Novel pseudomonas strain

Abstract

The present invention relates to an identified and isolated bacteria or biologically pure bacterial culture of the genus Pseudomonas. 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 PSEUDOMONAS STRAIN

[0002] Technical field of the invention

[0003] The present invention relates to an isolated bacteria or biologically pure bacterial culture of the genus Pseudomonas. 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] WO 2020 / 214843 Al discloses a method of incorporating Pseudomonas bacteria into a plant seed for increasing plant growth, improving nutrient supply, enhancing plant defence against plant pathogens such as fungi and viruses, and improving tolerance to abiotic stress. The growth temperatures of the plant seeds are disclosed as 19-25°C. However, the Pseudomonas bacteria have not been reported to be active under low-temperature conditions, such as below 20°C and even below 7°C, representing the climate in Scandinavia during mild winter or spring seasons. Rafikova G. F. et al. ("New Bacterial Strains of Pseudomonas laurentiana: Promising Agents for Agrobiotechnology", MOSCOW UNIVERSITY BIOLOGICAL SCIENCES BULLETIN, 2020, vol. 75, no. 4, pages 206-211} discloses Pseudomonas laurentiana strains, which possess a set of properties of plant growth-promoting traits, e.g. antifungal activity against phytopathogenic fungi. However, the disclosed Pseudomonas strains have not been reported to exert plant growth-promoting capabilities under low-temperature conditions such as 5°C.

[0010] WO 2008 / 130701 Al discloses a Pseudomonas antarctica and a Pseudomonas trivialis strain, which are able to solubilize inorganic phosphorus sources, as well as seeds coated with the strains and the use of the strains as biofertilizers.

[0011] Hence, expanding the portfolio of bacteria and providing a bacteria capable of improving plant growth, such as at low temperatures, would be advantageous, and in particular, more efficient and / or reliable compositions comprising such bacteria would be advantageous.

[0012] Summary of the invention

[0013] The inventing team has isolated, identified, characterized and propagated a newly identified strain of bacteria, namely the Pseudomonas strain DSM 34653 deposited with deposit number DSM 34653. 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.

[0014] 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.

[0015] 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.

[0016] 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 be used in mining, recycling, or urban mining.

[0017] Example 1 shows how bacterial strains with biocontrol properties were isolated from the soil of a Danish grassland and screened to identify the best- performing bacterial strain. The microbeTRAP enabled capture of multiple potential bacterial biocontrol strains from Danish grassland soil. Of the isolated strains, TF-D (DSM 34653) was chosen as the most promising candidate for a biocontrol agent based on the initial screening for biocontrol properties on three common phytopathogenic fungi. Example 2 shows that DSM 34653 is a novel bacterial species belonging to the genus Pseudomonas. Interestingly, DSM 34653 encodes several properties that are beneficial to agricultural farming, including phosphate solubilizing capabilities. As DSM 34653 is classified as a novel bacterial species, virulence and antimicrobial resistance properties were assessed. These analyses revealed the presence of few virulence and antimicrobial resistance genes.

[0018] Several DNA sequences containing open reading frames and that have high specificity for DSM 34653 were identified; three long genomic regions (14,515- 16,414 nucleotides, SEQ ID NOs: 2-4) wherein short DNA sequences (303-2,779 nucleotides) were extracted (SEQ ID NOs: 5-13) as well as four short DNA sequences (714-1,077 nucleotides) identified outside of the three long genomic regions (SEQ ID NOs: 14-17). Combined or alone, these DNA sequences may be used to identify DSM 34653 at the DNA sequence level.

[0019] Example 3 shows the ability of DSM 34653 to control growth of common fungal plant pathogens. DSM 34653 was shown to have antifungal activity against four common phytopathogenic fungi Gaeumannomyces graminis var. tritici (isolate CBS 450.77), Pyrenophora teres f. teres (isolate CP2189), Zymoseptoria tritici (isolate IPO323), Fusarium oxysporum (isolate CBS 619.87) when evaluated in a dual culture assay. Based on these results DSM 34653 could hold potential for mitigating crop losses caused by fungal diseases, thereby enhancing agricultural productivity and sustainability.

[0020] Example 4 shows the efficacy of DSM 34653 as a biostimulant in increasing the availability of common plant nutrients under low, medium, and high temperature conditions. Only DSM 34653 was able to grow and display bacterial activity at 5°C, demonstrating that DSM 34653 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.

[0021] Example 5 shows the stimulatory properties of DSM 34653, when applied as a seed coat, on early plant growth in vitro in spring barley. Treating spring barley seeds with a bacterial suspension containing DSM 34653 before sowing demonstrates that DSM 34653 can enhance the emergence of seedlings, also when seeds have been treated with a commercially available fungicide, suggesting that an increased yield also may be obtained.

[0022] Example 6 shows that DSM 34653 can produce secondary metabolites that are important for plant health and development, including hydrogen cyanide (HCN), siderophores, and indole-3-acetic acid (IAA), thus strongly indicating that DSM 34653 has a high capacity for crop management in the field.

[0023] Example 7 shows that DSM 34653 has phytate-degrading capabilities throughout the entire experimental period with a maximum solubilization index (SI) at day 7. These results suggest that DSM 34653 has a high capacity for mineralizing organic phosphate by which inorganic phosphorous is released, which would otherwise remain unavailable to plants.

[0024] Example 8 shows that DSM 34653 exhibits the ability to both survive in a zinc-rich environment and solubilize a highly insoluble form of zinc. These capabilities are absent in the commercial biofungicide strain that was included for benchmarking purposes. These findings support that DSM 34653 has potential for supplying crops with otherwise unavailable nutrients in the field.

[0025] Example 9 shows that treating spring barley seeds or plants with a bacterial suspension of DSM 34653, as either a seed coat, a foliar spray or as a combination, leads to spring barley plants being less affected by brown leaf rust or net blotch. Applying DSM 34653 to seeds before sowing results in a similar or greater decrease in disease presence compared to chemical fungicides that is applied as either a seed coat or a foliar spray while also resulting in an increased harvest yield when DSM 34653 is applied as a combination of a seed coat and a spray. Thus, DSM 34653 exhibits biocontrol properties in the field.

[0026] Example 10 shows a comparative genome analysis revealing that DSM 34653 and the remaining isolated strains presented in Example 1 (TF-A, TF-B, TF-C, TF-E, TF-F and TF-G) are variants of the same type of Pseudomonas strain. In particular, comparing TF-A, TF-B, TF-C, TF-E, TF-F and TF-G to each other and to the genome representing DSM 34653 through average nucleotide identity (ANI) analyses reveals that DSM 34653 as well as the bacterial strains TF-A, TF-B, TF-C, TF-E, TF-F and TF-G represent the same Pseudomonas strain which should further be considered as novel Pseudomonas species.

[0027] Example 11 shows that an increased emergence of spinach plants can be obtained in the field when spinach seeds are treated with a bacterial suspension containing DSM 34653 as a seed coat before sowing. The increase in emergence is comparable or significantly increased compared to the emergence stimulated by a chemical fungicide that was included as a benchmarking control. Thus, DSM 34653 stimulates growth of spinach plants which may lead to an increased yield.

[0028] Example 12 shows that symptoms of Septoria tritici blotch in winter wheat can be reduced under controlled conditions with foliar spray application of suspensions containing DSM 34653. Accordingly, DSM 34653 shows potential as a biocontrol agent for managing fungal diseases in cereal crops.

[0029] The aforementioned deposit was made by Bioomix A / S on 24 May 2023. The deposit was given the following reference number: DSM 34653.

[0030] Thus, an object of the present invention relates to the provision of novel bacterial strains / biostimulants for improving plant growth, such as at low temperatures.

[0031] 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.

[0032] 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: 5-17; 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: 5-17; and / or c) a fragment of one or more of the sequences of a) or b), having a length of at least 300 nucleotides. Another aspect of the present invention relates to a bacteria or biologically pure bacterial culture being Pseudomonas, DSM 34653 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.

[0033] Yet another 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.

[0034] A further 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 the invention and / or the composition according to the invention.

[0035] Another aspect of the present invention relates to a plant seed coated with the composition according to the invention or coated with a coating composition according to the invention.

[0036] Yet another aspect of the present invention relates to use of a 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.

[0037] Another aspect of the present invention relates to a method for stimulating plant growth comprising 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 to a plant, plant seed, a sowing furrow, soil and / or plant growth medium.

[0038] A further aspect of the present invention relates to a kit of parts for stimulating plant growth comprising • a first container comprising the 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

[0039] • instructions for applying the 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.

[0040] 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.

[0041] 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.

[0042] A yet an 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.

[0043] Brief description of the figures

[0044] Figure 1

[0045] Figure 1 shows a histogram showing genome-to-genome comparisons from an average nucleotide identity (ANI) analysis of the genome assembly representing DSM 34653 and all Pseudomonas RefSeq reference genomes available at NCBI (n=368, excluding atypical genomes, as of April 3, 2024). The dotted vertical line represents the boundary for which new species are defined (at 95% ANI).

[0046] Figure 2

[0047] Figure 2 shows phylogenetic trees representing evolutionary relatedness between DSM 34653 and Pseudomonas references at species level (all Pseudomonas RefSeq reference genomes available at the National Center for Biotechnology Information (NCBI) (n = 368, excluding atypical genomes, as of April 3, 2024)) (circular tree to the left). The tree to the right represents a zoomed view of the clade containing DSM 34653 (further highlighted within the grey area).

[0048] Figure 3

[0049] Figure 3 shows a phylogenetic tree representing evolutionary relatedness between DSM 34653 and all available RefSeq strain genomes at the National Center for Biotechnology Information (NCBI) belonging to the following Pseudomonas species: wadenswilerensis, donghuensis, tructae and rubra (n = 14, excluding atypical genomes, as of April 5, 2024). DSM 34653 is highlighted within the grey area.

[0050] Figure 4

[0051] Figure 4 shows a heatmap representing the presence of virulence factors (VFs) in the genome assembly of DSM 34653 and four pathogenic Pseudomonas aeruginosa strains (PAO1, UCBPP PA14, LESB58 or PA7). Dark grey bars indicate significant hits (presence of a VF) and light grey bars indicate no hit (no presence of VF in strain). 329 significant and unique VFs were found across all genomes analyzed.

[0052] Figure 5

[0053] Figure 5 shows the ability of DSM 34653 to inhibit the growth of four common phytopathogenic fungi. Dual cultures in a spot on lawn design with DSM 34653 and a commercial biofungicide (Biol, Bacillus amyloliquefaciens QST 713) on plates covered with Gaeumannomyces graminis (G. graminis), Zymoseptoria tritici (Z. tritici), Pyrenophora teres f. teres (P. teres), or Fusarium oxysporum (F. oxysporum). Control plates are shown to the left. G. graminis and F. oxysporum were cultivated on oatmeal agar (OA), P. teres on grass agar (GA), and Z. tritici on potato dextrose agar (PDA).

[0054] Figure 6

[0055] Figure 6 shows in vitro mineral-dissolving properties of DSM 34653 and of two strains isolated from commercially available microbial plant products at low, medium and high temperatures. A) Colony size of DSM 34653 and of two bacterial strains (Biol, Bacillus amyloliquefaciens QST 713 and Bio2, Bacillus atrophaeus Abi05) isolated from one biofungicide and one biostimulant commercially available product, respectively, 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), and C) Solubilization index (SI, Areanaio / Areacoiony) for DSM 34653, Biol and Bio2 grown on Pikovskaya medium (insoluble phosphate agar) for 7 days (B), Aleksandrow medium (insoluble potassium agar) for 10 days (C) at low (5°C), medium (15°C) or high (25°C) temperatures. Experiments were carried out in three replicates. Bars in each plot represent the mean across replicates and error bars represent standard deviations across replicates in each experimental group.

[0056] Figure 7

[0057] Figure 7 shows the emergence of seedlings from spring barley seeds five days following sowing. 70 seeds were sowed in each experimental group: DSM 34653; seeds coated with a bacterial suspension containing DSM 34653, Control; untreated seeds; Fungicide + DSM 34653; seeds coated with a commercially available chemical fungicide (containing tebuconazol (20 g / L) and prothioconazol (150 g / L) as active components) and DSM 34653.

[0058] Figure 8

[0059] Figure 8 shows an image of a filter paper showing hydrogen cyanide (HCN) production in DSM 34653. Grey circular areas represent detection of volatile HCN produced by liquid bacterial cultures. Increasing darkness of grey correlates with higher levels of hydrogen cyanide being produced. Biol; A bacterial strain isolated from a commercial microbial biofungicide product, Positive control; a Pseudomonas strain that produces HCN, Negative control; a mutant derivative of the positive control that cannot produce HCN.

[0060] Figure 9 Figure 9 shows a line plot showing siderophore production in DSM 34653. Siderophore production was quantified on a halo index (HI) scale by normalizing the halo areas, corresponding to siderophore production, to the bacterial colony areas on chrome azurol S (CAS) agar plates (related to Figure 10). HI was calculated using the following equation: HI — AreaHaio / Areacoiony

[0061] Siderophore production was measured over the course of seven days (n=9 per day) and HI quantified at Day 2 to Day 7 were compared statistically to the HI quantified at Day 1 using a paired Wilcoxon signed rank test (* p-value <0.005, ** p-value <0.002). Siderophore production is represented as mean HI and the error bars represent the standard error of the mean (SEM).

[0062] Figure 10

[0063] Figure 10 shows representative images of siderophore production on chrome azurol S (CAS) agar plates from Day 1, Day 3 and Day 7 (related to Figure 9). The inner white circles represent bacterial colonies and the light grey circles surrounding the bacterial colonies represent halos corresponding to siderophore production.

[0064] Figure 11

[0065] Figure 11 shows bar plots showing the production of indole-3-acetic acid (IAA) in DSM 34653. IAA production was quantified in liquid bacterial cultures using Salkowski's reagent and spectrophotometry. Relative IAA concentrations (pg / mL) represent IAA concentrations in cultures normalized to the bacterial cell density. IAA were quantified in cultures grown with 5mM L-tryptophan (with Trp, n = 3) or without L-tryptophan (w / o Trp, n=3). Biol; A bacterial strain isolated from a commercial microbial biofungicide product was included as a benchmarking control.

[0066] Figure 12

[0067] Figure 12 shows quantifications of in vitro organic phosphate (phytate) mineralization properties of DSM 34653 and a strain isolated from a commercially available biofungicide product (Biol) (related to Figure 13). DSM 34653 and Biol were cultivated on standardized modified Pikowskaya agar plates with calcium phosphate (Ca3(PO4)2) substituted for phytate for seven days at 15°C. Colony and halo sizes were monitored throughout a week and quantified by calculating a solubilization index (SI) using the following equation:

[0068] SI = AreaHaio / Areacoiony Datapoints represent technical replicates (n = 3 per day).

[0069] Figure 13

[0070] Figure 13 shows representative images of in vitro organic phosphate (phytate) mineralization properties of DSM 34653 and a strain isolated from a commercially available biofungicide product (Biol) (related to Figure 12). DSM 34653 and Biol were cultivated on standardized modified Pikowskaya agar plates with calcium phosphate (Ca3(PO4)2) substituted for phytate for seven days at 15°C and their colony and halo sizes were monitored throughout a week. Pictures are shown for Day 1, 4 and 7.

[0071] Figure 14

[0072] Figure 14 shows guantifications of in vitro zinc solubilization properties of DSM 34653 (related to Figure 15). DSM 34653 were cultivated on zinc-solubilizing agar for seven days at 15°C. Colony and halo sizes were monitored throughout a week and guantified by calculating a solubilization index (SI) using the following eguation:

[0073] SI = AreaHaio / Areacoiony

[0074] Datapoints represent mean SI values across three technical replicates from one (Days 3 and 5) or two (Days 2, 6 and 7) biological replicates.

