Novel rahnella strain

Abstract

The present invention relates to an identified and isolated bacteria or biologically pure bacterial culture of the species Rahnella aceris. The invention further relates to compositions such as fertilizers and seed coatings comprising said bacteria and uses thereof, such as plant growth promoting agents.

Description

[0001] NOVEL RAHNELLA STRAIN

[0002] Technical field of the invention

[0003] The present invention relates to isolated bacteria or biologically pure bacterial culture of the species Rahnella aceris. The invention also relates to compositions such as fertilizers, probiotics, medicines and seed coatings comprising said bacteria and uses thereof, in particular uses as plant growth promoting agents in colder regions.

[0004] Background of the invention

[0005] The United Nations estimate that we will reach 8 billion people this year, and nearly 10 billion people by 2050. This population growth, and a parallel rise in wealth, will increase food demand by 50% in 2050. In the same period, the agricultural area per capita will decrease by about 25%. The lack of food is already urgent, the World Food Programme estimates that, in 2022, 828 million people do not have enough food and 50 million are facing emergency levels of hunger around the world. The amount of people that are already malnourished, the projected growth in population and food consumption and the decrease in farmland per capita highlights the need for a more efficient and productive agricultural industry.

[0006] Agrochemicals, such as fertilizers and pesticides, are the primary means by which agricultural productivity is increased currently. The widespread use of these chemicals has negative environmental consequences. They can affect the health of humans, animals, plants, soils and microbiomes, they can degrade ecosystems, promote anti-microbial resistance and their use contributes to climate change. Further increasing the use of these chemicals to support the demands for higher food productivity would therefore be highly problematic.

[0007] The germination, emergence, growth, development, yield quantity and quality of a plant may be beneficially affected by microorganisms that enhance the availability of macronutrients and micronutrients, strength plant defences and protect them against biotic and abiotic stress factors. Due to global warming and past agricultural practices, there is an increase in more extreme weather, drought, floods, heat waves, salt stress, pollution of soil and water sources and a loss of soil fertility and health. There is also an increase and spread of new plant pathogens and anti-microbial resistance. The addition of beneficial bacteria to plants may be a method for increasing food productivity under normal conditions, but it may also be a method for decreasing losses due to extreme weather and other biotic and abiotic stress conditions.

[0008] There are many mechanisms by which beneficial microorganisms may promote plant growth or survival. Beneficial bacteria help the plant access heavily bound nutrients in the soil that would otherwise not be accessible. They may increase root size or branching and improve the availability of water and nutrients for the plant. The bacteria may also bind water and nutrients and provide it to the plant during periods of drought or increased nutrient needs. Bacteria may also colonize the surface of plants and seeds such as their leaves and roots and protect it against unfriendly microorganisms through specific mechanisms or by simply taking the space and nutrients that pathogenic bacteria and fungi may have used. Not all bacteria are beneficial to plants, however, some may be directly detrimental to plant growth whereas others may not damage the plant directly but still affect them indirectly by taking space and nutrients that would otherwise be available to beneficial microorganisms. Making sure that beneficial bacteria are present on, in and near plants has potential as a solution for increasing agricultural productivity of a given area of farmland without adding further agrochemicals. Bacteria that are beneficial to plants may also have applications in other fields than agriculture such as in horticulture, vertical farming, gardens, landscaping, reforestation and in the restoration of degraded areas. In addition, bacteria that are capable of solubilizing nutrient-containing minerals may have applications outside agriculture such as in promoting the health of humans and animals as well as in mining, ore extraction, and waste reuse / recycling.

[0009] Several bacteria belonging to the genus Rahnella have been described as rhizobacteria with capabilities for promoting plant growth and health. A novel species declared as R. aceris was added to the genus Rahnella in 2020. Xu et al. 2022 (Comparative Genomics Assisted Functional Characterization of Rahnella aceris ZF458 as a Novel Plant Growth Promoting Rhizobacterium) and Guardiola- Marquez et al. 2023 (Identification and characterization of beneficial soil microbial strains for the formulation of biofertilizers based on native plant growth promoting microorganisms isolated from northern Mexico) disclose three R. aceris strains (ZF458 (complete genome), B14KEA1 (16S rRNA gene sequence) and A210R1 (16S rRNA gene sequence)) and these have been considered as potential plant growth-promoting bacteria, either as a potential biocontrol agent (ZF458) or as potential biofertilizers (B14KEA1 and A210R1). However, none of these R. aceris strains have been reported to be active under low-temperature conditions, representing the climate in Scandinavia during mild winter or spring seasons.

[0010] Hence, expanding the portfolio of bacteria and providing a bacteria capable of improving plant growth would be advantageous, and in particular, more efficient and / or reliable compositions comprising such bacteria would be advantageous.

[0011] Summary of the invention

[0012] The inventing team has isolated, identified, characterized and propagated a newly identified strain of bacteria, namely the Rahnella aceris strain DSM 34654 deposited with deposit number DSM 34654. The strain may be used to promote a wide variety of plant health parameters. Such parameters may for example be the speed by which seeds germinate, the percentage of seeds that germinate, the speed by which root or shoot elongates and grows, the speed by which sown seeds emerge, the percentage of sown seeds that emerge, the speed by which a plant grows, the speed by which a plant develops on the BBCH scale, the size and shape of particular parts of the plant e.g. roots, stem, leaves, flowers and seeds, the colors of a plant or parts of the plant, the resistance of the plant towards biotic and abiotic stresses, the appearance, taste and smell of a plant as well as the yield (mass / area) and yield properties (e.g. protein, starch, oil or toxin content). Promoting these parameters may have application in agriculture, horticulture, landscaping, gardening, and indoor plants, users may be business or private individuals and the plants may be food crops, fiber crops, fuel crops or plants that serve aesthetic or functional purposes such as flowers, grass and trees. Promoting these parameters may have application in the restoration of degraded land whereon plant cover is needed, such as degraded mine sites or degraded agricultural or pastoral land. It may also have applications in reforestation or revegetation and in carbon capture / storage based on growing biomass.

[0013] The strain may also be used to inhibit the growth of pathogens, such as bacteria and fungi. This may have application in promoting the health of plants and potentially also humans. The strain may also be used to dissolve otherwise poorly soluble substances such as minerals and salts. These minerals and salts may be present naturally in soil or other plant growth media, examples are calcium phosphate, iron phosphate, aluminium phosphate and potassium alumino silicate. They may also form when macronutrients or micronutrients, possibly in the form of fertilizer, are added to the soil or other plant growth media. The minerals and salts may also be components of nutrient bearing materials destined for agricultural use such as wastewater, sludge, biochar, manure, green manures, compost, biogas and pyrolysis residues, biowaste, household waste and other biomasses. The purpose of adding the strain would in these cases be to dissolve the minerals or salts and thereby release nutrients such as phosphate, potassium, calcium, magnesium, sulfate, manganese and iron that can enhance plant growth.

[0014] The strain may also be used to dissolve otherwise poorly soluble substances such as minerals and salts for applications outside plants. It may be used to bioleach ores or waste materials. It may therefore by used in mining, recycling, or urban mining.

[0015] Example 1 shows the capture of a range of microorganisms from the soil of a Danish grassland with potential beneficial properties. The subsequent screening of these novel strains indicated that especially six strains (TP1, TP2, TP3, TP4, TCI, and TC3) stood out as efficient nitrogen fixers and phosphate solubilizers, and therefore hold a large potential for the use as biofertilizers in colder climate regions.

[0016] Example 2 shows the bacterial strains TCI, TC3, TP1, TP2 and TP3, which were selected as best- performers from a large screening setup (n = 28), represent at least two different groups of strains belonging to Rahnella aquatilis / aceris, where TCI and TC3 represent one group of strain and TP1, TP2 and TP3 represent another group of strain.

[0017] Example 3 shows the bacterial strain DSM 34654, which was selected amongst the top performing bacterial strains, is a novel biofertilizer strain. A comparative genome analysis of the bacterial strain DSM 34654 revealed that DSM 34654 is a novel Rahnella aceris strain. Example 4 shows the ability of DSM 34654 to increase the availability of common plant nutrients specifically under low, medium, and high temperature conditions. In particular, only DSM 34654 was able to grow and display bacterial activity at 5°C, demonstrating that DSM 34654 has a high potential for supplying crops in the field with otherwise unavailable nutrients through the entire plant life cycle from as early as germination in cold soil until maturation and harvest.

[0018] Example 5 shows the antifungal activity of DSM 34654 against fungal plant pathogens including phytopathogenic fungi Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres, and Fusarium oxysporum when evaluated in two dual culture assays.

[0019] Example 6 shows the stimulatory properties of DSM 34654, when applied as a seed coat, on early plant growth in vitro in two typical agricultural crops. Coating seeds with DSM 34654 seed coat stimulates early germination processes by increasing the emergence of both the radicle and the plumule which may be attributed to the increased phosphate activity in the vicinity of seeds. Collectively, these data demonstrate the plant growth-promoting properties of DSM 34654 seed coat.

[0020] Example 7 shows the positive effects of DSM 34654 on the emergence and growth of oil radish crops harvested from Danish fields when applied as a seed coat onto seeds before sowing. Applying the bacterial strain DSM 34654 as a seed coat onto oil radish seeds before sowing increases the emergence of oil radish plants in the field. Plants that have developed from DSM 34654-treated seeds have bigger roots compared to control plants, demonstrating that DSM 34654 adds to the beneficial characteristics of oil radish plants when used as a cover crop by for example increasing the storage capacity of nitrogen.

[0021] Example 8 shows the ability of DSM 34654 to affect the yield of root and cereal crops, such as sugar beet, potato, and maize, in outdoor conditions. Treating sugar beet seeds, seed potatoes and maize seeds with a bacterial solution containing DSM 34654 before sowing in fields demonstrates that DSM 34654 can affect the harvest yield of root and cereal crops as diverse as sugar beets, potatoes, and maize by a minimum of 14%.

[0022] Example 9 shows the ability of DSM 34654 to affect the growth of winter crops, such as winter wheat, when applied as a seed coat before sowing. Treating winter wheat seeds with a bacterial solution containing DSM 34654 before sowing demonstrates that DSM 34654 can enhance the emergence of winter wheat crops in the field, suggesting that an increased yield can be obtained.

[0023] Example 10 shows definition of DNA sequences that collectively characterize the best-performing Rahnella aceris strains, identified in Example 1 and 2, including DSM 34654. All TC and TP strains contain genes that are important for plant growth, such as the iron-scavenging siderophore desferrioxamine E gene cluster, indicating that all strains DSM 34654, TP1, TP3, TCI, and TC3 have a high potential to perform as plant growth promoters. Further, TP1, TP3, TCI, and TC3 comprise several sequence overlaps with the deposited DSM 34654, and thus it is expected that TP1, TP3, TCI, and TC3 comprise similar functional properties as DSM 34654

[0024] Example 11 shows the ability of DSM 34654 to affecti the harvest yield of maize when applied directly into the seed furrow during sowing. Applying a bacterial solution comprising DSM 34654 to the seed furrow during sowing leads to an increase in the emergence of maize plants and most importantly also an increase in the harvest yield of 17.37%, clearly demonstrating that DSM 34654 affects the harvest yield positively.

[0025] Example 12 shows that DSM 34654 exhibits a great ability to mineralize organic phosphate, such as phytate, compared to a commercial biostimulant strain, demonstrating that DSM 34654 has a high potential for supplying crops with otherwise unavailable nutrients in the field. Additionally, when coupled with its previously demonstrated ability to solubilize inorganic phosphate (Example 4), DSM 34654 emerges as a versatile biostimulant.

[0026] The aforementioned deposit was made by Bioomix Aps on 24 May 2023. The deposit was given the following reference number: DSM 34654. Thus, an object of the present invention relates to the provision of novel bacterial strains / biostimulants for improving plant growth.

[0027] In particular, it is an object of the present invention to provide novel bacterial strains / biostimulants for improving plant growth that can be effective at colder soil or air temperatures such as below 10°C.

[0028] Thus, one aspect of the invention relates to a bacteria, preferably isolated, or biologically pure bacterial culture comprising a) a genomic sequence according to any of SEQ ID NOs: 9-19, preferably SEQ ID NOs: 9-14; b) a genomic sequence having at least 66%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as preferably at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to any of SEQ ID NO: 9-19, preferably SEQ ID NOs: 9- 14; and / or c) a fragment of one or more of the sequences of a) or b), having a length of at least 400 nucleotides.

[0029] A second aspect of the present invention relates to a bacteria or biologically pure bacterial culture being Rahnella aceris, DSM 34654 deposited with the DSMZ [LEIBNIZ- INSTITUT DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH, Inhoffenstr. 7 B, D-38124 Braunschweig, Germany] on 24 May 2023.

[0030] A third aspect of the invention relates to a fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition comprising the bacteria or biologically pure bacterial culture according to the first and / or second aspect.

[0031] A fourth aspect of the present invention relates to a coating composition, preferably a seed coating composition, comprising the bacteria or biologically pure bacterial culture according to any of first and / or second aspect and / or the composition according to the third aspect.

[0032] A fifth aspect of the present invention relates to a plant seed coated with the composition according to the third aspect or coated with a coating composition according to the fourth aspect.

[0033] A sixth aspect of the present invention relates to use of a Rahnella bacteria for plant growth, such as a fertilizer or inoculum or biostimulant, at a temperature of between 2°C and 8°C, preferably around 5°C.

[0034] A seventh aspect of the present invention relates to use of a bacteria or biologically pure bacterial culture according to any of the first and / or second aspect, the composition according to the third aspect or the coating composition according to the fourth aspect, as a plant growth promoting agent, such as a fertilizer or inoculum or biostimulant.

[0035] An eighth aspect of the present invention relates to a method for stimulating plant growth comprising applying the bacteria or biologically pure bacterial culture according to any of the first and / or second aspect, the composition according to the third aspect, or the coating composition according to the fourth aspect to a plant, plant seed, a sowing forrow, soil and / or plant growth medium.

[0036] A nineth aspect of the present invention relates to a kit of parts for stimulating plant growth comprising

[0037] • a first container comprising the bacteria or biologically pure bacterial culture according to any of the first and / or second aspect, the composition according to the third aspect and / or the coating composition according to the fourth aspect; and

[0038] • instructions for applying the inoculum to plants, plant seeds, or a plant growth medium.

[0039] A further aspect of the invention relates to use of the (isolated) bacteria or biologically pure bacterial culture according to the invention or the composition according to the invention for solubilizing minerals in ores. A yet an aspect of the present invention relates to a bacteria or biologically pure bacterial culture according to the invention, a composition according to the invention or a coating composition according to the invention for use as a medicament.

[0040] Another aspect relates to a bacteria or biologically pure bacterial culture according to the invention or a composition according to the invention use in the treatment, alleviation and / or prevention of fungal infections.

[0041] Brief description of the figures

[0042] Figure 1 shows heatmaps and bar plots representing the area of colonies on Jensen medium (graphs to the left) or the solubilization index (S / I) on Pikovskaya medium (graphs to the right). Heatmaps represent scaled colony areas or scaled S / I values on day 1-4. Bar plots represent the increase in colony area on Jensen medium or S / I from day 1 to day 4,

[0043] Figure 2 shows a phylogenetic tree representing the genetic relatedness between DSM 34654 and all publicly available Rahnella aceris (n=8) and Rahnella aquatilis (n=4) genomes (as of October 11 2023, excluding atypical genomes). The grey area marks the node containing DSM 34654 (strain DSM 34654 is indicated with bold font),

[0044] Figure 3 shows the absence of a long genomic region (13,537 nucleotides, annotated as SEQ ID NO: 8) in the type strain of Rahnella aceris (SAP-19). The bottom part (light grey) represents a part of the assembled chromosome in strain DSM 34654. The middle part represents genomic regions that either overlap (black bars) or do not overlap (white space) with the type strain of Rahnella aceris (SAP-19). The top part represents genes (CDS; coding sequences in dark grey bars) and their associated annotations,

[0045] Figure 4 shows in vitro mineral-dissolving properties of DSM 34654 and of two strains isolated from commercially available microbial plant products at low, medium and high temperatures. A) Colony size of DSM 34654 and of two bacterial strains (Biol and Bio2) isolated from one biofungicide and one biostimulant commercially available product that were grown on Jensen medium (nitrogen-free agar) for 7 days at low (5°C), medium (15°C) or high (25°C) temperatures. Colony sizes were quantified on a log scale. B), C) and D) Solubilization index (SI, Areanaio / Areacoiony) for DSM 34654, Biol and Bio2 grown on Aleksandrow medium (insoluble potassium agar) for 10 days (B), Pikovskaya medium (insoluble phosphate agar) for 7 days (C) or iron phosphate medium for 15 days (D) at low (5°C), medium (15°C) or high (25°C) temperatures. Experiments were carried out in three replicates. Bars in each plot of A)-C) represent the mean across replicates and error bars represent standard deviations across replicates in each experimental group. Bars in D) represent binary data of iron-phosphate solubilization; l=solubilization, 0 = no solubilization,

[0046] Figure 5 shows the ability of DSM 34654 to inhibit the growth of phytopathogenic fungi. A) Spot on lawn assay: Dual cultures of DSM 34654 and Gaeumannomyces graminis, Pyrenophora teres f. teres, or Fusarium oxysporum (right column). Fungal negative control plates (left column). B) Plug assay: Dual cultures of DSM 34654 and Pyrenophora teres f. teres and Fusarium oxysporum (right column). Fungal negative control plates (left column).

