiotic genes in the candidate soybean nodulators

[0119] Whole-genome sequencing analysis was performed on all the Bradyrhizobium, Rhizobium and Tardiphaga strains that were tested for their nodulation capacity (Fig. 1A). The aim was to evaluate the phylogenetic relationships between these isolates and assess the presence of genes involved in the symbiosis, to provide a genetic basis for the performances of these native strains. The genomes of two commercially used Bradyrhizobium strains, G49 and 532C, were included to compare them with our isolates. The assessed genes were subdivided into categories based on their function: nitrogenase synthesis (nif genes), nodulation factor synthesis and transport (nod genes), symbiosis-specific respiration (fix genes), and genes encoding components of the type III secretion systems (T3SS).

[0120] Most of the strains only contained genes involved in respiration under microaerobic conditions (fix), and either belonged to the species B. baranii, T. robiniae, R. lusitanum, or did not belong to any described species (Table 1). A few strains, including B. baranii 604_D8_N2_3, R. leguminosarum 862_C5_N1_2, 814_E9_N1_1 and 62_C5_N11_2 and R. redzepovicii 969_B3_N1, presented most of the main genes involved in symbiosis but lacked the T3SS (Table 1). For strain 604_D8_N2_3, this might explain its inability to efficiently form red nodules (Fig. 1C). Conversely, the Bradyrhizobium strains that appeared to perform well in terms of induction of nodulation, including the commercial strains, presented all the main genes involved in symbiosis (nif, nod, and fix) as well as the T3SS . These strains belonged either to the species B. diazoefficiens (G49, 590_E5_N4_2, 1200_D9_Nl_l, 1200_D9_Nl_2, and 1200_B8_Nl_2) or the species B. japonicum (532C, 521_C7_N1_3) (Table 1 ). Interestingly, 590_E5_N4_2 and 1200_D9_Nl_2 shared a high genetic similarity (ANI > 99%) with G49 (Table 1). SoGo / BRADY-2 / 853

[0121] 5. R-85129 is a novel strain

[0122] BLAST analyses based on the 16S rRNA sequence (depicted SEQ ID NO: 1) of Bradyrhizobium sp. R85129 (also designated herein as 521_C7_N1.3) revealed 100% similarity with Bradyrhizobium japonicum strains S04E-Bj and Bradyrhizobium diazoefficiens strain 41.

[0123] When the whole genome of R-85129 was compared with the Bradyrhizobium japonicum USDA110 strain the obtained ANI value was 89.76%.

[0124] When the whole genome of R-85129 was compared with the Bradyrhizobium japonicum USDA6 strain the obtained ANI value was 96.93%. We therefore conclude that the R-85129 strain is a Bradyrhizobium japonicum species since the threshold normally used for a species is 95%.

[0125] SEQ ID NO: 1

[0126] 16S rRNA sequence of strain R-85129

[0127] GTCCAACTTGAGAGTTTGATCCTGGCTCAGAGCGAACGCTGGCGGCAGGCTTAACACATG

[0128] CAAGTCGAGCGGGCGTAGCAATACGTCAGCGGCAGACGGGTGAGTAACGCGTGGGAACGT

[0129] ACCTTTTGGTTCGGAACAACACAGGGAAACTTGTGCTAATACCGGATAAGCCCTTACGGG

[0130] GAAAGATTTATCGCCGAAAGATCGGCCCGCGTCTGATTAGCTAGTTGGTGAGGTAATGGC

[0131] TCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGA

[0132] CACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCT

[0133] GATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTTGTGCGGG

[0134] AAGATAATGACGGTACCGCAAGAATAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTA

[0135] ATACGAAGGGGGCTAGCGTTGCTCGGAATCACTGGGCGTAAAGGGTGCGTAGGCGGGTCT

[0136] TTAAGTCAGGGGTGAAATCCTGGAGCTCAACTCCAGAACTGCCTTTGATACTGAGGATCT

[0137] TGAGTTCGGGAGAGGTGAGTGGAACTGCGAGTGTAGAGGTGAAATTCGTAGATATTCGCA

[0138] AGAACACCAGTGGCGAAGGCGGCTCACTGGCCCGATACTGACGCTGAGGCACGAAAGCGT

[0139] GGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCCAGCCG

[0140] TTAGTGGGTTTACTCACTAGTGGCGCAGCTAACGCTTTAAGCATTCCGCCTGGGGAGTAC

[0141] GGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG

[0142] GTTTAATTCGACGCAACGCGCAGAACCTTACCAGCCCTTGACATGTCCAGGACCGGTCGC

[0143] AGAGATGTGACCCTCTCTTCGGAGCCTGGAACACAGGTGCTGCATGGCTGTCGTCAGCTC

[0144] GTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCCGTCCTTAGTTGCTAC

[0145] CATTTAGTTGAGCACTCTAAGGAGACTGCCGGTGATAAGCCGCGAGGAAGGTGGGGATGA

[0146] CGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGCGGTGACAA

[0147] TGGGATGCTAAGGGGCGACCCTTCGCAAATCTCAAAAAGCCGTCTCAGTTCGGATTGGGC SoGo / BRADY-2 / 853

[0148] TCTGCAACTCGAGCCCATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGT GAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCTG AAGACGGTGCGCTAACCCGCAAGGGAGGCAGCCGGCCACGGTAGGGTCAGCGACTGGGGT GAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTTTCTA

[0149] 6. Measuring the protein content of a soybean variety after inoculation with five different strains

[0150] Five different strains (indicated in figure 6) described in the instant invention where inoculated on a soybean variety. The effect of the inoculation in the protein content of the seeds was determined for each of the five different strains. The nulobject (first row in figure 6 is the soybean variety without inoculant) and the second row in figure 6 is the Bradyrhizobium strain 521_C7_N1.3 of the present invention.