[0075] Figure 15

[0076] Figure 15 shows representative images of in vitro zinc solubilization properties of DSM 34653 and a strain isolated from a commercially available biofungicide product (Biol) (related to Figure 14). DSM 34653 and Biol were cultivated on zinc-solubilizing agar, which contains zinc oxide (ZnO), for seven days at 15°C. Colony and halo sizes were monitored throughout the duration of the experiment. Biol was additionally cultivated on LB agar which were overlaid with zinc- solubilizing agar on multiple timepoints with pictures taken after 24h. The black arrowhead indicates the bacterial colony and the white arrowhead indicates the halo corresponding to zinc solubilization. Figure 16

[0077] Figure 16 shows box plots showing disease assessment of brown leaf rust (A) and net blotch (B) caused by the plant pathogens Puccinia hordei and Pyrenophora teres, respectively (n=80 plants per treatment group). Plant leaves were visually scored along a disease index scale (0-100%) corresponding to the leaf area affected by disease symptoms. Treatment groups: Ctrl; Control, Chemical fungicide Coat; Chemical fungicide #1 applied as a seed coat, Chemical fungicide W2; Chemical fungicide #2 applied as a foliar spray in window 2 (W2), DSM 34653 Coat; DSM 34653 applied as a seed coat, DSM 34653 Coat + W2; DSM 34653 applied as a seed coat followed by a foliar spray in W2, DSM 34653 Wl; DSM 34653 applied as a foliar spray in window 1 (Wl), DSM 34653 W2; DSM 34653 applied as a foliar spray in W2. Statistics were computed using a one-sided Wilcoxon rank sum test.

[0078] Figure 17

[0079] Figure 17 shows box plots showing the harvest yield from spring barley crops. The harvest yield is corrected for water content and is represented as ton per hectare (t / ha). Treatment groups: Ctrl; Control, Chemical fungicide Coat; Chemical fungicide #1 applied as a seed coat, Chemical fungicide W2; Chemical fungicide #2 applied as a foliar spray in window 2 (W2), DSM 34653 Coat; DSM 34653 applied as a seed coat, DSM 34653 Coat + W2; DSM 34653 applied as a seed coat followed by a foliar spray in W2, DSM 34653 Wl; DSM 34653 applied as a foliar spray in window 1 (Wl), DSM 34653 W2; DSM 34653 applied as a foliar spray in W2. Statistics were computed using a one-sided Wilcoxon rank sum test.

[0080] Figure 18

[0081] Figure 18 shows box plots showing the early emergence of spinach plants in a field. Treatment groups: Ctrl; Control, DSM 34653; concentrated fermentation broth containing DSM 34653, DSM 34653F; concentrated fermentation broth containing DSM 34653 and additives, Chemical fungicide; A commercial chemical fungicide containing fludioxonil as the active substance (included as a benchmarking control). Statistics were computed using a one-sided Wilcoxon rank sum test (* p-value <0.05 comparing each seed coat treatment to control, # p- value <0.05 comparing DSM 34653 seed coat treatment with seeds treated with the chemical fungicide).

[0082] Figure 19

[0083] Figure 19 shows the plants at day 19 after inoculation with Zymoseptoria tritici spores. Treatment groups are indicated horizontally: W; water, E; DSM 34653 high dose (109CFU / mL), B; DSM 34653F normal dose (2xl07CFU / mL), and A; DSM 34653 normal dose (2xl07CFU / mL). Repetitions are indicated vertically (n = 4). The treatments were applied as foliar spray 24 hours prior to inoculation with Zymoseptoria tritici.

[0084] The present invention will now be described in more detail in the following.

[0085] Detailed description of the invention

[0086] Definitions

[0087] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0088] Effective amount

[0089] 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.

[0090] Inoculant

[0091] The term "inoculant" as described in this invention is defined in several Federal, or State regulations as:

[0092] (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); (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);

[0093] (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

[0094] (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).

[0095] Biostimulant

[0096] 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.

[0097] In here the terms "fertilizer", "inoculant" and "biostimulant" may be used interchangeably.

[0098] Fungicide

[0099] In the present context a "fungicide" is a substance used to kill or prevent the growth of fungi and their spores. They can be used to control fungi that damage plants, including but not limited to rusts, mildews, wilts, canker, smuts, damping- off, rot, anthracnose, scab, mould, and blights.

[0100] Fungicides may be classified as chemical fungicides or biofungicides. Biofungicide

[0101] In the present context a "biofungicide" are formulations of one or more living organisms that are used to control the activity of plant pathogenic fungi and bacteria. The mechanisms used to control plant pathogens include competition, parasitism, antibiosis, induction of plant defense responses, and promotion of plant growth.

[0102] Isolated bacteria and biologically pure bacterial culture

[0103] 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.

[0104] 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.

[0105] Preferably the bacteria according to the invention is an isolated bacteria, such as forming part of an isolated composition.

[0106] Plant growth promoting agent

[0107] 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.

[0108] 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

[0109] (Nref-Nd.f'jlOO sequences. The sequence identity can be calculated as Nref > wherein

[0110] 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)).

[0111] 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.

[0112] 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%.

[0113] Solubilization

[0114] 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.

[0115] The level of solubilization can be determined by: providing solid media such as agar plates comprising insoluble mineral complexes,

[0116] - cultivating bacteria of interest,

[0117] - and calculating a solubilization index (SI) using the formula:

[0118] SI = AreaHaio / Areacoiony

[0119] The "Area" is determined by measuring the diameter of the bacterial colonies and the corresponding clear / halo zones.

[0120] Secondary metabolite

[0121] Secondary metabolites are also known as specialized metabolites or natural products, that are organic compounds produced by various organisms, including bacteria. Unlike primary metabolites, which are directly involved in growth, development, and reproduction, secondary metabolites are not necessarily essential for these processes. Instead, secondary metabolites play roles in ecological interactions, such as defence mechanisms against predators, pathogens, and herbivores, as well as in competition and mutualistic relationships.

[0122] Foliar spray

[0123] Foliar spray is a method of applying pesticides, biostimulants, microbial agents liquid fertilizers, or other nutrients directly to the leaves of plants. This technique allows plants to absorb nutrients more quickly through their leaves compared to their roots. They are typically applied using a trigger or pump sprayer and can include a variety of substances, such as water-soluble fertilizers, insecticides, and fungicides.

[0124] Seed coat

[0125] In the present context "seed coat" or "plant seed coating" refer to seed surface applications such as; seed treatment, seed inoculation, film coat, seed dressing, seed coat, seed pelleting, slurry coating, seed priming. Seed coating involves applying a protective (outer) layer or (outer) layer of treatment to plant seeds before planting the coated seeds.

[0126] Protective seed coating shields the seeds from environmental stressors like extreme temperatures, drought, pathogens, and pests.

[0127] Another seed coating can further include nutrients, hormones, or beneficial microbes that promote germination and improve seedling vigor.

[0128] Some seed coatings can contain fungicides, insecticides, or nematicides to protect seeds from soil-borne pathogens and pests.

[0129] Bacteria or biologically pure bacterial culture

[0130] As outlined above, the inventing team has identified, isolated and propagated a newly identified strain of bacteria, namely the Pseudomomas strain DSM 34653 deposited with deposit number DSM 34653. The strain may be used to promote a wide variety of plant health parameters.

[0131] The bacterial strain of the present invention was identified by isolating bacterial strains using microbeTRAP to capture multiple potential bacterial biocontrol strains from Danish grassland soil and screened to identify the best-performing bacterial strain. A comparative screening test was conducted to identify the most effective candidate among the isolated strains (n=7). Of the isolated strains, DSM 34653 was chosen as the most promising candidate for a biocontrol agent based on the initial screening for biocontrol properties on three common phytopathogenic fungi (Example 1).

[0132] 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: 5-17; 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: 5-17; and / or c) a fragment of one or more of the sequences of a) or b), having a length of at least 300 nucleotides. Another 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: 5-17; b) a genomic sequence having 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: 5- 17; and / or c) a fragment of one or more of the sequences of a) or b), having a length of at least 300 nucleotides.

[0133] In a preferred embodiment, the bacteria is an isolated bacteria.

[0134] As shown in Example 2, comparative genome analysis of the bacterial strain DSM 34653 reveals that DSM 34653 is a novel Pseudomonas strain.

[0135] Further, as seen in Example 10, comparing TF-A, TF-B, TF-C, TF-E, TF-F and TF-G to each other and to the genome representing DSM 34653 through average nucleotide identity (ANI) analyses reveals no ANI values below the putative strain threshold at 99.5% ANI (strains compared to DSM 34653: ANI range=99.9809- 99.9989%, strains compared to each other: ANI range=99.9952-99.9995%), demonstrating that DSM 34653 as well as the bacterial strains TF-A, TF-B, TF-C, TF-E, TF-F and TF-G represent the same Pseudomonas strain which are thus considered to be novel Pseudomonas species.

[0136] 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: 5, 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: 5.

[0137] 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: 6, 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: 6.

[0138] 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: 7, 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: 7.

[0139] 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: 8, 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] In an embodiment, the bacteria or biologically pure bacterial culture according to the invention comprising any of SEQ ID NO: 5-17, such as one or more of SEQ ID NO: 5-17, such as one or more of SEQ ID NO: 5-17, such as two or more of SEQ ID NO: 5-17, such as three or more of SEQ ID NO: 5-17, such as four or more of SEQ ID NO: 5-17, such as five or more of SEQ ID NO: 5-17, such as six or more of SEQ ID NO: 5-17, such as eight or more of SEQ ID NO: 5-17, such as nine or more of SEQ ID NO: 5-17, such as ten or more of SEQ ID NO: 5-17, such as eleven or more of SEQ ID NO: 5-17, such as twelve or more of SEQ ID NO: 5-17.

[0150] In an embodiment, the bacteria or biologically pure bacterial culture according to the invention comprises any of SEQ ID NO's: 5-17, such as one or more of SEQ ID NO's: 5-17. 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, such as eleven or more, such as twelve or more, or preferably such as all of SEQ ID NO: 5-17, or with a sequence identity for SEQ ID NO: 5-17 as defined above. SEQ ID NOs: 5-13 form part of the long genomic regions SEQ ID NOs: 2-4.

[0151] SEQ ID NOs: 14-17 are identified outside of the long genomic regions SEQ ID NOs: 2-4.

[0152] SEQ ID NO: 18 forms part of SEQ ID NO: 3.

[0153] Thus, in another embodiment, the bacteria or biologically pure bacterial culture according to the invention, comprising, a) a genomic sequence according to any of SEQ ID NO: 2-4 or 18; b) a genomic sequence having at least 90% sequence identity to any of SEQ ID NO: 2-4 or 18, 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: 2-4 or 18, 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 14000 nucleotides.

[0154] The DSM 34653 strain according to the invention has been annotated as being a Pseudomonas genus. Thus, in an embodiment, the bacteria or biologically pure bacterial culture is of the genus Pseudomonas, such as Pseudomonas wadenswilerensis, Pseudomonas donghuensis, Pseudomonas tructae, or Pseudomonas rubra.

[0155] In an embodiment, the bacteria or biologically pure bacterial culture comprises a 16S rRNA gene encoded by SEQ ID NO: 1.

[0156] As shown in Example 2, six 16S ribosomal RNA (rRNA) genes were identified in the assembled genome of DSM 34653. These six 16S rRNA gene sequences are 100% identical to each other, the DNA sequence corresponding to one of these is encoded by SEQ ID NO: 1.

[0157] In an embodiment, the isolated bacteria or biologically pure bacterial culture being Pseudomonas, DSM 34653 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] .

[0158] In the present patent application, this strain is also named TF-D or TF2. In yet an aspect the invention relates to an (isolated) bacteria or biologically pure bacterial culture being Pseudomonas, DSM 34653 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.

[0159] 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

[0160] - 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

[0161] - capable of inhibiting one or more phytopathogenic fungi, such as phytopathogenic fungi specific for (colder) regions.

[0162] As seen in Example 4, it is evident that the bacteria is capable of solubilizing calcium phosphate (Cas PC )?) and potassium alumino-silicate at low (5°C), medium (15°C), and high (25°C) temperatures. As seen in Example 7, the bacteria are capable of solubilizing phytate at a temperature of 15°C. Hence, the bacteria exhibit a great ability to mineralize organic phosphate.

[0163] Especially, the bacteria of the present invention is able to grow and display bacterial activity at temperatures down to 5°C.

[0164] As seen in Example 3, the bacteria has antifungal activity against phytopathogenic fungi Gaeumannomyces graminis var. tritici, Pyrenophora teres, Zymoseptoria tritici, and Fusarium oxysporum. In Example 9, the bacteria further have antifungal activity against Puccinia hordei.

[0165] Thus, in an embodiment, the one or more phytopathogenic fungi are selected from the group consisting of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres, Zymoseptoria tritici, Puccinia hordei, and Fusarium oxysporum.

[0166] 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 40% to, equal to and / or above a level of solubilization at 15°C and / or 25°C. As seen in Figure 6A-6C, the solubilization of minerals by the bacteria of the present invention is indeed works at a temperature of 5°C and at higher temperatures at 15°C and 25°C.

[0167] 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.

[0168] In an embodiment, the level of solubilization is determined by a solubilization index (SI) calculated by the formula:

[0169] SI = AreaHaio / Areacoiony.

[0170] As seen in Example 4, the solubilization index (SI) is calculated using the above formula.

[0171] In an embodiment, the SI is at least 2, such as at least 4, such as between 2 and 10, preferably the SI is between 2 and 9.

[0172] As seen in Example 4 the solubilization index (SI) of DSM 34653 on day 7 on Pikovskaya agar was in the range 2 and 9, wherein the SI was about 2.3 at 5°C, about 8.6 at 15°C, and about 4.7 at 25°C (Figure 6B). The SI of DSM 34653 on day 10 on Aleksandrow agar was in the range 2 and 5, wherein the SI was about 2.2 at 5°C, about 2.2 at 15°C, and about 4.8 at 25°C (Figure 6C).

[0173] In a further embodiment, the SI depends on temperature after cultivation start and / or incubation time.

[0174] 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.

[0175] 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:

[0176] 2.3 at 5°C,

[0177] 8.6 at 15°C, and

[0178] 4.7 at 25°C. The SI on Aleksandrow agar at each temperature at day 10 was:

[0179] • 2.2 at 5°C,

[0180] • 2.2 at 15°C, and

[0181] • 4.8 at 25°C.

[0182] 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.

[0183] As seen in Example 7, DSM 34653 exhibits an improved ability to mineralize organic phosphate, such as phytate, at day 1 to 10, such as day 1 to day 7 compared to a commercial biofungicide strain. The mean SI for DSM 34653 on a modified Pikovskaya agar containing sodium phytate was 8.96 at at 15°C.

[0184] This demonstrates that DSM 34653 has a higher capacity for mineralizing organic phosphate, which would otherwise remain unavailable to plants, and thus a high potential for supplying crops with otherwise unavailable nutrients in the field.

[0185] In an embodiment, the bacteria or biologically pure bacterial culture comprises a capacity for solubilizing zinc, such as zinc oxide, at day 1 to day 10, such as day 2 to day 7 at 15°C.

[0186] As seen in Example 8, DSM 34653 exhibits an improved ability to mineralize zinc, such as zinc oxide (ZnO), which is a source of insoluble zinc, at day 2 to 7 compared to a commercial biofungicide strain. The mean SI DSM 34653 on a zinc- solubilizing agar containing zinc oxide was 3.61 at 15°C.

[0187] This demonstrates that DSM 34653 exhibits the ability to both survive in a zinc- rich environment and solubilize a highly insoluble form of zinc. These capabilities are absent in a commercial biofungicide strain.

[0188] In an embodiment, the bacteria or biologically pure bacterial culture is dehydrated, such as spray-dried.

[0189] 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 2, this gene cluster may be toxic to human health. In an embodiment, the bacteria or biologically pure bacterial culture according to the invention further comprises one or more of a prebiotic, stabilizer, antibacterial agent, antifungal agent, preservative, and / or media component.