[0047] Figure 6 shows the early germination performance of DSM 34654 seed coat in vitro. A) Emergence of the radicle from oilseed rape and winter wheat seeds that were either uncoated (Control) or coated with DSM 34654 seed coat (Treated). Seeds were placed in petri dishes on top of filter paper after treatment and emergence was quantified after one day. B) Emergence of the plumule from oilseed rape and winter wheat seeds that were either uncoated (Control) or coated with DSM 34654 seed coat (Treated). Seeds were placed in petri dishes on top of filter paper after treatment and emergence was quantified after two days. Bar plots are depicted as mean percentage emergence from seeds across 4 individual replicates. Error bars represent the standard error of the mean (SEM) across replicates. Asterisks indicate statistical significance (p<0.05) of DSM 34654 seed coat vs control using a one-sided Wilcoxon test,

[0048] Figure 7 shows the capacity of DSM 34654 seed coat to solubilize inorganic phosphate in vitro. Oilseed rape and winter wheat seeds that were either uncoated (Control) or coated with DSM 34654 seed coat (Treated) were placed on top of Pikovskaya medium. Pictures of petri dishes were taken two days following coating and plating and show significant clearing zones formed around DSM 34654-coated seeds (exemplified by arrows),

[0049] Figure 8 shows the effect of DSM 34654 seed coat on the emergence and root yield of oil radish crops from a Danish field (performed by an independent third- party contract research organization (VKST, Denmark)). A) Boxplots show the emergence of oil radish plants (quantified as plants per m2) that have developed from non-treated control seeds (left) or DSM 34654-treated seeds (right) across 4 replicates. The p-value indicates statistical significance (p=0.0142) of DSM 34654 treatment vs control seeds using a one-sided Wilcoxon test. B) Boxplots show the root yield from oil radish plants (quantified as fresh weight of roots in grams per 50 oil radish plants) that have developed from non-treated control seeds (left) or DSM 34654-treated seeds (right) across four replicates. The p-value indicates statistical significance (p=0.02857) of plants developed from DSM 34654-treated seeds vs plants developed from non-treated control seeds using a one-sided Wilcoxon test. C) Representative picture of oil radish plants that have developed from non-treated control (plants to the left) or DSM 34654-treated seeds (plants to the right),

[0050] Figure 9 shows the effect of DSM 34654 on sugar beet, potato and maize yields from a Danish field. A) Boxplots show the yield of sugar beet roots per plant that have developed from control or DSM 34654-treated sugar beet seeds (10 seeds planted in total per treatment). The p-value indicates statistical significance (p=0.001799) of DSM 34654 treatment vs control seeds using a one-sided Wilcoxon test. B) Representative picture of sugar beet plants that have developed from non-treated control (plants to the left) or DSM 34654-treated seeds (plants to the right). C) Boxplots show the yield of potatoes per plant that have developed from control or DSM 34654-treated seed potatoes (50 seed potatoes planted in total per treatment). The p-value indicates statistical significance (p=0.02854) of DSM 34654 treatment vs control seeds using a one-sided Wilcoxon test. D) Boxplots show the yield of maize cobs per plant that have developed from control or DSM 34654-treated maize seeds (10 maize seeds planted in total per treatment). The p-value indicates statistical significance (p=0.00278) of DSM 34654 treatment vs control seeds using a one-sided

[0051] Wilcoxon test, and

[0052] Figure 10 shows the effect of DSM 34654 on the early and late emergence of winter wheat plants from a Danish field (performed by an independent third-party contract research organization (VKST, Denmark)). Boxplots to the left of the vertical line represent early emergence (21 days after sowing) of winter wheat plants. Boxplots to the right of the vertical line represent late emergence (42 days after sowing) of winter wheat plants. The p-value indicates statistical significance (p=0.009644) in the late emergence of winter wheat plants comparing plants developed from seeds treated with a DSM 34654 seed coat to plant developed from non-treated control seeds using a one-sided Wilcoxon test.

[0053] Figure 11 shows emergence and harvest yield data from maize field trials performed by Ytteborg I / S - an independent third-party contract research organization placed in Denmark. A) Boxplots representing plants per m2(n=7 field parcels per experimental group). Boxplots to the left represent emergence data from fields where no fertilizer was applied (w / o start fertilizer). Boxplots to the right represent emergence data from fields that had received fertilization during sowing (with start fertilizer). B) Boxplots representing harvest yield of maize as ton per ha (t / ha) (n=7 field parcels per experimental group). Boxplots to the left represent yield data from fields where no fertilizer was applied (w / o start fertilizer). Boxplots to the right represent yield data from fields that had received fertilization during sowing (with start fertilizer). Control; untreated maize seeds, DSM 34654; applying DSM 34654 to the seed furrow during sowing. Start fertilizer represents an NP 19-8 fertilizer containing 23 kg / ha nitrogen (N) and 10 kg / ha phosphorous (P). * Indicates statistical significance (p<0.05) of DSM34654 treatment vs control (comparing fields received fertilization or not) using a onesided Wilcoxon rank sum test.

[0054] Figure 12 shows in vitro organic phosphate mineralization properties of DSM 34654 and a strain isolated from a commercially available biofungicide product (Biol). DSM 34654 and Biol were cultivated on standardized modified Pikowskaya agar plates with calcium phosphate (Cas PC )?) substituted for phytate for 7 days at 15°C and their colony and halo size were monitored throughout a week. Figure 13 shows quantifications of in vitro organic phosphate mineralization properties of DSM34654 and a strain isolated from a commercially available biofungicide product (Biol). DSM 34654 and Bio 1 were cultivated on standardized modified Pikowskaya agar plates with calcium phosphate (Cas PCk)?) substituted for phytate for 7 days at 15°C. Colony and halo size were monitored throughout a week and quantified by calculating the solubilization index (SI, SI = Areanaio / Areacoiony). Datapoints represent technical replicates (n = 3).

[0055] The present invention will now be described in more detail in the following.

[0056] Detailed description of the invention

[0057] Definitions

[0058] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0059] Effective amount

[0060] In the present context, the term "effective amount" refers to a quantity which is sufficient to result in a statistically significant increase in a desirable plant property such as germination, emergence, growth and / or of protein yield and / or of grain / crop yield of a plant as compared to the germination, emergence growth, protein yield and grain yield of the control-treated plant.

[0061] Inoculant

[0062] The term "inoculant" as described in this invention is defined in several Federal, or State regulations as:

[0063] (1) "soil or plant inoculants shall include any carrier or culture of a specific microorganism or mixture of micro-organisms represented to improve the soil or the growth, quality, or yield of plants, and shall also include any seed or fertilizer represented to be inoculated with such a culture" (New York State 10-A Consolidated Law);

[0064] (2) "substances other than fertilizers, manufactured, sold or represented for use in the improvement of the physical condition of the soil or to aid plant growth or crop yields" (Canada Fertilizers Act); (3) "a formulation containing pure or predetermined mixtures of living bacteria, fungi or virus particles for the treatment of seed, seedlings or other plant propagation material for the purpose of enhancing the growth capabilities or disease resistance or otherwise altering the properties of the eventual plants or crop" (Ad hoc European Working Group, 1997); or

[0065] (4) "meaning any chemical or biological substance of mixture of substances or device distributed in this state to be applied to soil, plants or seeds for soil corrective purposes; or which is intended to improve germination, growth, quality, yield, product quality, reproduction, flavor, or other desirable characteristics of plants or which is intended to produce any chemical, biochemical, biological or physical change in soil" (Section 14513 of the California Food and Agriculture Code).

[0066] Biostimulant

[0067] In the present context a "biostimulant" or plant biostimulant is any substance or microorganism applied to plants with the aim to enhance nutrition efficiency, abiotic stress tolerance and / or crop quality traits, regardless of its nutrients content. By extension, plant biostimulants also designate commercial products containing mixtures of such substances and / or microorganisms.

[0068] In here the terms "fertilizer", "inoculant" and "biostimulant" may be used interchangeably.

[0069] Isolated bacteria and biologically pure bacterial culture

[0070] In the present context, the terms "isolated bacteria" and "biologically pure bacterial culture" refer to isolated bacteria or a culture of bacteria containing no other bacterial species in quantities sufficient to interfere with the replication or function of the culture or be detected by normal bacteriological techniques. Stated another way, it is a culture wherein virtually all of the bacterial cells present are of the selected strain.

[0071] Phrased in a different way, the "biologically pure bacterial culture" is at least 90% pure, such as at least 95% pure, such as at least 98% pure, such as at least 99% pure, such as 99.5% pure. Percentage is to be determined by a number of bacteria in the culture.

[0072] Preferably the bacteria according to the invention is an isolated bacteria, such as forming part of an isolated composition.

[0073] In the present context, the term "plant growth promoting agent" refers to the ability to enhance or increase at least one desirable plant trait or property such as the plant's height, weight, leaf size, root size, or stem size, to increase protein yield from the plant or to increase grain / crop yield of the plant.

[0074] In the present context, the term "sequence identity" indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length or between two nucleic acid sequences of substantially equal length. The two sequences to be compared must be aligned to best possible fit with the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as , wherein

[0075] Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Ndif=2 and Nref=8). A gap is counted as non-identity of the specific residue(s), i.e. the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (Ndif=2 and Nref=8). Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program for protein alignment (W.R Pearson and D.J. Lipman (1988)).

[0076] For calculations of sequence identity when comparing polypeptide fragments with longer amino acid sequences, the polypeptide fragment is aligned with a segment of the longer amino acid sequence. The polypeptide fragment and the segment of the longer amino acid sequence may be of substantially equal length. Thus, the polypeptide fragment and the segment of the longer amino acid sequence may be of equal length. After alignment of the polypeptide fragment with the segment of the longer amino acid sequence, the sequence identity is computed as described above.

[0077] A preferred minimum percentage of sequence identity is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.

[0078] Solubilization

[0079] In the present context, the term "solubilization" in relation to mineral solubilization is to be understood as the dissolution of insoluble minerals such as calcium phosphate (Cas PC )?), iron phosphate (FePC>4-4 H2O), and potassium alumino-silicate and thereby increase the availability of common plant nutrients. Soil phosphate and potassium exist to a large extent in insoluble complexes bound to insoluble inorganic minerals, which are unavailable for plants. Thus, solubilization / dissolution of these minerals may improve mineral availability for plant uptake.

[0080] The level of solubilization can be determined by: providing solid media such as agar plates comprising insoluble mineral complexes,

[0081] - cultivating bacteria of interest,

[0082] - and calculating a solubilization index (SI) using the formula:

[0083] SI = AreaHaio / Areacoiony

[0084] The "Area" is determined by measuring the diameter of the bacterial colonies and the corresponding clear / halo zones.

[0085] Bacteria or biologically pure bacterial culture

[0086] As outlined above, the inventing team has identified, isolated and propagated a newly identified strain of bacteria, namely the Rahnella aceris strain DSM 34654 deposited with deposit number DSM 34654. The strain may be used to promote a wide variety of plant health parameters. The bacterial strain of the present invention was identified by isolating bacterial strains with mineral-dissolving properties from the soil of a Danish grassland and screened to identify the best-performing bacterial strains. A comparative screening test was conducted to identify the most effective candidates among the isolated strains (n = 28). The bacterial strain of the present invention was selected amongst the top performing bacterial strains having an increased ability to solubilize phosphorous (Example 1).

[0087] Thus, an aspect of the invention relates to a bacteria, preferably isolated, or biologically pure bacterial culture comprising a) a genomic sequence according to any of SEQ ID NOs: 9-19, preferably SEQ ID NOs: 9-14; b) a genomic sequence having at least 66%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as preferably at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to any of SEQ ID NO: 9-19, preferably SEQ ID NOs: 9- 14; and / or c) a fragment of one or more of the sequences of a) or b), having a length of at least 400 nucleotides.

[0088] In an embodiment, the bacteria further comprises d) SEQ ID NO. 15 or a sequence having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as preferably at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 15.

[0089] SEQ ID NO. 15 is comprised in / of plasmid DNA of the strains TP1, TP2 (DSM 34654), TP3, TCI and TC3. Thus, in an embodiment the sequence under d) is positioned in non-genomic DNA, such as in a plasmid.

[0090] In a preferred embodiment, the bacteria is an isolated bacteria.

[0091] As shown in Example 3, comparative genome analysis of the bacterial strain DSM 34654 reveals that DSM 34654 is a novel Rahnella aceris strain. In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 9, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 9.

[0092] In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 10, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 10.

[0093] In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 11, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 11.

[0094] In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 12, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 12.

[0095] In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 13, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 13.

[0096] In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 14, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 14.

[0097] In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 15, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 15. In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 16, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 16.

[0098] In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 17, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 17.

[0099] In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 18, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 18.

[0100] In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 19, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 19.

[0101] In an embodiment, the bacteria or biologically pure bacterial culture according to the invention comprises any of SEQ ID NO's: 9-19, such as one or more of SEQ ID NO's: 9-19, such as one or more of SEQ ID NO: 9-14. Thus, it is to be understood that the bacteria may also comprise two or more, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, or preferably such as all of SEQ ID NO: 9-19, or with a sequence identity for SEQ ID NO: 9-19 as defined above.

[0102] Thus, in an embodiment, the bacteria or biologically pure bacterial culture according to the invention comprising SEQ ID NO: 9-19, such as SEQ ID NO: 9- 14.

[0103] SEQ ID No's: 9-12 form part of SEQ ID NO: 8, wherein short DNA sequences SEQ ID Nos: 13-15 are identified outside of the long genomic region. Example 3 demonstrates that this identified large genomic region consisting of 13,537 nucleotides (SEQ ID NO: 8) showing <66% coverage in other prokaryotic genomes and importantly no identity in R. aceris strains.

[0104] The bacterial strains TP1 and TP3 as described in the Examples comprise SEQ ID NOs: 9-15.

[0105] The bacterial strains TCI and TC3 as described in the Examples comprise SEQ ID NO: 15.

[0106] As seen in Example 10, TP1, TP3, TCI, TC3 further comprise SEQ ID NOs: 16-19.

[0107] Thus, in another embodiment, the bacteria or biologically pure bacterial culture according to the invention, comprising, a) a genomic sequence according to SEQ ID NO: 8; b) a genomic sequence having at least 90% sequence identity to SEQ ID NO: 8, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 8; and / or c) a fragment of the sequence of a) or b), having a length of at least 5000 nucleotides, such as at least 8000 nucleotides, such as at least 10000 nucleotides, such as at least 13000 nucleotides.

[0108] The DSM 34654 strain according to the invention has been annotated as being a Rahnella aceris species. Thus, in an embodiment, the bacteria or biologically pure bacterial culture is of the genus Rahnella, such as species Rahnella aceris.

[0109] In an embodiment, the bacteria or biologically pure bacterial culture comprises 16S rRNA genes encoded by one or more of SEQ ID NOs: 1-7, such as two or more, such as three or more, such as four or more, such as five or more, such as six or more of SEQ ID NOs: 1-7. Preferably, the bacteria or biologically pure bacterial culture comprises SEQ ID NOs: 1-7.

[0110] As shown in Example 3, the strain of the invention has been identified to comprise 16S rRNA genes encoded by SEQ ID NOs: 1-7.

[0111] In an embodiment, the isolated bacteria or biologically pure bacterial culture being Rahnella aceris, DSM 34654 deposited with the DSMZ [(LEIBNIZ-INSTITUT DSMZ- DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH, Inhoffenstr. 7 B, D-38124, Braunschweig, Germany) on 24 May 2023] . In here this strain is also named TP4121.

[0112] In yet an aspect the invention relates to an (isolated) bacteria or biologically pure bacterial culture being Rahnella aceris, DSM 34654 deposited with the DSMZ [(LEIBNIZ-INSTITUT DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH, Inhoffenstr. 7 B, D-38124, Braunschweig, Germany) on 24 May 2023] .

[0113] For colder regions it would be an advantage if the bacteria is active under colder conditions. Thus, in an embodiment, the bacteria or biologically pure bacterial culture according to the invention is

[0114] - capable of solubilizing calcium phosphate at 5°C, and / or iron phosphate at 5°C and / or potassium aluminum silicate at 5°C, and / or insoluble organic phosphate, such as phytate, at 15°C; and / or

[0115] - capable of inhibiting one or more phytopathogenic fungi, such as phytopathogenic fungi specific for (colder) regions.

[0116] As seen in Example 4, it is evident that the bacteria is capable of solubilizing calcium phosphate (Cas PC )?), iron phosphate (FePC>4-4 H2O), and potassium alumino-silicate at low (5°C), medium (15°C), and high (25°C) temperatures. Especially, the bacteria of the present invention is able to grow and display bacterial activity at temperatures down to 5°C.

[0117] As seen in Example 5, the bacteria has antifungal activity against phytopathogenic fungi Gaeumannomyces graminis var. tritici, Pyrenophora teres and Fusarium oxysporum.

[0118] As seen in Example 12, the bacteria are capable of solubilizing phytate at a temperature of 15°C. Hence, the bacteria exhibit a great ability to mineralize organic phosphate.

[0119] In an embodiment, the solubilization of calcium phosphate, and / or iron phosphate, and / or potassium aluminum silicate at 5°C is at a level of at least 80% to, equal to and / or above a level of solubilization at 15°C and / or 25°C.

[0120] As seen in Figure 4, the solubilization of minerals by the bacteria of the present invention is approximately the same level at a temperature of 5°C as at higher temperatures at 15°C and 25°C. Hence, lower temperatures at 5°C do not cause a decreased activity, such as ability to solubilize minerals, of the bacteria of the invention.

[0121] This demonstrates that the bacteria of the present invention has a high potential for supplying crops in the field with otherwise unavailable nutrients through the entire plant life cycle from as early as germination in cold soil until maturation and harvest.

[0122] In an embodiment, the bacteria or biologically pure bacterial culture according to the invention comprises a capacity for transforming soil-bound iron into forms that plants can utilize for growth.

[0123] As seen in Example 3, DSM 34654 contains a genomic region encoding the siderophore desferrioxamine E (MIBiG accession BGC0001572 found in Pantoea agglomerans); an iron-scavenging bioactive molecule secreted by a variety of microorganisms, including plant growth-promoting bacteria, that allows for increased iron uptake in plant cells which thereby promotes plant growth.

[0124] In an embodiment, the level of solubilization is determined by a solubilization index (SI) calculated by the formula:

[0125] SI = AreaHaio / Areacoiony.

[0126] As seen in Example 4, the solubilization index is calculated using the above formula.

[0127] In an embodiment, the SI is at least 7, such as at least 10, such as between 10 and 23. Preferably the SI is between 10 and 21.

[0128] As seen in Example 1 the SI of the strains were measured between 11.0 and 19.6, and in Example 4 the SI of the strains were measured between 11.7 and 20.6.

[0129] In a further embodiment, the SI depends on temperature after cultivation start and / or incubation time.

[0130] In yet an embodiment, the temperature is between 2°C and 30°C, such as between 5°C and 25°C, such as between 5°C and 10°C, preferably between 5°C and 8°C. As seen in Example 1, the strains were cultivated at 8°C for measuring the SI.

[0131] As seen in Example 4, the strains were cultivated at 5°C, 15°C, and 25°C for measuring the SI, wherein the SI on Pikovskaya at each temperature at day 7 was:

[0132] • 20.6 at 5°C,

[0133] • 11.7 at 15°C, and

[0134] • 19.3 at 25°C.

[0135] In an embodiment, the bacteria or biologically pure bacterial culture comprises a capacity for solubilizing insoluble organic phosphate, such as phytate, at day 1 to day 10, such as day 1 to day 7 at 15°C.

[0136] As seen in Example 12, DSM 34654 displayed phytate-degrading activities at day 1 and 4 and still maintained phytate-degrading activities at day 7, wherein the SI on modified Pikovskaya was:

[0137] • 4.99 at 15°C.

[0138] These results demonstrate that DSM 34654 exhibits a high capacity for phytate mineralization. The results suggest that DSM 34654 has a higher capacity for mineralizing organic phosphate, which would otherwise remain unavailable to plants.