[0151] MATERIALS AND METHODS

[0152] Participant recruitment and selection

[0153] Participants were recruited via 'Mijn Tuinlab', an interactive platform where citizen science projects linked to gardens are gathered (https: / / miintuinlab.be)19. Next to this collaboration, the project launch was accompanied by a press moment in March 2021, attended by the Flemish minister of Agriculture and Fisheries. People could register via a website (www.soiainl000tuinen.be) for which a visual identity was created that helped with marketing and communication. The selection of participants from the pool of interested citizens was based on garden location, whether their garden was chemically / organically fertilised (25%) or not (75%), and demographic characteristics (e.g. age, gender and educational level). A total of 1,200 candidates were selected out of 5,335 applicants. All candidates received a unique ID number and were asked to create an account on the project website to confirm their participation and to ensure they had the digital skills required for online data submission throughout the project.

[0154] Garden set-up

[0155] In April 2021, each participant received a participation package containing soybeans (Glycine max cv. Acardia), a 10L plastic flowerpot with participant ID sticker, ten plant labels, a flag to mark the northwestern corner of the sowing grid and a standardized colour card with a ruler. In each garden one square meter was set up in a 6 x 10 grid to plant 60 soybeans. SoGo / BRADY-2 / 853

[0156] Soil samples

[0157] Taking samples

[0158] Soil samples were taken in April 2021, when the participation packages were delivered, and the garden grid was prepared. Soil samples of the top 30 cm were collected in each garden by mixing 20 to 30 auger samples that were randomly taken throughout the plot. Sub samples were then stored for physicochemical (4°C), phospholipid-derived fatty acids (PLFA) (-20°C) and 16S and ITS rRNA microbial (- 70°C) analysis.

[0159] Analysis of physicochemical soil characteristics

[0160] The PSC analysis was performed by the Soil Service of Belgium using a standardized soil analysis protocol (KEMA analysis) including an ammonium lactate extraction (AL). Measurements consisted of potassium (K-AL, mg / lOOg), phosphorus (P-AL, mg / lOOg), sodium (Na-AL, mg / lOOg), magnesium (Mg-AL, mg / lOOg), calcium (Ca-AL, mg / lOOg), total mineral nitrogen (mg / kg), nitrate (kg N / ha), ammonium (kg N / ha), conductivity (EC, mS / cm), salt (mg / L soil), bulk density (kg / L), soil texture, total organic carbon (TOC, %) and pH-KCI (pH)47. During the PSC data processing, the measurements that had a value below the detection limit were set to half the limit. This approach could only be applied to variables that had a known detection limit, which was not the case for ammonium. Consequently, ammonium values that fell below the detection limit were set to 0.

[0161] PLFA soil analysis

[0162] PLFA isolation and analysis was performed based on a previously described protocol48. Briefly, PLFAs were extracted from 0.75 g freeze-dried material by adding extraction buffer (3 mL ethyl acetate, 1,5 mL 0.8% (v / v) 19:0 PC diluted in ethanol and 0,1 mL ultrapure water) and subsequent sonication for 15 min. After incubation for 15 min at room temperature, 5 mL KCI 0,58% (w / v) was added, samples were vortexed for 10 s, and centrifuged at 2500 rpm and 20°C. Finally, the supernatant was collected and dried under Nj at 45°C. Next, PLFAs were purified by solid phase extraction (SPE) using a Discovery DSC-Si (50 mg) SPE 96-well Plate (Merck, Darmstadt, Germany) that was prepared by rinsing with ethanol (3x1 mL), methanol (3x1 mL) and chloroform (3x1 mL). Samples were dissolved in 0,5 mL chloroform, added to the columns, rinsed with 0,5 mL chloroform and 0,5 mL acetone, and finally eluted with methanol (2x1 mL). The samples were dried under Nj at 45°C. Next, PLFAs were methylated by adding 0.2 M methanolic KOH to form fatty acid methyl esters (FAME). These were analysed using a capillary gas chromatograph with flame ionization detector (Thermo Scientific Trace 1300, Thermo Scientific, Waltham, MA, USA) with a Supelco SP-2560 column. PLFAs were identified by retention time using an external FAME (RESTEK Corporation, Bellefonte, PA, USA) and bacterial acid methyl ester BAME mix (Sigma Aldrich, St Louis, MO, USA) and quantified with a C19:0 internal standard. Total microbial biomass was calculated as the sum SoGo / BRADY-2 / 853 of 18 PLFAs (i-C15:0, a-C15:0, i-C16:0, i-C17:0, C16:lc9, C17:0cy, C19:0cy, C14:0, C15:0, C16:0, C17:0, C18:0, 10Me-C16:0, 10Me-C18:0, C18:2c9,12, C16:lcll and C18:lc9). To cope with the range of starting material contents and bulk density, total microbial biomass was expressed per g starting material.