[0190] In a further embodiment, the prebiotic is selected from the group consisting of inulin, fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, lactulose, or a combination thereof.

[0191] In a further embodiment, the stabilizer is selected from the group consisting of a sugar, a sugar alcohol, an amino acid, a lipid, or any combination thereof.

[0192] In a further embodiment, the stabilizer is selected from the group consisting of glucose, sucrose, trehalose, lactose, maltodextrin, polydextrose, dextran, inulin, fructose, oligofructose, cellulose, glycerol, adonitol, inositol, mannitol, sorbitol, gums, hydrolyzed protein, casein, skim milk powder, milk powder, glycerin, gelatine, gum arabic, L-leucin, gel beads, or any combination thereof.

[0193] In an embodiment, the antibacterial agent is selected from the group consisting of bacteriocin, amoxicillin, ampicillin, azithromycin, cefaclor, cefdinir, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, cephalexin, cephalosporin, ciprofloxacin, clarithromycin, clavulanate, clindamycin, clotrimazole, dalbavancin, demeclocycline, dicloxacillin, doxycycline, eravacycline, erythromycin, fluconazole, furazolidone, lansoprazole, levofloxacin, lincomycin, metronidazole, minocycline, moxifloxacin, nitroimidazole, omadacycline, oritavancin, oxacillin, penem, penicillin, penicillin V potassium, rifabutin, sulfamethoxazole, sulfasalazine, telavancin, tetracycline, tinidazole, trimethoprim, vancomycin, an antimicrobial peptide, or any combination thereof.

[0194] In yet an embodiment, the antifungal agent is amphotericin B, clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, natamycin, nystatin, posaconazole, terconazole, terbinafine, voriconazole, or any combination thereof. In an embodiment, the bacteria or biologically pure bacterial culture according to the invention is lyophilized, spray dried, or freeze-dried.

[0195] In an embodiment, the bacteria or biologically pure bacterial culture according to the invention comprises a complete biosynthetic gene cluster.

[0196] In a further embodiment, the complete biosynthetic gene cluster is a hcnABC gene cluster.

[0197] As seen in Example 2, DSM 34653 contains a complete biosynthetic gene cluster matching towards the hcnABC gene cluster encoding the secondary metabolite hydrogen cyanide, which has been suggested to act as a broad-spectrum biocontrol agent.

[0198] In an embodiment, the bacteria or biologically pure bacterial culture produces one or more secondary metabolites, such as hydrogen cyanide (HCN), siderophore, and / or indole-3-acetic acid (IAA).

[0199] As seen in Example 6, DSM 34653 can produce secondary metabolites, including hydrogen cyanide (HCN), siderophores and indole-3-acetic acid (IAA).

[0200] In an embodiment, the bacteria or biologically pure bacterial culture comprises one or more genes, such as one or more gene clusters, encoding one or more secondary metabolites, such as hydrogen cyanide (HCN), siderophore, and / or indole-3-acetic acid (IAA).

[0201] Example 2 provides evidence that DSM 34653 contains a complete biosynthetic gene cluster matching towards the hcnABC gene cluster encoding a secondary metabolite hydrogen cyanide. Further, Example 6 shows that DSM 34653 can produce secondary metabolites that are important for plant health and development, including hydrogen cyanide (HCN), siderophores and indole-3-acetic acid (IAA), thus strongly indicating that DSM 34653 has a high capacity for crop management in the field.

[0202] In an embodiment, the level of production of secondary metabolites, such as siderophore, is determined by a halo index (HI) calculated by the formula:

[0203] HI = AreaHaio / Areacoiony. As seen in Example 6, the halo index (HI), such as for siderophore production, is calculated using the above formula.

[0204] In an embodiment, the HI is at least 2, such as at least 4, such as between 2 and 10, preferably between 4 and 7, more preferably the HI is between 4.5 and 6.5.

[0205] As seen in Example 6, the halo index (HI) for siderophore production is 4.66-6.30 across all experimental days (seven days), wherein a maximum HI is obtained at day 3 of HI=6.30.

[0206] In a further embodiment, the HI depends on temperature after cultivation start and / or during incubation time.

[0207] 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, such as between 20°C and 25°C, preferably between 5°C and 8°C.

[0208] Fertilizer or inoculant or biostimulant

[0209] 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.

[0210] 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.

[0211] In an embodiment, the fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition is a biofungicide.

[0212] As seen in Example 6, DSM 34653 can produce secondary metabolites that are important for biostimulant and biocontrol activities. Example 9 further confirms that DSM 34653 can manage fungal diseases in spring barley when applied as a seed coat, as a foliar spray or a combination hereof in the field. In an embodiment, the composition comprises the bacteria or biologically pure bacterial culture according to the invention in a concentration in the range 1.5xl09to 4.0xl09colony-forming unit (CFU) / kg, such as 2.0xl09to 3.5.0xl09CFU / kg, preferably 2.2xl09to 3.0xl09CFU / kg, more preferably 2.3xl09to 2.7xl09CFU / kg, most preferably 2.5xl09CFU / kg plant seed.

[0213] Example 11 provides a bacterial suspension of DSM 34653 containing 2.5xl09CFU / kg plant seed.

[0214] In an embodiment, the composition further comprises one or more agriculturally acceptable carriers.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] In yet another embodiment, the composition is formulated as a granular formulation or a powder formulation.

[0219] 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. 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, zeolite, or a combination thereof.

[0220] 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.

[0221] In an embodiment, the composition is free of fungicide different from the bacteria or biologically pure bacterial culture according to the invention.

[0222] In an embodiment, the composition is formulated as a foliar spray.

[0223] As seen in Example 9, DSM 34653 can manage fungal diseases when applied as a seed coat, as a foliar spray or a combination hereof in the field.

[0224] In a further embodiment, the foliar spray comprises one or more of agents selected from the group consisting of tween, lecithin, saponins, plant oils, xanthan, guar gum, cellulose, carboxymethyl cellulose, agar, alginate, diutan, gellan gum, tara gum, ethoxylated fatty alcohols or ethoxylated vegetable oils, alkyl polyglucosides, organosilicones, and polyalkykeneoxide modified heptamethyltrisiloxane. The agents increase the dispersion (surfactants), adherence to the plant surface, and / or protection.

[0225] In an embodiment, the foliar spray comprises the bacteria or biologically pure bacterial culture according to the invention in a concentration in the range lxlO6to 2xl09colony-forming unit (CFU) / mL, such as 2xl06to 1.5xl09CFU / mL, such as lxlO7to 1.5xl09CFU / mL, 1.5xl07to 1.5xl09CFU / mL, preferably 2xl07to lxlO9CFU / mL, most preferably 2xl07or lxlO9CFU / mL.

[0226] As seen in Example 12, the foliar spray comprises DSM 34653 in a concentration of 109CFU / mL (high dose) or 2xl07CFU / mL (normal dose).

[0227] Coating composition

[0228] 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.

[0229] In an embodiment, the coating composition comprises a biopolymer promoting adherence to a plant seed.

[0230] In an embodiment, the coating composition is formulated as an aqueous or oilbased solution for application to seeds, preferably aqueous.

[0231] In an embodiment, the coating composition (for application to seeds) is formulated as a concentrated fermentation broth.

[0232] In another embodiment, the coating composition is formulated as a powder or granular formulation for application to seeds.

[0233] In an embodiment, the coating composition is free of fungicide different from the bacteria or biologically pure bacterial culture according to the invention. In an embodiment, the coating composition (for application to seeds) comprises the bacteria or biologically pure bacterial culture according to the invention in a concentration in the range 1.5xl09to 4.0xl09colony-forming unit (CFU) / kg, such as 2.0xl09to 3.5.0xl09CFU / kg, preferably 2.2xl09to 3.0xl09CFU / kg, more preferably 2.3xl09to 2.7xl09CFU / kg, most preferably 2.5xl09CFU / kg plant seed.

[0234] Example 11 provides a bacterial suspension of DSM 34653 containing 2.5xl09CFU / kg plant seed for coating spinach plant seeds.

[0235] In another preferred embodiment the coating composition is in the form of a foliar spray as mentioned above.

[0236] In an embodiment, the coating composition (in form of a foliar spray) comprises the bacteria or biologically pure bacterial culture according to the invention in a concentration in the range lxlO6to 2xl09colony-forming unit (CFU) / mL, such as 2xl06to 1.5xl09CFU / mL, such as lxlO7to 1.5xl09CFU / mL, 1.5xl07to 1.5xl09CFU / mL, preferably 2xl07to lxlO9CFU / mL, most preferably 2xl07or lxlO9CFU / mL.

[0237] As seen in Example 12, the foliar spray comprises DSM 34653 in a concentration of 109CFU / mL (high dose) or 2xl07CFU / mL (normal dose).

[0238] In yet an embodiment, the foliar spray comprises one or more of agents selected from the group consisting of tween, lecithin, saponins, plant oils, xanthan, guar gum, cellulose, carboxymethyl cellulose, agar, alginate, diutan, gellan gum, tara gum, ethoxylated fatty alcohols or ethoxylated vegetable oils, alkyl polyglucosides, organosilicones, and polyalkykeneoxide modified hepta methyltrisiloxane.

[0239] In an embodiment, the coating composition further comprises one or more agriculturally acceptable carriers.

[0240] 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.

[0241] In yet an embodiment, the coating 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.

[0242] It is to be understood that embodiments mentioned for one aspect of the invention may also be combinable with other aspects of the invention. For example, embodiments described under "Fertilizer or inoculant or biostimulant" may be combined with aspects under coating compositions.

[0243] Coated plant seed

[0244] The present invention also relates to seeds coated with the 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.

[0245] In an embodiment, the plant seed is a dicotyledon, monocotyledon or a gymnosperm seed.

[0246] 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.

[0247] As seen in Example 5, spring barley seeds coated with the composition according to the invention or coated with a coating composition according to the invention are shown to result in an enhanced emergence of seedlings, thus increased growth and yield. 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.

[0248] In yet an embodiment, the plant seed being selected from the group consisting of trees, bushes or grasses.

[0249] In an embodiment, the plant seed is free of fungicide different from the bacteria or biologically pure bacterial culture according to the invention.

[0250] In an embodiment, the composition according to the invention or the coating composition according to the invention comprises the bacteria or biologically pure bacterial culture according to the invention in a concentration in the range 1.5xl09to 4.0xl09colony-forming unit (CFU) / kg, such as 2.0xl09to 3.5.0xl09CFU / kg, preferably 2.2xl09to 3.0xl09CFU / kg, more preferably 2.3xl09to 2.7xl09CFU / kg, most preferably 2.5xl09CFU / kg plant seed.

[0251] As seen in Example 11, the plant seeds are coated with a bacterial suspension of DSM 34653 containing 2.5xl09CFU / kg seed.

[0252] Coated plant leaves

[0253] Similar to coating plant seeds, the present invention also relates to coated plant leaves. Thus, yet an aspect of the invention relates to plant leaves coated with the composition according to the invention or coated with a coating composition according to the invention (such as a foliar spray).

[0254] Uses and methods

[0255] 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.

[0256] Another aspect of the invention relates to 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 biofungicide, preferably at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C.

[0257] As seen in Example 6, the DSM 34653 can produce secondary metabolites that are important for biocontrol activities. Example 9 further confirms that DSM 34653 can manage fungal diseases in spring barley. Hence, the DSM 34653 can indeed be used as a biofungicide.

[0258] In an embodiment, the temperature is a surface temperature.

[0259] In an embodiment, the use as a plant growth-promoting agent is for:

[0260] • 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

[0261] • chelating inorganic minerals, such as iron, zinc, manganese, copper; and / or

[0262] • allowing for growth under low soil nitrogen conditions; and / or

[0263] • fixating atmospheric nitrogen; and / or

[0264] • increasing plant growth, such as plant length, leaf diameter and / or root length; and / or

[0265] • improving germination of the seeds; and / or

[0266] • improving emergence of the plants; and / or

[0267] • increasing biomass; and / or

[0268] • increasing growth; and / or

[0269] • increasing crop yield; and / or

[0270] • inducing anti-fungal effects; and / or

[0271] • increasing harvest yield; and / or

[0272] • increasing hectoliter weight; and / or

[0273] • increasing protein content; and / or

[0274] • increasing protein yield; and / or

[0275] • increasing oil content; and / or

[0276] • increasing oil yield; and / or

[0277] • reducing mycotoxin contamination; and / or

[0278] • reducing seed-borne fungal or bacterial contamination; and / or

[0279] • 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.

[0280] 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 average 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.

[0281] In an embodiment, the inorganic minerals or salts solubilized are selected from the group consisting of

[0282] • minerals, such as rock phosphate, potash, lime, clay, ground rocks, sand, silt, sediment and natural deposits;

[0283] • 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

[0284] • 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.

[0285] 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.

[0286] 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.

[0287] In an 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 as a seed coat, as a foliar spray, or a combination hereof.

[0288] 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.

[0289] In an embodiment, the plant is a dicotyledon, monocotyledon or a gymnosperm.

[0290] 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.

[0291] The dicotyledon can be selected from the group consisting of bean, pea, tomato, pepper, squash, alfalfa, almond, aniseseed, apple, apricot, arracha, artichoke, avocado, bambara 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, culantro, arugula, cardoon, caigua, mitsuba, 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.

[0292] 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 (basella), 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 (elaeocarpus), Elatinaceae (waterwort), Empetraceae (crowberry), Epacridaceae (epacris), Eremolepidaceae (catkin-mistletoe), Ericaceae (heath), Erythroxylaceae (coca), Eucommiaceae, Eucryphiaceae, Euphorbiaceae (spurge), Eupomatiaceae, Eupteleaceae, Fabaceae (pea or legume), Fagaceae (beech), Flacourtiaceae (flacourtia), Fouquieriaceae (ocotillo), Frankeniaceae (frankenia), 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 (barbados 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).

[0293] 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.

[0294] 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 (posidonia), Potamogetonaceae (pondweed), Rapateaceae, Restionaceae, Ruppiaceae (ditch grass), Scheuchzeriaceae (scheuchzeria), 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).

[0295] 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.

[0296] Method for stimulating plant growth

[0297] 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, preferably at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C.

[0298] 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 :

[0299] - to a plant growth medium, such as sphagnum; and / or

[0300] - 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

[0301] - to plant leaves, roots, or stems; and / or

[0302] - to plant seeds, and / or

[0303] - to seed furrows, and / or

[0304] - 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.

[0305] Kit

[0306] In yet a further aspect, the invention relates to a kit of parts for stimulating plant growth comprising

[0307] • 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

[0308] • 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.

[0309] In an embodiment, the kit of parts further comprises one or more containers comprising fertilizers, nutrients, and / or other microorganisms.

[0310] Ores

[0311] 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.

[0312] 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.

[0313] Medical uses

[0314] 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.

[0315] In a further embodiment, the fungal infection is selected from the group consisting of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres, Zymoseptoria tritici, Fusarium oxysporum, Puccinia hordei, or a combination thereof.

[0316] As can be seen in Example 3, the bacteria or biologically pure bacterial culture according to the invention comprises antifungal activity.

[0317] 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.

[0318] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0319] The invention will now be described in further details in the following non-limiting examples.

[0320] Examples

[0321] Example 1 - Isolation of bacterial strains from a Danish grassland with biocontrol properties

[0322] Aim of study

[0323] The scope of this example is to demonstrate how bacterial strains with biocontrol properties were isolated from the soil of a Danish grassland and screened to identify the best-performing bacterial strain.

[0324] Materials and methods 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.

