WO2024104961A1 - Novel pseudomonas strain - Google Patents

Novel pseudomonas strain Download PDF

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Publication number
WO2024104961A1
WO2024104961A1 PCT/EP2023/081608 EP2023081608W WO2024104961A1 WO 2024104961 A1 WO2024104961 A1 WO 2024104961A1 EP 2023081608 W EP2023081608 W EP 2023081608W WO 2024104961 A1 WO2024104961 A1 WO 2024104961A1
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Prior art keywords
bacteria
plant
vip
biologically pure
bacterial culture
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PCT/EP2023/081608
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French (fr)
Inventor
Morten Østergaard ANDERSEN
Julia Magdalena Østergaard ANDERSEN
Cecilie Toft VANGSØE
Elvira VAN HAUWAERT
Rasmus Christian Andersen
Zahra SALIMI
Signe Hagelsø SCHULTZ
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Bioomix Aps
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Publication of WO2024104961A1 publication Critical patent/WO2024104961A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

Definitions

  • the present invention relates to isolated bacteria or biologically pure bacterial culture of the species Pseudomonas protegens.
  • 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.
  • Agrochemicals such as fertilizers and pesticides
  • fertilizers and pesticides are the primary means by which agricultural productivity is increased currently.
  • the widespread use of these chemicals has negative consequences for our environment and climate. 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.
  • 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.
  • beneficial microorganisms may promote plant growth or survival.
  • beneficial bacteria may help the plant access strongly bound nutrients in the soil that would otherwise not be plant-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 or interior of plants and seeds such as their leaves, stem 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.
  • bacteria that are beneficial to plants may also have applications in other fields than agriculture such as in horticulture, vertical farming, green houses, gardens, landscaping, reforestation, carbon capture and storage and in the restoration of degraded areas.
  • bacteria that are capable of solubilizing nutrient containing minerals or of inhibiting pathogenic microorganisms 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.
  • WO 2020/214843 Al discloses a method of incorporating 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.
  • the inventing team has isolated, identified, characterized and propagated a newly identified strain of bacteria, namely the Pseudomonas protegens strain VIP deposited with deposit number DSM 34378 (otherwise indicated as VIP).
  • 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.
  • 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 desert land. It may also have applications in reforestation or revegetation and in carbon capture/storage based on growing biomass.
  • 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, animals and humans. This may be done by placing the strain on the surface of the body, by placing it in a cavity in the body or by injecting it into the body of the recipient plant, animal or human.
  • the strain may also be used as a probiotic for ingestion. Specifically, the strain may be used to treat extended spectrum beta-lactamase (ESBL)-producing bacteria, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin- resistant enterococci (VRE) infections in animals and humans.
  • ESBL beta-lactamase
  • MRSA Methicillin-resistant Staphylococcus aureus
  • VRE vancomycin- resistant enterococci
  • the strain may also be used to dissolve otherwise poorly soluble substances such as minerals and salts.
  • 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.
  • the strain may also be used to dissolve otherwise poorly soluble substances such as minerals and salts for applications outside plants. It may be used to bioleach ores or waste materials. It may therefore by used in mining, recycling, or urban mining.
  • Example 1 shows how the VIP strain has been identified and isolated.
  • Example 2 shows, using bioinformatic tools, that the VIP strain has a unique genomic signature and therefore is considered a newly identified strain.
  • Example 3 shows that the VIP strain can increase the availability of common plants nutrients.
  • Example 4 demonstrates the stimulatory effects of a VIP seed coat on early plant growth in vitro in two typical agricultural crops, namely winter wheat and oilseed rape.
  • Example 5 shows how the VIP strain positively alters plant properties in the field.
  • Example 6 shows that the VIP strain may also be relevant for medical purposes.
  • Example 7 demonstrates that VIP can enhance the emergence of crops as diverse as rapeseed (belonging to the plant family Brassicaceae, a dicot) and winter wheat (belonging to the plant family Poaceae, a monocot).
  • VIP is also capable of increasing the biomass of a crop and therefore the rate of photosynthesis, carbon capture and carbon storage in biomass.
  • Example 8 shows the ability of VIP to affect the growth of various spring crops. Treating seeds, including grain and legume crops, with a bacterial solution containing VIP before sowing demonstrates that VIP can enhance the emergence of crops in the field and lead to a higher harvest yield.