[0163] Soil microbiome analysis: 16S rRNA sequencing

[0164] Soil samples were stored in -70°C in 2 mL Eppendorfs. DNA was extracted with the DNeasy PowerSoil Pro kit (QIAGEN, Hilden, Germany). The V4 region of the 16S rRNA gene was PCR amplified as described previously49. The amplicons of all soils were pooled and sequenced on the Illumina NovaSeq 6000 instrument with the following parameters: NovaSeq6000 flowcell SP 500 kit vl.5., paired-end reads (251- 12-12-251), 130pM / 120pM + 1% PhiX (VIB, Nucleomics Core, Leuven, Belgium). After sequencing, the samples were demultiplexed and the primers were removed. The reads were trimmed by means of the DADA2 package in Rstudio, by truncation at 200 and 180 bp for the forward and reverse reads respectively, and with the default quality parameters [trunQ = 2; maxEE = c(3,3)]. After filtering, the amplicon sequence variants (ASVs) were generated as described previously50. Subsequently, the taxonomy was assigned using the function IdTaxa (DECIPHER), and the default minimum bootstrap confidence (100%). The database used in this study was the Silva vsl38 database51-52. The reads belonging to chloroplast (Class Chloroplast) and mitochondrial (Order Rickettsiales) DNA were removed, reducing the read count by 0.109% on average. Samples that had less than 10,000 reads per sample were removed from the analysis. Also, ASVs that did not represent four reads in at least one sample were discarded. The final mean read count per sample was 149,275.4, and the library yielded 172,114,563 reads corresponding to a total of 19,235 ASVs across the full dataset. All the ASVs that were not classified at phylum level, were subsequently removed from the analysis. Moreover, there were 175 ASVs belonging to Archaea (phylum Crenarchaeota, Euryarchaeota, Halobacterota and Thermoplasmatota), which were kept during the analysis.

[0165] Soil microbiome analysis: ITS sequencing

[0166] Soil samples were stored in -70°C in 2 mL Eppendorfs. DNA was extracted with the DNeasy PowerSoil Pro kit (QIAGEN, Hilden, Germany). The fungal rDNA-ITS2 region was PCR amplified as described previously53, with some adjustments of the protocol: Mastermixes for all PCRs were prepared using the iProof High-Fidelity PCR Kit (Bio-rad, Hercules, USA). The total reaction volume of the first PCR was 20 pL (1 pL Template, 4 pL Buffer, 1 pL dNTP, 1 pL FW primer, 1 pL REV primer, 0.5 pL Polymerase, 11.5 pL distilled water) and for the second PCR 40 pL (2 pL Template, 8 pL Buffer, 2 pL dNTP, 10 pL FW primer, 10 pL REV primer, 1 pL Polymerase, 7 pL distilled water). Each PCR was followed by a PCR product cleanup using magnetic bead purification with HighPrep PCR beads (CleanNGS) (Cleanna, Waddinxveen, The Netherlands). The amplicons of the soils were pooled and sequenced in 4 runs on an Illumina MiSeq instrument, using the Reagent Kit v2 for 500 cycles (4.5 pM + 24.10% PhiX v3), and generating paired- SoGo / BRADY-2 / 853 end reads (251-8-8-251) (VIB, Nucleomics Core, Leuven, Belgium). After sequencing, the samples were demultiplexed. Primers and adapters were trimmed using the cutadapt tool54, DADA255, ShortReads56and Biostrings57R packages (R version 3.6.058R core team, 2019). Sequences with ambiguous bases and low-quality tails were removed by means of the DADA2 filterAndTrim function55. The minimum length of this function was set to 50 bases and the truncation length (204 to 150) was determined with the Figaro tool59, which analyses the error rates in the FastQ. files to determine the optimal trimming parameters per run. The quality parameters were set at trunQ. = 2 and maxEE = 5. The generation of the ASVs was performed as described previously50. The taxonomy of the ITS data was assigned with the naive Bayesian classifier method (assignTaxonomy function in the DADA2 package55) using the UNITE database (V960). The confidence intervals of the assignments were determined using a default minimum bootstrap sample of 50. Samples that had less than 5000 reads were not included in the analysis. Chimeras, sequences of which the phylum was not identified and ASVs that did not represent four reads in at least one sample were discarded from the dataset. The final average read count was 25,089, the total library had 27,071,472 reads corresponding to a total of 40,619 ASVs across 1,079 samples. Some samples were re-sequenced to improve read count, in these cases the samples with the highest read numbers were used for modelling to allow the evaluation of batch effects.

[0167] Nodule analysis

[0168] Nodule sterilisation

[0169] After harvest, nodules were stored at 4°C on silica. Before sterilization, desiccated nodules were rehydrated individually in sterile tubes with sterile distilled water overnight at 4°C. Sterile glass beads were added and the tubes were vortexed for 1 min. Nodules were transferred to a fresh sterile tube, glass beads and 70% ethanol were added and tubes were vortexed for 1 to 3 min, depending on the nodule size. Next the nodules were washed 3 times with sterile distilled water. Subsequently, glass beads were added and nodules were sterilised with 2.5% - 3% NaCIO for 3 to 5 min by vortexing. Next, nodules were washed 4 times with sterile distilled water. Finally, nodules were again vortexed in 70% ethanol for 2 min and washed 3 times with sterile distilled water. As a control for sterility, the last wash was plated on TSA medium and the sterilized nodule was rolled on to a TSA plate that was incubated. Large nodules were cut into equal halves, each placed in a separate 2 mL tube. One tube was immediately snap-frozen with liquid nitrogen and stored at -80°C for use in the nodule microbiome experiment. The other nodule half was used for bacterial isolation. For small nodules that could not be cut in two equal parts, separate nodules from the same plant were used for bacterial isolation and the microbiome experiment, respectively. SoGo / BRADY-2 / 853