[0325] A microbeTRAPs were prepared to select for microorganisms with biocontrol properties: prior to incubation the capture pills were soaked in a LB broth containing Saccharomyces cerevisiae (S. cerevisiae) for 24 hours to ensure cultivation of this organism within the capture pill. Nutrient pills and capture pills were then placed in each microbeTRAP and it was then sealed with a lid. Soil from an old grassland on southern Funen, Denmark (coordinates: 55°02'34.8"N 10°24'43.3"E) was collected in December 2021. The soil was placed in a biosafety plastic box indoor to ensure a temperature of approximately 20°C during incubation. The microbeTRAP was placed in the middle of the box and covered with around 10 cm of soil. The soil was moistened with 1 L of sterile water during the incubation period to create a moist environment that would promote nutrient exchange and chemotaxi. The microbeTRAPs were excavated after 14 days and the capture pills retrieved. Capture pills were cultured on LB agar plates covered with S. cerevisiae (100 pL, 5 McF solution / suspension). The plates were incubated at 20°C for 2 days. Capture pills with clearing zones of S. cerevisiae were sampled for microorganisms using an inoculation loop that were swept from the capture pill out to the edge of the colony (n = 12). The microorganisms were purified on LB agar using the 4-streak method. This step was repeated until the colonies were pure and only contained one bacterial strain. Pure cultures were transferred to new LB plates covered with S. cerevisiae to confirm inhibition was retained. Inhibition was confirmed in 7 of the 12 strains.

[0326] A dual culture assay was used to screen for the most efficient biocontrol candidate among the strains (n = 7). Three phytopathogenic fungi were used in the assay: Bipolaris maydis (isolate CP2050), Pyrenophora teres f. teres (isolate CP2189), and Zymoseptoria tritici (isolate IPO323). A spot on lawn setup was employed: petri dishes containing different nutrient agar were covered with a solution / suspension of fungal spores. Subsequently a solution / suspension of bacteria was spotted onto the plate in three droplets. A solution / suspension of fungal spores was prepared in 1: 10 PBS and 100 pL were spread on nutrient agar. Bipolaris maydis were cultivated on 1 / 3 strength potato dextrose agar (PDA) and regular PDA, Pyrenophora teres f. teres was cultivated on grass agar and PDA, and Zymoseptoria tritici was cultivated on PDA. Solutions / suspensions of bacterial strains were prepared in a concentration of 0.5 McF in 1: 10 PBS and 10 pL was spotted onto the plates in replicates of three. The plates were incubated at 20°C for 7 days. Inhibition was semi-qualitatively evaluated by scoring the degree of inhibition from 0-4, where 0 indicates no inhibition and 1-4 indicates increasing inhibition zones around the bacterial colonies.

[0327] Results

[0328] From the initial screening of capture pills on plates covered with S. cerevisiae 12 of 32 capture pills were identified with clearing zones around them. In the following screening of purified cultures 7 strains (TF-A, TF-B. TF-C, TF-D, TF-E, TF-F, TF-G) were identified to maintain this capacity. These 7 strains were used dual culture screening test with 3 common phytopathogenic fungi (Table 1). All strains inhibited growth of all fungi on at least one of the tested nutrient agars. TF-B, TF-D, and TF-G had the highest total inhibition score. As TF-D was the only strain inhibiting growth of Pyrenophora teres f. teres on grass, and furthermore exhibited largely similar inhibition score as the other two, this strain was selected for further analysis. TF-D was deposited at DSMZ with the accession number DSM 34653 and the strain was from here on referred to as DSM 34653.

[0329] Table 1 : Fungal inhibition by the 7 bacterial strains isolated from the MicrobeTRAP. Three phytopathogenic fungi were tested Bipolaris maydis (isolate CP2050), Pyrenophora teres f. teres (isolate CP2189), and Zymoseptoria tritici (isolate IP0323). The fungal inhibition was scored from 0-4, where 0 indicates no inhibition and 1-4 indicates increasing inhibition zones around tested bacterial strains.

[0330] Conclusion

[0331] The microbeTRAP enabled capture of multiple potential bacterial biocontrol strains from Danish grassland soil. Of the isolated strains, TF-D (DSM 34653) was chosen as the most promising candidate for a biocontrol agent based on the initial screening for biocontrol properties on three common phytopathogenic fungi.

[0332] Example 2 - Characterizing DSM 34653 as a novel Pseudomonas species Aim of study

[0333] The scope of this example is to demonstrate that DSM 34653, which was identified as the best-performing biocontrol strain from a screening setup (TF-D in Example 1), is a novel bacterial species belonging to the genus Pseudomonas.

[0334] Materials and methods

[0335] Illumina short-read DNA sequencing

[0336] DNA was extracted from a bacterial culture of DSM 34653, 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 5,950,818 reads were obtained.

[0337] ONT long-read DNA sequencing

[0338] DNA was extracted from a bacterial culture of DSM 34653 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.

[0339] Quality control and processing of Illumina data

[0340] 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. A total of 5,950,066 reads were retained.

[0341] Quality control and processing of ONT data

[0342] 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 (9.2% of all 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 512,878,87 bp.

[0343] Taxonomic classification of sequencing reads

[0344] 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 October 09, 2023, downloaded from https: / / benlangmead.github.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.

[0345] Hybrid de novo genome assembly and assembly statistics

[0346] A complete de novo genome for DSM 34653 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. Genome annotation

[0347] Genomic features present in the assembled chromosome were annotated using

[0348] Prokka (vl.14.6).

[0349] Average nucleotide identity (ANI) analysis

[0350] FastANI (vl.34) was used for computing average nucleotide identity (ANI) scores of the assembled genome of DSM 34653 compared to all Pseudomonas RefSeq reference genomes available at NCBI (n = 368 excluding atypical genomes, as of April 3, 2024).

[0351] Whole genome-based phylogenetic reconstruction

[0352] SANS ambages (v2.3_9A) was used for determining genome-to-genome evolutionary relationships of DSM 34653 and all Pseudomonas RefSeq reference genomes available at NCBI (n = 368, excluding atypical genomes, as of April 3 2024) or all P. wadenswilerensis , P. donghuensis, P. tructae and P. rubra RefSeq strain genomes available at NCBI (n = 14, excluding atypical genomes, as of April 5, 2024). The computed phylogenetic splits were subsequently visualized as phylogenetic trees using R (v4.3.3) and ggtree (v3.8.2).

[0353] Secondary metabolite analysis antiSMASH (v7.0.0) was used for the identification and annotation of secondary metabolite biosynthesis gene clusters within the genome assembly of DSM 34653. The Minimum Information about a Biosynthetic Gene cluster (MIBiG) repository containing experimentally characterized biosynthetic gene clusters was used for annotation.

[0354] Virulence factor analysis

[0355] RefSeq genomes of four P. aeruginosa strains (PAO1, UCBPP PA14, LESB58 and PA7) were downloaded from NCBI (on April 16, 2024) and annotated using Prokka (see the Genome annotation section). Translated nucleotide sequences corresponding to genes identified by Prokka from each P. aeruginosa strain as well as DSM 34653 were subjected to a protein similarity search against a database containing 4,236 experimentally validated virulence factors (protein core dataset downloaded from the Virulence Factor Database (VFDB) on April 16, 2024) using the Basic Local Alignment Search Tool (BLAST). The blastp algorithm (v2.12.0+) with a word-size of 7 was used for the alignment search.

[0356] Antimicrobial resistance analysis

[0357] RefSeq genomes of the four P. aeruginosa strains included in the virulence analysis (see the Virulence factor analysis section) and the genome assembly of DSM 34653 were subjected to antimicrobial resistance analysis using the Resistance Gene Identifier tool (rgi v6.0.3) and the Comprehensive Antibiotic Resistance Database (CARD v3.2.9 containing 5,194 reference sequences, downloaded on April 17, 2024). rgi main with default parameters (Strict and Perfect hits were reported) was performed.

[0358] 16S rRNA extraction and mapping

[0359] Six 16S ribosomal RNA (rRNA) genes were identified in the assembled genome of DSM 34653. The DNA sequences corresponding to the six 16S rRNA genes were extracted from the Prokka genome annotation output and compared to each other and 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 April 3, 2024.

[0360] As the six 16S rRNA gene sequences are 100% identical to each other, the DNA sequence corresponding to one of these is encoded by SEQ ID NO: 1 (1,532 nucleotides).

[0361] Extraction and analysis of DSM 34653-specific DNA sequences

[0362] DNA sequences with no or little overlap with the strain genomes of P. wadenswilerensis were extracted from the DSM 34653 genome assembly using samtools (v.1.10) and subsequently compared to either the complete nucleotide collection (nt database) from NCBI containing

[0363] 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 April 9-11, 2024.

[0364] Three long extracted DNA sequences 14,515-16,414 nucleotides) are annotated as SEQ ID NOs: 2-4.

[0365] Short DNA sequences (303-2,779 nucleotides) extracted from the genome assembly are annotated as SEQ ID NOs: 5-17.

[0366] Results

[0367] Initial taxonomic classification of Illumina and ONT sequencing reads obtained from the bacterial strain DSM 34653 revealed that 99.89% and 99.93% of all quality-controlled reads were assigned to the genus Pseudomonas, respectively. Analysis at species level revealed assignments to several characterized and uncharacterized Pseudomonas species (Table 2) where the majority of the sequencing data were assigned towards the latter (representing a total assignment of 44.84% and 41.37% of Illumina and ONT data, respectively).

[0368] Amongst the characterized Pseudomonas species, top assignments were observed towards P. wadenswilerensis , P. donghuensis and P. amygdali (Table 2). Collectively, these data suggest that DSM 34653 belongs to the genus Pseudomonas and that it further shares taxonomic and genetic relatedness with several Pseudomonas species. Further insight is therefore needed to delineate DSM 34653.

[0369] Table 2: Top taxonomic assignments of all classified Illumina or ONT reads at species level. The uncharacterized Pseudomonas spp. row indicates assignments towards several Pseudomonas species of uncharacterized status.

[0370] To obtain further biological insight into the taxonomic designation of DSM 34653, the genome of strain DSM 34653 was reconstructed de novo. A hybrid assembly approach was applied where long ONT reads were assembled into a nonfragmented 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 34653. The complete genome is composed of one circular chromosome with characteristics depicted in Table 3.

[0371] Table 3: Number of features associated with the genome assembly of DSM 34653. CDS=coding sequence; rRNA=ribosomal RNA; tRNA=transfer RNA; tmRNA=transfer-messenger RNA.

[0372] 16S ribosomal RNA (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. Six identical 16S rRNA genes were identified in DSM 34653. As these are 100% identical to each other, only the DNA sequence corresponding to one of the 16S rRNA genes identified in DSM 34653 is included here as SEQ ID NO: 1. Comparing SEQ ID NO: 1 to the NCBI 16S ribosomal type strain database revealed a sequence identity of 99.80% along with 100% sequence coverage towards the 16S rRNA gene identified in the type strain of Pseudomonas donghuensis (HYS), indicating that DSM 34653 may be related to P. donghuensis which the taxonomic assignment initially also suggested.

[0373] 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 34653, average nucleotide identity (ANI) values were computed for DSM 34653 in a comparative analysis against all available Pseudomonas reference genomes located at NCBI (n = 368 as of April 4, 2024, excluding atypical genomes). 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. Comparing the assembled genome of DSM 34653 to all reference Pseudomonas genomes revealed a maximum ANI value of 93.07% towards the type strain of Pseudomonas wadenswilerensis (strain CCOS 864) (Table 4, Figure 1). Thus, the comparative analysis revealed no ANI values at or above the species threshold at 95% ANI, demonstrating that DSM 34653 should be considered as a novel bacterial species belonging to the genus Pseudomonas.

[0374] Table 4: Average nucleotide identity (ANI) analysis of DSM 34653 genome assembly compared to all Pseudomonas reference genomes downloaded from NCBI (n=368, excluding atypical genomes, as of April 3, 2024). Scores >90% ANI are depicted here.

[0375] 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 gene regions might contribute with additional information on the specification of DSM 34653, whole genome phylogenies, representing measures of evolutionary distantness, were inferred on the basis of all available Pseudomonas reference genomes (n = 368) and visualized as phylogenetic trees (Figure 2). Zooming into the phylogenetic clade containing DSM 34653 revealed that DSM 34653 is genetically closest to P. wadenswilerensis, P. donghuensis, P. tructae and P. rubra (Figure 2, tree to the right), which is in line with the ANI analysis (Table 4). Phylogenetic analysis of DSM 34653 and all available P. wadenswilerensis, P. donghuensis, P. tructae and P. rubra strain genomes (n = 14) moreover revealed that DSM 34653 forms a separate phylogenetic branch, strongly indicating that DSM 34653 has diverged into a new bacterial lineage representative of a novel Pseudomonas species (Figure 3). To further define DSM 34653 at the genome level, genome-to- genome comparisons were performed between DSM 34653 and strain genomes of P. wadenswilerensis (n = 2). Genomic regions in the genome assembly of DSM 34653 with no or low coverage in the strains of P. wadenswilerensis were extracted and subjected to a broad sequence similarity search. This identified several longer genomic regions matching <67% towards regions found in other prokaryotic genomes (Table 5, SEQ ID NOs: 2-4). Thus, these regions seem highly specific for DSM 34653.

[0376] Table 5: Maximum percent sequence coverage and maximum percent sequence identity (relating to the max. seq. coverage percentage) from a nucleotide sequence similarity search of SEQ ID NOs: 2-4 against the nucleotide database from the National Center for Biotechnology Information (NCBI). Search was performed on April 9, 2024. nt; nucleotides.

[0377] Importantly, several long open reading frames encoding hypothetical proteins were discovered within these long genomic regions, indicating that SEQ ID NOs: 2-4 might be of functional importance to DSM 34653. Such regions are included for each longer region (SEQ ID NOs: 2-4) as SEQ ID NOs: 5-13 (Table 6).

[0378] Table 6: Maximum percent sequence coverage and maximum percent sequence identity (relating to the max. seq. coverage percentage) from a nucleotide sequence similarity search of SEQ ID NOs: 5-13 against the nucleotide database from the National Center for Biotechnology Information (NCBI). Search was performed on April 9-10, 2024. nt; nucleotides. Four additional, and randomly selected, DNA sequences, encoding hypothetical proteins and located outside of SEQ ID NOs: 2-4 are further enclosed as SEQ ID NOs: 14-17 (Table 7). Comparing each of SEQ ID NOs: 5-17 to the nucleotide database at NCBI using BLAST revealed that 4-85% of the extracted sequences could be aligned to other prokaryotic genomic regions with sequence identities of 65.28-95.45% (Table 6 and 7). Thus, SEQ ID NOs: 5-17 seem highly selective for DSM 34653.

[0379] Table 7: Maximum percent sequence coverage and maximum sequence identity (relating to the max. seq. coverage percentage) from a nucleotide sequence similarity search of SEQ ID NOs: 14-17 against the nucleotide database from the National Center for Biotechnology Information (NCBI). Search was performed on April 11, 2024. nt; nucleotides.