[0139] In an embodiment, the bacteria or biologically pure bacterial culture is dehydrated, such as spray-dried.

[0140] In an embodiment, the bacteria or biologically pure bacterial culture does not contain the rhizoxin-biosynthetic gene cluster. In a related embodiment the bacteria or biologically pure bacterial culture does not contain the rhi and / or rzx gene loci. As explained in example 3, this gene cluster may be involved in producing carcinogenic secondary metabolites.

[0141] Fertilizer or inoculant or biostimulant

[0142] The isolated bacteria or biologically pure bacterial culture according to the invention may form part of different compositions. Thus, an aspect of the invention relates to a fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition comprising the bacteria or biologically pure bacterial culture according to the invention. The composition may comprise other microorganisms. Thus, in an embodiment, the fertilizer and / or inoculant and / or biostimulant composition further comprises other microorganisms, such as other microorganisms able to function as a biostimulant.

[0143] In an embodiment, the composition further comprises one or more agriculturally acceptable carriers.

[0144] In a related embodiment, the agriculturally acceptable carrier is selected from the group consisting of a dispersant, a surfactant, an additive, water, a thickener, an anti-caking agent, residue breakdown, a composting formulation, a granular application, diatomaceous earth, an oil, a coloring agent, a stabilizer, a preservative, a polymer, biopolymer, a coating, or a combination thereof.

[0145] In yet an embodiment, the composition comprises a biopolymer, oligosaccharide, disaccharide or monosaccharide selected from the group consisting of pectin, alginate, chitosan, cellulose, a cellulose derivative, starch, maltodextrin, chitin, glucose, trehalose, sucrose, xanthan gum, guar gum, diutan gum or a biopolymer derived from a natural source, possibly chemically modified afterwards.

[0146] In another embodiment, the composition is formulated as a liquid formulation for application to plants or to a plant growth medium, or a solid formulation for application to plants or to a plant growth medium.

[0147] In yet another embodiment, the composition is formulated as a granular formulation or a powder formulation.

[0148] In an embodiment, the composition further comprises 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.

[0149] In an embodiment, the composition comprises one or more of 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, calcium nitrate, dolomitic limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, urea ammonium nitrate, sulfur- coated urea, polymer- coated urea, isobutylidene diurea, K2S04-2MgS04, 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, biochar, sludge, green manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion, or a combination thereof.

[0150] In an embodiment, the micronutrient fertilizer material comprises one or more of 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 carbonate, a zinc frit, zinc phosphate, a zinc chelate, or a combination thereof.

[0151] Coating composition

[0152] The composition according to the invention may be a coating composition, such as a seed coating composition. Thus, a further aspect of the invention relates to a coating composition, preferably a seed coating composition, comprising the bacteria or biologically pure bacterial culture according to the invention and / or the composition according to the invention.

[0153] In an embodiment, the coating composition comprises a biopolymer promoting adherence to a plant seed. As shown in Example 6, pectin has been tested as biopolymers promoting adherence to plant seeds. In an embodiment, the coating composition is formulated as an aqueous or oilbased solution for application to seeds, preferably aqueous.

[0154] In another embodiment, the coating composition is formulated as a powder or granular formulation for application to seeds.

[0155] Coated plant seed

[0156] The present invention also relates to seeds coated with compositions according to the invention. Thus, an aspect relates to a plant seed coated with the composition according to the invention or coated with a coating composition according to the invention.

[0157] In an embodiment, the plant seed is a dicotyledon, monocotyledon or a gymnosperm seed.

[0158] In yet an embodiment, the plant seed being selected from the group consisting of a crop seed, such as barley seed, such as spring or winter barley, oilseed, such as rapeseed or oil radish seeds, wheat, such as winter or spring wheat, oats, triticale, maize, rye, grass, clover, broad bean, lupines, strawberries, tomatoes, cucumber, peas, potatoes, onions, carrots and sugar beets.

[0159] As seen in Examples 6-9, the plant seeds listed above coated with the composition according to the invention or coated with a coating composition according to the invention are shown to result in an increased growth and yield.

[0160] Particularly, it is seen in Example 9, that the plant seeds coated with the composition according to the invention or coated with a coating composition according to the invention are able to grow and provide an increased yield at low temperatures during the winter in Denmark.

[0161] In yet an embodiment, the plant seed being selected from the group consisting of a cover crop seed, such as fodder radish, clover, yellow mustard or Phacelia.

[0162] In yet an embodiment, the plant seed being selected from the group consisting of trees, bushes or grasses. Uses and methods

[0163] An aspect of the invention relates to the use of an (isolated) Rahnella bacteria for plant growth, such as a fertilizer or inoculum or biostimulant, at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C.

[0164] In an embodiment, the temperature is a surface temperature.

[0165] An aspect of the invention relates to the use of an (isolated) bacteria or biologically pure bacterial culture according to the invention, the composition according to the invention or the coating composition according to the invention, as a plant growth promoting agent, such as a fertilizer or inoculum or biostimulant, preferably at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C.

[0166] In an embodiment, the use as a plant growth-promoting agent is for:

[0167] • solubilizing inorganic minerals or salts, such as phosphate, and / or potassium, and / or iron, and / or aluminium, and / or zinc, and / or manganese, and / or organic phosphate, such as phytate; and / or

[0168] • chelating inorganic minerals, such as iron, zinc, manganese, copper; and / or

[0169] • allowing for growth under low soil nitrogen conditions; and / or

[0170] • fixating atmospheric nitrogen; and / or

[0171] • increasing plant growth, such as plant length, leaf diameter and / or root length; and / or

[0172] • improving germination of the seeds; and / or

[0173] • improving emergence of the plants; and / or

[0174] • increasing biomass; and / or

[0175] • increasing growth; and / or

[0176] • increasing crop yield; and / or

[0177] • inducing anti-fungal effects; and / or

[0178] • increasing harvest yield; and / or

[0179] • increasing hectoliter weight; and / or

[0180] • increasing protein content; and / or • increasing protein yield; and / or

[0181] • increasing oil content; and / or

[0182] • increasing oil yield; and / or

[0183] • reducing mycotoxin contamination; and / or

[0184] • reducing seed-borne fungal or bacterial contamination; and / or

[0185] • improving taste, smell or appearance; and / or

[0186] • replacing or substituting any chemical product used in plant production while retaining one or more desirable plant parameters.

[0187] The stimulation of plant growth achieved by the present methods and uses can be measured in a number of ways. Stimulation of plant growth can be determined by increase in the average height of the plant, such as an increase of at least 5%, by at least 10%, by at least 15% or by at least 20% as compared to the average height of plants grown under the same conditions but that have not been treated according to the present invention. Also, stimulation of plant growth can be determined by an increase in the average leaf diameter of the leaves of plant, such as an increase of at least 5%, of at least 10%, of at least 15% or of at least 20% as compared to the average leaf diameter of plants grown under the same conditions but that have not been treated according to the present invention. Similarly, stimulation of plant growth can be shown by an increase in instances the Root length of the plant, such as an increase of at least 5%, of at least 10%, of at least 15% or of at least 20% as compared to the average root length of the plants grown under the same conditions but that have not been treated according to the present invention. As outlined above, stimulation of plant growth can also be estimated using other parameters.

[0188] In an embodiment, the inorganic minerals or salts solubilized are selected from the group consisting of

[0189] • minerals, such as rock phosphate, potash, lime, clay, ground rocks, sand, silt, sediment and natural deposits;

[0190] • salts, such as ammonium phosphate, potassium phosphate, potassium nitrate, potassium chloride, ammonium sulfate, calcium phosphate; calcium sulphate, magnesium phosphate, iron phosphate, potassium alumino silicate (feldspar, mica), and salts that contain potassium or phosphate • fertilizing substances, such as mineral fertilizer, NPK fertilizer, NS fertilizer, K fertilizer, P fertilizer, N fertilizer, organic fertilizer, manure, sludge, compost, biowaste, biochar, biogas residue, ash, wood ash, bone ash, bone meal, urine, faeces or a plant-based fertilizer.

[0191] As shown in the example section, the isolated bacteria or biologically pure bacterial culture can grow under low temperature conditions. Thus, in an embodiment, the use takes place at field temperatures (measured as the soil surface temperature) in the range -10°C to 15°C, such as -5°C to 15°C, preferably 0 to 15°C, more preferably such as 2 to 10°C, such 2 to 8°C.

[0192] In another embodiment, the (isolated) bacteria or biologically pure bacterial culture according to the invention, the composition according to the invention or the coating composition according to the invention is applied in an effective amount.

[0193] In yet another embodiment, the use takes place in Scandinavia, such as Norway, Sweden, Finland, Denmark, such as in Jutland, Fyn, Zealand, and / or Great Britain, and / or Germany.

[0194] In an embodiment, the plant is a dicotyledon, monocotyledon or a gymnosperm.

[0195] In another embodiment, the plant is selected from the group consisting of a crop seed, such as barley seed, such as spring barley, oilseed, such as rapeseed, wheat, such as winter wheat.

[0196] The dicotyledon can be selected from the group consisting of bean, pea, tomato, pepper, squash, alfalfa, almond, aniseseed, apple, apricot, arracha, artichoke, avocado, 29cheuch groundnut, beet, bergamot, black pepper, black wattle, blackberry, blueberry, bitter orange, bok- choi, Brazil nut, breadfruit, broccoli, broad bean, Brussels sprouts, buckwheat, cabbage, camelina, Chinese cabbage, cacao, cantaloupe, caraway seeds, cardoon, carob, carrot, cashew nuts, cassava, castor bean, cauliflower, celeriac, celery, cherry, chestnut, chickpea, chicory, chili pepper, chrysanthemum, cinnamon, citron, Clementine, clove, clover, coffee, cola nut, colza, corn, cotton, cottonseed, cowpea, crambe, cranberry, cress, cucumber, currant, custard apple, drumstick tree, earth pea, eggplant, endive, fennel, fenugreek, fig, filbert, flax, geranium, gooseberry, gourd, grape, grapefruit, guava, hemp, hempseed, henna, hop, horse bean, horseradish, indigo, jasmine, Jerusalem artichoke, jute, kale, kapok, kenaf, kohlrabi, kumquat, lavender, lemon, lentil, lespedeza, lettuce, lime, liquorice, litchi, loquat, lupine, macadamia nut, mace, mandarin, mangel, mango, medlar, melon, mint, mulberry, mustard, nectarine, niger seed, nutmeg, okra, olive, opium, orange, papaya, parsnip, pea, peach, peanut, pear, pecan nut, persimmon, pigeon pea, pistachio nut, plantain, plum, pomegranate, pomelo, poppy seed, potato, sweet potato, prune, pumpkin, quebracho, quince, trees of the genus Cinchona, quinoa, radish, ramie, rapeseed, raspberry, rhea, rhubarb, rose, rubber, rutabaga, safflower, sainfoin, salsify, sapodilla, Satsuma, scorzonera, sesame, shea tree, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, swede, sweet pepper, tangerine, tea, teff, tobacco, tomato, trefoil, tung tree, turnip, urena, vetch, walnut, watermelon, yerba mate, wintercress, shepherd's purse, garden cress, peppercress, watercress, pennycress, star anise, laurel, bay laurel, cassia, jamun, dill, tamarind, peppermint, oregano, rosemary, sage, soursop, pennywort, calophyllum, balsam pear, kukui nut, Tahitian chestnut, basil, huckleberry, hibiscus, passionfruit, star apple, sassafras, cactus, St. John's wort, loosestrife, hawthorn, cilantro, curry plant, kiwi, thyme, zucchini, ulluco, jicama, waterleaf, spiny monkey orange, yellow mombin, starfruit, amaranth, wasabi, Japanese pepper, yellow plum, mashua, Chinese toon, New Zealand spinach, bower spinach, ugu, tansy, chickweed, jocote, Malay apple, paracress, sowthistle, Chinese potato, horse parsley, hedge mustard, campion, agate, cassod tree, thistle, burnet, star gooseberry, saltwort, glasswort, sorrel, silver lace fern, collard greens, primrose, cowslip, purslane, knotgrass, terebinth, tree lettuce, wild betel, West African pepper, yerba santa, tarragon, parsley, chervil, land cress, burnet saxifrage, honeyherb, butterbur, shiso, water pepper, perilla, bitter bean, oca, kampong, Chinese celery, lemon basil, Thai basil, water mimosa, cicely, cabbagetree, moringa, mauka, ostrich fern, rice paddy herb, yellow sawah lettuce, lovage, pepper grass, maca, bottle gourd, hyacinth bean, water spinach, catsear, fishwort, Okinawan spinach, lotus sweetjuice, gallant soldier, 30cheuchz, arugula, cardoon, caigua, 30cheuch, chipilin, samphire, mampat, ebolo, ivy gourd, cabbage thistle, sea kale, chaya, huauzontle, Ethiopian mustard, magenta spreen, good king henry, epazole, lamb's quarters, centella plumed cockscomb, caper, rapini, napa cabbage, mizuna, Chinese savoy, kai-lan, mustard greens, Malabar spinach, chard, marshmallow, climbing wattle, China jute, paprika, annatto seed, spearmint, savory, marjoram, cumin, chamomile, lemon balm, allspice, bilberry, cherimoya, cloudberry, damson, pitaya, durian, elderberry, feijoa, jackfruit, jambul, jujube, physalis, purple mangosteen, rambutan, redcurrant, blackcurrant, salal berry, satsuma, ugli fruit, azuki bean, black bean, black-eyed pea, borlotti bean, common bean, green bean, kidney bean, lima bean, mung bean, navy bean, pinto bean, runner bean, mangetout, snap pea, broccoflower, calabrese, nettle, bell pepper, raddichio, daikon, white radish, skirret, tat soi, broccolini, black radish, burdock root, fava bean, broccoli raab, lablab, lupin, sterculia, velvet beans, winged beans, yam beans, mulga, ironweed, umbrella bush, tjuntjula, wakalpulka, witchetty bush, wiry wattle, chia, beech nut, candlenut, colocynth, mamoncillo, Maya nut, mongongo, ogbono nut, paradise nut, and cempedak.