[0170] Bacterial isolation

[0171] The nodule material was squashed with 50 pL of sterile distilled water using a sterile plastic pestle. Nodule juice was plated on R2A and YMA. In addition, a lOx, 20x and lOOx dilution was made and plated on R2A. Plates were incubated at 28°C. The remaining nodules juice was mixed with 50% glycerol and stored at -20°C. Plates were regularly checked for growth and after 14 days, single colonies were selected and replated for purification. For temporary storage, a single colony from pure isolate was inoculated in 5 mL sterile R2A broth. After incubation at 28°C under constant shaking for 24h (or until bacteria had grown well) 0.9 mL of the culture was combined with 0.9 mL of sterile 50% glycerol and stored at -20°C until further use.

[0172] MALDI-TOF MS

[0173] Pure isolates were grown on R2A plates at 28°C. Extracts for MALDI-TOF mass spectrometry analysis were prepared as described before61. All extracts were spotted in duplicate as technical replicates. Each sample spot was overlaid with 1 pL of matrix solution (10 mg mL-1 a-cyano-4-hydroxycinnamic acid in acetonitrile:water:trifluoroacetic acid 50:47:5:2:5) and MALDI-TOF MS profiles were acquired on a Bruker MicroflexTM LT / SH (Bruker Daltonik, Bremen, Germany) as previously described61. For identification, spectra were compared to the Bruker BDAL (9607 MSP) and the LM-UGent in house databases using MBT Compass Explorer according to the manufacturer's guidelines (Bruker Daltonik, Bremen, Germany). Identification scores were interpreted following Broker's instructions as 'highly probable species identification' (2.300-3.000), 'secure genus identification' (2.000-2.299), 'low- confidence identification' (1.700-2.000) or 'no identification possible' (<1.700). For dereplication (i.e. grouping of strains according to similarity of MALDI-TOF MS profiles and selection of representative isolates) the SPeDE software tool was used as described previously61. For cluster analysis, profile similarities were calculated using the curve-based Pearson product-moment correlation coefficient and the unweighted pair group method using arithmetic average (UPGMA) clustering algorithm was used in BioNumerics 7.6.3 (Applied Maths).

[0174] Nodule microbiome analysis: 16S rRNA sequencing

[0175] Nodules that were stored in -70°C in 2 mL Eppendorfs after sterilisation were kept on dry ice. Two 5mm stainless-steel balls were added and the Epps were flash frozen in liquid nitrogen before homogenization using a bead beater for 2 times 1 minute at 25 Hz. Subsequently, DNA was extracted with the DNeasy PowerSoil Pro kit (QIAGEN, Hilden, Germany). First, the Powerbeads from the kit were added to the Epps before following the provided protocol starting from the vortexing step. The V4 region of the 16S rRNA gene was PCR amplified as described previously49. The amplicons of all nodules were pooled and sequenced on an Illumina NovaSeq 6000 instrument following these parameters: NovaSeq6000 flowcell SP 500 kit vl.5., paired-end reads (251-12-12-251), 130pM / 120pM + 1% PhiX (VIB, Nucleomics Core, SoGo / BRADY-2 / 853

[0176] Leuven, Belgium). After sequencing, the samples were demultiplexed and the primers were removed. The reads were trimmed by means of the DADA2 package in Rstudio, by truncation at 230 and 180 bp for the forward and reverse reads respectively, and with the default quality parameters [trunQ. = 2; maxEE = c(3,3)]. After filtering, the amplicon sequence variants (ASVs) were generated as described previously50. Subsequently, the taxonomy was assigned using the function IdTaxa (DECIPHER), and the default minimum bootstrap confidence (100%). The database used in this study was the Silva vsl38 database51-52. The reads belonging to chloroplast (Class Chloroplast) and mitochondrial (Order Rickettsiales) DNA were removed, reducing the read count by 83.86% on average. Samples with a read count below 1000 reads were eliminated, and those ASVs that did not represent four reads in at least one sample were discarded from the dataset. The final mean read count was 67,288.63, and the library yielded 73,546,471 reads corresponding to a total of 23,766 ASVs across the full dataset. All the ASVs that were not classified at phylum level were subsequently removed from the analysis. Moreover, there were 39 ASVs belonging to Archaea (phylum Crenarchaeota and Thermoplasmatota), which were kept during the analysis.

[0177] Statistical analysis of the nodule microbiome

[0178] For the samples' beta diversity, a dissimilarity matrix based on the Bray-Curtis dissimilarity index was generated with the function vegdist in the vegan package62using the ASV count tables as input. The differences between the samples due to colour phenotype were checked by means of the Permutational Multivariate Analysis of Variance (PERMANOVA), using the function adonis2 in the vegan package. Homogeneity of variances between groups was verified by calculating the Multivariate Homogeneity of Groups Dispersions with the betadisper function within the vegan package. Principal Coordinate Analysis (PCoA) plots were generated to illustrate the data with the package MicrobiotaProcess63. The analyses were done at ASV level.