[0380] To assess the potential properties of DSM 34653 that may be beneficial for agriculture, the genome of DSM 34653 was further explored. Interestingly, the assembled genome of DSM 34653 contains genes relating to the solubilization and mineralization of both inorganic gcd, ppx and ppa) and organic aphA) phosphate sources, phosphate transportation pitA, pstB, pstS) and phosphate-deprivation responses phoB, phoR, phoLT), indicating a strong genomic potential of DSM 34653 to increase the pool of available phosphate, even under phosphate-scarce conditions, that plants can utilize for growth. Phosphate-solubilizing capabilities of DSM 34653 are supported by in vitro assays presented in Example 4. Interestingly, DSM 34653 contains the hcnABC gene cluster encoding the enzyme involved in the synthesis of hydrogen cyanide (HCN); a secondary metabolite that has been linked to biocontrol properties by suppressing plant pathogens. The ability of DSM 34653 to suppress the growth of phytopathogenic fungi in vitro is presented in Example 3. Several pseudomonads are known to cause a variety of opportunistic infections and diseases in humans, including P. aeruginosa, P. fluorescens and P. putida. P. aeruginosa strains account for most infections, and many of these are also resistant to commonly used antibiotics. P. aeruginosa produces several virulence factors, including toxins that via secretion and injection systems can enter host cells and cause cellular death. Comparing translated nucleotide sequences of DSM 34653 and four virulent P. aeruginosa strains (PAO1, UCBPP PA14, LESB58 and PA7) to a protein database containing experimentally validated virulence factors (core dataset containing 4,236 virulence factors, downloaded from the Virulence Factor Database (VFDB) on April 16, 2024) revealed that gene products of DSM 34653 showed a significant (>90% sequence overlap and >90% sequence identity) overlap with seven putative virulence factors (Table 8) while gene products of the included P. aeruginosa strains matched towards 215-326 virulence factors (Figure 4). Importantly, the maximum sequence identity between a gene encoding a virulence factor and genes annotated in DSM 34653 was 94.36%, whereas 100% sequence identities were reported for virulence hits in all four P. aeruginosa strains (10-98.50% of all hits were associated with 100% sequence identities) (Figure 4), suggesting that virulence hits in P. aeruginosa strains are more valid. The virulence factors discovered in DSM 34653 relate to flagellar motility, scavenging of iron (via pyoverdine secretion), biofilm formation and secretory systems (Table 8). While such properties are relevant for pathogenic microorganisms, the total number of virulence factors found in DSM 34653 is low, at least compared to hypervirulent microorganisms, such as P. aeruginosa strains. Moreover, and importantly, the gene encoding exotoxin A, which has been reported as the most toxic virulence factor and is secreted by many clinically relevant P. aeruginosa strains, is not present in the genome assembly of DSM 34653, suggesting a non-pathogenic nature of DSM 34653. In addition, the virulence factors listed in Table 8 can also be viewed as important factors for improving plant growth by for example facilitating iron uptake in plants and secretion of plant-beneficial biomolecules such as HCN, demonstrating that the function of individual virulence factors are multifaceted. Plant growth-promoting capabilities of DSM 34653 are presented in Example 5. Finally, genome mining further revealed no presence of the rhizoxin-biosynthetic gene cluster, including both rhi and rzx gene loci, which encode metabolites that may be toxic to human health. Table 8: Virulence factor analysis of DSM 34653 by comparing translated nucleotide sequences to the protein core dataset from the Virulence Factor Database (VFDB, downloaded on April 16, 2024). The listed virulence factors represent significant hits in DSM 34653. VF=virulence factor.

[0381] Drug-resistant infections caused by antimicrobial resistance are a global burden and one of the leading threats to public health. Antimicrobial resistance occurs when microorganisms no longer respond to antibiotic drugs through the acquirement of antibiotic resistance traits. Performing an antimicrobial resistance analysis of DSM 34653 identified seven putative antimicrobial resistance genes of which none importantly were perfect hits, i.e., having a complete sequence overlap along with 100% identity towards a resistance gene (Table 9). Comparing this to the pattern of antimicrobial resistance genes present in the four virulent P. aeruginosa strains (PAO1, UCBPP PA14, LESB58 and PA7), that were also included in the virulence analysis, 55-62 resistance genes were detected in each strain. A heatmap was obtained representing sequence similarities (% identity) of antimicrobial resistance (AMR) genes and the genome assembly of DSM 34653 and four pathogenic Pseudomonas aeruginosa strains (PAO1, UCBPP PA14, LESB58 or PA7). 78 significant and unique AMR genes were found across all genomes analyzed (Data not shown).

[0382] Of these, rsmA and soxR (the top2 hits in DSM 34653 (Table 9)) were detected as perfect hits in three-four P. aeruginosa strains. Moreover, 92-95% of all resistance genes identified in the included P. aeruginosa strains had sequence identities above the best hit identity (80.65%) in DSM 34653. In addition, studies have shown the presence of rsmA, encoding a global posttranscriptional regulator, in non-pathogenic bacteria. While prediction of a phenotype solely on the basis of genome information is still in its infancy, the virulence factor and antimicrobial resistance analyses collectively suggest that DSM 34653 can be regarded as a commensal bacterium.

[0383] Table 9: Antimicrobial resistance analysis of DSM 34653 by comparing translated nucleotide sequences to The Comprehensive Antibiotic Resistance Database (CARD, downloaded on April 17, 2024). The listed hits represent the corresponding antimicrobial resistance genes (ARGs), percent overlap (coverage) between translated nucleotide sequences and protein sequences from CARD along with percent identity and the associated antibiotic drug class.

[0384] Conclusion Phylogenetic analyses and genome comparisons revealed that DSM 34653 can be considered as a novel bacterial species belonging to the genus Pseudomonas. Interestingly, DSM 34653 encodes several properties that are beneficial to agricultural farming, including phosphate solubilizing capabilities. In addition, DSM 34653 contains a complete biosynthetic gene cluster matching towards the hcnABC gene cluster that encodes the secondary metabolite hydrogen cyanide which has been suggested to act as a broad-spectrum biocontrol agent. As DSM 34653 is classified as a novel bacterial species, virulence and antimicrobial resistance properties were assessed. These analyses revealed the presence of few virulence and antimicrobial resistance genes; however, these were associated with lower sequence identities compared to gene hits identified in pathogenic P. aeruginosa strains.

[0385] Several DNA sequences containing open reading frames and that have high specificity for DSM 34653 were identified; three long genomic regions (14,515- 16,414 nucleotides, SEQ ID NOs: 2-4) wherein short DNA sequences (303-2,779 nucleotides) were extracted (SEQ ID NOs: 5-13) as well as four short DNA sequences (714-1,077 nucleotides) identified outside of the three long genomic regions (SEQ ID NOs: 14-17). Combined or alone, these DNA sequences may be used to identify DSM 34653 at the DNA sequence level.

[0386] Example 3 - Assessing biocontrol properties of DSM 34653

[0387] Aim of study

[0388] To demonstrate the ability of DSM 34653 to control growth of common fungal plant pathogens.

[0389] Materials and methods

[0390] Dual culture assays were used to determine the antifungal activity of DSM 34653. DSM 34653 was co-cultured on potato dextrose agar (PDA), grass agar (GA), or oatmeal agar (OA) with three isolates of fungal pathogens; Gaeumannomyces graminis var. tritici (isolate CBS 450.77), Pyrenophora teres f. teres (isolate CP2189), Zymoseptoria tritici (isolate IPO323), Fusarium oxysporum (isolate CBS 619.87). These four fungi are causing some of the major diseases in agricultural crops; wheat root rot (take-all), barley net blotch, septoria tritici blotch, and vascular wilts in a variety of crops. A spot on lawn dual culture assay was used to determine the antifungal activity. Petri dishes containing OA, GA, or PDA 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 to the initial direction further enhancing the uniformity of the lawn. A solution / suspension with a concentration of 3 McF of DSM 34653 was prepared in 1: 10 PBS. 10 pL of the solution / suspension was spotted onto each petri dish already prepared with fungal spores in replicates of three. As benchmarking control the Bacillus amyloliquefaciens QST 713 strain was included (Biol). This strain is the active ingredient in a commercial microbial biofungicide. The active microorganisms in this product were isolated and included in the test on equal footing with DSM 34653. Inhibition was qualitatively evaluated by the presence of a halo zone with no fungal growth on the 'lawn'.

[0391] The assay was performed at 20°C, to ensure optimal conditions for fungal growth. Inhibition was evaluated on day 8, except DSM 34653 on Zymoseptoria tritici, which was evaluated on day 10.

[0392] Results

[0393] DSM 34653 inhibited the growth of all four phytopathogenic fungi (Gaeumannomyces graminis var. tritici (isolate CBS 450.77), Pyrenophora teres f. teres (isolate CP2189), Zymoseptoria tritici (isolate IPO323), Fusarium oxysporum (isolate CBS 619.87)) when grown in a dual culture assay with a spot on lawn design (Figure 5 and Table 10). Similar results were observed with the benchmarking control (Biol), a biofungicide product already on the market, except it did not inhibit growth of Pyrenophora teres f. teres. These findings underscore the potential of DSM 34653 as a biofungicide for mitigating fungal attacks on agricultural crops.

[0394] Table 10. Assessment of fungal growth inhibition by bacterial products. Inhibition was assessed qualitatively; indicates no inhibition of fungal growth, while '+' indicates inhibition of fungal growth around the bacterial colony. Fungi were cultivated on oatmeal agar (OA), grass agar (GA), or potato dextrose agar (PDA). Conclusion

[0395] Addressing fungal crop diseases is crucial for maintaining global food security and sustainable agricultural practices. DSM 34653 was shown to have antifungal activity against four common phytopathogenic fungi Gaeumannomyces graminis var. tritici (isolate CBS 450.77), Pyrenophora teres f. teres (isolate CP2189), Zymoseptoria tritici (isolate IPO323), Fusarium oxysporum (isolate CBS 619.87) when evaluated in a dual culture assay. Based on these results DSM 34653 could hold potential for mitigating crop losses caused by fungal diseases, thereby enhancing agricultural productivity and sustainability.

[0396] Without being bound by theory, DSM 34653 may be used in the treatment of fungal infections in humans, and thus suitable for use as a medicament.

[0397] Example 4 - DSM 34653 has mineral-dissolving properties at low, medium and high temperatures

[0398] Aim of study

[0399] The aim is to demonstrate the efficacy of DSM 34653 as a biostimulant in increasing the availability of common plant nutrients under low, medium, and high temperature conditions.

[0400] Materials and methods

[0401] To demonstrate the ability of DSM 34653 to increase the availability of nitrogen, phosphorous, and potassium it was cultivated on different solid media: nitrogen- free agar; Jensen medium, insoluble phosphate agar; Pikovskaya agar, and insoluble potassium agar; Aleksandrow agar). These three solid media are recognized as 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, and Aleksandrow agar indicates solubilization of calcium phosphate (Cas(PO4)2 and potassium alumino-silicate respectively. The solubilization capacity was evaluated by calculating the solubilization index (SI, Areanaio / Areacoiony). To demonstrate their activity dependent on temperature the experiment was performed at low (5°C), medium (15°C), and high (25°C) temperatures. Typically, spring seeding will occur at soil temperatures around 8°C in Northern Europe, and activity at lower temperatures is therefore of great importance at this altitude.

[0402] As benchmarking control the Bacillus amyloliquefaciens QST 713 strain was included (Biol). This strain is the active ingredient in a commercial microbial biofungicide

[0403] The active strains of two well known biofungicide and biostimulant products were included in the experiment as benchmarking controls on equal footing with DSM 34653. These strains were Bacillus amyloliquefaciens QST 713 (Biol) and Bacillus atrophaeus Abi05 (Bio2). A fluid culture of each bacterial strain was prepared in a concentration of 0.5 McFarland. The solution / suspension was then spotted onto plates containing the different four 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 image!.

[0404] Results

[0405] The ability of DSM 34653 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 6). DSM 34653 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 34653 exerted mineral-dissolving properties at all temperatures (Figure 6B-C) while the bacterial control strains isolated from commercial products only demonstrated little (Figure 6B) or no (Figure 6C) capacity towards dissolving phosphate or potassium, respectively. The solubilization index (SI) of DSM 34653 on day 7 on Pikovskaya agar was in the range 2 and 9, wherein the SI was about 2.3 at 5°C, about 8.6 at 15°C, and about 4.7 at 25°C (Figure 6B). The SI of DSM 34653 on day 10 on Aleksandrow agar was in the range 2 and 5, wherein the SI was about 2.2 at 5°C, about 2.2 at 15°C, and about 4.8 at 25°C (Figure 6).

[0406] This suggests that DSM 34653 has a better capacity towards dissolving minerals which are otherwise unavailable for plants.

[0407] Conclusion 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 34653 was able to grow and display bacterial activity at 5°C, demonstrating that DSM 34653 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.

[0408] Example 5 - Seed coat containing DSM 34653 promotes early plant growth

[0409] Aim of study

[0410] The scope of this example is to demonstrate the stimulatory properties of DSM 34653, when applied as a seed coat, on early plant growth in vitro in spring barley; a typical agricultural crop.

[0411] Materials and methods

[0412] Spring barley seeds (Hordenum vulgare 'Skyway') were manually coated with a bacterial solution / suspension containing 1.47xlO9CFU DSM 34653 per mL (n = 70 seeds). The following was used for coating 5 g of seeds: 12.5 pL DSM 34653 (1.47xl09CFU / mL) + 62.5 pL of demineralized water. Untreated seeds were used as a control (n=70 seeds). In a third experimental group, seeds were treated with both DSM 34653 and a commercial chemical fungicide containing the following active substances: tebuconazol (20 g / L) and prothioconazol (150 g / L) (n = 70 seeds).

[0413] The coating process was performed in a 50 mL conical tube where a bacterial solution / suspension containing DSM 34653 (diluted in water) or a commercial chemical fungicide were added to the seeds. Tubes were thoroughly shaken to ensure even distribution of bacteria or fungicide on seeds. Seeds were afterwards sown into trays and stored indoors with a day-night cycle of 16h / 8h at 20±2°C. The emergence percentage of seedlings was quantified five days following sowing as (# of emerged seedlings / # seeds sown) x 100%. Results

[0414] Treating spring barley seeds with a seed coat containing a bacterial solution / suspension of DSM 34653 before sowing increased the emergence at five days following sowing with 19% compared to non-treated seeds (Figure 7). Compared to seeds that were treated with both a commercial chemical fungicide and DSM 34653, DSM 34653 coating alone increased the emergence with 41%. Interestingly, the emergence of seedlings was higher among non-treated seeds compared to seeds that were treated with both commercial chemical fungicide and DSM 34653 (16%), suggesting that fungicide treatment prior to sowing is associated with lower emergence percentages.

[0415] Conclusion

[0416] Spring barley is among the most common grown cereal crops in Denmark.

[0417] Treating spring barley seeds with a bacterial solution / suspension containing DSM 34653 before sowing demonstrates that DSM 34653 can enhance the emergence of seedlings, also when seeds have been treated with a commercially available chemical fungicide, suggesting that an increased yield also may be obtained.

[0418] Example 6 - DSM 34653 produces secondary metabolites important for plant health

[0419] Aim of study

[0420] The scope of this example is to demonstrate that DSM 34653 can produce secondary metabolites that are important for biostimulant and biocontrol activities in the field.

[0421] Materials and methods

[0422] Hydrogen cyanide production

[0423] Production of hydrogen cyanide (HCN) was detected by the formation of a blue color on a filter paper soaked in a HCN detection reagent (20mg / ml Cu(II)- ethylacetoacetate in 96% ethanol and 20mg / ml 4,4'-methylenebis(N,N- dimethylaniline) in methanol). Bacterial cultures of DSM 34653, a positive control (a Pseudomonas strain that produces HCN) and a negative control (a mutant derivative of the positive control that cannot produce HCN) were incubated overnight in LB media at 30°C and 300 rpm. A bacterial strain isolated from a commercial microbial biofungicide product was included for benchmarking purposes (Biol) and was handled in the same manner as DSM 34653 and the controls. Overnight cultures were diluted to ODeoo = 0.02, transferred to a 96-well plate and incubated for 48h at 30°C and 600 rpm (n = 3; DSM 34653 and benchmarking control, n = l; positive and negative controls) with a filter paper containing HCN detection reagent placed on top of the 96-well plate along with a breathable membrane to seal the plate. Following the 48h, the 96-well plate was allowed to dry overnight at 30°C. A color change from colorless to blue on the filter paper indicates that volatile HCN has been produced and released from the liquid cultures.