[0197] The dicotyledon can be from a family selected from the group consisting of Acanthaceae (acanthus), Aceraceae (maple), Achariaceae, Achatocarpaceae (achatocarpus), Actinidiaceae (Chinese gooseberry), Adoxaceae (moschatel), Aextoxicaceae, Aizoaceae (fig marigold), Akaniaceae, Alangiaceae, Alseuosmiaceae, Alzateaceae, Amaranthaceae (amaranth), Amborellaceae, Anacardiaceae (sumac), Ancistrocladaceae, Anisophylleaceae, Annonaceae (custard apple), Apiaceae (carrot), Apocynaceae (dogbane), Aquifoliaceae (holly), Araliaceae (ginseng), Aristolochiaceae (birthwort), Asclepiadaceae (milkweed), Asteraceae (aster), Austrobaileyaceae, Balanopaceae, Balanophoraceae (balanophora), Balsaminaceae (touch-me- not), Barbeyaceae, Barclayaceae, Basellaceae (31cheuch), Bataceae (saltwort), Begoniaceae (begonia), Berberidaceae (barberry), Betulaceae (birch), Bignoniaceae (trumpet creeper), Bixaceae (lipstick tree), Bombacaceae (kapok tree), Boraginaceae (borage), Brassicaceae (mustard, also Cruciferae), Bretschneideraceae, Brunelliaceae (brunellia), Bruniaceae, Brunoniaceae, Buddlejaceae (butterfly bush), Burseraceae (frankincense), Buxaceae (boxwood), Byblidaceae, Cabombaceae (water shield), Cactaceae (cactus), Caesalpiniaceae, Callitrichaceae (water starwort), Calycanthaceae (strawberry shrub), Calyceraceae (calycera), Campanulaceae (bellflower), Canellaceae (canella), Cannabaceae (hemp), Capparaceae (caper), Caprifoliaceae (honeysuckle), Cardiopteridaceae, Caricaceae (papaya), Caryocaraceae (souari), Caryophyllaceae (pink), Casuarinaceae (she-oak), Cecropiaceae (cecropia), Celastraceae (bittersweet), Cephalotaceae, Ceratophyllaceae (hornwort), Cercidiphyllaceae (katsura tree), Chenopodiaceae (goosefoot), Chloranthaceae (chloranthus), Chrysobalanaceae (cocoa plum), Circaeasteraceae, Cistaceae (rockrose), Clethraceae (clethra), Clusiaceae (mangosteen, also Guttiferae), Cneoraceae, Columelliaceae, Combretaceae (Indian almond), Compositae (aster), Connaraceae (cannarus), Convolvulaceae (morning glory), Coriariaceae, Cornaceae (dogwood), Corynocarpaceae (karaka), Crassulaceae (stonecrop), Crossosomataceae (crossosoma), Crypteroniaceae, Cucurbitaceae (cucumber), Cunoniaceae (cunonia), Cuscutaceae (dodder), Cyrillaceae (cyrilla), Daphniphyllaceae, Datiscaceae (datisca), Davidsoniaceae, Degeneriaceae, Dialypetalanthaceae, Diapensiaceae (diapensia), Dichapetalaceae, Didiereaceae, Didymelaceae, Dilleniaceae (dillenia), Dioncophyllaceae, Dipentodontaceae, Dipsacaceae (teasel), Dipterocarpaceae (meranti), Donatiaceae, Droseraceae (sundew), Duckeodendraceae, Ebenaceae (ebony), Elaeagnaceae (oleaster), Elaeocarpaceae (32cheuchzeri), Elatinaceae (waterwort), Empetraceae (crowberry), Epacridaceae (epacris), Eremolepidaceae (catkinmistletoe), Ericaceae (heath), Erythroxylaceae (coca), Eucommiaceae, Eucryphiaceae, Euphorbiaceae (spurge), Eupomatiaceae, Eupteleaceae, Fabaceae (pea or legume), Fagaceae (beech), Flacourtiaceae (flacourtia), Fouquieriaceae (ocotillo), Frankeniaceae (32cheuchze), Fumariaceae (fumitory), Garryaceae (silk tassel), Geissolomataceae, Gentianaceae (gentian), Geraniaceae (geranium), Gesneriaceae (gesneriad), Globulariaceae, Gomortegaceae, Goodeniaceae (goodenia), Greyiaceae, Grossulariaceae (currant), Grubbiaceae, Gunneraceae (gunnera), Gyrostemonaceae, Haloragaceae (water milfoil), Hamamelidaceae (witch hazel), Hernandiaceae (hernandia), Himantandraceae, Hippocastanaceae (horse chestnut), Hippocrateaceae (hippocratea), Hippuridaceae (mare's tail), Hoplestigmataceae, Huaceae, Hugoniaceae, Humiriaceae, Hydnoraceae, Hydrangeaceae (hydrangea), Hydrophyllaceae (waterleaf), Hydrostachyaceae, Icacinaceae (icacina), Idiospermaceae, Illiciaceae (star anise), Ixonanthaceae, Juglandaceae (walnut), Julianiaceae, Krameriaceae (krameria), Lacistemataceae, Lamiaceae (mint, also Labiatae), Lardizabalaceae (lardizabala), Lauraceae (laurel), Lecythidaceae (brazil nut), Leeaceae, Leitneriaceae (corkwood), Lennoaceae (lennoa), Lentibulariaceae (bladderwort), Limnanthaceae (meadow foam), Linaceae (flax), Lissocarpaceae, Loasaceae (loasa), Loganiaceae (logania), Loranthaceae (showy mistletoe), Lythraceae (loosestrife), Magnoliaceae (magnolia), Malesherbiaceae, Malpighiaceae (32cheuchz cherry), Malvaceae (mallow), Marcgraviaceae (shingle plant), Medusagynaceae, Medusandraceae, Melastomataceae (melastome), Meliaceae (mahogany), Melianthaceae, Mendonciaceae, Menispermaceae (moonseed), Menyanthaceae (buckbean), Mimosaceae, Misodendraceae, Mitrastemonaceae, Molluginaceae (carpetweed), Monimiaceae (monimia), Monotropaceae (Indian pipe), Moraceae (mulberry), Moringaceae (horseradish tree), Myoporaceae (myoporum), Myricaceae (bayberry), Myristicaceae (nutmeg), Myrothamnaceae, Myrsinaceae (myrsine), Myrtaceae (myrtle), Nelumbonaceae (lotus lily), Nepenthaceae (East Indian pitcherplant), Neuradaceae, Nolanaceae, Nothofagaceae, Nyctaginaceae (four- o'clock), Nymphaeaceae (water lily), Nyssaceae (sour gum), Ochnaceae (ochna), Olacaceae (olax), Oleaceae (olive), Oliniaceae, Onagraceae (evening primrose), Oncothecaceae, Opiliaceae, Orobanchaceae (broom rape), Oxalidaceae (wood sorrel), Paeoniaceae (peony), Pandaceae, Papaveraceae (poppy), Papilionaceae, Paracryphiaceae, Passifloraceae (passionflower), Pedaliaceae (sesame), Pellicieraceae, Penaeaceae, Pentaphragmataceae, Pentaphylacaceae, Peridiscaceae, Physenaceae, Phytolaccaceae (pokeweed), Piperaceae (pepper), Pittosporaceae (pittosporum), Plantaginaceae (plantain), Platanaceae (plane tree), Plumbaginaceae (leadwort), Podostemaceae (river weed), Polemoniaceae (phlox), Polygalaceae (milkwort), Polygonaceae (buckwheat), Portulacaceae (purslane), Primulaceae (primrose), Proteaceae (protea), Punicaceae (pomegranate), Pyrolaceae (shinleaf), Quiinaceae, Rafflesiaceae (rafflesia), Ranunculaceae (buttercup orranunculus), Resedaceae (mignonette), Retziaceae, Rhabdodendraceae, Rhamnaceae (buckthorn), Rhizophoraceae (red mangrove), Rhoipteleaceae, Rhynchocalycaceae, Rosaceae (rose), Rubiaceae (madder), Rutaceae (rue), Sabiaceae (sabia), Saccifoliaceae, Salicaceae (willow), Salvadoraceae, Santalaceae (sandalwood), Sapindaceae (soapberry), Sapotaceae (sapodilla), Sarcolaenaceae, Sargentodoxaceae, Sarraceniaceae (pitcher plant), Saururaceae (lizard's tail), Saxifragaceae (saxifrage), Schisandraceae (schisandra), Scrophulariaceae (figwort), Scyphostegiaceae, Scytopetalaceae, Simaroubaceae (quassia), Simmondsiaceae (jojoba), Solanaceae (potato), Sonneratiaceae (sonneratia), Sphaerosepalaceae, Sphenocleaceae (spenoclea), Stackhousiaceae (stackhousia), Stachyuraceae, Staphyleaceae (bladdernut), Sterculiaceae (cacao), Stylidiaceae, Styracaceae (storax), Surianaceae (suriana), Symplocaceae (sweetleaf), Tamaricaceae (tamarix), Tepuianthaceae, Tetracentraceae, Tetrameristaceae, Theaceae (tea), Theligonaceae, Theophrastaceae (theophrasta), Thymelaeaceae (mezereum), Ticodendraceae, Tiliaceae (linden), Tovariaceae, Trapaceae (water chestnut), Tremandraceae, Trigoniaceae, Trimeniaceae, Trochodendraceae, Tropaeolaceae (nasturtium), Turneraceae (turnera), Ulmaceae (elm), Urticaceae (nettle), Valerianaceae (valerian), Verbenaceae (verbena), Violaceae (violet), Viscaceae (Christmas mistletoe), Vitaceae (grape), Vochysiaceae, Winteraceae (wintera), Xanthophyllaceae, and Zygophyllaceae (creosote bush).

[0198] The monocotyledon can be selected from the group consisting of corn, wheat, oat, rice, barley, millet, banana, onion, garlic, asparagus, ryegrass, millet, fonio, raishan, nipa grass, turmeric, saffron, galangal, chive, cardamom, date palm, pineapple, shallot, leek, scallion, water chestnut, ramp, Job's tears, bamboo, ragi, spotless watermeal, arrowleaf elephant ear, Tahitian spinach, abaca, areca, bajra, betel nut, broom millet, broom sorghum, citronella, coconut, cocoyam, maize, dasheen, durra, durum wheat, edo, fique, formio, ginger, orchard grass, esparto grass, Sudan grass, guinea corn, Manila hemp, henequen, hybrid maize, jowar, lemon grass, maguey, bulrush millet, finger millet, foxtail millet, Japanese millet, proso millet, New Zealand flax, oats, oil palm, palm palmyra, sago palm, redtop, sisal, sorghum, spelt wheat, sweet corn, sweet sorghum, taro, teff, timothy grass, triticale, vanilla, wheat, and yam.

[0199] Alternatively, the monocotyledon can be selected from a family selected from the group consisting of Acoraceae (calamus), Agavaceae (century plant), Alismataceae (water plantain), Aloeaceae (aloe), Aponogetonaceae (cape pondweed), Araceae (arum), Arecaceae (palm), Bromeliaceae (bromeliad), Burmanniaceae (burmannia), Butomaceae (flowering rush), Cannaceae (canna), Centrolepidaceae, Commelinaceae (spiderwort), Corsiaceae, Costaceae (costus), Cyanastraceae, Cyclanthaceae (Panama hat), Cymodoceaceae (manatee grass), Cyperaceae (sedge), Dioscoreaceae (yam), Eriocaulaceae (pipewort), Flagellariaceae, Geosiridaceae, Haemodoraceae (bloodwort), Hanguanaceae (hanguana), Heliconiaceae (heliconia), Hydatellaceae, Hydrocharitaceae (tape grass), Iridaceae (iris), Joinvilleaceae (joinvillea), Juncaceae (rush), Juncaginaceae (arrow grass), Lemnaceae (duckweed), Liliaceae (lily), Limnocharitaceae (water poppy), Lowiaceae, Marantaceae (prayer plant), Mayacaceae (mayaca), Musaceae (banana), Najadaceae (water nymph), Orchidaceae (orchid), Pandanaceae (screw pine), Petrosaviaceae, Philydraceae (philydraceae), Poaceae (grass), Pontederiaceae (water hyacinth), Posidoniaceae (35cheuchze), Potamogetonaceae (pondweed), Rapateaceae, Restionaceae, Ruppiaceae (ditch grass), Scheuchzeriaceae (35cheuchzeria), Smilacaceae (catbrier), Sparganiaceae (bur reed), Stemonaceae (stemona), Strelitziaceae, Taccaceae (tacca), Thurniaceae, Triuridaceae, Typhaceae (cattail), Velloziaceae, Xanthorrhoeaceae,, Xyridaceae (yellow-eyed grass), Zannichelliaceae (horned pondweed), Zingiberaceae (ginger), and Zosteraceae (eelgrass).

[0200] The gymnosperm can be selected from a family selected from the group consisting of Araucariaceae, Boweniaceae, Cephalotaxaceae, Cupressaceae, Cycadaceae, Ephedraceae, Ginkgoaceae, Gnetaceae, Pinaceae, Podocarpaceae, Taxaceae, Taxodiaceae, Welwitschiaceae, and Zamiaceae.

[0201] Method for stimulating plant growth

[0202] In a further aspect, the invention relates to a method for stimulating plant growth comprising applying the (isolated) bacteria or biologically pure bacterial culture according to the invention, the composition according to the invention or the coating composition according to the invention to a plant, plant seed, a sowing furrow, soil and / or plant growth medium.

[0203] In an embodiment, the method comprises applying the bacteria or biologically pure bacterial culture according to the invention, the composition according to the invention or the coating composition according to the invention :

[0204] - to a plant growth medium, such as sphagnum; and / or

[0205] - to a plant growth medium prior to, concurrently with, or after planting of seeds, seedlings, cuttings, bulbs, or plants in the plant growth medium; or

[0206] - to plant leaves, roots, or stems; and / or

[0207] - to plant seeds, and / or

[0208] - to seed furrows, and / or

[0209] - to a watering system for the plants, such as hydroponics, aeroponics and / or aquaponics. In yet an embodiment, the bacteria or biologically pure bacterial culture according to the invention, the composition according to the invention or the coating composition according to the invention is sprayed or irrigated onto plants or fields.

[0210] Kit

[0211] In yet a further aspect, the invention relates to a kit of parts for stimulating plant growth comprising

[0212] • a first container comprising the (isolated) bacteria or biologically pure bacterial culture according to the invention, the composition according to the invention and / or the coating composition according to the invention; and

[0213] • instructions for applying the (isolated) bacteria or biologically pure bacterial culture according to the invention, the composition according to the invention and / or the coating composition according to the invention to plants, plant seeds, or a plant growth medium, preferably at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C.

[0214] In an embodiment, the kit of parts further comprises one or more containers comprising fertilizers, nutrients, and / or other microorganisms.

[0215] Ores

[0216] A further aspect of the invention relates to use of the (isolated) bacteria or biologically pure bacterial culture according to the invention or the composition according to the invention for solubilizing minerals in ores.

[0217] The bacteria may be added to an ore containing e.g. insoluble calcium phosphate, dissolving the mineral and releasing soluble calcium and phosphate that may be extracted from the ore. This process is also known as bioleaching and biomining.

[0218] Medical uses

[0219] A yet an aspect of the present invention relates to a bacteria or biologically pure bacterial culture according to the invention, a composition according to the invention or a coating composition according to the invention for use as a medicament. Another aspect relates to a bacteria or biologically pure bacterial culture according to the invention or a composition according to the invention use in the treatment, alleviation and / or prevention of fungal infections.

[0220] In a further embodiment, the fungal infection is selected from the group consisting of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres, Fusarium oxysporum or a combination thereof.

[0221] As can be seen in Example 5, the bacteria or biologically pure bacterial culture according to the invention comprises antifungal activity.

[0222] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0223] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0224] The invention will now be described in further details in the following non-limiting examples.

[0225] Examples

[0226] Example 1 - Isolation of bacterial strains from a Danish grassland and screening of their mineral dissolving properties

[0227] Aim of study

[0228] The scope of this example is to demonstrate how bacterial strains with mineraldissolving properties were isolated from the soil of a Danish grassland and screened to identify the best-performing bacterial strains.

[0229] Materials and methods

[0230] The microbeTRAP [W02021 / 180941 Al] was 3D printed from PA (SLS, Materialize), it contained 32 paired chambers, where 16 of the chambers contained a nutrient pill and the other 16 a capture pill. Each of the nutrient pills were connected to a separate chamber containing a capture pill.

[0231] Three microbeTRAPs were prepared and each contained a different type of nutrient pill (TRAP1 was designed to select for nitrogen fixing species, TRAP2 for phosphate solubilizing species, and TRAP3 functioned as a control). Nutrient pills and capture pills were placed in each microbeTRAP and it was then sealed with a lid. The microbeTRAPs were placed in an old grassland on southern Funen. The microbeTRAPs were excavated after 14 days and the capture pills retrieved. These were placed on agar plates containing either Pikovskaya or Jensen medium. Pikovskaya medium is recognized for detection of phosphate solubilizing bacterial species and contains phosphate only as insoluble calcium phosphate. A halo zone on this medium indicates solubilization of calcium phosphate. The ratio between halo zone and area is a measure of the solubilization efficiency of the strain and denoted solubilization index (S / I). Jensen agar is a nitrogen-free medium often used to select for nitrogen-fixing bacterial species. Growth on this medium indicates the ability to fix nitrogen. The plates were incubated at 12°C for 2 days. Plates with capture pills were then investigated for fast growing colonies with either of the two abilities. Colonies of interest were swept using an inoculation loop from the pill and out to the edge of the colony and transferred to a new plate of either Pikovskaya or Jensen media. The plates were incubated at 12°C for 1-2 days again. Single fast-growing colonies with ability to solubilize calcium phosphate or fix nitrogen were isolated again using an inoculation loop and transferred by 4-streak method to a new plate. This step was repeated until the colonies were pure and only contained one bacterial strain.

[0232] A comparative screening test was conducted to identify the most effective candidates for a novel biofertilizer product among the isolated strains (n = 28). This test was performed at controlled temperature and concentration conditions: A fluid culture of each strain was prepared with a concentration of 0.5 McFarland (McF). 10 pL of the solution was then spotted onto plates containing either 15 mL of Pikovskaya or Jensen media in repetitions of three. The plates were incubated at 8°C. This temperature was chosen to mimic the conditions during spring seeding in northern Europe. Colony and halo zone area were measured each day the following 4 days using imageJ. Results

[0233] From the 3 MicrobeTRAPs a total of 28 bacterial strains were isolated during the initial screening process: 4 on Pikovskaya medium (from TRAP2) and 24 on Jensen medium (from TRAP1 and TRAP3).

[0234] The capacity of isolated strains to augment nitrogen and phosphorus accessibility was assessed through plate assays, during which colony and / or halo zones size were measured. The results of the comparative plate assay on media with an insoluble phosphate source (Pikovskaya) or free of nitrogen (Jensen) are shown in Figure 1.

[0235] Especially 6 strains exhibited large potential as phosphate solubilizers: TP1, TP2, TP3, TP4, TCI, and TC3. They yielded significantly larger halo zones (ranging from: 446-706 mm2) and solubilization indices above 10 (ranging from 11.0-19.6) on Pikovskaya compared to the remaining strains on day 4. In agricultural soils, phosphorous often exists in a bound form, associated with insoluble inorganic minerals, making it inaccessible to plants. Thus, the ability of these strains to solubilize phosphorous may enhance its availability to plants and promote their growth.

[0236] Moreover, the 6 best phosphate solubilizing strains also ranked within the top 12 of best-performing strains on Jensen medium. As this medium is free of nitrogen, increased growth indicates greater nitrogen fixing abilities. Nitrogen is essential to plant growth and present in limited amount in soil.

[0237] Collectively, the most promising strains identified across both screenings were TP1, TP2, TP3, TP4, TCI, and TC3. These top 6-performing strains may have the potential to promote plant growth through nitrogen fixation and phosphate solubilization.

[0238] Conclusion

[0239] The microbeTRAP technology enabled the capture of a range of microorganisms from the soil of a Danish grassland with potential beneficial properties. The subsequent screening of these novel strains indicated that especially 6 strains (TP1, TP2, TP3, TP4, TCI, and TC3) stood out as efficient nitrogen fixers and phosphate solubilizers, and therefore hold a large potential for the use as biofertilizers in colder climate regions such as northern Europe. Example 2 - Delineating the best-performing bacterial strains as Rahnella species

[0240] Aim of study

[0241] The scope of this example is to delineate the best-performing bacterial strains among the best-performing bacterial strains identified in Example 1.

[0242] Materials and methods

[0243] DNA extraction, library preparation, and Illumina whole genome DNA sequencing DNA was extracted from the remaining top 5 best-performing bacterial strains (TCI, TC3, TP1, TP2 and TP3) using the Dneasy UltraClean Microbial Kit from Qiagen following the instructions from the manufacturer. 50 ng of DNA from each bacterium was prepared for Illumina paired-end sequencing (2x150 bp reads) using the TWIST Library Preparation EF 2.0 enzymatic fragmentation kit following the instructions provided by the manufacturer. DNA libraries were sequenced on an Illumina MiniSeq machine. A total of 1,351,824-2,624,372 reads were obtained for each strain.

[0244] Quality control and processing of sequencing data

[0245] Adapter and barcode contaminants originating from library preparation steps were removed from the obtained sequencing data using Trim Galore (vO.6.7) while retaining nucleotides with a Phred score >20. Only quality-controlled reads were considered for downstream analyses.

[0246] Taxonomic classification of sequencing reads

[0247] Sequencing reads obtained from TCI, TC3, TP1, TP2 and TP3 were taxonomically classified according to the National Center for Biotechnology Information (NCBI) Taxonomy using Kraken2 (v2.1.2). A comprehensive pre-built database containing complete (as of 12 / 09 / 2022, downloaded from https : / / ben la nQmead.Qithub.io / aws-indexes / k2) RefSeq genomes mapping to archea, bacteria, virures, plasmids, humans, fungi, protozoans as well as vector sequences was used as a reference for the taxonomic assignment. Bracken (v2.8) was subsequently used for abundance re-estimation at genus and species level (Table 1).

[0248] De novo genome assembly and assembly statistics SPAdes (v3.15.5) was used for assembling trimmed sequencing reads into de novo contigs that were subsequently joined into putative scaffolds. A read errorcorrection step (BayesHammer module) was performed prior to the genome assembly step to minimize the number of mismatches in the resulting contigs. Following the genome assembly step, QUAST (v5.0.2) was used for computing assembly quality metrics (Table 2).

[0249] Genome annotation

[0250] Genomic features present in the assembled genomes were annotated using Prokka (vl.14.6) (Table 3).

[0251] Average nucleotide identity (ANI) analysis

[0252] FastANI (vl.l) was used for computing average nucleotide identity (ANI) scores where each assembled genome was iteratively compared to each other (Table 4).

[0253] Results

[0254] Strain TP4 was excluded from further analysis as initial 16S rDNA sequencing (performed by Novogene) revealed that the strain annotated as a Serratia strain and several Serratia species have been implemented in multiple human clinical diseases. As a precaution, strain TP4 was therefore excluded from additional studies.