[0179] For analysis of the samples' alpha diversity, the ASV count tables were rarefied to the sample with the smallest read countr, with the function rarefy_even_depth in the phyloseq package. The Shannon- Wiener index was calculated with the function alpha in the package microbiome. We performed a Kruskal Wallis test by using the function kruskal.test in R, followed by a Wilcoxon signed-rank test with the function pairwise. wilcox.test to explore whether the nodule colour phenotype was associated with differences in the alpha diversity of the samples. The p values were adjusted with the Holm-Bonferroni method. To confirm these results we also created mixed models with the Ime4 package in Rstudio64, integrating as random effects the soil and the plants the nodules came from and Colour as fixed effect. Subsequently we performed a pairwise analysis with the emmeans package in Rstudio65. SoGo / BRADY-2 / 853

[0180] Pot trials

[0181] Trials in sterile vermiculite

[0182] Soybean seeds (Glycine max cv. Acardia) were surface sterilized by first washing 5 minutes with sterile water, followed by a 2-minute 70% ethanol washing step. Next, the seeds were washed for 10 minutes in 30% NaCIO (12%-13%) stock solution and then washed for 1 minute in sterile water. Finally, the seeds were washed 5 times more for 2 minutes in sterile water. During each washing step, the seeds were shaken thoroughly. The sterile seeds were pre-germinated on 1% plant agar plates at 22°C in the dark. After 4 days, seedlings were sown in sterilized vermiculite in 13-cm round pots (3 plants / pot) and grown under a 16-h light / 8-h dark photoperiod at 22°C. Plants were watered twice a week with 50 mL nitrogen poor SOLi solution66. After 1 week, plants were inoculated at the shoot / root transition with 1 mL of the indicated bacterial strain when in the exponential growth phase (OD 0.01). Non-inoculated mock plants and plants inoculated with the Bradyrhizobium diazoefficiens strain G49 were used as negative and positive controls, respectively. Four weeks post-inoculation, nodules were counted and nodule colour was noted.

[0183] Trials in non-sterile substrate

[0184] Five soybean varieties, Glycine max cv. Hermes (Protealis), Acardia (Saaten-Union), Lenka (Prograin), Aurelina (Saatbau Linz) and Gallec (Agroscope / DSP), were grown in an 8-week pot trial, using three repeats per three blocks in a 16-h light / 8-h dark photoperiod at 20°C / 10°C. Pots were filled with 4 L substrate, consisting of 50% (v / v) fresh agricultural soil + 50% (v / v) (0 / 2) sand (Leus N.V.) and soil samples were taken to check pH-KCI, %TOC, NO3 / NH4, P, K, Ca, Mg, and Na in ammonium-lactate. On the day of sowing, each pot received 200 mL water and 3 sowing holes of 1 - 2 cm deep were prepared to sow 9 soybean seeds (3x3) of the same variety per pot. Per variety, 35 seeds were soaked in 15 mL of the indicated bacterial strain when in the exponential growth phase (OD 0.01). Non-inoculated mock plants and plants inoculated with the Bradyrhizobium diazoefficiens strain G49 were used as negative and positive controls, respectively. After sowing the seeds, water was given three times a week, with a starting weight of the whole pot of 3750 g to avoid overwatering and adding 50 mL on the third and fifth day, which was increased upon plant growth. Two weeks after sowing, seedlings were reduced to 3 plants per pot. At 8 weeks, plant height was measured and plants were harvested. Per plant, dry weight of the above-ground biomass was measured, the number of nodules was counted and nodule dry weight was determined. Chlorophyll content was measured using a CCM-200 m (Opti-Sciences Inc., Hudson, USA), as described previously6. The output was expressed as chlorophyll content index (CCI), defined as the ratio of transmission at 931 to 653 nm through a leaf. Measurements were conducted on the youngest fully expanded leaf of each of the plants. Data analysis was performed using R version 4.1.2. Results were analysed using a linear mixed model with the Ime4 package. SoGo / BRADY-2 / 853

[0185] The following base model was considered for the pot trials (1):

[0186] With "Y" and "Yl' being the response variables, " V the soybean variety (fixed effect), "B' the Bradyrhizobium strain (fixed effect), “P’ the respective pot trial (random effect). "Repl" and "Rep2" are random effects which represent the spatial components within the experimental design of the trials.

[0187] Data distribution was assessed using QQ-plots and outliers were removed if necessary. Pairwise comparison was done using the Tukey-test (P<0.05).

[0188] N-fixation capacity measurements

[0189] To check the N-fixation capacity of the selected strains isolated from nodules, soybean plants were grown as discussed in the previous section (cf. 'Pot trials'). For each treatment, at least 9 plants were harvested 4 weeks post-inoculation. For each plant, nodules were counted, and nodule fresh weight and dry weight were measured. Additionally, the nitrogenase activity of each root system was determined by measuring the acetylene reduction activity (ARA), as described previously67. Briefly, ethylene production is quantified using a Hewlett-Packard 5890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a PLOT fused silica column, with propane as an internal standard. Finally, ARA is reported as pmol of ethylene produced per hour. Significant differences between the treatments were determined by performing One-way ANOVA with Tukey multiple comparison correction, using GraphPad Prism version 9.3.1.