  • Example 9 shows the ability of VIP to affect the yield of vegetables such as onions, potatoes and maize. Treating onion sets, seed potatoes and maize seeds with a bacterial solution containing VIP before sowing in fields demonstrates that VIP can affect the harvest yield of vegetables as diverse as onions, potatoes and maize by a minimum of 18%.
  • Example 10 shows the ability of VIP to increase the availability of common plant nutrients, such as nitrogen, phosphate and potassium, specifically under low, medium, and high temperature conditions. This demonstrates that VIP 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.
  • common plant nutrients such as nitrogen, phosphate and potassium
  • an object of the present invention relates to the provision of novel bacterial strains/biostimulants for improving plant growth.
  • 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: 1-7; b) a genomic sequence having at least 84% sequence identity to any of SEQ ID NOs: 1-7, preferably such as at least 90%, more preferably 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: 1-7; and/or c) a fragment of one or more of the sequences of a) or b), having a length of at least 500 nucleotides.
  • Another aspect of the present invention relates to a fertilizer and/or inoculant and/or biostimulant composition comprising the bacteria or biologically pure bacterial culture according to the invention.
  • Yet another aspect of the present invention is to provide 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.
  • Still another aspect of the present invention is to provide a plant seed coated with the composition according to the invention or coated with a coating composition according to the invention.
  • An aspect of the invention relates to the use of 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, as a plant growth promoting agent, such as a fertilizer or inoculum or biostimulant.
  • the 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 forrow, soil and/or plant growth medium.
  • the 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;
  • an aspect of the invention relates to 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 for use as a medicament.
  • the bacteria according to the invention may have anti-bacterial properties. It may also be used to promote animal or human health, such as by dissolving or freeing nutrients.
  • a further aspect of the invention relates to the use of the bacteria or biologically pure bacterial culture according to the invention or the composition according to the invention for solubilizing minerals in ores.
  • Figure 1 shows initial screening of retrieved capture pills from the microbeTRAP on agar plated with VRE.
  • Figure 2 shows de novo genome assembly of VIP. Scaffolds of VIP genome assembly (arrow bars) depicted along with BLAST results against the type strain of Pseudomonas protegens CHAO (top bars). White areas in the bars at the top indicate no sequence overlap between VIP and the type strain. A gradient from grey to black indicates percent identity, where black represents 100% sequence similarity. Note: The assembly has been divided into two overlapping sections.
  • Figure 3 shows bacterial growth and solubilization halo of VIP, Serenade, and FloraGro on solid nitrogen-free medium (top row) or medium with insoluble phosphate source (calcium phosphate) (bottom row) at day 4, 8°C.
  • Figure 4 shows bacterial growth and solubilization halo of VIP, Serenade, and FloraGro on solid nitrogen-free medium (top row) or medium with insoluble phosphate source (calcium phosphate) (bottom row) at day 4, 8°C.
  • Figure 4 shows bacterial growth and solubilization halo of VIP, Serenade, and FloraGro on solid nitrogen-free medium (top row) or medium with insoluble phosphate source (calcium phosphate) (bottom row) at day 4, 8°C.
  • Figure 4 shows A) Colony area of VIP, Serenade, and FloraGro when grown on nitrogen-free medium; and B) Colony area and C) halo area of VIP, Serenade, and FloraGro when grown on medium with insoluble phosphate source (calcium phosphate) at day 4, 8°C.
  • Figure 5 shows bacterial growth and solubilization halo of VIP and FloraGro on solid medium with insoluble potassium source (potassium alumino silicate) at day 15, 8°C.
  • Figure 6 shows A) colony area and B) halo area of VIP and FloraGro when grown on a medium containing insoluble potassium (potassium alumino silicate) at day 15, 8°C.
  • Figure 7 shows early germination performance of VIP seed coat in crops in vitro.
  • Figure 8 shows in vitro solubilization of inorganic phosphate using VIP seed coat. Uncoated (control) and VIP-coated (VIP seed coat) seeds from oilseed rape and winter wheat crops, respectively, on top of Pikowskaya medium. Pictures of petri dishes were taken two days following coating and plating and show significant clearing zones formed around VIP-coated seeds (exemplified by white arrow heads).