[0255] Taxonomic classification of sequencing data obtained from the remaining strains; TCI, TC3, TP1, TP2 and TP3, revealed that 98.88-99.16% of all quality-controlled reads were assigned to the genus Rahnella. Analysis at species level revealed diverse assignments to R. aquatilis and R. aceris with a slightly higher proportion of data matching towards R. aquatilis (Table 1). The diversity in the fractional distribution towards R. aquatilis and R. aceris suggests that TCI, TC3, TP1, TP2 and TP3 are not identical microorganisms, although TCI and TC3 do seem to be more similar (Table 1). R. aquatilis is described as the type species of Rahnella, but in 2020 phylogenetic analyses revealed a novel Rahnella species designated as R. aceris. While R. aquatilis and R. aceris seem to be genetically related at species level, it is currently unknown how R. aquatilis / aceris strains are related. Table 1: Percent taxonomic assignment of all classified reads at species level.

[0256] To further exploit the genetic diversity of TCI, TC3, TP1, TP2 and TP3, de novo genomes were assembled for each individual best-performing strain and compared extensively to each other. Evaluating size and contiguity of assemblies, the average GC content (DNA base composition relating to guanine-cytosine content) (Table 2) and the presence of genes encoding proteins or RIMA products (Table 3) support that TCI, TC3, TP1, TP2 and TP3 are genetically diverse, however, TCI and TC3 seem to display similar characteristics at the genome level and the same goes for TP1, TP2 and TP3.

[0257] Table 2: Genome assembly statistics. Assembly sizes are based on contigs > = 500 bp. Table 3: Genome annotation. CDS=coding sequence; rRNA=ribosomal RNA; tRNA=transfer RNA; tmRNA=transfer-messenger RNA.

[0258] Average nucleotide identity (ANI) is a measure of the mean nucleotide identity of orthologous gene pairs that are shared between two microbial genomes and has been shown to be a robust measure when delineating bacterial genomes at species level. Large-scale studies have shown that microorganisms showing -95% ANI belong to the same species taxonomy. Although consensus criteria for defining boundaries at the strain level remain controversial, recent studies suggest that a threshold at 99.5% ANI is consistent with the diversification of clonal complexes; clusters of genotypes sharing an unique allelic profile that can be linked to for example disease outbreaks. To explore the diversity of the bestperforming strains at strain taxonomy level, ANI values were computed and compared for TCI, TC3, TP1, TP2 and TP3. Comparing the individual TC strains against the individual TP strains and vice versa revealed ANI values around 99.3% which strongly suggests that TC strains and TP strains collectively represent two different groups of strains belonging to R. aquatilis / aceris (Table 4). The observed ANI values also suggest that none of the bacteria within each strain group are identical at the DNA level; however, whether this variance can be translated into functional differences, is currently unknown.

[0259] Table 4: Comparative average nucleotide identity (ANI) analysis. Grey areas indicate groups of ANI values representing different strain groupings (TCI and TC3 and TP1-3).

[0260] Comparing the capacity of TCI, TC3, TP1, TP2 and TP3 to either grow on media free of nitrogen, which is a proxy for the capability to fixate nitrogen, or to solubilize phosphorous (Example 1 and Figure 1) reveals that TP1 and TP2 perform equally good at solubilizing phosphorous while TP2 demonstrates a greater capacity towards fixing nitrogen. Thus, TP2 was selected as the bestperforming Rahnella strain.

[0261] Conclusion

[0262] The bacterial strains TCI, TC3, TP1, TP2 and TP3, which were selected as bestperformers from a large screening setup (n = 28, Example 1), represent at least two different groups of strains belonging to Rahnella aquatilis / aceris, where TCI and TC3 represent one group of strain and TP1, TP2 and TP3 represent another group of strain. Of the best-performers, TP2 demonstrated high capacity towards fixing nitrogen and solubilizing phosphorous. TP2 was therefore selected as the best-performing Rahnella strain and is deposited at DSMZ with accession number DSM 34654.

[0263] Example 3 - DSM 34654 is a novel Rahnella aceris strain

[0264] Aim of study

[0265] The scope of this example is to demonstrate that DSM 34654 is a novel bacterial strain belonging to Rahnella aceris.

[0266] Materials and methods

[0267] Illumina short-read DNA sequencing

[0268] DNA was extracted from a bacterial culture of TP2, from here on referred to as DSM 34654, using the Dneasy UltraClean Microbial Kit from Qiagen following the instructions from the manufacturer. 50 ng of DNA was prepared for Illumina paired-end sequencing (2x150 bp reads) using the TWIST Library Preparation EF 2.0 enzymatic fragmentation kit following the instructions provided by the manufacturer. The DNA library was sequenced on an Illumina MiniSeq machine. A total of 1,351,824 reads were obtained.

[0269] ONT long-read DNA sequencing

[0270] DNA was extracted from a bacterial culture of DSM 34654 using the Dneasy UltraClean Microbial Kit from Qiagen following the instructions from the manufacturer. 400 ng of DNA was prepared for Oxford Nanopore Technologies (ONT) whole genome sequencing using the Native Barcoding Kit 24 V14 (SQK- NBD114.24). The DNA library was sequenced on a MinlON Mklb device using a R10.4.1 flow cell until a sequencing depth of minimum lOOx was obtained. Quality control and processing of Illumina data

[0271] Adapter and barcode contaminants originating from library preparation steps were removed from the obtained sequencing data using Trim Galore (vO.6.7) while retaining nucleotides with a Phred score >20. Only quality-controlled reads were considered for downstream analyses.

[0272] Quality control and processing of ONT data

[0273] Adapter and barcode contaminants originating from library preparation steps were removed while basecalling raw ONT data with the super accuracy (SUP) model using Guppy (v6.4.6). To obtain ONT data with high accuracy, duplex basecalling was subsequently performed using Guppy (v6.4.6) and Duplex Tools (vO.2.17) which utilizes both complement and template DNA strands to generate consensus basecalled data with a high confidence (10% of data was identified as good read pairs). Combining duplex data with the SUP basecalled ONT data (where only the template DNA strand was basecalled), high-accuracy and high-coverage ONT data was obtained. To ensure that only high-quality reads were kept, chopper (v0.2.0) was used for retaining sequencing reads with an average Phred score >20 and a read length of minimum 500 bp. Only quality-controlled reads were considered for downstream analyses corresponding to a total of 498,315,168 bp.

[0274] Taxonomic classification of sequencing reads

[0275] Kraken2 (v2.1.2) was used for assigning Illumina and ONT sequencing data according to the National Center for Biotechnology Information (NCBI) Taxonomy. A comprehensive pre-built database containing complete (as of December 09 2022, downloaded from https: / / ben la no mead .cjithub.io / aws-indexes / k2) RefSeq genomes mapping to archea, bacteria, viruses, plasmids, humans, fungi, protozoans as well as vector sequences was used as a reference for the taxonomic assignment. Bracken (v2.8) was subsequently used for abundance re-estimation at genus and species level.

[0276] Hybrid de novo genome assembly and assembly statistics

[0277] A complete de novo genome for DSM 34654 was obtained through a hybrid assembly approach utilizing both short Illumina and long ONT quality-controlled reads. The long ONT reads were used for generating a non-fragmented de novo genome using Flye (v2.9.2) and the short Illumina reads were subsequently used for error-correction at the nucleotide level using Pilon (vl.24). QUAST (v5.0.2) was used for computing assembly quality metrics.

[0278] Genome annotation

[0279] Chromosome and plasmid assemblies were extracted from the complete de novo genome and genomic features present in each assembly were annotated using Prokka (vl.14.6).

[0280] Average nucleotide identity (ANI) analysis

[0281] FastANI (vl.l) was used for computing average nucleotide identity (ANI) scores of the assembled genome compared to all Rahnella aquatilis and Rahnella aceris GenBank genomes available at NCBI (as of October 11, 2023). All atypical genomes were excluded, resulting in 4 R. aquatilis and 8 R. aceris reference genomes (Table 5).

[0282] Table 5: GenBank IDs of Rahnella aceris and Rahnella aquatilis genomes (n = 12, as of October 11, 2023) used in comparative analyses. Type strains are indicated in grey.

[0283] Whole genome-based phylogenetic reconstruction SANS serif (v2.2_12A) was used for determining evolutionary relationships among DSM 34654 and all of the Rahnella strains used here (Table 5). The computed phylogenetic splits were subsequently visualized in a circular phylogenetic tree using R (v4.2.3) and ggtree (v3.6.2).

[0284] Secondary metabolite analysis antiSMASH (v7.0.0) was used for the identification and annotation of secondary metabolite biosynthesis gene clusters within DSM 34654 chromosome and plasmids. The MIBiG repository containing experimentally characterized biosynthetic gene clusters was used for annotation.

[0285] Virulence factor analysis

[0286] Genes annotated using Prokka {Genome annotation section) were subjected to a sequence similarity search against DNA sequences corresponding to experimentally validated virulence factors from the Virulence Factor Database (VFDB, core dataset downloaded on September 26 2023) using the Basic Local Alignment Search Tool (BLAST). The blastn algorithm (v2.14.1+) with a word-size of 7 was used for the nucleotide alignment search.

[0287] 16S rRNA extraction and mapping

[0288] Seven 16S ribosomal RNA (rRNA) genes were identified in the assembled genome of DSM 34654. The DNA sequences corresponding to the seven 16S rRNA genes were extracted from the Prokka output and compared to a database from NCBI containing curated 16S rRNA gene sequences from bacteria and archaea type strains using BLAST. The blastn algorithm (v2.14.1 + ) with a word-size of 11 was used for the nucleotide alignment search. Search was performed on November 7 2023.

[0289] The DNA sequences corresponding to the identified 16S rRNA genes are encoded by SEQ ID NO: 1-7 (each 1,539 nucleotides).

[0290] Extraction and analysis of DSM 34654-specific DNA sequences

[0291] DNA sequences with no or little overlap with the genome of the type strain of R. aceris (SAP-19) were extracted from the DSM 34654 genome assembly using samtools (v.1.10) and subsequently compared to either the prokaryotic nucleotide collection (Prokaryota (bacteria and archaea) nt database) or the complete nucleotide collection (nt database) from NCBI containing

[0292] GenBank+EMBL+DDBJ + PDB+RefSeq sequences using BLAST. The blast search using SEQ ID NO: 8 as a query was restricted to the prokaryotic database due to the sequence length of SEQ ID NO: 8 (13,537 nucleotides). The blastn algorithm (v2.14.1+) with a word-size of 11 was used for all nucleotide alignment searches. Searches were performed on October 10 2023.

[0293] One long extracted DNA sequence (13,537 nucleotides) is annotated as SEQ ID NO: 8.

[0294] Short DNA sequences (438-1,197 nucleotides) extracted from the genome assembly are annotated as SEQ ID NOs: 9-15.

[0295] Visualization of genomic regions

[0296] Proksee (available at https: / / proksee.ca / ) was used for visualizing genomic regions in DSM 34654 along with BLAST results comparing DSM 34654 to the type strain of Rahnella aceris (SAP-19).

[0297] Results

[0298] Initial taxonomic classification of Illumina and ONT sequencing reads obtained from the bacterial strain DSM 34654 revealed that 99.16% and 99.64% of all quality-controlled reads were assigned to the genus Rahnella, respectively. Analysis at species level further revealed assignments to both R. aquatilis and R. aceris with a slightly higher proportion of data matching towards R. aquatilis (Table 6).

[0299] Table 6: Percent taxonomic assignment of all classified Illumina or ONT reads at species level. To obtain further biological insight into the genome of strain DSM 34654, a hybrid assembly approach was applied. Here, long ONT reads were assembled into a non-fragmented de novo genome and the short Illumina reads were subsequently used for error-correction of the genome assembly at the nucleotide level. This approach allowed for generating a high-quality and complete de novo genome of strain DSM 34654. The complete genome is composed of one circular chromosome and 3 circular (putative) plasmids; each containing protein-encoded genes while all RNA products are encoded on the chromosome alone (Table 7).

[0300] Table 7: Number of features associated with each chromosome and plasmid assembly. CDS=coding sequence; rRNA=ribosomal RNA; tRNA=transfer RNA; tmRNA=transfer-messenger RNA.

[0301] Interestingly, DSM 34654 contains a genomic region displaying a strong similarity to a biosynthetic gene cluster encoding the siderophore desferrioxamine E (MIBiG accession BGC0001572 found in Pantoea agglomerans') (Table 8); an iron- scavenging bioactive molecule secreted by a variety of microorganisms, including plant growth-promoting bacteria, that allows for increased iron uptake in plant cells which thereby promotes plant growth. This finding suggests that DSM 34654 might have capacity for transforming soil-bound iron into forms that plants can utilize for growth. In addition, the assembled genome of DSM 34654 also contains several genes relating to the fixation of atmospheric nitrogen nifABDFHJKLSW') and phosphate solubilization, including high-affinity phosphate transporters pstABC) as well as gcd; a biomarker for phosphate-solubilizing bacteria, which support the in vivo assays in Example 1. Some Rahnella strains, e.g. R. aquatilis strains, have been associated with infections and diseases in humans. A similarity search of the genes identified in DSM 34654 and genes encoding experimentally verified virulence factors importantly revealed no complete overlaps (maximum overlap = 30%, i.e. 30% of a gene in DSM 34654 could be aligned to a virulence gene), strongly indicating that DSM 34654 is a non-virulent Rahnella strain. Additional analyses importantly showed no presence of the rhizoxin-biosynthetic gene cluster, including both rhi and rzx gene loci, which may be involved in producing carcinogenic secondary metabolites.

[0302] Table 8: Similarity to the siderophore desferrioxamine E biosynthetic gene cluster. Percent sequence coverage and identity between genes in DSM 34654 and the corresponding BGC genes. BGC=biosynthetic gene cluster.

[0303] 16S ribosomal RIMA (rRNA) sequences have been extensively used to classify bacteria. Here, a threshold of 98.65% similarity at the 16S rRNA level has been recognized as the cutoff for delineating bacterial species. Seven 16S rRNA genes were identified in DSM 34654 (SEQ ID NOs: 1-7). Extracting the DNA sequences corresponding to each of the 16S rRNA genes and comparing them to the NCBI 16S ribosomal type strain database revealed sequence identities of 99.86-100% along with 96% sequence coverage matching towards a 16S rRNA gene in the type strain of Rahnella aceris (SAP-19), indicating that DSM 34654 might be related to R. aceris.

[0304] Although the 16S rRNA gene region contains hypervariable regions that can be used for taxonomic discrimination, the analysis of 16S rRNA alone ignores the genome-wide variability. Thus, to further delineate the taxonomic identity of DSM 34654, average nucleotide identity (ANI) values were computed for DSM 34654 in a comparative analysis against all available R. aceris and R. aquatilis genomes (n = 12 as of October 11 2023, excluding atypical genomes) (Table 5). This revealed four comparisons with ANI values below the species boundary at 95% (Table 9). All of these comparisons relate to R. aquatilis strain genomes, including the type strain R. aquatilis CIP 78.65, while the remaining comparisons revealed >98% ANI, demonstrating that DSM 34654 is more closely related to R. aceris (Table 9). Interestingly, no ANI values were reported above the putative strain threshold at 99.5%, suggesting that DSM 34654 might be a novel strain belonging to R. aceris.

[0305] Table 9: Average nucleotide identity (ANI) analysis of DSM34654 genome assembly compared to Rahnella aquatilis and Rahnella aceris genome assemblies. Type strains are indicated in grey. Comparisons with reported ANI values >95% are highlighted in bold.

[0306] While ANI is a robust measure for delineating organisms at species, or even strain, level, the methodology is based on a pair-wise comparison of orthologous gene pairs that are shared between microbial genomes. To explore whether DNA sequence information located outside of orthologous regions might contribute to additional specification of DSM 34654, whole genome phylogenies, representing measures of evolutionary distantness, were inferred and visualized as a phylogenetic tree (Figure 2). While hitherho phylogenetic methods are commonly based on pairwise comparisons of single or multiple marker genes, e.g., 16S rRNA, whole genome phylogenomics do not rely on the identification of marker genes and do therefore not ignore the total genome-wide variability that forms the basis for evolutionary distinctness. The resulting phylogenetic tree shows that DSM 34654 is genetically closest to R. aceris AR20 and together they form a cluster that is separated from the remaining tree nodes (Figure 2, grey area). Thus, both the ANI analysis and the phylogenetic reconstruction of R. aceris, R. aquatilis and DSM 34654 relatedness, collectively demonstrate that strain DSM

[0307] 34654 can be considered as a novel R. aceris strain.

[0308] To identify DNA sequences with high specificity for strain DSM 34654, genomic regions within the DSM 34654 genome assembly, showing no or low concordance with the type strain of R. aceris (SAP-19), were extracted and subjected to a broad sequence similarity search. This identified a large genomic region consisting of 13,537 nucleotides (SEQ ID NO: 8, Figure 3) showing <66% coverage in other prokaryotic genomes (Table 10) and importantly no identity in R. aceris strains. Thus, this region seems highly specific for DSM 34654.

[0309] Table 10: Percent sequence coverage and identity of Top5 alignments from a BLAST search of SEQ ID NO: 8 against the Prokaryotic nucleotide database from NCBI (performed. Alignments were sorted according to sequence coverage (high- to-low).

[0310] Importantly, several long open reading frames encoding hypothetical proteins were discovered within this genomic region (Figure 3), indicating that SEQ ID NO: 8 might be of functional importance to DSM 34654. Four of such regions are included as SEQ ID NOs: 9-12 (Table 11). Three additional, and randomly selected, DNA sequences, encoding hypothetical proteins and located outside of SEQ ID NO: 8 are further enclosed as SEQ ID NOs: 13-15 (Table 11). SEQ ID NOs: 13-14 are present on the chromosome while SEQ ID NO: 15 is present on Plasmid2. Comparing each of these sequences to the nucleotide database at NCBI using BLAST revealed sequence identities at 66.31-92.50% across 4-88% of the extracted sequences (Table 11) and importantly no hits from any Rahnella aceris strains (as of November 7, 2023). Thus, SEQ ID NOs: 9-15 are highly selective for DSM 34654. Table 11: Reported maximum percent sequence coverage and sequence identity of SEQ ID Nos: 9-15 from a BLAST search against the nucleotide database from NCBI. Nt; nucleotides.

[0311] Conclusion

[0312] The comparative genome analysis of the bacterial strain DSM 34654 revealed that DSM 34654 is a novel Rahnella aceris strain. Several R. aquatilis strains have been shown to promote plant growth through processes such as nitrogen fixation, phosphate solubilization and biosynthesis of plant growth regulators, such as pyrroloquinoline quinone; however, no studies have to date demonstrated plantgrowth promoting effects of R. aceris strains. Interestingly, the assembled genome of DSM 34654 contains a genomic region with high similarity towards a biosynthetic gene cluster encoding the iron-scavenging siderophore desferrioxamine E, indicating that DSM 34654 might have capacity for transforming soil-bound iron into forms that plants can utilize for growth.