[0190] Field trials

[0191] Two field trials were set up at the beginning of May 2022 at nearby locations (Merelbeke and Bottelare, Flanders, Belgium), both featuring sandy-loam soils. Two soybean varieties were tested, Glycine max cv. Lenka (Prograin) and RGT Shouna (RAGT), to examine potential variety x rhizobia interactions. Seeds were inoculated by coating them in a cement mixer with the indicated bacterial strain when in the exponential growth phase (OD 0.01) and an adhesive (IMPF Signum Soy) with 0.001 mL bacterial culture per seed (estimated concentration of 10A6 CFU / mL or 1000 CFU / seed). Non-inoculated mock plants and plants inoculated with the Bradyrhizobium diazoefficiens strain G49 were used as negative and positive controls, respectively. Seeds were sown within 24h after inoculation with a sowing machine at a depth of + / - 3.5 cm with a row distance of 25 cm (5 rows per plot). Detailed descriptions of soil characteristics, fertiliser, sowing and harvesting are provided in Table 2. Field trials were set up in a randomised complete block design (RCBD) with three replicates, at both locations. Each plot comprised a net surface area of 9.375 m2. After sowing, pre-emergence herbicides were applied (IL Arundo (720 g / L SoGo / BRADY-2 / 853 dimethenamide-p), 1.5L Proman (500 g / L metobromuron) and 0.2 L Centium 360 CS (360 g / L clomazon) per hectare). At the reproductive growth stage R5, chlorophyll content was measured using a CCM-200 m (Opti-Sciences Inc., Hudson, USA), as described previously6. The output was expressed as chlorophyll content index (CCI), defined as the ratio of transmission at 931 to 653 nm through a leaf. Measurements were conducted on the youngest fully expanded leaf of 10 plants of each plot and the average was calculated. Five randomly chosen plants per plot were harvested in the front and the back of the plot (ten plants per treatment). When nodules were present, they were counted and weighed. Nodule dry weight was determined after drying the nodules for 72 h at 70°C. The remaining plants were harvested at full plant maturity (growth stage R8) with a trial field thresher. After harvest and per plot, seed yield was measured in kg ha-1and adjusted to 0.150 kg H2O kg-1seed. Thousand-grain weight was determined for dry seeds after drying for 72 h at 70°C. Finally, protein content of the seeds was determined, as described previously6, on dry seed material milled over a 1-mm screen using a cutting mill (Peppink Type 200AN). Protein content was estimated using near-infrared reflectance spectroscopy (NIRS). Calibration was based on soybean samples from 2014 to 2017 from different soybean trials in Belgium. Data analysis was performed using R version 4.1.2. Results were analysed using a linear mixed model with the Ime4 package.

[0192] The following base model was considered for the field trials (2):

[0193] (2) Y2= V * B + (l |Repl)

[0194] With "Y2' being the response variables, " V the soybean variety (fixed effect), "B' the Bradyrhizobium strain (fixed effect). "Repl" is a random effect which represent the spatial components within the experimental design of the trials.

[0195] Data distribution was assessed using QQ-plots and outliers were removed if necessary. Pairwise comparison was done using the Tukey-test (p < 0.05).

[0196] Whole-genome sequencing, assembly, and annotation

[0197] DNA from the selected strains was extracted from colonies grown on R2A plates using Maxwell® RSC Cultured Cells DNA Kit (Promega) and the quantity and quality was checked as described previously68. The DNA of the strains was then sequenced using Illumina NovaSeq 6000 (Oxford Genomics Center, University of Oxford, Oxford, UK), preparing the library with an in-house adapted protocol of the NEB prep kit (New England Biolabs, Ipswich, MA, USA). We also used Oxford Nanopore Technologies (PromethlON P24) (Neuromics Support Facility VIB-UAntwerp, Antwerp, Belgium), preparing the library with native barcoding kit 96 V14. The genomes were then assembled using the Unicycler pipeline for SoGo / BRADY-2 / 853 hybrid assembly69. Subsequently, they were annotated by the Pathosystems Resource Integration Center (PATRIC)70.

[0198] To investigate the phylogeny of the isolates, the type strains from the genus Bradyrhizobium, Tardiphaga and Rhizobium were downloaded from the NCBI database and analysed as described previously71. The phylogenetic tree was performed using bcgTree with a 1000 bootstraps as described previously72, and visualised with the ggtree package in RStudio73.

[0199] Genes that were assessed based on their importance for nodulation and nitrogen fixation include the following. For the nitrogenase synthesis (nif genes), we included structural genes (nifHDK), regulators of nitrogen fixation (nifA), genes involved in the formation of the iron-sulphur cluster (nifUS) and the synthesis of the iron-molybdenum cofactor (nifB, nifEN, nifOQ.). For nodulation factors (nod genes), we determined the presence of genes involved in their synthesis (nodABC), transport (nodlJ), and the regulation of nod gene expression (nodD). For symbiosis-specific respiration (fix genes), we also included genes involved in electron transfer to the nitrogenase (fixABCX), in respiration under the microaerobic conditions (fixGHIS and fixNOQP), and in low oxygen-dependent induction of nitrogen fixation genes (fixU). Finally, we also assessed the presence of genes encoding components of the type III secretion systems (T3SS) .