[0424] Siderophore production

[0425] Siderophore production was assessed using an universal siderophore assay containing chrome azurol S (CAS) and hexadecyltrimethylammonium bromide (HDTMA) that form tight complexes with ferric ions. A color change is observed from blue to orange when ferric ions are scavenged as a result of siderophore production. 10 pL of an overnight culture of DSM 34653 diluted to OD600 = 0.2 was spotted onto three CAS agar plates each containing three spots (n=9 in total) and the plates were incubated for seven days at room temperature. The area of orange halos observed around the bacterial colonies, corresponding to siderophore production, as well as the area of the bacterial colonies were measured daily using Image! and a halo index (HI), representing siderophore production, was calculated using the following equation:

[0426] HI = AreaHaio / Areacoiony

[0427] An agar overlay assay was additionally performed for Biol, since Biol could not grow on the CAS agar. 10 pL of overnight culture of Biol diluted to OD600 = 0.2 was spotted onto LB agar plates in three individual replicates; each containing three spots (n=9 spots in total per day). Plates were incubated for seven days at room temperature. One plate from each replicate was overlaid with a standardized volume of CAS agar for each day during the experiment period. Plates were visually inspected 24h and 48h post overlay.

[0428] Indole-3-acetic acid production

[0429] The biosynthesis of indole-3-acetic acid (IAA) was assessed using Salkowski's reagent which contains ferric chloride (FeCh) and perchloric acid (HCIO4) that upon complex formation with IAA yields a pink color. As IAA is a product of L- tryptophan metabolism, bacterial cultures of DSM 34653 and Biol were grown in LB media for 48h at 30°C and 300rpm with 5 mM L-tryptophan (n=3) or without L-tryptophan (n=3). Bacterial cell densities were quantified spectrophotometrically at 600 nm. To quantify IAA production, the supernatant was harvested from the bacterial cultures by sterile filtration and subsequently incubated with Salkowski's reagent in a 1:2 ratio for 30 min in the dark at room temperature and afterward quantified spectrophotometrically at 530 nm. A standard curve was generated from known IAA concentrations (0-100 pg / mL) and IAA concentrations, corresponding to IAA produced by each bacterium, were estimated using linear regression. IAA concentrations were normalized to the bacterial cell densities (ODeoo values) in order to compare IAA production across the two bacterial strains.

[0430] Results Computational mining of the genome of DSM 34653 revealed the presence of the hcnABC gene cluster encoding the enzyme involved in the synthesis of hydrogen cyanide (HCN); a secondary metabolite that has been suggested to act as a broad-spectrum biocontrol agent (presented in Example 2). The ability of DSM 34653 to synthesize and secrete HCN was importantly validated using a biochemical assay (Figure 8). DSM 34653 moreover produced higher levels of HCN than the bacterial strain isolated from a commercial biofungicide product (Figure 8, Biol), suggesting that DSM 34653 might have a high capacity for controlling fungal plant diseases through HCN action. The ability of DSM 34653 to suppress the growth of phytopathogenic fungi in vitro is presented in Example 3.

[0431] To investigate whether DSM 34653 could produce additional secondary metabolites, siderophore and indole-3-acetid acid (IAA) production was assessed as these represent some of the most important compounds benefitting plant growth and health. Siderophores are a group of iron-scavenging compounds that can transform iron, which is often found as insoluble complexes in soil, into forms that are available for plant uptake. In addition, plant pathogens, such as phytopathogens, require iron in order to grow. Thus, siderophore-producing bacteria can deprive plant pathogens which affect not only plant health but also growth indirectly as plant pathogens often impact plant development negatively. IAA is the most abundant and naturally occurring plant hormone and is essential for plant development and defense mechanisms against plant pathogens. DSM 34653 importantly demonstrated the ability to produce both siderophores (Figure 9 and Figure 10) with a maximum HI at Day 3 (mean HI=6.30, mean range across all experimental days =4.66-6.30) and IAA (Figure 11) in vitro. Siderophore production from Biol was assessed using an agar overlay assay as Biol was not viable on CAS agar. Here, Biol was cultured on LB agar and then overlaid with CAS agar which revealed that Biol possessed capacity for producing siderophores (data not shown). However, comparing the capacity for siderophore production from DSM 34653 and Biol, respectively, was not feasible as siderophore production in each strain was evaluated using two different solid medium strategies.

[0432] IAA is synthesized using tryptophan (Trp) as a precursor, but research has also shown synthesis of IAA in a Trp-independent manner using pathways that have not yet been fully elucidated. Adding Trp to the growth media revealed an induction (corresponding to a fold change of 1.7) of IAA synthesis in DSM 34653 (Figure 11, bars to the left). IAA production was also assessed in Biol which synthesized higher levels of IAA in a Trp-independent manner compared to DSM 34653 (Figure 11, bars to the right). While IAA synthesis was also upregulated in Biol upon Trp addition (corresponding to a fold change of 1.2), DSM 34653 seems more responsive towards Trp.

[0433] Conclusion

[0434] Biochemical and solid medium-based assays importantly confirmed that DSM 34653 can produce secondary metabolites that are important for plant health and development, including hydrogen cyanide (HCN), siderophores and indole-3-acetic acid (IAA), thus strongly indicating that DSM 34653 has a high capacity for crop management in the field.

[0435] Example 7 - DSM 34653 has phytate-degrading activity

[0436] Aim of study

[0437] The aim of this example is to demonstrate the ability of DSM 34653 to mineralize phytate (myo-inositol hexakisphosphate) representing one of the major forms of organic phosphate in soil. Materials and methods

[0438] To demonstrate the ability of DSM 34653 to mineralize phytate, DSM 34653 was cultured on a modified Pikovskaya agar containing sodium phytate; a source of insoluble organic phosphate. 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 using the following equation:

[0439] SI = AreaHaio / Areacoiony

[0440] A bacterial strain isolated from a commercial microbial biofungicide product was included as a benchmarking (Biol). 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 seven days. Colony and halo zone areas were quantified at each day throughout the duration of the experiment using Image!.

[0441] Results

[0442] The ability of DSM 34653 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 34653 and Biol were able to grow on the phytate-containing medium, however, only DSM 34653 displayed phytate-degrading capabilities throughout the entire experimental period with a maximum solubilization index (SI) at day 7 (mean SI=8.96) while Biol started to mineralize phytate at day 5 reaching a maximum SI at day 7 (mean SI=2.25) (Figure 12 and 13). These results suggest that DSM 34653 has a high capacity for mineralizing organic phosphate by which inorganic phosphorous is released, which would otherwise remain unavailable to plants.

[0443] Conclusion

[0444] Organic phosphate can be found as aggregated forms, known as phytate, in soil. Phytate is inaccessible to plants and its mineralization to inorganic phosphorous can enhance nutrient availability, thereby promoting plant growth. In this study, DSM 34653 exhibited a greater ability to mineralize organic phosphate, here represented as phytate, compared to a commercial biofungicide strain (Biol), demonstrating that DSM 34653 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 34653 emerges as a versatile biostimulant.

[0445] Example 8 - DSM 34653 has zinc-solubilizing potential

[0446] Aim of study

[0447] The aim of this example is to demonstrate the ability of DSM 34653 to solubilize zinc; a micronutrient essential for normal plant development.

[0448] Materials and methods

[0449] To demonstrate the ability of DSM 34653 to solubilize zinc, DSM 34653 was cultured on zinc-solubilizing agar containing zinc oxide (ZnO); a source of insoluble zinc. The formation of translucent halo zones surrounding the bacterial colonies corresponds to enzymatic activities leading to the solubilization of zinc. The solubilization capacity was evaluated by calculating a solubilization index using the following equation:

[0450] SI = AreaHaio / Areacoiony

[0451] A bacterial strain isolated from a commercial microbial biofungicide was included in the experiment as a benchmarking control (Biol). An overnight culture was prepared for each strain and adjusted to OD600 = 1 the following day. Equal volumes of the bacterial suspensions were spotted (n = 3) onto ZnO-containing agar plates which were incubated at 15°C for seven days. Colony and halo zone areas were quantified at each day throughout the duration of the experiment using Image!. An agar overlay assay was additionally performed for Biol, since Biol could not grow on the zinc-solubilizing agar. Here, Biol was cultured on LB agar plates and then overlaid with a standardized volume of zinc-solubilizing agar at three different timepoints. Overlaid plates were otherwise cultured in the same manner as non-overlaid plates and quantified the following day.

[0452] Results

[0453] The ability of DSM 34653 to solubilize zinc was evaluated using a solid medium strategy where bacterial colony and halo zone areas on zinc-solubilizing agar were quantified at 15°C and furthermore compared to a bacterial strain that was isolated from a commercial biofungicide product (Biol). DSM 34653 displays zinc- solubilizing activity from day 2 with a maximum solubilization index (SI) at day 7 (mean SI=3.61) (Figure 14) while Biol was not viable on the zinc-solubilizing agar (Figure 15, pictures in the middle column). To account for the lack of viability, Biol was cultivated on LB plates and then overlaid with a standardized volume of zinc-solubilizing agar at three different timepoints throughout the experiment, however, no zinc solubilization was observed for Biol (Figure 15, pictures in the third column). Importantly, no solubilization was observed even after several days post overlay, strongly suggesting that Biol does not have a capacity for solubilizing zinc. Collectively, these results suggest that DSM 34653 has a capacity for solubilizing zinc, which would otherwise remain unavailable to plants.

[0454] Conclusion

[0455] Zinc belongs to the category of essential micronutrients and is therefore pivotal for normal and healthy growth of plants. It can be bound in different complexes in soil, of which zinc oxide is a highly insoluble compound. Accordingly, solubilization of zinc oxide can enhance nutrient availability, thereby promoting plant growth. In this study, DSM 34653 exhibited the ability to both survive in a zinc-rich environment and solubilize a highly insoluble form of zinc. These capabilities were absent in the commercial biofungicide strain that was included in the study for benchmarking purposes. These findings support that DSM 34653 has potential for supplying crops with otherwise unavailable nutrients in the field.

[0456] Example 9 - Biocontrol applications of DSM 34653 in the field

[0457] Aim of study

[0458] The aim of this study was to test the ability of DSM 34653 to manage common fungal diseases in spring barley when applied as a seed coat, as a foliar spray or a combination hereof in the field.

[0459] Materials and methods

[0460] Spring barley (Hordeum vulgare, variety Prospect) seeds were used for field trials listed in Table 11. All experiments were performed by an independent third-party contract research organization (VKST Field Trials, Denmark) and seeds were sown on April 16, 2024, in 8 field parcels (2x16m per parcel) per treatment group with a planting rate of 173 kg / ha (the soil temperature was 14°C at the time of sowing). Fields were harvested on August 8, 2024.

[0461] Table 11: Experimental overview of field trials in spring barley. Trials were carried out by an independent third-party contract research organization (VKST Field Trials, Denmark). DSM 34653 were applied either as a seed coat, as a foliar spray or as a combination of both. Two chemical fungicides were included and applied as either a seed coat or as a foliar spray. Spring barley plants were sprayed in two windows: Window 1; Before appearance of disease symptoms, Window 2; At the presence of 0-5% disease symptoms.

[0462] Seed coat experiments

[0463] Seed treatments were carried out by applying either a bacterial suspension of DSM 34653 (containing 1.47xl09CFU / mL) or a commercial chemical fungicide (Chemical fungicide #1, containing fludioxonil as the active substance) to seeds at a rate of 2.5 mL / kg. Seeds were stirred during the seed coating process to ensure an even distribution onto seeds. Following seed coating, seeds were dried over night at 8C° before sowing.

[0464] Foliar spray experiments

[0465] Spring barley plants were sprayed in two windows: Window 1; Before appearance of any disease symptoms, Window 2; At the appearance of 0-5% disease symptoms using either a bacterial suspension of DSM 34653 (containing 2xl05- 4.1xl08CFU / mL) or a commercial chemical fungicide (Chemical fungicide #2, containing pyraclostrobin as the active substance) at a rate of 200 L / ha (4 L / ha of DSM 34653 and 0.2 L / ha of Chemical fungicide #2 to a total application concentration of 200 L / ha). Each window contains two consecutive rounds of spraying: Window 1; May 23, 2024, and June 21, 2024, Window 2; June 4, 2024, and July 1, 2024.

[0466] Assessment of crop disease

[0467] Spring barley plants were assessed for a final presence of Brown leaf rust (caused by the fungal plant pathogen Puccinia horde!) and Net blotch (caused by the fungal plant pathogen Pyrenophora teres) on July 12, 2024. 10 plants were randomly selected from each parcel (n=80 per treatment group) and plant leaves were visually scored along a disease index scale (0-100%) corresponding to the leaf area affected by disease symptoms (as recommended by SEGES Innovation P / S, an independent agricultural research and consulting company based in Denmark).

[0468] Results

[0469] The ability of DSM 34653 to inhibit common fungal plant pathogens in vitro was demonstrated in Example 3, including the inhibition of Pyrenophora teres causing net blotch in barley crops. The presence of net blotch and brown leaf rust, two common barley crop diseases, was assessed in spring barley crops treated with either DSM 34653 or two chemical fungicides as a seed coat, a foliar spray or as a combination (Table 11). All treatments were effective in diminishing disease symptoms caused by either brown leaf rust (72.64-86.05% decrease, p- value<2.2e-16) or net blotch (74.83-86.08% decrease, p-value<2.2e-16) compared to untreated (control) spring barley plants.

[0470] Applying DSM 34653 as a foliar spray to plants before the appearance of any disease symptoms (in Window 1) was more effective in managing the final disease presence of both brown leaf rust (20.07% decrease, p-value= 6e-04) and net blotch (40.43% decrease, p-value= 5.477e-08) compared to spraying plants after disease symptoms have appeared (in Window 2) (Figure 16A and Figure 16B). Interestingly, no significant differences were observed in the final assessment of net blotch when comparing DSM 34653 spray treatment in Window 1 with the chemical fungicide that was sprayed onto plants in Window 2, indicating that DSM 34653 might decrease the presence of certain plant diseases to the same extent as a chemical fungicide when sprayed directly onto plants (Figure 16B). Interestingly, coating seeds with DSM 34653 or combining the seed coat with a spray treatment in Window 2 were as, or even more, effective as both chemical fungicides in reducing overall disease symptoms in spring barley plants (Figure 16A and Figure 16B). Interestingly, no significant differences were observed across experiments involving DSM 34653 as a seed coat and moreover, these experiments were similarly or even more effective in managing crop disease compared to spraying plants with DSM 34653 before the presence of disease symptoms. However, out of all DSM 34653 treatments, only the seed coat treatment that was combined with a spray treatment in Window 2 resulted in an increased harvest yield, compared to either control plants (12.95% increase, p- value = 0.002331) or plants developed from seeds coated with a chemical fungicide (9.18% increase, p-value=0.01033) (Figure 17). These data suggest that treating spring barley seeds with a bacterial suspension containing DSM 34653 before sowing might be an efficient strategy for managing diseases in crops while also increasing the harvest yield.

[0471] Conclusion

[0472] Brown leaf rust and net blotch are two diseases commonly found in barley crops which can have a negative impact on harvest yields. Treating spring barley seeds or plants with a bacterial suspension of DSM 34653, as either a seed coat, a foliar spray or as a combination, led to spring barley plants being less affected by brown leaf rust or net blotch. Interestingly, applying DSM 34653 to seeds before sowing resulted in a similar or greater decrease in disease presence compared to chemical fungicides that was applied as either a seed coat or a foliar spray while also resulting in an increased harvest yield when DSM 34653 was applied as a combination of a seed coat and a spray. In conclusion, DSM 34653 exhibits biocontrol properties in the field.

[0473] Example 10 - A genomic definition of novel Pseudomonas strains with biocontrol properties

[0474] Aim of study

[0475] The scope of this example is to demonstrate that DSM 34653 and the remaining TF strains presented in Example 1 (TF-A, TF-B, TF-C, TF-E, TF-F and TF-G) can collectively be identified on the basis of shared DNA sequences.

[0476] Materials and methods DNA extraction, library preparation and whole genome sequencing

[0477] DSM 34653 was previously sequenced using (Oxford Nanopore Technology) ONT technology (see Example 1). DNA was extracted from the remaining bacterial strains (TF-A, TF-B, TF-C, TF-E, TF-F and TF-G) 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 3,992,948- 5,126,384 reads were obtained for each strain.

[0478] Quality control and processing of sequencing data

[0479] Adapter and barcode contaminants originating from Illumina 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.