  • Figure 9 shows in vitro solubilization of inorganic phosphate using VIP seed coat. Uncoated (control) and VIP-coated (VIP seed coat) seeds from oilseed rape and winter wheat crops, respectively, on top of Pikowskaya medium. Pictures of petri dishes were taken two days following coating and plating and show significant clearing zones formed around VIP-coated seeds (exemplified by white arrow heads).
  • Figure 9 shows in vitro solubilization of inorganic phosphate using VIP seed coat. Uncoated (control) and VIP-coated (VIP seed coat) seeds from oilseed rape and winter wheat crops
  • Figure 9 shows anti-fungi properties of VIP seed coat in vitro. Uncoated (control) and VIP-coated (VIP seed coat) seeds from oilseed rape and winter wheat crops, respectively, on top of Pikowskaya medium. Pictures of petri dishes were taken four (oilseed rape) and five (winter wheat) days following plating. Black arrowheads indicate fungal contamination. White arrowheads indicate clearing zones formed around seeds as a result of phosphate solubilization.
  • Figure 10 shows the effect of VIP on early and late emergence of spring crop plants in the field.
  • VKST independent third-party contract research organization
  • Field areas were fertilized with either a combination of nitrogen-sulfur (NS; 30% nitrogen, 10% sulfur) + phosphate-potassium-sulfur (PKS; 10% phosphate, 20% potassium, 3% nitrogen) fertilizers (C and F) or a recycled organic fertilizer (0GRO; 10% nitrogen 3% phosphate, 1% potassium) (D and E) at the day before sowing (faba bean and pea fields were not fertilized (A and B)). Fertilizers were applied at two rates; 100% and 75%. Asterisks indicate statistical significance (p ⁇ 0.05) of VIP seed coat vs control within early emergence, late emergence and fertilizer groups using a one-sided t-test.
  • Figure 11 shows the effect of VIP on onion, potato and maize yields from fields.
  • Figure 12 shows in vitro mineral-dissolving properties of VIP and commercially available biostimulants at low, medium and high temperatures.
  • 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.
  • inoculant as described in this invention is defined in several Federal, or State regulations as:
  • 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);
  • 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.
  • plant biostimulants also designate commercial products containing mixtures of such substances and/or microorganisms.
  • 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.
  • 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 number of bacteria in the culture.
  • the bacteria according to the invention is an isolated bacteria, such as forming part of an isolated composition.
  • Plant growth promoting agent Plant growth promoting agent
  • 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.
  • sequence identity indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length or between two nucleic acid sequences of substantially equal length.
  • the two sequences to be compared must be aligned to best possible fit with the insertion of gaps or alternatively, truncation at the ends of the protein sequences.
  • sequence identity can be calculated as , wherein
  • Ndif is the total number of non-identical residues in the two sequences when aligned and wherein N re f is the number of residues in one of the sequences.
  • Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program for protein alignment (W.R Pearson and D . Lipman (1988)).
  • 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.
  • the polypeptide fragment and the segment of the longer amino acid sequence may be of equal length.
  • 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%.
  • Hectoliter weight relates to the harvest yield, which is also provided as kilogram (kg) per 100 liter (L) or kg per hectoliter (kg/hL).
  • the inventing team has identified, isolated and propagated a newly identified strain of bacteria, namely the Pseudomonas protegens strain VIP deposited with deposit number DSM 34378 (otherwise indicated as VIP).
  • the strain may be used to promote a wide variety of plant health parameters.
  • 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: 1-7; b) a genomic sequence having at least 84% sequence identity to any of SEQ ID NOs: 1-7, preferably at least 90%, such as at least 95%, more preferably 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: 1-7; and/or c) a fragment of one or more of the sequences of a) or b), having a length of at least 500 nucleotides.
  • the bacteria is an isolated bacteria.
  • the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 1, 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: 1.
  • the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 97% sequence identity to SEQ ID NO: 2, 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: 2.
  • the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 85% sequence identity to SEQ ID NO: 3, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 3.
  • the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 95% sequence identity to SEQ ID NO: 4, 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: 4.
  • the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 90% sequence identity to SEQ ID NO: 5, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 5.