[0200] Bulk soil data exploration

[0201] All soil data analyses were carried out using R, versions 4.0.3 and 4.1.374. The datasets analysed included the participant / garden level PSC, PLFA, ITS and 16S data and information on previous fertilization practices of the citizen scientists, the presence of other legumes in their gardens and the presence of nodules on the harvested soybean roots. Initial exploration of the 16S and ITS data was performed on ASV level, while for the correlation analyses and models (see below) only genus level data was used.

[0202] Data exploration included a PCA analysis to evaluate batch effects, spatial autocorrelation and the presence of potential confounding factors and outliers (prcomp function, stats R package74). The analysis was performed on z-scored PSC, PLFA, ITS and 16S data separately (Supplementary Fig. S6). Spatial patterns were also visualized on a map of Flanders (available at www.geopunt.be) using the tmap and sf packages75-76(Supplementary Fig. SI). Total biomass and batch effects were present in the PLFA data (Supplementary Fig. S6). These effects were removed by normalizing the data points of a given sample for the total biomass of the sample and by performing a batch correction, respectively. For batch correction, linear regression models were used with the total biomass-normalized PLFA variables as independent variables and the batch number as dependent variable (stats R package). The residuals of these regression models were used as batch-corrected data. Spearman correlations were calculated for SoGo / BRADY-2 / 853 the combined soil PSC, corrected PLFA, 16S and ITS data using the stats R package (Supplementary Fig.

[0203] S7).

[0204] Nodule presence prediction modelling

[0205] Models were developed to investigate whether bulk soil data can be used to predict the presence of nodules in a given garden and, if so, which soil factors potentially influence nodule presence. Soil PSC data, corrected PLFA data, ITS and 16S genus-level data, data on the previous fertilization practices of the citizen scientists and the presence of other legumes in the gardens, and the Shannon index were used to train single- and multi-variable models. The Shannon index (microbiome R package77) was calculated using data that was rarefied to an even depth of 786 (16S) and 1000 (ITS) reads (phyloseq R package78). Only gardens that had a complete dataset were taken into account. As most data layers are only available on the garden level (rather than the individual plant level), the models were trained on the garden level, and a garden was categorized as 'nodules present' if at least one plant from the garden exhibited at least one bona-fide root nodule. Gardens with an ambiguous nodule status, i.e. that contained nodule-like structures, but no confirmed nodules , were removed (nnOduie: 247, nnOnoduie: 401). Different modelling approaches were evaluated, including auto-logistic regression (auto-log), elastic net logistic regression (eln-log), random forest classification (RF) and partial least squares auto-logistic regression (pls-log).

[0206] As the PCA analyses and variable maps indicated that a spatial structure was present in the data (Supplementary Fig. SI and S7), a distance-weighted auto-covariate variable was included in all models to take spatial autocorrelation effects into account. This variable was calculated using the autocov_dist function of the spdep R package79, using the presence / absence of nodules and garden coordinates as input, inverse squared distance weights, a neighbourhood style B and a neighbourhood radius of 0.2.

[0207] Single-variable models

[0208] Single-variable auto-logistic regression models predicting nodule presence were developed for each data layer. Individual PSC soil variables were supplemented with information on the citizen scientists' fertilization practices and the presence of other legumes in the gardens, while the ITS and 16S variables included the corresponding Shannon indices. The models for PLFA variables also included TOC and soil type as potential confounding factors. The auto-logistic models were built using the glm function (family:binomial, stats package), with nodule presence as dependent variable and the factor of interest, potential confounders for this factor and the distance weighted auto-covariate as independent variables. False Discovery Rate (FDR) p-value correction was performed on the p-values of the coefficients. SoGo / BRADY-2 / 853

[0209] Multi-variable models

[0210] Three algorithms were evaluated to predict nodule presence as a function of multiple variables and data layers, and the results of the different algorithms were combined to identify a robust set of important predictors. The model algorithms used in this analysis were elastic net logistic regression (eln-log), random forest classification (RF) and partial least squares auto-logistic regression (pls-log). All three algorithms can cope with multicollinearity, which is common in high-dimensional data. The models were trained on different data layer combinations supplemented by the spatial-autocovariate. The combinations included each separate data layer, all available data, all data except 16S, PSC+PLFA+div and PSC+div. The potential confounders TOC and soil type were also included in the multi-variable PLFA models. The training data of the eln-log and pls-log models were z-scored, and for the regression methods, the categorical variables were converted to dummy variables.

[0211] The eln-log models were developed using the glmnet function (glmnet R package80) with a conditional response variable distribution of the binomial family. The alpha and lambda parameters of the final model were determined through a grid search in a 10-fold cross-validation (CV) setup (caret package81; Supplementary Table S4). The optimal values were selected based on the 10-fold CV area under the receiver operating characteristic curve (AUC).

[0212] The pls-log models combined partial least square (pls) dimensionality reduction with auto-logistic regression models. The pls algorithm removes multicollinearity by calculating latent variables that maximize the covariance between the features and the response variable. By calculating these latent variables no features are removed. However, the interpretation of the model predictors can become more complicated. Pls dimensionality reduction was performed with the tidymodels and learntidymodels R packages. The number of pls components was determined using the 10-fold CV AUC (caret package, Supplementary Table S4). These components were subsequently used in an auto-logistic model which had nodule presence as dependent variable and the pls components and the spatial auto-covariate as independent variables (glm function, stats package).