[0480] Genome assemblies

[0481] A complete de novo genome was previously generated for DSM 34653 (see Example 2). SPAdes (v4.0.0) was used for assembling trimmed sequencing reads obtained from TF-A, TF-B, TF-C, TF-E, TF-F and TF-G into de novo contigs that were subsequently joined into putative scaffolds. A read error-correction step (BayesHammer module) was performed prior to the genome assembly step to minimize the number of mismatches in the resulting contigs. Only scaffolds >500 bp along with a coverage of >2 were kept following the assembly step. QUAST (v5.0.2) was used for computing assembly quality metrics.

[0482] Genome annotation

[0483] The complete genome representing DSM 34653 was previously annotated (see Example 2). Genomic features present within the genome assemblies representing TF-A, TF-B, TF-C, TF-E, TF-F and TF-G were annotated using Prokka (vl.14.6).

[0484] Average nucleotide identity (ANI) analysis

[0485] FastANI (vl.33) was used for computing average nucleotide identity (ANI) scores where each assembled genome belonging to strain TF-A, TF-B, TF-C, TF-E, TF-F and TF-G was iteratively compared to the genome representing DSM 34653 (Table 4). DNA sequence analysis

[0486] The Basic Local Alignment Search Tool (BLAST) was used for evaluating the presence of SEQ ID NO: 2-17 (defined in Example 2) in the genome assemblies representing TF-A, TF-B, TF-C, TF-E, TF-F and TF-G. The blastn algorithm (v2.12.0+) were used for the alignment searches along with the following parameters set: evalue=0.0001, num_alignments = 10000.

[0487] SEQ ID NO: 18 (10,536 nt, forms part of SEQ ID NO: 3) was furthermore included. Results

[0488] DSM 34653 was selected as the most promising biocontrol candidate out of seven isolated bacterial strains (see Example 1). Example 2 demonstrates that DSM 34653 should be considered a novel Pseudomonas species and that the genome representing DSM 34653 encodes properties that are beneficial to agricultural farming. To exploit the genetic relatedness of DSM 34653 and the remaining isolated strains presented in Example 1 (n = 6; TF-A, TF-B, TF-C, TF-E, TF-F and TF-G), de novo genomes were assembled for each strain. Comparing size, contiguity, GC content (average guanine and cytosine (GC) content based on the DNA nucleotide composition) (Table 12) and the presence of genes encoding proteins or RNA products (Table 13), indicate that TF-A, TF-B, TF-C, TF-E, TF-F and TF-G are very similar at the genome level.

[0489] Table 12: Genome assembly statistics for strains TF-A, TF-B, TF-C, TF-E, TF-F and TF-G. Statistics are based on scaffolds >500 bp.

[0490] Table 13: Genome features associated with genome assemblies for strains TF-A, TF-B, TF-C, TF-E, TF-F and TF-G. CDS=coding sequence; rRNA=ribosomal RNA; tRNA=transfer RNA; tmRNA=transfer-messenger RNA.

[0491] Comparing TF-A, TF-B, TF-C, TF-E, TF-F and TF-G to each other and to the genome representing DSM 34653 through average nucleotide identity (ANI) analyses revealed no ANI values below the putative strain threshold at 99.5% ANI (strains compared to DSM 34653: ANI range=99.9809-99.9989%, strains compared to each other: ANI range=99.9952-99.9995%), demonstrating that DSM 34653 as well as the bacterial strains TF-A, TF-B, TF-C, TF-E, TF-F and TF-G represent the same Pseudomonas strain which should further be considered as a novel Pseudomonas species.

[0492] The bacterial strains TF-A, TF-B, TF-C, TF-E, TF-F and TF-G are very similar and almost identical to DSM 34653, which may explain the common ability for fungal inhibition as seen in Table 1 in Example 1. In addition, genome assemblies representing TF-A, TF-B, TF-C, TF-E, TF-F and TF-G also contain the hcnABC gene cluster encoding the enzyme involved in the synthesis of hydrogen cyanide which was also found in DSM 34653 (presented in Example 2).

[0493] Thus, it can be expected that the said bacterial strains TF-A, TF-B, TF-C, TF-E, TF- F and TF-G exhibit similar or identical properties as DSM 34653, such as improved biocontrol properties, mineral dissolving properties, promoting early plant growth, production of secondary metabolites, phytate degrading properties, and zinc- solubilizing properties.

[0494] Several DNA sequences were identified as highly selective for DSM 34653 (SEQ ID NOs: 2-17). All of these, except for SEQ ID NO: 3, were importantly also identified as full-length fragments (99.96-100% coverage with 100% seq identities) in TF-A, TF-B, TF-C, TF-E, TF-F and TF-G. SEQ ID NO: 3 (16,414 nt) is found as several fragments spanning multiple scaffolds in the genome assemblies representing TF-A, TF-B, TF-C, TF-E, TF-F and TF-G with the largest intact fragment being 10,536 nt. Several open reading frames are however importantly located on this fragment which is further enclosed as SEQ ID NO: 18. Thus, SEQ ID NOs: 2 and 4-18 are highly selective not only for DSM 34653 but also for TF- A, TF-B, TF-C, TF-E, TF-F and TF-G.

[0495] Conclusion

[0496] A comparative genome analysis revealed that DSM 34653 and the remaining isolated strains presented in Example 1 (TF-A, TF-B, TF-C, TF-E, TF-F and TF-G) are variants of the same type of Pseudomonas strain. Combined or alone, SEQ ID NOs: 2 and 4-18 (where SEQ ID NO: 18 (16,414 nt) forms part of SEQ ID NO: 3), may be used to identify DSM 34653, TF-A, TF-B, TF-C, TF-E, TF-F and TF-G collectively at the DNA level.

[0497] Example 11 - Seed coat containing DSM 34653 promotes early emergence of spinach plants

[0498] Aim of study

[0499] The aim of this example is to demonstrate the stimulatory properties of DSM 34653, when applied as a seed coat, on the emergence of spinach plants in the field.

[0500] Materials and methods

[0501] Spinach Spinacia oleracea, female variety susceptible to Fusarium sp. seeds were used for field trials listed in Table 14. All experiments were performed by an independent seed production company (Jensen Seeds A / S, Denmark) and seeds were sown on fields on Funen on April 15th, 2025, in 24 field parcels (40 m2per parcel) per treatment group with a planting rate of 15,000 plants per plot (mean air temperature was 9.1°C for April, 2025). The early emergence of spinach plants was quantified on May 1st, 2025, as percent emerged plants per square meter, using the following equation:

[0502] (No. of emerged plants / No. of seeds sown) x 100% Table 14: Experimental overview of field trials in spinach. Trials were carried out by an independent seed production company (Jensen Seeds A / S, Denmark). All treatments, except controls, were conducted as seed coats. DSM 34653 were included as a concentrated fermentation broth with or without additives. A chemical fungicide was included for benchmarking purposes.

[0503] Seed coating details

[0504] Seed treatments were carried out by applying either a bacterial suspension of DSM 34653 (with or without additives, containing 2.5xl09CFU / kg seed) or a commercial chemical fungicide (containing fludioxonil as the active substance) to seeds at a rate of 0.48 g / kg, All seeds were treated using a Hege 11 Vorfiihrgerat seed coater, and stored in paper bags (approved for the food industry) following the procedure. Seeds were sown 4 days after coating.

[0505] Results

[0506] Treating spinach seeds with a seed coat containing a bacterial suspension of DSM 34653, either with or without additives, before sowing increased the emergence of spinach plants 16 days following sowing with 65.85% and 90.65%, respectively, in the field compared to non-treated seeds (Figure 18). Treating spinach seeds with a chemical fungicide containing fludioxonil as the active substance also led to an increase in the emergence of spinach plants compared to control plants by 50% (Figure 18). However, and interestingly, treating seeds with DSM 34653 resulted in a similar (DSM 34653 in the formulated form (DSM 34653F)) or significantly increased emergence (DSM 34653 as concentrated fermentation broth, p=0.03622) compared to seeds that were treated with the chemical fungicide (Figure 18). These data demonstrates that DSM 34653 exerts positive effects on early plant growth to at least the same extent as chemically derived fungicides.

[0507] Conclusion

[0508] Treating spinach seeds with a seed coat containing a bacterial suspension of DSM 34653 before sowing led to an increase in the emergence of spinach plants with 65.85-90.65% in the field which is similar or significantly increased compared to seeds treated with a commercial chemical fungicide. This demonstrates that DSM 34653 can stimulate plant growth in spinach, suggesting that an increased yield also may be obtained.

[0509] Example 12 - Foliar spray containing DSM 34653 reduces symptoms of Septoria tritici blotch in winter wheat under controlled climate chamber conditions

[0510] Aim of study

[0511] The aim of this example was to evaluate the ability of strain DSM 34653 to control Septoria tritici blotch in winter wheat, when applied as a foliar spray under controlled climate chamber conditions, followed by artificial inoculation with Zymoseptoria tritici.

[0512] Materials and methods

[0513] Control of septoria tritici blotch was evaluated in winter wheat Tri ti cum aestivum, Sevin Sejet) following foliar spray with different treatments containing DSM 34653 followed by inoculation with Zymoseptoria tritici spores. Treatment groups are described in detail in Table 15. The experiment was performed by Copenhagen University, Department of Plant and Environmental Sciences. Plants were grown in the potting mix Pindstrup substrate no. 2 (Pindstrup Mosebrug A / S, Denmark).

[0514] Growth conditions in the growth chamber were 16h / 8h light / darkness with 200 pE m-2 s-1 light intensity (fluorescent tubes, Osram L 36W / 11-860 Lumilux plus Eco Daylight). Temperature and relative humidity (RH) were approximately 19°C / 50- 60 % in light and 16°C / 80-90 % in darkness. When the plants were 14 days old, 12 leaves were mounted onto Plexiglass plates to allow foliar spray application and fungal spore inoculation. Each treatment was performed with four biological replicates (n = 4). Four different treatments were applied to the plants as a foliar spray and the plants were kept in the dark for 24 hours. The treatments were W) water (control) or E, B, A) Bacterial suspensions of DSM 34653: E) DSM 34653 high dose (lxlO9CFU / mL), B) DSM34653F normal dose (2xl07CFU / mL with additives), or A) DSM 34653 normal dose (2xl07CFU / mL). Subsequently the plants were inoculated with Zymoseptoria tritici spores (lxlO6spores / mL). The plants were kept in darkness for 72 hours. After this, the light was turned on in the chambers and the light / dark cycle was reestablished. The plants were photographed on day 14, 17, and 19 after inoculation (DAI). Percentage of leaf area with disease symptoms was determined using programme Assess 2.0 Image Analysis Software for Plant Disease Quantification (The American Phytopathological Society).

[0515] Table 15: Experimental overview of treatments used in the climate chamber trial. The trial was carried out at Copenhagen University, Department of Plant and Environmental Sciences, Denmark. All treatments were applied as foliar sprays.

[0516] Preliminary results

[0517] The ability of DSM 34653 to inhibit common fungal plant pathogens in vitro was demonstrated in Example 3, including the inhibition of Zymoseptoria tritici causing Septoria tritici blotch. In this example the ability of DSM 34653 to control Septoria tritici blotch in winter wheat was evaluated (Table 16 and Figure 19). Figure 19 shows visual reduction of disease symptoms on leaves treated with either of the three formulations containing DSM 34653 (E, B, and A). Table 16 shows mean values of the percentage of leaf area with symptoms of Septoria tritici blotch. All formulations containing DSM 34653 reduced disease symptoms at 17 (37-50% reduction) and 19 DAI (34-53% reduction). These data suggest that DSM 34653 can control Zymoseptoria tritici in winter wheat, and, thus, has potential to manage this major crop disease in the field. Table 16: Preliminary results from climate chamber pot trial with winter wheat inoculated with Zymoseptoria tritici. Symptoms are evaluated at 14, 17. And 19 days after inoculation (DAI) with a suspension of Zymoseptoria tritici spores.

[0518] Values indicate the mean percentage of leaf area with symptoms.

[0519] Conclusion

[0520] Preliminary data show that foliar application of DSM 34653 reduced symptoms of Septoria tritici blotch in winter wheat under controlled conditions. These results support the potential of DSM 34653 as a biocontrol agent for managing fungal diseases in cereal crops.

[0521] Sequence listing Items of the invention

[0522] 1. A bacteria, preferably isolated, or biologically pure bacterial culture comprising a) a genomic sequence according to any of SEQ ID NOs: 5-17; 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: 5-17; and / or c) a fragment of one or more of the sequences of a) or b), having a length of at least 300 nucleotides.

[0523] 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: 5, 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: 5.

[0524] 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: 6, 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: 6.

[0525] 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: 7, 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: 7.

[0526] 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: 8, 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.

[0527] 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: 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. 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: 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.

[0528] 8. 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.

[0529] 9. 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.

[0530] 10. 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.

[0531] 11. 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.

[0532] 12. 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: 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.

[0533] 13. 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: 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. 14. 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: 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.

[0534] 15. The bacteria or biologically pure bacterial culture according to item 1 comprising any of SEQ ID NO: 5-17, such as one or more of SEQ ID NO: 5-17.

[0535] 16. The bacteria or biologically pure bacterial culture according to item 1, comprising a) a genomic sequence according to any of SEQ ID NO: 2-4 or 18; b) a genomic sequence having at least 90% sequence identity to any of SEQ ID NO: 2-4, 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: 2-4, 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 14000 nucleotides.

[0536] 17. The bacteria or biologically pure bacterial culture according to any of the preceding items, being a Pseudomonas, such as Pseudomonas wadenswilerensis , Pseudomonas donghuensis, Pseudomonas tructae, or Pseudomonas rubra.

[0537] 18. A bacteria or biologically pure bacterial culture being Pseudomonas, DSM 34653 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.

[0538] 19. The bacteria or biologically pure bacterial culture according to any of the preceding items, being

[0539] - 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.

[0540] 20. The bacteria or biologically pure bacterial culture according to item 19, wherein the one or more phytopathogenic fungi are selected from the group consisting of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres, Zymoseptoria tritici, Puccinia hordei, and Fusarium oxysporum.

[0541] 21. The bacteria or biologically pure bacterial culture according to item 19, wherein the solubilization of calcium phosphate, iron phosphate, and / or potassium aluminum silicate at 5°C is at a level of at least 40% to, equal to and / or above a level of solubilization at 15°C and / or 25°C.

[0542] 22. The bacteria or biologically pure bacterial culture according to any of items 19 or 21, wherein the level of solubilization is determined by a solubilization index (SI) calculated by the formula:

[0543] SI = AreaHaio / Areacoiony.

[0544] 23. The bacteria or biologically pure bacterial culture according to item 22, wherein the SI is at least 2, such as at least 4, such as between 2 and 10, preferably the SI is between 2 and 9.

[0545] 24. The bacteria or biologically pure bacterial culture according to any of items 22 or 23, wherein the SI depends on temperature after cultivation start and / or incubation time.

[0546] 25. The bacteria or biologically pure bacterial culture according to item 24, 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.

[0547] Fertilizer:

[0548] 26. 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 1-25. 27. The fertilizer and / or inoculant and / or biostimulant composition according to item 26, further comprising other microorganisms, such as other microorganisms able to function as a biostimulant.

[0549] 28. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 26 or 27, further comprises one or more agriculturally acceptable carriers.

[0550] 29. The fertilizer and / or inoculant and / or biostimulant composition according to item 28, 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.

[0551] 30. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 26-29, 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.

[0552] 31. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 26-30, 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.

[0553] 32. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 26-30, the composition is formulated as a granular formulation or a powder formulation.

[0554] 33. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 26-32, 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.

[0555] 34. The fertilizer and / or inoculant and / or biostimulant composition according to any of items 26-33, 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.

[0556] 35. The fertilizer and / or inoculant and / or biostimulant composition according to item 33, 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.