  • the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 90% sequence identity to SEQ ID NO: 6, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 6.
  • 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.
  • the bacteria or biologically pure bacterial culture according to the invention comprises any of SEQ ID NO's: 1-7, such as one or more of SEQ ID NO's: 1-7.
  • 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, or preferably such as all of SEQ ID NO: 1-7, or with a sequence identity for SEQ ID NO: 1-7 as defined above.
  • SEQ ID No's: 1-5 forms part of SEQ ID NO: 8, and have been identified to comprise reading frames with low sequence identity to other known proteincoding genes as also illustrated in example 2.
  • SEQ ID NO's: 6 and 7 lies outside SEQ ID NO: 8 and have also been identified to comprise open reading frames.
  • the bacteria or biologically pure bacterial culture according to the invention comprises a) a genomic sequence according to SEQ ID NO: 8; b) a genomic sequence having at least 90% sequence identity to SEQ ID NO: 6, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 8; and/or c) a fragment of the sequence of a) or b), having a length of at least 5000 nucleotides, such as at least 8000 nucleotides, such as at least 10000 nucleotides, such as at least 14000 nucleotides.
  • the VIP strain according to the invention has been annotated as being a Pseudomonas protegens species.
  • the bacteria or biologically pure bacterial culture is of the genus Pseudomonas, such as species Pseudomonas protegens.
  • the bacteria or biologically pure bacterial culture comprises a 16S rRNA gene encoded by SEQ ID NO: 9.
  • the strain of the invention has been identified to comprise a 16S rRNA gene encoded by SEQ ID NO: 9.
  • the isolated bacteria or biologically pure bacterial culture being Pseudomonas protegens, DSM 34378 deposited with the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) on 15 September 2022]. In here this strain is also named VIP.
  • the invention relates to an (isolated) bacteria or biologically pure bacterial culture being Pseudomonas protegens, DSM 34378 deposited with the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) on 15 September 2022].
  • the bacteria or biologically pure bacterial culture according to the invention is
  • the bacteria or biologically pure bacterial culture is dehydrated, such as spray-dried.
  • 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 involved in producing carcinogenic secondary metabolites.
  • the isolated bacteria or biologically pure bacterial culture according to the invention may form part of different compositions.
  • an aspect of the invention relates to a fertilizer and/or inoculant and/or biostimulant and/or biofungicide and/or antimicrobial composition comprising the bacteria or biologically pure bacterial culture according to the invention.
  • the composition may comprise other microorganisms.
  • the fertilizer and/or inoculant and/or biostimulant composition further comprises other microorganisms, such as other microorganisms able to function as a biostimulant.
  • the composition further comprises one or more agriculturally acceptable carriers.
  • 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 cryoprotectant, a drying additive, a preservative, a polymer, biopolymer, a coating, or a combination thereof.
  • 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 or a biopolymer derived from a natural source, possibly chemically modified afterwards.
  • 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 or a biopolymer derived from a natural source, possibly chemically modified afterwards.
  • 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.
  • the composition is formulated as a granular formulation or a powder formulation.
  • the composition further comprises a fertilizer, a micronutrient fertilizer material, an insecticide, a herbicide, a plant growth amendment, a fungicide, a molluscicide, an algicide, abacterial inoculant, a fungal inoculant, or a combination thereof.
  • 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 ammonimonium
  • 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.
  • composition according to the invention may be a coating composition, such as a seed coating composition.
  • 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.
  • the coating composition comprises a biopolymer promoting adherent to a plant seed.
  • a biopolymer promoting adherent to a plant seed As shown in Example 3, pectin has been tested as biopolymers promoting adherence to plant seeds.
  • the coating composition is formulated as an aqueous or oilbased solution for application to seeds, preferably aqueous.
  • the coating composition is formulated as a powder or granular formulation for application to seeds.
  • the present invention also relates to seeds coated with the compositions according to the invention.
  • 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.
  • the plant seed is a dicotyledon, monocotyledon or a gymnosperm seed.
  • 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, strawberries, peas, potatoes, onions, carrots and sugar beets.
  • a crop seed such as barley seed, such as spring or winter barley
  • Oilseed such as rapeseed
  • wheat such as winter or spring wheat.