[0313] Several DNA sequences containing open reading frames and that have high specificity for DSM 34654 were identified; one long genomic region (13,537 nucleotides, SEQ ID NO: 8) wherein short DNA sequences (438-1,197 nucleotides) were extracted (SEQ ID NOs: 9-12) as well as three short DNA sequences (504-870 nucleotides) identified outside of the long genomic region (SEQ ID NOs: 13-15). Combined or alone, these DNA sequences may be used to identify DSM 34654 at the DNA sequence level.

[0314] Example 4 - DSM 34654 has mineral-dissolving properties at low, medium and high temperatures

[0315] Aim of study The aim of this study is to demonstrate the ability of DSM 34654 to make common plant nutrients available specifically under low, medium, and high temperature conditions.

[0316] Materials and methods

[0317] To demonstrate the ability of DSM 34654 to increase the availability of nitrogen, phosphate, and potassium, DSM 34654 was cultivated on different solid media: nitrogen-free agar; Jensen medium, insoluble phosphate agar; Pikovskaya agar and iron phosphate agar, and insoluble potassium agar; Aleksandrow agar. These four solid media are recognized for being specific for detection and cultivation of nitrogen-fixing, phosphate-solubilizing, and potassium-solubilizing soil microorganisms. Growth on Jensen agar indicates nitrogen-fixating abilities, as this medium is free of nitrogen. A halo zone on Pikovskaya, iron-phosphate and Aleksandrow agar indicates solubilization of calcium phosphate (Cas PC )?), iron phosphate (FePC>4-4 H2O), and potassium alumino-silicate respectively. The solubilization capacity was evaluated by calculating a solubilization index (SI, SI = Areanaio / Areacoiony). To demonstrate temperature-dependent activities, experiments were performed at low (5°C), medium (15°C), and high (25°C) temperatures. Typically, spring seeding will occur at soil temperatures around 6- 8°C in Northern Europe, and activity at lower temperatures is therefore of great importance at this altitude. A commercial microbial biofungicide and biostimulant, biol and bio2, respectively, were included in the experiment as benchmarking controls. The active microorganisms in these products were isolated and included in the test on equal footing with DSM 34654: A fluid culture of each bacterial strain was prepared in a concentration of 0.5 McFarland. The solution was then spotted onto plates containing the different media in repetitions of three. The plates were incubated at either 5°C, 15°C, or 25°C. Colony and halo zone areas were measured each day using imageJ.

[0318] Results

[0319] The ability of DSM 34654 to increase the availability of nitrogen, phosphate or potassium was evaluated using a solid media strategy where each bacterial colony and / or halo zone area was quantified and furthermore compared to bacterial strains isolated from commercial biostimulant and biofungicide products (Figure 4). DSM 34654 was actively growing on each respective media across the entire temperature span (low (5°C), medium (15°C, and high (25°C)) and was the only strain that could grow and / or exert activity at 5°C. Moreover, DSM 34654 exerted mineral-dissolving properties at all temperatures (Figure 4B-D) while the bacterial control strains isolated from commercial products only demonstrated little (Figure 4C) or no (Figure 4B and D) capacity towards dissolving potassium or phosphate (on iron-phosphate medium), respectively. The solubilization index was between 11.7 and 20.6 on Pikovskaya on day 7. The SI on Pikovskaya at each temperature at day 7 was:

[0320] • 20.6 at 5°C,

[0321] • 11.7 at 15°C, and

[0322] • 19.3 at 25°C.

[0323] This suggests that DSM 34654 has a better capacity towards dissolving minerals which are otherwise unavailable for plants.

[0324] Conclusion

[0325] Soil phosphate and potassium exist to a large extent in insoluble complexes bound to insoluble inorganic minerals, which are unavailable for plants. Thus, solubilization of these minerals may improve mineral availability and thereby has the potential to promote plant growth. Only DSM 34654 was able to grow and display bacterial activity at 5°C, demonstrating that DSM 34654 has a high potential for supplying crops in the field with otherwise unavailable nutrients through the entire plant life cycle from as early as germination in cold soil until maturation and harvest.

[0326] Example 5 - Assessing biocontrol properties against common fungal phytopathogens

[0327] Aim of study

[0328] To demonstrate the ability of DSM 34654 to control common fungal plant pathogens.

[0329] Materials and methods

[0330] Dual culture assays were used to determine the antimicrobial activity of DSM 34654. DSM 34654 was co-cultured on PDA and oatmeal agar with three isolates of fungal phytopathogens; Gaeumannomyces graminis var. tritici (CBS 450.77), Pyrenophora teres f. teres (CP2189), Fusarium oxysporum (CBS 619.87). These three fungi are causing some of the major diseases in agricultural crops; wheat root rot, barley net blotch, and vascular wilts in a variety of crops. Two dual culture assays were used to determine the antifungal activity: spot on lawn and plug assay. In the spot on lawn assay petri dishes containing PDA or oatmeal were prepared with a lawn of fungi using a cotton swab covered in fungal spores. The spores were distributed by striking the swab methodically across the agar surface in one direction ensuring close and even distribution of spores. Subsequently the swab was struck perpendicularly the initial direction further enhancing the uniformity of the lawn. A solution with a concentration of 3 McF of DSM 34654 was prepared in 1: 10 PBS. 10 pL of the solution was spotted onto each petri dish already prepared with fungal spores in replicates of three. Inhibition was qualitatively evaluated by the presence of a halo zone with no fungal growth on the 'lawn'. In the plug assay a mycelial agar plug was cut out from the growing edge of each fungus using a sterile cork borer with a 6 mm dia. The plug was placed in the center of another petri dish containing either PDA or oatmeal agar. Four spots of 10 pL of bacterial solution with a concentration of 3 McF were spotted onto the plate in a quadratic pattern. Inhibition was indicated by a decrease in fungal growth area relative to control.

[0331] The assays were performed at 20°C, to ensure optimal conditions for fungal growth. Inhibitions were evaluated on day 8 in the spot on lawn assay while Fusarium oxysporum and Pyrenophora teres f. teres was evaluated on day 7 and 12 in the plug assay.

[0332] Results

[0333] Inhibition of growth of the fungal phytopathogen was evaluated using two dual culture techniques: spot on lawn (Figure 5A) and plug (Figure 5B) assay. DSM 34654 was found to inhibit the growth of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres and Fusarium oxysporum in the spot on lawn assay (Figure 5A) and Pyrenophora teres and Fusarium oxysporum in the plug assay (Figure 5B).

[0334] Conclusions

[0335] DSM 34654 has antifungal activity against three phytopathogenic fungi Gaeumannomyces graminis var. tritici, Pyrenophora teres and Fusarium oxysporum when evaluated in two dual culture assays at an incubation temperature of 20°C.

[0336] Example 6 - Seed coat containing DSM 34654 promotes early plant growth

[0337] Aim of study

[0338] The scope of this example is to demonstrate the properties of DSM 34654, when applied as a seed coat, on early plant growth in vitro in two typical agricultural crops, namely winter wheat and oilseed rape.

[0339] Materials and methods

[0340] Seed germination

[0341] Winter wheat seeds (RGT Saki seeds from Danish Agro) and oilseed rape seeds (organic seeds from Danish Agro) were coated with a bacterial solution of DSM 34654 along with pectin, acting as a biopolymer, using the following preparations:

[0342] • Oilseed rape: 2 McFarland standards of DSM 34654 were diluted in lx PBS + pectin (final concentration of 1.5% (by volume)). 1 g of oilseed rape seeds were coated with 3 mL of a DSM 34654-pectin solution.

[0343] • Winter wheat: 2 McFarland standards of DSM 34654 were diluted in lx PBS + pectin (final concentration of 1.5% (by volume)). 5 g of winter wheat seeds were coated with 6 mL of a DSM 34654-pectin solution.

[0344] To obtain a homogenous seed coating, seeds were mixed with a DSM 34654- pectin solution (referred to as DSM 34654 seed coat from hereafter), as previously described, and stirred at 300 rpm for 30 min. Following stirring, seeds were sieved to remove excess coating solution.

[0345] For early germination performance experiments, coated seeds and control seeds (uncoated) were placed on top of presoaked sterile blue germination paper within 9.5 cm petri dishes (4 replicates, each containing 25 seeds). Petri dishes were kept at room temperature (23±2) with a 16 / 8 h light / dark regime. Early germination processes, here evaluated as the emergence of the primary root (radicle) and the young shoot (plumule), respectively, were recorded from the day after seeds were plated. Radicle and plumule emergence were averaged across replicates (n=4) and plotted as mean emergence for each crop and treatment in bar plots with standard error of the mean (SEM) as error bars. Statistical testing of DSM 34654 seed coat effects on radicle and plumule emergence were performed using a one-sided Wilcoxon test where an asterisk denotes a p-value <0.05.

[0346] Phosphate solubilization

[0347] The capacity of DSM 34654 seed coat to solubilize inorganic phosphate was determined by plating coated seeds from oilseed rape and winter wheat crops on Pikovskaya media Oilseed rape and winter wheat seeds were coated with DSM34654 seed coat, as previously described, and immediately plated on Pikovskaya media in 9.5 cm petri dishes (2 replicates, each containing 5 seeds). Petri dishes were kept in incubators at 24°C. The formation of clearing zones around plated seeds was examined at two days following seed plating.

[0348] Results

[0349] Seed germination

[0350] Early germination was evaluated by the emergence of the primary root (radicle) followed by the emergence of the young shoot (plumule) at one and two days following plating of oilseed rape and winter wheat seeds in petri dishes, respectively. Figure 6 shows the emergence of the radicle (Figure 6A) and the plumule (Figure 6B) in oilseed rape and winter wheat seeds, respectively. Coating winter wheat seeds with DSM 34654 seed coat resulted in a significant (p<0.05) increase of both radicle (8-fold on average) and plumule (1.8-fold on average) emergence, respectively, compared to control winter wheat seeds that have not received coating (Figure 6A and 6B, bars to the right). A similar tendency was also observed for radicle and plumule emergence in oilseed rape seeds that were coated with DSM 34654 seed coat (Figure 6A and 6B, bars to the left). These findings suggest that DSM 34654 seed coat has a stimulatory effect on early plant growth.

[0351] Phosphate availability

[0352] Plant growth is often limited by the availability of phosphate. Figure 7 shows the potential of DSM 34654, when applied as a seed coat, to solubilize inorganic phosphate employed in Pikovskaya medium. Formation of clearing zones around seeds indicate phosphate solubilization. Prominent clearing zones were observed around DSM 34654-coated oilseed rape and winter wheat seeds at two days following coating and plating on Pikovskaya media (Figure 7, arrows). Minor clearing zones were also observed around control winter wheat seeds, indicating that phosphate-solubilizing microbes are naturally present on winter wheat seeds. Clearing zones do however increase significantly in size around seeds that have been coated with DSM 34654 seed coat, suggesting that DSM 34654 seed coat has a high efficacy towards solubilizing inorganic phosphate and thereby increasing the availability of phosphate for plant growth.

[0353] Conclusion

[0354] Administration of beneficial microbes to crop seeds before sowing can benefit plant growth by for example increasing the availability of phosphate in the soil. Here, we show that DSM 34654 seed coat has multiple beneficial effects on early plant growth of agricultural crops in vitro. Coating seeds with DSM 34654 seed coat stimulates early germination processes by increasing the emergence of both the radicle and the plumule which may be attributed to the increased phosphate activity in the vicinity of seeds. Collectively, these data demonstrate the plant growth-promoting properties of DSM 34654 seed coat.

[0355] Example 7 - Seed coat containing DSM 34654 stimulates cover crop growth

[0356] Aim of study

[0357] The scope of this example is to demonstrate the effects of DSM 34654 on the emergence and growth of oil radish crops harvested from Danish fields when applied as a seed coat onto seeds before sowing.

[0358] Materials and methods

[0359] Treatment of oil radish seeds and harvest

[0360] A bacterial solution containing 2 McF DSM 34654 in 2% pectin was added to oil radish seeds Raphanus sativus subsp. oleiferus) at a ratio of 8 mljkg. The seeds were then air dried and stored in a bag until sowing. Coated seeds as well as control seeds (uncoated) were sown in August 2022 (mean air temperature for August 2022 was 18°C) on fields on Zealand, Denmark (performed by an independent third-party contract research organization (VKST, Denmark)). Emergence was counted 21 days after sowing and was quantified as plants / m2.

[0361] The fresh weight of roots was recorded as grams per 50 plants before fields were plowed in October 2022 (mean air temperature for October 2022 was 11.7°C).

[0362] Results

[0363] Treating oil radish seeds with a seed coat solution containing the bacterial strain DSM 34654 before sowing resulted in a significant (p-value = 0.01421, based on a one-sided Wilcoxon test) increase in the emergence of oil radish plants of 8.57% (median emergence of non-treated control seeds = 35 plants / m2, median emergence of DSM 34654-treated seeds = 38 plants / m2) (Figure 8A) as well as a significant (p-value = 0.02857, based on a one-sided Wilcoxon test) increase in root weight following harvest of 23.41% (median root weight of non-treated control plants = 530.50 g / 50 plants, median root weight of DSM 34654-treated plants = 654.70 g / 50 plants) (Figure 8B and C). This demonstrates that DSM 34654, when applied as a seed coat, can enhance both the emergence and root weight yield in oil radish crops from a field.

[0364] Conclusion

[0365] Oil radish is commonly used as a cover crop that aids against soil compaction and excessive nitrogen leaching. Oil radish plants develop thick, deep roots that penetrate compacted layers of soil and improve drainage and establishment of primary crops. The deep rooting ability of oil radish plants further allows for recycling nitrogen in the soil as the nitrogen trapped by the roots becomes available again after oil radish plants decompose.

[0366] Applying the bacterial strain DSM 34654 as a seed coat onto oil radish seeds before sowing increases the emergence of oil radish plants in the field. Plants that have developed from DSM 34654-treated seeds have bigger roots compared to control plants, demonstrating that DSM 34654 adds to the beneficial characteristics of oil radish plants when used as a cover crop by for example increasing the storage capacity of nitrogen.

[0367] Example 8 - DSM 34654 increases the yield of root and cereal crops

[0368] Aim of study

[0369] The aim of this study was to test the ability of DSM 34654 to affect the yield of root and cereal crops, such as sugar beet, potato and maize, in outdoor conditions.

[0370] Materials and methods

[0371] Sugar beet

[0372] Sugar beet seeds (Beta vulgaris 'Gahan') were manually sprayed (3 times per seed) with a bacterial solution containing 67.2 McF DSM 34654 (n = 10) or water (control, n = 10). Seeds were planted in a field on Funen, Denmark, on May 11, 2023 (mean air temperature for May 2023 was 11.2°C) in one long row (control seeds in one end of the row and treated seeds in the other end of the row) at a depth of 3-4 cm with 20 cm between each seed bed. Sugar beets were harvested on June 21, 2023 (mean air temperature for June 2023 was 16.4°C).

[0373] Potato

[0374] Seed potatoes Solanum tuberosum 'Bintje') were dipped in a bacterial solution containing 1 McF DSM 34654 (n = 50) or water (control, n = 50). Seed potatoes were air dried for 2 hours and planted in a field on Funen, Denmark, on April 21, 2023 (mean air temperature for April 2023 was 7°C) in 5 rows of 10 plants at a depth of 2.5 cm with 30 cm between each seed potato. Rows were placed 50 cm apart. Potatoes were harvested on August 12-13, 2023 (mean air temperature for August 2023 was 16.1°C).

[0375] Maize

[0376] Maize seeds (Zea mays, silage hybrid) were dipped in a bacterial solution containing 1 McF DSM 34654 (n = 10) or water (control, n = 10). Immediately following dipping, maize seeds were sown in a field on Funen, Denmark, on April 21, 2023 (mean air temperature for April 2023 was 7°C) in rows at a depth of 2 cm with 2 cm between each seed. Rows were placed 50 cm apart. Maize cobs plants were harvested on August 25, 2023 (mean air temperature for August 2023 was 16.1°C).

[0377] Results

[0378] Treating sugar beet seeds, seed potatoes and maize seeds with a bacterial solution of DSM 34654 before sowing led to significant increases in harvest yields across all tested crops (Figure 9). The fresh weight of roots from sugar beets that have developed from DSM 34654-treated seeds was on average 10.54 grams per plant compared to 3.31 grams per plant in the control group (Figure 9A and B), resulting in an increase in the yield of sugar beet roots of 218%. The fresh weight of potatoes harvested from DSM 34654-treated seed potatoes was on average 667 grams per plant compared to 586 grams per control plant (Figure 9C), resulting in an increase in potato yield of 14%. The fresh weight of maize cobs harvested from plants that have developed from DSM 34654-treated seeds was on average 516 grams per plant compared to 363 grams per control plant (Figure 9D), resulting in an increase in maize yield of 42%.

[0379] Conclusion

[0380] Treating sugar beet seeds, seed potatoes and maize seeds with a bacterial solution containing DSM 34654 before sowing in fields demonstrates that DSM 34654 can affect the harvest yield of root and cereal crops as diverse as sugar beets, potatoes and maize by a minimum of 14%.

[0381] Example 9 - Field applications of DSM 34654 in winter crops

[0382] Aim of study

[0383] The aim of this study was to test the ability of DSM 34654 to affect the growth of winter crops, such as winter wheat, when applied as a seed coat before sowing.

[0384] Materials and methods

[0385] Winter wheat seeds Triticum aestivum 'Momentum') were used for field trials (performed by an independent third-party contract research organization (VKST, Denmark)). Seed treatment was carried out by applying a bacterial seed coat solution containing 3.33xl08CFU / mL DSM 34654 to seeds at a rate of 30 mL / kg. Seeds were mixed thoroughly with the seed coat solution in a clean bag until an even distribution was assured. Untreated seeds were used as control seeds.

[0386] Following seed coat application, seeds were dried over night at maximum 8C° and sown on fields in repetitions of 16 on September 27, 2023 (mean air temperature for September 2023 was 16.3°C). Early and late emergence of winter wheat plants were quantified at 21 and 42 days following sowing as plants per m2.

[0387] Results

[0388] Treating winter wheat seeds with a seed coat containing a bacterial solution of DSM 34654 before sowing increased the late emergence (42 days after sowing) significantly (p-value=0.009644 using a one-sided Wilcoxon test) (Figure 10) with a mean increase in the emergence of 5.9%. This strongly suggests that DSM 34654 has the ability to increase plant emergence in fields when applied as a seed coat to seeds before sowing.

[0389] Conclusion

[0390] Winter wheat is one of the most common grown winter crops in Denmark.

[0391] Treating winter wheat seeds with a bacterial solution containing DSM 34654 before sowing demonstrates that DSM 34654 can enhance the emergence of winter wheat crops in the field, suggesting that an increased yield also may be obtained.