[0213] RF models were created using the randomForest R package. A grid search in a 10-fold CV setup was performed to determine the number of trees (ntree), number of variables randomly sampled at each split (mtry), the number of samples drawn (sampsize) and the minimum size of the terminal nodes (nodesize). The optimal values were selected based on the 10-fold CV AUC (mlr package82; Supplementary Table S4).

[0214] Model Accuracy

[0215] Model accuracy was calculated using nested leave-pair-out cross-validation (NLPO). The idea of NLPO is to hold out pairs of one positive and one negative case as a test set. The remaining data is used to train SoGo / BRADY-2 / 853 and finetune the model which is subsequently used to make predictions for the test pair. The hyperparameter finetuning approach of eln-log and pls-log was the same for NLPO as for the final model (grid search using 10-fold CV loop setup in inner NLPO loop), while for RF NLPO a computationally less intensive randomized search in a 10-fold CV setup was implemented in which 1000 hyperparameter combinations were randomly selected from the hyperparameter space. The NLPO approach applied in this study estimated an average AUC from the predictions of 1000 random test set pairs containing one garden with and one without nodules. AUC was calculated by averaging over the Heaviside step function with the following formula83(3):

[0216] Where Xi and Xj are the samples with and without nodules, respectively, denotes a model algorithm trained without the i and j sample test pair. | / +| | / _ | corresponds with the number of pairs, with 1+ representing the positive, and I. representing the negative instances. H is the Heaviside step function.

[0217] Confidence intervals of the AUC values were determined by bootstrapping the test set prediction pairs 2000 times and calculating AUCs for each of these bootstrap samples (boot R package84). The 95% confidence intervals were calculated under the assumption that the sampled AUC values are normally distributed.

[0218] Predictor importance

[0219] To determine the variables that were most strongly related to nodule presence, the importance of the NLPO model predictors was evaluated by calculating the median importance score. The eln-log coefficients of the independent variables were taken as variable importances of the eln-log models. Importance values for the pls-log models were calculated by multiplying the coefficients of the significant PLS components with the loadings of the variables. In case a variable had loadings for multiple significant components the sum of these products was taken. The conditional permutation accuracy was used as a variable importance metric for the RF models (permimp R package85). The importance values were calculated using an RF model without resampling and 1000 trees. The other finetuned hyperparameters of the NLPO models stayed the same. To facilitate model comparison, the importance values of the eln- log and pls-log models were scaled between -1 and 1 before the median value was calculated, while the RF importance values were scaled between 0 and 1. SoGo / BRADY-2 / 853

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Claims

SoGo / BRADY-2 / 853CLAIMS1. An isolated Bradyrhizobium strain Bradyrhizobium sp. R-85129 having the deposit accession number LMG P-33693.

2. An enriched culture of the Bradyrhizobium strain according to claim 1.

3. A biologically pure culture of the Bradyrhizobium strain according to claim 1.

4. A composition comprising the Bradyrhizobium strain according to claim 1 or the culture according to claims 2-3.

5. The composition according to claim 4, wherein the Bradyrhizobium strain is lyophilized, freeze-dried to a powder or present as an aqueous slurry.

6. The composition according to claim 4 further comprising growth medium appropriate for Bradyrhizobium species and / or a cryoprotectant.

7. The composition according to any of claims 4-6 further comprising an agriculturally compatible carrier.

8. A plant seed coated with the Bradyrhizobium strain according to claim 1 or with the culture according to any of claims 2-3.

9. The plant seed according to claim 8, where the plant seed is a leguminous plant seed.

10. The plant seed according to claim 9, wherein the leguminous plant seed is a soybean seed.

11. Use of the Bradyrhizobium strain according to claim 1, the culture according to any of claims 2-3 or the composition according to any of claims 4-7 to enhance yield and / or nodulation of a leguminous plant.

12. Use of the Bradyrhizobium strain according to claim 1, the culture according to any of claims 2-3 or the composition according to any of claims 4-7 to enhance the protein content of seeds of a leguminous plant.

13. The use according to any of claims 11-12, wherein the leguminous plant is soybean.

14. A method for enhancing yield and / or nodulation of a leguminous plant or enhancing the protein content of the seeds of a leguminous plant, the method comprising the steps of: inoculating a plant growth medium with a microbial population, said population comprises the Bradyrhizobium strain according to claim 1, the culture according to any of claims 2-3 or the composition according to any of claims 4-7; and growing the leguminous plant in said plant growth medium.

15. The method of claim 14, wherein the microbial population is applied to the plant growth medium as a powder, as a pellet, as a granule or as a liquid.

16. A method for enhancing yield and / or nodulation of a leguminous plant or enhancing the protein content of the seeds of a leguminous plant, said method comprising growing the coated plant seedSoGo / BRADY-2 / 853 according to any of claims 8-10, to obtain enhanced yield and / or nodulation of said plant or an enhanced protein content of the seeds of said plant.

17. A method for enhancing yield and / or nodulation of a leguminous plant or enhancing the protein content of the seeds of a leguminous plant comprising: - growing said plant in an environment that supports plant growth; and administering a sprayable formulation to said environment or to said plant, said formulation comprising the Bradyrhizobium strain according to claim 1, the culture according to any of claims 2-3 or the composition according to any of claims 4-7; to obtain enhanced yield and / or nodulation of said plant or enhanced protein content of the seeds of said plant.

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