  • the plant seed being selected from the group consisting of a cover crop seed, such as fodder radish, clover, yellow mustard or Phacelia.
  • the plant seed being selected from the group of trees, bushes or grasses.
  • 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.
  • the use as a plant growth-promoting agent is for:
  • 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.
  • 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.
  • 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.
  • stimulation of plant growth can also be estimated using other parameters.
  • the inorganic minerals or salts solubilized 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, 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.
  • the isolated bacteria or biologically pure bacterial culture can grow under low temperature conditions.
  • 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.
  • the use takes place at field temperatures in the range 2 to 8°C. It is shown in the examples, such as examples 3, 5, and 10, the isolated bacteria or biologically pure bacterial culture are used and grown at temperatures at 8°C and below.
  • 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.
  • the use takes place in Scandinavia, such as Norway, Sweden, Finland or Denmark, such as in Jutland, Fyn, and/or Zealand.
  • the plant is a dicotyledon, monocotyledon or a gymnosperm.
  • 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, and vegetables, such as onion.
  • a crop seed such as barley seed, such as spring barley
  • Oilseed such as rapeseed
  • wheat such as winter wheat
  • vegetables such as onion.
  • the (isolated) bacteria or biologically pure bacterial culture according to the invention have been used on the above-mentioned plant varieties and demonstrates increased growth and yield.
  • 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,
  • 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
  • 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,
  • 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, Ha
  • 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.
  • 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 forrow, soil and/or plant growth medium.
  • 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:
  • a plant growth medium such as sphagnum
  • 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.
  • the invention relates to a kit of parts for stimulating plant growth comprising
  • 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;
  • the kit of parts further comprises one or more containers comprising fertilizers, nutrients, and/or other microorganisms.
  • the invention may also have medical uses.
  • an aspect of the invention relates to 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 for use as a medicament.
  • the bacteria according to the invention may have anti-bacterial properties. It is shown in Example 6 that VIP inhibits a diverse selection of multi-resistant bacteria that cause diseases.
  • VIP may therefore find use as a probiotic, a cosmetic ingredient, a food additive, a feed additive, a veterinary medicine, a biocide, an antibiotic, a pharmaceutical, or a biotechnological product.
  • an aspect relates to the (isolated) bacteria or biologically pure bacterial culture according to the invention or composition according to the invention for use in the treatment, alleviation and/or prevention of bacterial infections, such as E. coli, spectrum beta-lactamase (ESBL)-producing bacteria, Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).
  • bacterial infections such as E. coli, spectrum beta-lactamase (ESBL)-producing bacteria, Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).
  • a further aspect of the invention relates to the 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.
  • 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.
  • Example 1 Isolation of the strain and assessment of its anti-bacterial properties
  • Tricalcium phosphate (TCP), myristic acid (MA) and LB medium was from Sigma Aldrich.
  • the microbeTRAP was 3D printed in polyamide (PA12) by Materialize.
  • MRSA, ESBL and VRE were donated by the Department of Clinical Microbiology at Odense University Hospital.
  • Strain VIP was isolated from the soil of an old beech wood in October 2019 (coordinates: 55°21'57.6"N 10°25'55.2"E) using a microbeTRAP [W02021/180941 Al].
  • the microbeTRAP was 3D printed from PA (SLS, Materialize) with the overall dimensions of a microtiterplate, it contained 16 central chambers that contained a nutrient pills, each of these chambers were connected to 4 separate outer chambers that contained a capture pills. Nutrient and capture pills were made as previously described. Nutrient pills were made by compressing LB medium into a pellet using a pill press. Capture pills were made by first suspending 25g TCP in 6g MA that had been melted to 70°C on a hot-plate, the suspension was then cast to pills using a silicone mold and the pills were then carbonized for 1 hours at 400°C and sintered for 2 hours at 1100°C resulting in porous ceramic cylinders.
  • Nutrient pills and capture pills were placed in the microbeTRAP and it was sealed with microtiter plate seal.
  • Forest soil was collected from an old beech forest in Odense, Denmark (coordinates 55°21'57.6"N 10°25'55.2"E) in October 2020.
  • the soil was placed in a biosafety plastic box, the microbeTRAP was placed in the middle of the soil and the soil was added IL of sterile water to create a moist environment that would promote nutrient exchange and chemotaxis.