[0392] Example 10 - Genomic sequences broadly defining Rahnella aceris strains with biofertilizer potential

[0393] Aim of study

[0394] The scope of this example is to define DNA sequences that collectively characterize the best-performing Rahnella aceris strains, identified in Example 1 and 2, including DSM 34654.

[0395] Materials and methods

[0396] Strains, genome assemblies and genome annotations

[0397] A complete de novo genome was previously generated for DSM 34654 (see Example 3) and assemblies at contig level were previously generated for strains TCI, TC3, TP1 and TP3 (the remaining top- performers from Example 2) de novo (see Example 2). Genome annotations for all strains were previously generated (see Example 2 and 3).

[0398] Secondary metabolite analysis

[0399] A secondary metabolite profile was previously generated for DSM 34654 (see Example 3). antiSMASH (v7.0.0) was used for the identification and annotation of secondary metabolite biosynthesis gene clusters in TC and TP strains. The MIBiG repository containing experimentally characterized biosynthetic gene clusters was used for annotation. Extraction and analysis of DNA sequences defining Rahnella aceris strains DSM 34654, TP1, TP3, TCI and TC3

[0400] Samtools (vl.6) was used to extract DNA sequences with no overlap between DSM 34654, TP1, TP3, TCI and TC3 strains and Rahnella aceris strains listed in Table 12 representing publicly available R. aceris genomes. DNA sequences were subsequently compared to the complete nucleotide collection (nt database) from NCBI containing GenBank+EMBL+DDBJ+PDB+RefSeq sequences using BLAST. The blastn algorithm (v2.14.1 + ) with a word-size of 11 was used for all nucleotide alignment searches. Searches were performed on February 4, 2025. Gene product predictions were confirmed using BLAST and the blastx algorithm (v2.14.1 + ) using the non-redundant protein database. Searches were performed on February 4, 2025.

[0401] 4 DNA sequences (3,774-10,937 nucleotides) are annotated as SEQ ID NOs: 16- 19.

[0402] Table 12: GenBank IDs of Rahnella aceris genomes downloaded from NCBI (n=10 excluding atypical genomes, as of February 4, 2025) used for identifying DNA sequences that specify R. aceris strains DSM 34654, TP1, TP3, TCI and TC3.

[0403] Results

[0404] A pair-wise genome comparison analysis revealed that the best-performing TC and TP strains (TP1, TP3, TCI, and TC3), including DSM 34654, represent two different groups of Rahnella aceris strains (see Example 2). Besides possessing capacities for nitrogen fixation and solubilization of phosphorous (see Example 1), all TC and TP strains moreover contain genes that are important for plant growth, such as the iron-scavenging siderophore desferrioxamine E gene cluster, indicating that strains DSM 34654, TP1, TP3, TCI and TC3 have a high potential to perform as plant growth promoters. Genome assemblies representing DSM 34654, TP1, TP3, TCI and TC3 strains were compared to all publicly available R. aceris genomes (Table 12) and DNA sequences highly specific for DSM 34654, TP1, TP3, TCI and TC3 strains were extracted on the basis of the genome of DSM 34654 and subjected to a broad sequence similarity search. This identified 4 DNA sequences (SEQ ID NOs: 16-19 where SEQ ID NO: 15 comprises a minor part of SEQ ID NO: 19) showing 100% overlap with TC and TP strains with sequence identities of 99.91-100%, showing <99% overlap with other prokaryotic DNA sequences (Table 13) and importantly no identity in any Rahnella aceris species. Importantly, SEQ ID NOs: 16-19 contain genes encoding proteins that are involved in a broad spectrum of physiological processes such as bacterial immunity, gene regulation, enzymatic activities and DNA modification (Table 13). Thus, SEQ ID NOs: 16-19 are not only highly selective for DSM 34654, TP1, TP3, TCI and TC3 strains but do also represent DNA sequences with important functional properties.

[0405] Table 13: Reported maximum percent sequence coverage and sequence identity of SEQ ID Nos: 16-19 from a BLAST search against the nucleotide database from NCBI (performed on February 4, 2025). Hits were ranked according to 1) sequence coverage and 2) sequence identity. Encoded gene products indicate the predicted functions of coding sequences encoded by SEQ ID NO: 16-19. nt; nucleotides. Conclusion

[0406] DNA sequences characterizing Rahnella aceris strains DSM 34654, TP1, TP3, TCI and TC3 were identified on the basis of sequence alignment analyses (SEQ ID NOs: 16-19). Combining SEQ ID NOs displaying high specificity towards DSM 34654 (SEQ ID NOs: 8-15, as defined in Example 3) with SEQ ID NOs: 16-19 allow for defining combinations of SEQ ID NOs representing DSM 34654, TP1, TP3, TCI and TC3 strains. Combined or alone, these DNA sequences may be used to identify DSM 34654, TP1, TP3, TCI or TC3 strains at the DNA level. Thus, SEQ ID NOs: 16-19 are not only highly selective for DSM 34654, TP1, TP3, TCI and TC3 strains but do also represent DNA sequences with important functional properties. Hence, the study shows that several Rahnella aceris strains, isolated and identified in Examples 1-2, comprise several sequence overlaps with the deposited DSM 34654, and thus it is expected that TP1, TP3, TCI, and TC3 strains comprise similar functional properties as DSM 34654 (Table 14). Importantly, none of SEQ ID NOs: 16-19 demonstrate identity towards any other known Rahnella aceris species.

[0407] Table 14: List of combinations of SEQ ID NOs defining R. aceris strains DSM 34654, TP1, TP3, TCI and TC3.

[0408] Example 11 - Field applications of DSM 34654 in maize

[0409] Aim of study

[0410] The aim of this study was to test the ability of DSM 34654 to affect the harvest yield of maize when applied directly into the seed furrow during sowing. Materials and methods

[0411] Organic maize seeds (Zea mays L., variety KWS Autens) were used for field trials listed in Table 15. All experiments were performed by an independent third-party contract research organization (Ytteborg I / S, Denmark) and seeds were sown on May 14, 2024, in fields placed in the southwestern part of Jutland, Denmark (n = 7 per treatment group, where each n represents a field parcel of 13.5 m2). Seeds were sown with a planting rate of 9.5 seeds per m2and the soil temperature was 8°C at the time of sowing. Half of the field parcels received a NP 19-8 fertilizer treatment (130 kg / ha, containing 23 kg / ha nitrogen (N) and 10 kg / ha phosphorous (P)) directly into the seed furrow during sowing (Table 15). A bacterial solution of DSM 34654 containing 2.36xl010CFU / mL was sprayed directly into the seed furrow during sowing (seeds used for control experiments did not receive any treatment). The emergence of maize plants was quantified on September 18, 2024, and fields were harvested on September 23, 2024.

[0412] Table 15: Experimental overview of maize field trials (n=7 per treatment group, where each n represents a field parcel of 13.5 m2). Trials were carried out by an independent third-party contract research organization (Ytteborg I / S, Denmark). DSM 34654 were applied to the seed furrow during sowing. Seeds used for control experiments were untreated. Half of the field parcels received a NP fertilizer treatment (130 kg / ha, containing 23 kg / ha N and 10 kg / ha P) during sowing.

[0413] Results

[0414] The ability of DSM 34654 to increase the harvest yield of root and cereal crops, including maize, when applied as a seed coat, was demonstrated in Example 8. Emergence of maize plants along with harvest yield were assessed from fields were DSM 34654 was applied directly to the seed furrow during sowing of maize seeds. Interestingly, increased emergences of maize plants were observed for fields that were treated with DSM 34654 compared to control fields, irrespective of whether fields received fertilization during sowing (significant increase of 14.42%) or not (significant increase of 4.78%) (evaluated using a one-sided Wilcoxon rank sum test) (Figure 11A). Applying DSM 34654 to the seed furrow during sowing importantly led to a significant increase in the harvest yield of 17.37% from fields that received a fertilization treatment during sowing (Figure 11B, boxplots to the right). This demonstrates that DSM 34654 can affect harvest yields positively when applied either as a seed coat or a seed furrow treatment.

[0415] Conclusion

[0416] Applying a bacterial solution containing DSM 34654 to the seed furrow during sowing led to an increase in the emergence of maize plants and most importantly also an increase in the harvest yield of 17.37% (the later from fields that received fertilization during sowing), clearly demonstrating the ability of DSM 34654 to affect the harvest yield positively.

[0417] Example 12 - DSM 34654 has phytate-degrading activity

[0418] Aim of study

[0419] The aim of this study is to demonstrate the ability of DSM 34654 to mineralize phytate (myo-inositol hexakisphosphate) representing one of the major forms of organic phosphorous in soil.

[0420] Materials and methods

[0421] To demonstrate the ability of DSM 34654 to mineralize phytate, DSM 34654 was cultured on a modified Pikovskaya agar containing sodium phytate (insoluble organic phosphate) as sole phosphate source. The formation of translucent halo zones surrounding the bacterial colonies corresponds to enzymatic activities leading to the solubilization of phytate. The solubilization capacity was evaluated by calculating a solubilization index (SI, SI = Areanaio / Areacoiony). A commercial microbial biostimulant, Biol, was included in the experiment as a benchmarking control. An overnight culture was prepared for each strain and adjusted to OD600 = 1 the following day. Equal amounts of the bacterial suspensions were spotted (n=3) onto phytate-containing Pikovskaya agar plates, which were incubated at 15°C for 7 days. Colony and halo zone areas were quantified at day 1, day 4, day 5, day 6 and day 7 using Image!. Results

[0422] The ability of DSM 34654 to mineralize phytate was evaluated using a solid medium strategy where bacterial colony and halo zone areas were quantified at 15°C and furthermore compared to a bacterial strain that was isolated from a commercial biofungicide product (Biol). Both DSM 34654 and Biol were able to grow actively on the medium, however, only DSM 34654 displayed phytatedegrading activities at day 1 and 4 while Biol started to mineralize phytate at day 5 (Figure 12 and 13). These results demonstrate that DSM 34654 exhibits a high capacity for phytate mineralization highlighted by DSM 34654 reaching its maximum solubilization index (SI) the same day as Biol begins to show signs of mineralization (Figure 13). Thereafter, the bacterial colony size increases more relative to the halo size for DSM 34654 (Figure 12 and 13), however, a high capacity for phytate degradation is still maintained at day 7 (mean SI=4.99) compared to Biol (mean SI = 1.76). The SI on modified Pikovskaya at day 7 was:

[0423] • 4.99 at 15°C.

[0424] These results suggest that DSM 34654 has a higher capacity for mineralizing organic phosphate, which would otherwise remain unavailable to plants.

[0425] Conclusion

[0426] Organic phosphate can be found as aggregated forms, known as phytate, in soil. Phytate is inaccessible to plants and its mineralization can enhance nutrient availability, thereby promoting plant growth. In this study, DSM 34654 exhibited a greater ability to mineralize organic phosphate, here represented as phytate, compared to a commercial biofungicide strain (Biol), demonstrating that DSM 34654 has a high potential for supplying crops with otherwise unavailable nutrients in the field. Additionally, when coupled with its previously demonstrated ability to solubilize inorganic phosphate (Example 4), DSM 34654 emerges as a versatile biostimulant.

[0427] Sequence listing

[0428] Items of the invention

[0429] 1. A bacteria, preferably isolated, or biologically pure bacterial culture comprising a) a genomic sequence according to any of SEQ ID NOs: 9-19, preferably SEQ ID NOs:9-14; b) a genomic sequence having at least 66%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as preferably at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9

[0430] % sequence identity to any of SEQ ID NO: 9-19, preferably SEQ ID NOs: 9- 14; and / or c) a fragment of one or more of the sequences of a) or b), having a length of at least 400 nucleotides.

[0431] 2. The bacteria or biologically pure bacterial culture according to item 1 comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 9, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 9.

[0432] 3. The bacteria or biologically pure bacterial culture according to item 1 comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 10, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 10.

[0433] 4. The bacteria or biologically pure bacterial culture according to item 1 comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 11, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 11.

[0434] 5. The bacteria or biologically pure bacterial culture according to item 1 comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 12, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 12.

[0435] 6. The bacteria or biologically pure bacterial culture according to item 1 comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 13, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 13.

[0436] 7. The bacteria or biologically pure bacterial culture according to item 1 comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 14, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 14.

[0437] 8. The bacteria or biologically pure bacterial culture according to item 1 comprising any of SEQ ID NO: 9-14, such as one or more of SEQ ID NO: 9-14. 9. The bacteria or biologically pure bacterial culture according to item 1, comprising a) a genomic sequence according to SEQ ID NO: 8; b) a genomic sequence having at least 90% sequence identity to SEQ ID NO: 8, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 8; and / or c) a fragment of the sequence of a) or b), having a length of at least 5000 nucleotides, such as at least 8000 nucleotides, such as at least 10000 nucleotides, such as at least 13000 nucleotides.

[0438] 10. The bacteria or biologically pure bacterial culture according to any of the preceding items, being a Rahnella, such as Rahnella aceris.

[0439] 11. A bacteria or biologically pure bacterial culture being Rahnella aceris, DSM 34654 deposited with the DSMZ [LEIBNIZ- INSTITUT DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH, Inhoffenstr. 7 B, D-38124, Braunschweig, Germany] on 24 May 2023.

[0440] 12. The bacteria or biologically pure bacterial culture according to any of the preceding items, being

[0441] - capable of solubilizing calcium phosphate at 5°C, and / or iron phosphate at 5°C and / or potassium aluminum silicate at 5°C, and / or insoluble organic phosphate, such as phytate, at 15°C; and / or

[0442] - capable of inhibiting one or more phytopathogenic fungi.

[0443] 13. The bacteria or biologically pure bacterial culture according to item 12, wherein the one or more phytopathogenic fungi are selected from the group consisting of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres and Fusarium oxy s porum.

[0444] 14. The bacteria or biologically pure bacterial culture according to item 12, wherein the solubilization of calcium phosphate, iron phosphate, and / or potassium aluminum silicate at 5°C is at a level of at least 80% to, equal to and / or above a level of solubilization at 15°C and / or 25°C.

[0445] 15. The bacteria or biologically pure bacterial culture according to any of item 12 or 14, wherein the level of solubilization is determined by a solubilization index (SI) calculated by the formula:

[0446] SI = AreaHaio / Areacoiony.

[0447] 16. The bacteria or biologically pure bacterial culture according to item 15, wherein the SI is at least 7, such as at least 10, such as between 10 and 23, preferably the SI is between 10 and 21.

[0448] 17. The bacteria or biologically pure bacterial culture according to any of items 15 or 16, wherein the SI depends on temperature after cultivation start and / or incubation time.

[0449] 18. The bacteria or biologically pure bacterial culture according to any of items 15-17, wherein the temperature is between 2°C and 30°C, such as between 5°C and 25°C, such as between 5°C and 10°C, preferably between 5°C and 8°C.

[0450] Fertilizer:

[0451] 19. A fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition comprising the bacteria or biologically pure bacterial culture according to any of items 1-18.

[0452] 20. The fertilizer and / or inoculant and / or biostimulant composition according to item 19, further comprising other microorganisms, such as other microorganisms able to function as a biostimulant.

[0453] 21. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 19 or 20, further comprises one or more agriculturally acceptable carriers.

[0454] 22. The fertilizer and / or inoculant and / or biostimulant composition according to item 21, the agriculturally acceptable carrier is selected from the group consisting of a dispersant, a surfactant, an additive, water, a thickener, an anti-caking agent, residue breakdown, a composting formulation, a granular application, diatomaceous earth, an oil, a coloring agent, a stabilizer, a preservative, a polymer, biopolymer, a coating, or a combination thereof.

[0455] 23. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 19-22, the composition comprises a biopolymer, oligosaccharide, disaccharide or monosaccharide selected from the group consisting of pectin, alginate, chitosan, cellulose, a cellulose derivative, starch, maltodextrin, chitin, glucose, trehalose, sucrose, xanthan gum, guar gum, diutan gum, or a biopolymer derived from a natural source, possibly chemically modified afterwards.

[0456] 24. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 19-23, the composition is formulated as a liquid formulation for application to plants or to a plant growth medium, or a solid formulation for application to plants or to a plant growth medium.

[0457] 25. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 19-23, the composition is formulated as a granular formulation or a powder formulation.

[0458] 26. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 19-25, the composition further comprises 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.

[0459] 27. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 19-26, the composition comprises one or more of 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, calcium nitrate, dolomitic limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, urea ammonium nitrate, sulfur- coated urea, polymer- coated urea, isobutylidene diurea, K2S04-2MgS04, 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, biochar, sludge, green manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion, or a combination thereof.

[0460] 28. The fertilizer and / or inoculant and / or biostimulant composition according to item 26, the micronutrient fertilizer material comprises one or more of 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 carbonate, a zinc frit, zinc phosphate, a zinc chelate, or a combination thereof.

[0461] Coating composition

[0462] 29. A coating composition, preferably a seed coating composition, comprising the bacteria or biologically pure bacterial culture according to any of the items 1-18 and / or the composition according to any of item 19-28.

[0463] 30. The coating composition according to item 29, the coating composition comprises a biopolymer promoting adherent to a plant seed.

[0464] 31. The coating composition according to any of items 29 or 30, the coating composition is formulated as an aqueous or oil-based solution for application to seeds, preferably aqueous.

[0465] 32. The coating composition according to any of items 29-31, the coating composition is formulated as a powder or granular formulation for application to seeds. Plant seed

[0466] 33. A plant seed coated with the composition according to any of item 19-28 or coated with a coating composition according to any of items 29-32.

[0467] 34. The plant seed according to item 33, the plant seed is a dicotyledon, monocotyledon or a gymnosperm seed.

[0468] 35. The plant seed according to any of items 33 or 34, the plant seed being selected from the group consisting of a crop seed, such as barley seed, such as spring or winter barley, oilseed, such as rapeseed, wheat, such as winter or spring wheat, oats, triticale, maize, rye, grass, clover, broad bean, lupines, peas, strawberries, tomatoes, cucumber, peas, potatoes, onions, carrots, and sugar beets.

[0469] 36. The plant seed according to any of items 33-35, the plant seed being selected from the group consisting of a cover crop seed, such as fodder radish, clover, yellow mustard, or Phacelia.

[0470] 37. The plant seed according to any of items 33-36, the plant seed being selected from the group of trees, bushes, or grasses.

[0471] Use

[0472] 38. Use of a Rahnella bacteria for plant growth, such as a fertilizer or inoculum or biostimulant, at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C, preferably the temperature is a surface temperature.