[0557] Coating composition

[0558] 36. A coating composition, preferably a seed coating composition, comprising the bacteria or biologically pure bacterial culture according to any of the items 1-25 and / or the composition according to any of item 26-35. 37. The coating composition according to item 36, the coating composition comprises a biopolymer promoting adherence to a plant seed.

[0559] 38. The coating composition according to any of items 36 or 37, the coating composition is formulated as an aqueous or oil-based solution for application to seeds, preferably aqueous.

[0560] 39. The coating composition according to any of items 36-38, the coating composition is formulated as a powder or granular formulation for application to seeds.

[0561] Plant seed

[0562] 40. A plant seed coated with the composition according to any of items 26-35 or coated with a coating composition according to any of items 36-39.

[0563] 41. The plant seed according to item 40, the plant seed is a dicotyledon, monocotyledon or a gymnosperm seed.

[0564] 42. The plant seed according to any of items 40 or 41, 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.

[0565] 43. The plant seed according to any of items 40-42, the plant seed being selected from the group consisting of a cover crop seed, such as fodder radish, clover, yellow mustard, or Phacelia.

[0566] 44. The plant seed according to any of items 40-43, the plant seed being selected from the group of trees, bushes, or grasses.

[0567] Use

[0568] 45. Use of a Pseudomonas 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.

[0569] 46. Use of a bacteria or biologically pure bacterial culture according to any of items 1-25, the composition according to any of items 26-35 or the coating composition according to any of items 36-39, 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.

[0570] 47. The use according to items 45 or 46, wherein the use as a plant growthpromoting agent is for:

[0571] • solubilizing inorganic minerals or salts, such as phosphate, and / or potassium, and / or iron, and / or aluminium, and / or zinc, such as zinc oxide, and / or manganese, and / or organic phosphate, such as phytate; and / or

[0572] • chelating inorganic minerals such as iron, zinc, manganese, copper; and / or

[0573] • allowing for growth under low soil nitrogen conditions; and / or

[0574] • fixating atmospheric nitrogen; and / or

[0575] • increasing plant growth, such as plant length, leaf diameter and / or root length; and / or

[0576] • improving germination of the seeds; and / or

[0577] • improving emergence of the plants; and / or

[0578] • increasing biomass; and / or

[0579] • increasing growth; and / or

[0580] • increasing crop yield; and / or

[0581] • inducing anti-fungal effects; and / or

[0582] • increasing harvest yield; and / or

[0583] • increasing hectoliter weight; and / or

[0584] • increasing protein content; and / or

[0585] • increasing protein yield; and / or

[0586] • increasing oil content; and / or

[0587] • increasing oil yield; and / or

[0588] • reducing mycotoxin contamination; and / or

[0589] • reducing seed-borne fungal or bacterial contamination; and / or

[0590] • 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.

[0591] 48. The use according to item 47, wherein the inorganic minerals or salts are selected from the group consisting of

[0592] • minerals, such as rock phosphate, potash, lime, clay, ground rocks, sand, silt, sediment and natural deposits;

[0593] • 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;

[0594] • 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.

[0595] 49. The use according to any of items 45-48, 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.

[0596] 50. The use according to any of items 45-49, the bacteria or biologically pure bacterial culture according to any of items 1-25, the composition according to any of items 26-35 or the coating composition according to any of items 36-39 is applied in an effective amount.

[0597] 51. The use according to any of items 45-50, 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.

[0598] 52. The use according to any of items 45-51, the plant is a dicotyledon, monocotyledon or a gymnosperm. 53. The use according to item 52, 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.

[0599] Method

[0600] 54. A method for stimulating plant growth comprising applying the bacteria or biologically pure bacterial culture according to any of items 1-25, the composition according to any of items 26-35 or the coating composition according to any of items 36-39 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.

[0601] 55. The method for stimulating plant growth according to item 54, the method comprises applying the bacteria or biologically pure bacterial culture according to any of items 1-25, the composition according to any of items 26-35 or the coating composition according to any of items 36-39:

[0602] - to a plant growth medium, such as sphagnum; and / or

[0603] - 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

[0604] - to plant leaves, roots, or stems; and / or

[0605] - to plant seeds, and / or

[0606] - to a watering system for the plants, such as hydroponics, aeroponics and / or aquaponics.

[0607] 56. The method for stimulating plant growth according to any of items 54 or 55, the bacteria or biologically pure bacterial culture according to any of items 1-25, the composition according to any of items 26-35 or the coating composition according to any of items 36-39 is sprayed or irrigated onto plants or fields.

[0608] Kit

[0609] 57. A kit of parts for stimulating plant growth comprising

[0610] • a first container comprising the bacteria or biologically pure bacterial culture according to any of items 1-25, the composition according to any of items 26-35 and / or the coating composition according to any of items 36- 39; and • instructions for applying the bacteria or biologically pure bacterial culture according to any of items 1-25, the composition according to any of items 26-35 and / or the coating composition according to any of items 36-39 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.

[0611] 58. The kit of parts according to item 57 further comprises one or more containers comprising fertilizers, nutrients, and / or other microorganisms.

[0612] Medical uses

[0613] 59. A bacteria or biologically pure bacterial culture according to any of items 1-25, a composition according to any of items 26-35 or a coating composition according to any of items 36-39 for use as a medicament.

[0614] 60. A bacteria or biologically pure bacterial culture according to any of items 1-25 or a composition according to any of items 26-35 use in the treatment, alleviation and / or prevention of fungal infections.

[0615] 61. A bacteria or biologically pure bacterial culture for use according to item 60 wherein the fungal infection is selected from the group consisting of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres, Zymoseptoria tritici, Fusarium oxysporum, Puccinia hordei, or a combination thereof.

[0616] Ores

[0617] 62. Use of the bacteria or biologically pure bacterial culture according to any of items 1-25 or the composition according to any of items 26-35 for solubilizing minerals in ores.

Claims

Claims1. A bacteria, preferably isolated, or biologically pure bacterial culture comprising a) a genomic sequence according to any of SEQ ID NOs: 5-17; 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: 5-17; and / or c) a fragment of one or more of the sequences of a) or b), having a length of at least 300 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: 5, 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: 5.

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: 6, 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:6.

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: 7, 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: 7.

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: 8, 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.

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: 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.

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: 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.

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: 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.

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: 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.

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: 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.

11. 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 atleast 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 14.

12. 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.

13. 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.

14. 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.

15. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising any of SEQ ID NO: 5-17, such as one or more of SEQ ID NO: 5-17, such as one or more of SEQ ID NO: 5-17, such as two or more of SEQ ID NO: 5-17, such as three or more of SEQ ID NO: 5-17, such as four or more of SEQ ID NO: 5-17, such as five or more of SEQ ID NO: 5-17, such as six or more of SEQ ID NO: 5-17, such as eight or more of SEQ ID NO: 5-17, such as nine or more of SEQ ID NO: 5-17, such as ten or more of SEQ ID NO: 5-17, such as eleven or more of SEQ ID NO: 5-17, such as twelve or more of SEQ ID NO: 5- 17.

16. The bacteria or biologically pure bacterial culture according to any of the preceding claims comprising any of SEQ ID NO: 5-17, such as one or more of SEQ ID NO: 5-17.

17. The bacteria or biologically pure bacterial culture according to any of the preceding claims, comprising a) a genomic sequence according to any of SEQ ID NO: 2-4 or 18; b) a genomic sequence having at least 90% sequence identity to any of SEQ ID NO: 2-4 or 18, 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: 2-4 or 18, 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 14000 nucleotides.

18. The bacteria or biologically pure bacterial culture according to any of the preceding claims, being a Pseudomonas, such as Pseudomonas wadenswilerensis , Pseudomonas donghuensis, Pseudomonas tructae, or Pseudomonas rubra.

19. A bacteria or biologically pure bacterial culture being Pseudomonas, DSM 34653 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.

20. 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.

21. The bacteria or biologically pure bacterial culture according to claim 20, wherein the one or more phytopathogenic fungi are selected from the group consisting of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres, Zymoseptoria tritici, Puccinia hordei, and Fusarium oxysporum.

22. The bacteria or biologically pure bacterial culture according to claim 20, wherein the solubilization of calcium phosphate, iron phosphate, and / or potassiumaluminum silicate at 5°C is at a level of at least 40% to, equal to and / or above a level of solubilization at 15°C and / or 25°C.

23. The bacteria or biologically pure bacterial culture according to any of claims 20-22, wherein the level of solubilization is determined by a solubilization index (SI) calculated by the formula:SI = AreaHaio / Areacoiony.

24. The bacteria or biologically pure bacterial culture according to claim 23, wherein the SI is at least 2, such as at least 4, such as between 2 and 10, preferably the SI is between 2 and 9.

25. The bacteria or biologically pure bacterial culture according to any of claims 23 or 24, wherein the SI depends on temperature after cultivation start and / or incubation time.

26. The bacteria or biologically pure bacterial culture according to claim 25, 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.

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

28. The bacteria or biologically pure bacterial culture according to any of the preceding claims, wherein the bacteria or biologically pure bacterial culture comprises a capacity for solubilizing insoluble zinc, such as zinc oxide (ZnO), such as at 15°C.

29. The bacteria or biologically pure bacterial culture according to any of the preceding claims, wherein the bacteria or biologically pure bacterial culture produces one or more secondary metabolites, such as hydrogen cyanide (HCN), siderophore, and / or indole-3-acetic acid (IAA).

30. Bacteria or biologically pure bacterial culture according to any of the preceding claims, wherein the bacteria or biologically pure bacterial culture comprises one or more genes, such as one or more gene clusters, encoding one or more secondary metabolites, such as hydrogen cyanide (HCN), siderophore, and / or indole-3-acetic acid (IAA).

31. 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 1-30.

32. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to claim 31, wherein the composition is a biofungicide.

33. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to any of claims 31 or 32, further comprising other microorganisms, such as other microorganisms able to function as a biostimulant.

34. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to any of claims 31-33, further comprises one or more agriculturally acceptable carriers.

35. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to claim 34, 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.

36. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to any of claims 31-35, the composition comprises a biopolymer, oligosaccharide, disaccharide or monosaccharide selected from the group consisting of pectin, alginate, chitosan, cellulose, a cellulosederivative, starch, maltodextrin, chitin, glucose, trehalose, sucrose, xanthan gum, guar gum, diutan gum, or a biopolymer derived from a natural source, possibly chemically modified afterwards.

37. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to any of claims 31-36, 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.

38. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to any of claims 31-37, the composition is formulated as a granular formulation or a powder formulation.

39. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to any of claims 31-38, 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.

40. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to any of claims 31-39, 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, ureaformaldehydes, 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, woodash, manure, biochar, sludge, green manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion, or a combination thereof.

41. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to claim 39, 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.

42. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to any of claims 31-41, wherein the composition is formulated as a foliar spray.

43. The fertilizer and / or inoculant and / or biostimulant and / or biofungicide and / or antimicrobial composition according to claim 42, wherein the foliar spray comprises one or more of agents selected from the group consisting of tween, lecithin, saponins, plant oils, xanthan, guar gum, cellulose, carboxymethyl cellulose, agar, alginate, diutan, gellan gum, tara gum, ethoxylated fatty alcohols or ethoxylated vegetable oils, alkyl polyglucosides, organosilicones, and polyalkykeneoxide modified heptamethyltrisiloxane. The agents increase the dispersion (surfactants), adherence to the plant surface, and / or protection.

44. A coating composition, preferably a seed coating composition, comprising the bacteria or biologically pure bacterial culture according to any of the claims 1-30 and / or the composition according to any of claims 31-43.

45. The coating composition according to claim 4, the coating composition comprises a biopolymer promoting adherence to a plant seed.

46. The coating composition according to any of claims 44 or 45, the coating composition is formulated as an aqueous or oil-based solution for application to seeds, preferably aqueous.

47. The coating composition according to any of claims 44-46, the coating composition is formulated as a powder or granular formulation for application to seeds.

48. The coating composition according to any of claims 44-47, wherein the coating composition is in the form of a foliar spray.

49. A plant seed coated with the composition according to any of claims 31-43 or coated with a coating composition according to any of claims 44-48.

50. The plant seed according to claim 49, the plant seed is a dicotyledon, monocotyledon or a gymnosperm seed.

51. The plant seed according to any of claims 49 or 50, 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.

52. The plant seed according to any of claims 49-51, the plant seed being selected from the group consisting of a cover crop seed, such as fodder radish, clover, yellow mustard, or Phacelia.

53. The plant seed according to any of claims 49-52, the plant seed being selected from the group of trees, bushes, or grasses.

54. A plant leaf coated with the composition according to any of claims 31-43 or coated with a coating composition according to any of claims 44-48.

55. Use of a Pseudomonas 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.

56. Use of a Pseudomonas bacteria as a biofungicide, 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. Use of a bacteria or biologically pure bacterial culture according to any of claims 1-30, the composition according to any of claims 31-43, or the coating composition according to any of claims 44-48, 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.

58. Use of an bacteria or biologically pure bacterial culture according to any of claims 1-30, the composition according to any of claims 31-43, or the coating composition according to any of claims 44-48, as a biofungicide, preferably at a temperature below 15°C, such as below 10°C, preferably between 2°C and 8°C, more preferably around 5°C.

59. The use according to claim 57, 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, such as zinc oxide, 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.

60. The use according to claim 59, 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.

61. The use according to any of claims 57-60, 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.

62. The use according to any of claims 57-61, the bacteria or biologically pure bacterial culture according to any of claims 1-30, the composition according toany of claims 31-43, or the coating composition according to any of claims 44-48 is applied in an effective amount.

63. The use according to any of claims 57-62, the bacteria or biologically pure bacterial culture according to any of claims 1-30, the composition according to any of claims 31-43 or the coating composition according to any of claims 44-48 is applied as a seed coat, as a foliar spray, or a combination hereof.

64. The use according to any of claims 57-63, 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.

65. The use according to any of claims 57-64, the plant is a dicotyledon, monocotyledon or a gymnosperm.

66. The use according to claim 65, 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.

67. A method for stimulating plant growth comprising applying the bacteria or biologically pure bacterial culture according to any of claims 1-30, the composition according to any of claims 31-43, or the coating composition according to any of claims 44-48 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.

68. The method for stimulating plant growth according to claim 67, the method comprises applying the bacteria or biologically pure bacterial culture according to any of claims 1-30, the composition according to any of claims 31-43, or the coating composition according to any of claims 44-48:- 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 / orto a watering system for the plants, such as hydroponics, aeroponics and / or aquaponics.

69. The method for stimulating plant growth according to any of claims 67 or 68, the bacteria or biologically pure bacterial culture according to any of claims 1-30, the composition according to any of claims 31-43, or the coating composition according to any of claims 44-48 is sprayed or irrigated onto plants or fields.

70. 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 1-30, the composition according to any of claims 31-43, and / or the coating composition according to any of claims 44-48; and• instructions for applying the bacteria or biologically pure bacterial culture according to any of claims 1-30, the composition according to any of claims 31-43, and / or the coating composition according to any of claims 44-48 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.

71. The kit of parts according to claim 70 further comprises one or more containers comprising fertilizers, nutrients, and / or other microorganisms.

72. A bacteria or biologically pure bacterial culture according to any of claims 1- 30, a composition according to any of claims 31-43, or a coating composition according to any of claims 44-48 for use as a medicament.

73. A bacteria or biologically pure bacterial culture according to any of claims 1-30 or a composition according to any of claims 31-43 use in the treatment, alleviation and / or prevention of fungal infections.

74. The bacteria or biologically pure bacterial culture for use according to claim 73 wherein the fungal infection is selected from the group consisting of Gaeumannomyces graminis var. tritici, Pyrenophora teres f. teres, Zymoseptoria tritici, Fusarium oxysporum, Puccinia hordei, or a combination thereof.

75. Use of the bacteria or biologically pure bacterial culture according to any of claims 1-30 or the composition according to any of claims 31-43 for solubilizing minerals in ores.

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