[0473] 39. Use of a bacteria or biologically pure bacterial culture according to any of items 1-18, the composition according to any of items 19-28 or the coating composition according to any of items 29-32, as a plant growth promoting agent, such as a fertilizer or inoculum or biostimulant, preferably at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C. 40. The use according to item 39, wherein the use as a plant growth-promoting agent is for:

[0474] • solubilizing inorganic minerals or salts, such as phosphate, and / or potassium, and / or iron, and / or aluminium, and / or zinc, and / or manganese and / or organic phosphate, such as phytate; and / or

[0475] • chelating inorganic minerals such as iron, zinc, manganese, copper; and / or

[0476] • allowing for growth under low soil nitrogen conditions; and / or

[0477] • fixating atmospheric nitrogen; and / or

[0478] • increasing plant growth, such as plant length, leaf diameter and / or root length; and / or

[0479] • improving germination of the seeds; and / or

[0480] • improving emergence of the plants; and / or

[0481] • increasing biomass; and / or

[0482] • increasing growth; and / or

[0483] • increasing crop yield; and / or

[0484] • inducing anti-fungal effects; and / or

[0485] • increasing harvest yield; and / or

[0486] • increasing hectoliter weight; and / or

[0487] • increasing protein content; and / or

[0488] • increasing protein yield; and / or

[0489] • increasing oil content; and / or

[0490] • increasing oil yield; and / or

[0491] • reducing mycotoxin contamination; and / or

[0492] • reducing seed-borne fungal or bacterial contamination; and / or

[0493] • improving taste, smell or appearance; and / or

[0494] • replacing or substituting any chemical product used in plant production while retaining one or more desirable plant parameters.

[0495] 41. The use according to item 40, wherein the inorganic minerals or salts are selected from the group consisting of

[0496] • minerals, such as rock phosphate, potash, lime, clay, ground rocks, sand, silt, sediment and natural deposits;

[0497] • salts, such as ammonium phosphate, potassium phosphate, potassium nitrate, potassium chloride, ammonium sulfate, calcium phosphate; calcium sulphate, magnesium phosphate, iron phosphate, potassium alumino silicate (feldspar, mica) and salts that contain potassium or phosphate;

[0498] • fertilizing substances, such as mineral fertilizer, NPK fertilizer, NS fertilizer, K fertilizer, P fertilizer, N fertilizer, organic fertilizer, manure, sludge, compost, biowaste, biochar, biogas residue, ash, wood ash, bone ash, bone meal, urine, faeces or a plant-based fertilizer.

[0499] 42. The use according to any of items 38-41, wherein the use takes place at field temperatures in the range -10°C to 15°C, such as -5°C to 15°C, preferably 0 to 15°C, more preferably such as 2 to 10°C, such 2 to 8°C.

[0500] 43. The use according to any of items 38-42, the bacteria or biologically pure bacterial culture according to any of items 1-18, the composition according to any of items 19-28 or the coating composition according to any of items 29-32 is applied in an effective amount.

[0501] 44. The use according to any of items 38-43, the use takes place in Scandinavia, such as Norway, Sweden, Finland, Denmark, such as in Jutland, Fyn, Zealand, and / or Great Britain, and / or Germany.

[0502] 45. The use according to any of items 38-44, the plant is a dicotyledon, monocotyledon or a gymnosperm.

[0503] 46. The use according to item 45, the plant is selected from the group consisting of a crop seed, such as barley seed, such as spring barley, oilseed, such as rapeseed, wheat, such as winter wheat.

[0504] Method

[0505] 47. A method for stimulating plant growth comprising applying the bacteria or biologically pure bacterial culture according to any of items 1-18, the composition according to any of items 19-28 or the coating composition according to any of items 29-32 to a plant, plant seed, a sowing forrow, soil and / or plant growth medium, preferably at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C. 48. The method for stimulating plant growth according to item 47, the method comprises applying the bacteria or biologically pure bacterial culture according to any of items 1-18, the composition according to any of items 19-28 or the coating composition according to any of items 29-32:

[0506] - to a plant growth medium, such as sphagnum; and / or

[0507] - to a plant growth medium prior to, concurrently with, or after planting of seeds, seedlings, cuttings, bulbs, or plants in the plant growth medium; or

[0508] - to plant leaves, roots, or stems; and / or

[0509] - to plant seeds, and / or

[0510] - to a watering system for the plants, such as hydroponics, aeroponics and / or aquaponics.

[0511] 49. The method for stimulating plant growth according to any of items 47 or 48, the bacteria or biologically pure bacterial culture according to any of items 1-18, the composition according to any of items 19-28 or the coating composition according to any of items 29-32 is sprayed or irrigated onto plants or fields.

[0512] Kit

[0513] 50. A kit of parts for stimulating plant growth comprising

[0514] • a first container comprising the bacteria or biologically pure bacterial culture according to any of items 1-18, the composition according to any of items 19-28 and / or the coating composition according to any of items 29- 32; and

[0515] • instructions for applying the inoculum to plants, plant seeds, or a plant growth medium, preferably at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C.

[0516] 51. The kit of parts according to item 50 further comprises one or more containers comprising fertilizers, nutrients, and / or other microorganisms.

[0517] 52. Use of the bacteria or biologically pure bacterial culture according to any of items 1-18 or the composition according to any of items 19-28 for solubilizing minerals in ores. Medical uses

[0518] 53. A bacteria or biologically pure bacterial culture according to any of items 1-18, a composition according to any of items 19-28 or a coating composition according to any of items 29-32 for use as a medicament.

[0519] 54. A bacteria or biologically pure bacterial culture according to any of items 1-18 or a composition according to any of items 19-28 use in the treatment, alleviation and / or prevention of fungal infections. 55. A bacteria or biologically pure bacterial culture for use according to item 54 wherein the fungal infection is selected from the group consisting of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres, Fusarium oxysporum or a combination thereof.

Claims

Claims1. A bacteria, preferably isolated, or biologically pure bacterial culture comprising a) a genomic sequence according to any of SEQ ID NOs: 9-19, preferably SEQ ID NOs: 9-14; b) a genomic sequence having at least 66%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as preferably at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to any of SEQ ID NO: 9-19, preferably SEQ ID NOs: 9- 14; and / or c) a fragment of one or more of the sequences of a) or b), having a length of at least 400 nucleotides.

2. The bacteria or biologically pure bacterial culture according to claim 1 comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 9, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 9.

3. The bacteria or biologically pure bacterial culture according to any of claims 1 or 2 comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 10, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 10.

4. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 11, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 11.

5. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 12, such as at least 97%, such as at least 98%, such asat least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 12.

6. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 13, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 13.

7. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 14, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9% sequence identity to SEQ ID NO: 14.

8. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 15, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 15.

9. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 16, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 16.

10. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising a genomic sequence having at least 95% sequence identity to SEQ ID NO: 17, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 17.

11. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising a genomic sequence having at least 95% sequenceidentity to SEQ ID NO: 18 and / or 19, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 18 and / or 19.

12. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising any of SEQ ID NO: 9-19, such as one or more of SEQ ID NO: 9-19, such as one or more of SEQ ID NO: 9-14, such as two or more of SEQ ID NO: 9-19, such as three or more of SEQ ID NO: 9-19, such as four or more of SEQ ID NO: 9-19, such as five or more of SEQ ID NO: 9-19, such as six or more of SEQ ID NO: 9-19, such as eight or more of SEQ ID NO: 9-19, such as nine or more of SEQ ID NO: 9-19, such as ten or more of SEQ ID NO: 9-19.

13. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising SEQ ID NO: 9-19, such as SEQ ID NO: 9-14.

14. The bacteria or biologically pure bacterial culture according to any of the preceding claims, comprising a) a genomic sequence according to SEQ ID NO: 8; b) a genomic sequence having at least 90% sequence identity to SEQ ID NO: 8, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 8; and / or c) a fragment of the sequence of a) or b), having a length of at least 5000 nucleotides, such as at least 8000 nucleotides, such as at least 10000 nucleotides, such as at least 13000 nucleotides.

15. The bacteria or biologically pure bacterial culture according to any of the preceding claims, being a Rahnella, such as Rahnella aceris.

16. A bacteria or biologically pure bacterial culture being Rahnella aceris, DSM 34654 deposited with the DSMZ [LEIBNIZ- INSTITUT DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH, Inhoffenstr. 7 B, D-38124, Braunschweig, Germany] on 24 May 2023.

17. The bacteria or biologically pure bacterial culture according to any of the preceding claims, being- capable of solubilizing calcium phosphate at 5°C, and / or iron phosphate at 5°C and / or potassium aluminum silicate at 5°C, and / or insoluble organic phosphate, such as phytate, at 15°C; and / or- capable of inhibiting one or more phytopathogenic fungi.

18. The bacteria or biologically pure bacterial culture according to claim 17, wherein the one or more phytopathogenic fungi are selected from the group consisting of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres and Fusarium oxy s porum.

19. The bacteria or biologically pure bacterial culture according to claim 17, wherein the solubilization of calcium phosphate, iron phosphate, and / or potassium aluminum silicate at 5°C is at a level of at least 80% to, equal to and / or above a level of solubilization at 15°C and / or 25°C.

20. The bacteria or biologically pure bacterial culture according to any of claim 17- 19, wherein the level of solubilization is determined by a solubilization index (SI) calculated by the formula:SI = AreaHaio / Areacoiony.

21. The bacteria or biologically pure bacterial culture according to claim 20, wherein the SI is at least 7, such as at least 10, such as between 10 and 23, preferably the SI is between 10 and 21.

22. The bacteria or biologically pure bacterial culture according to any of claims 20 or 21, wherein the SI depends on temperature after cultivation start and / or incubation time.

23. The bacteria or biologically pure bacterial culture according to any of claims 20-22, wherein the temperature is between 2°C and 30°C, such as between 5°C and 25°C, such as between 5°C and 10°C, preferably between 5°C and 8°C.

24. The bacteria or biologically pure bacterial culture according to any of claims 17-23, wherein the bacteria or biologically pure bacterial culture comprises a capacity for solubilizing insoluble organic phosphate, such as phytate, such as at 15°C.

25. A fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition comprising the bacteria or biologically pure bacterial culture according to any of claims 1-24.

26. The fertilizer and / or inoculant and / or biostimulant composition according to claim 25, further comprising other microorganisms, such as other microorganisms able to function as a biostimulant.

27. The fertilizer and / or inoculant and / or biostimulant composition according to any of claims 25 or 26, further comprises one or more agriculturally acceptable carriers.

28. The fertilizer and / or inoculant and / or biostimulant composition according to claim 27, the agriculturally acceptable carrier is selected from the group consisting of a dispersant, a surfactant, an additive, water, a thickener, an anti-caking agent, residue breakdown, a composting formulation, a granular application, diatomaceous earth, an oil, a coloring agent, a stabilizer, a preservative, a polymer, biopolymer, a coating, or a combination thereof.

29. The fertilizer and / or inoculant and / or biostimulant composition according to any of claims 25-28, the composition comprises a biopolymer, oligosaccharide, disaccharide or monosaccharide selected from the group consisting of pectin, alginate, chitosan, cellulose, a cellulose derivative, starch, maltodextrin, chitin, glucose, trehalose, sucrose, xanthan gum, guar gum, diutan gum, or a biopolymer derived from a natural source, possibly chemically modified afterwards.

30. The fertilizer and / or inoculant and / or biostimulant composition according to any of claims 25-29, the composition is formulated as a liquid formulation for application to plants or to a plant growth medium, or a solid formulation for application to plants or to a plant growth medium.

31. The fertilizer and / or inoculant and / or biostimulant composition according to any of claims 25-30, the composition is formulated as a granular formulation or a powder formulation.

32. The fertilizer and / or inoculant and / or biostimulant composition according to any of claims 25-31, the composition further comprises 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.

33. The fertilizer and / or inoculant and / or biostimulant composition according to any of claims 25-32, the composition comprises one or more of 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, calcium nitrate, dolomitic limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, urea ammonium nitrate, sulfur- coated urea, polymer- coated urea, isobutylidene diurea, K2S04-2MgS04, 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, biochar, sludge, green manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion, or a combination thereof.

34. The fertilizer and / or inoculant and / or biostimulant composition according to claim 32, the micronutrient fertilizer material comprises one or more of 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 sodiummolybdate, molybdic acid, a zinc sulfate, a zinc oxide, a zinc carbonate, a zinc frit, zinc phosphate, a zinc chelate, or a combination thereof.

35. A coating composition, preferably a seed coating composition, comprising the bacteria or biologically pure bacterial culture according to any of the claims 1-24 and / or the composition according to any of the claims 25-34.

36. The coating composition according to claim 35, the coating composition comprises a biopolymer promoting adherent to a plant seed.

37. The coating composition according to any of claims 35 or 36, the coating composition is formulated as an aqueous or oil-based solution for application to seeds, preferably aqueous.

38. The coating composition according to any of claims 35-37, the coating composition is formulated as a powder or granular formulation for application to seeds.

39. A plant seed coated with the composition according to any of claims 25-34 or coated with a coating composition according to any of claims 35-38.

40. The plant seed according to claim 39, the plant seed is a dicotyledon, monocotyledon or a gymnosperm seed.

41. The plant seed according to any of claims 39 or 40, the plant seed being selected from the group consisting of a crop seed, such as barley seed, such as spring or winter barley, oilseed, such as rapeseed, wheat, such as winter or spring wheat, oats, triticale, maize, rye, grass, clover, broad bean, lupines, peas, strawberries, tomatoes, cucumber, peas, potatoes, onions, carrots, and sugar beets.

42. The plant seed according to any of claims 39-41, the plant seed being selected from the group consisting of a cover crop seed, such as fodder radish, clover, yellow mustard, or Phacelia.

43. The plant seed according to any of claims 39-42, the plant seed being selected from the group of trees, bushes, or grasses.

44. Use of a Rahnella bacteria for plant growth, such as a fertilizer or inoculum or biostimulant, at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C.

45. Use of a bacteria or biologically pure bacterial culture according to any of claims 1-24, the composition according to any of claims 25-34 or the coating composition according to any of claims 35-38, as a plant growth promoting agent, such as a fertilizer or inoculum or biostimulant, preferably at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C.

46. The use according to claim 45, wherein the use as a plant growth-promoting agent is for:• solubilizing inorganic minerals or salts, such as phosphate, and / or potassium, and / or iron, and / or aluminium, and / or zinc, and / or manganese and / or organic phosphate, such as phytate; and / or• chelating inorganic minerals such as iron, zinc, manganese, copper; and / or• allowing for growth under low soil nitrogen conditions; and / or• fixating atmospheric nitrogen; and / or• increasing plant growth, such as plant length, leaf diameter and / or root length; and / or• improving germination of the seeds; and / or• improving emergence of the plants; and / or• increasing biomass; and / or• increasing growth; and / or• increasing crop yield; and / or• inducing anti-fungal effects; and / or• increasing harvest yield; and / or• increasing hectoliter weight; and / or• increasing protein content; and / or• increasing protein yield; and / or• increasing oil content; and / or• increasing oil yield; and / or• reducing mycotoxin contamination; and / or• reducing seed-borne fungal or bacterial contamination; and / or• improving taste, smell or appearance; and / or• replacing or substituting any chemical product used in plant production while retaining one or more desirable plant parameters.

47. The use according to claim 46, wherein the inorganic minerals or salts are selected from the group consisting of• minerals, such as rock phosphate, potash, lime, clay, ground rocks, sand, silt, sediment and natural deposits;• salts, such as ammonium phosphate, potassium phosphate, potassium nitrate, potassium chloride, ammonium sulfate, calcium phosphate; calcium sulphate, magnesium phosphate, iron phosphate, potassium alumino silicate (feldspar, mica) and salts that contain potassium or phosphate;• fertilizing substances, such as mineral fertilizer, NPK fertilizer, NS fertilizer, K fertilizer, P fertilizer, N fertilizer, organic fertilizer, manure, sludge, compost, biowaste, biochar, biogas residue, ash, wood ash, bone ash, bone meal, urine, faeces or a plant-based fertilizer.

48. The use according to any of claims 44-47, wherein the use takes place at field temperatures in the range -10°C to 15°C, such as -5°C to 15°C, preferably 0 to 15°C, more preferably such as 2 to 10°C, such 2 to 8°C.

49. The use according to any of claims 44-48, the bacteria or biologically pure bacterial culture according to any of claims 1-24, the composition according to any of claims 25-34 or the coating composition according to any of claims 35-38 is applied in an effective amount.

50. The use according to any of claims 44-49, the use takes place in Scandinavia, such as Norway, Sweden, Finland, Denmark, such as in Jutland, Fyn, Zealand, and / or Great Britain, and / or Germany.

51. The use according to any of claims 44-50, the plant is a dicotyledon, monocotyledon or a gymnosperm.

52. The use according to claim 51, the plant is selected from the group consisting of a crop seed, such as barley seed, such as spring barley, oilseed, such as rapeseed, wheat, such as winter wheat.

53. A method for stimulating plant growth comprising applying the bacteria or biologically pure bacterial culture according to any of claims 1-24, the composition according to any of claims 25-34 or the coating composition according to any of claims 35-38 to a plant, plant seed, a sowing forrow, soil and / or plant growth medium, preferably at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C.

54. The method for stimulating plant growth according to claim 53, the method comprises applying the bacteria or biologically pure bacterial culture according to any of claims 1-24, the composition according to any of claims 25-34 or the coating composition according to any of claims 35-38:- to a plant growth medium, such as sphagnum; and / or- to a plant growth medium prior to, concurrently with, or after planting of seeds, seedlings, cuttings, bulbs, or plants in the plant growth medium; or- to plant leaves, roots, or stems; and / or- to plant seeds, and / or- to a watering system for the plants, such as hydroponics, aeroponics and / or aquaponics.

55. The method for stimulating plant growth according to any of claims 53 or 54, the bacteria or biologically pure bacterial culture according to any of claims 1-24, the composition according to any of claims 25-34 or the coating composition according to any of claims 35-38 is sprayed or irrigated onto plants or fields.

56. A kit of parts for stimulating plant growth comprising• a first container comprising the bacteria or biologically pure bacterial culture according to any of claims 1-24, the composition according to any of claims 25-34 and / or the coating composition according to any of claims 35-38; andinstructions for applying the inoculum to plants, plant seeds, or a plant growth medium, preferably at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C.

57. The kit of parts according to claim 56 further comprises one or more containers comprising fertilizers, nutrients, and / or other microorganisms.

58. A bacteria or biologically pure bacterial culture according to any of claims 1- 24, a composition according to any of claims 25-34 or a coating composition according to any of claims 35-38 for use as a medicament.

59. A bacteria or biologically pure bacterial culture according to any of claims 1-24 or a composition according to any of claims 25-34 use in the treatment, alleviation and / or prevention of fungal infections.

60. A bacteria or biologically pure bacterial culture for use according to claim 59 wherein the fungal infection is selected from the group consisting of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres, Fusarium oxysporum or a combination thereof.

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