  • the soil box was then incubated for 5 days at 37°C.
  • the microbeTRAP was excavated and the capture pills retrieved. These were placed on agar plates covered with either vancomycin resistant enterococci (VRE) or extended spectrum beta-lactamase (ESBL) expressing E. coli. After 24 hours of incubation at 37°C, the plates were placed at 4°C for 5 days. They were then investigated for clearing zones surrounding the capture pills after which they were incubated for 24 hours more at 37°C followed by 3 days at 4C before being investigated for clearing zones again.
  • VRE vancomycin resistant enterococci
  • ESBL extended spectrum beta-lactamase
  • the microorganisms were then spread on 5% blood agar and CSPA agar plates. These plates were incubated at 37°C and the colonies that developed were further isolated on subsequent plates. The anti-microbial properties of these colonies were then assessed using the "spot on lawn” technique.
  • the colonies were spotted onto Mueller-Hinton or 5% blood agar plates containing a lawn of either VRE, MRSA, ESBL or C. albicans. The plates were incubated for 24 hours at 37°C.
  • Colonies that successfully displayed an inhibition zone were reisolated from the inhibition zone onto fresh 5% blood agar plates. There the microorganism responsible for the inhibition was identified using MALDI-TOF mass spectrometry (Biotyper, Bruker). Another round of inhibition zone assessment was conducted to confirm their inhibitory ability, in this round 2/3 strains failed to recreate the inhibition zone. The remaining inhibiting colony was then re-cultured on CSPA agar, its identity reconfirmed using mass spectrometry and finally DNA was extracted from the colonies using a kit (Qiagen). The DNA was sent to sequencing at Novogene and a partial genome assembly was carried out. The strain was tested for antibiotics susceptibility against a panel of compounds commonly used to treat infections by bacteria in the Pseudomonas genus at Odense University Hospital.
  • microbeTRAP technology enabled the capture of an anti-bacterial microorganism from Danish forest soil that was later identified to be a new Pseudomonas protegens strain (see Example 2).
  • the strain proved capable of inhibiting ESBL E. coli, MRSA and VRE and was susceptible to many antibiotics.
  • Example 2 - VIP is a novel plant-protecting Pseudomonas protegens strain
  • VIP is a novel bacterial strain belonging to the plant-protecting Pseudomonas protegens species.
  • DNA was extracted from a bacterial culture of VIP using the DNease UltraClean Microbial Kit from Qiagen following the instructions from the manufacturer. 50 ng of bacterial 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. DNA libraries were sequenced on an Illumina MiniSeq machine. A total of 4,681,350 reads were obtained, corresponding to a genome coverage of lOOx (compared to the reference genome size of Pseudomonas protegens CHAO (Table 1)).
  • Adapter contaminants from the library preparation step as well as nucleotides with a Phred score ⁇ 20 were removed from raw sequencing reads using Trim Galore (vO.6.7). Only trimmed reads were used for downstream analyses.
  • the metagenomic classification tool KrakenUniq (vl.0.1) was used for taxonomic assignment of trimmed sequencing reads according to the National Center for Biotechnology Information (NCBI) Taxonomy.
  • KrakenUniq utilizes an advanced k- mer-based approach where short DNA sequences of k length are matched to a nucleotide reference database.
  • a pre-built database containing all current (as of 6/16/2022) and completely assembled bacterial, archaeal, viral and human RefSeq genomes as well as vector sequences (downloaded from https://benlangmead.github.io/aws-indexes/k2) was used.
  • Trimmed sequencing reads were aligned against three Pseudomonas protegens genomes (Table 1) using the burrows-wheeler aligner (BWA) (vO.7.17) with the BWA-MEM algorithm.
  • Pseudomonas protegens genomes were downloaded from NCBI (Table 1).
  • Table 1 Pseudomonas protegens reference genomes.
  • SPAdes (v3.15.5) was used for assembling trimmed sequencing reads into de novo contigs that were subsequently joined into putative scaffolds.
  • a read errorcorrection step (BayesHammer module) was performed prior to the genome assembly step to minimize the number of mismatches in the resulting contigs.
  • QUAST (v5.0.2) was used for computing assembly quality metrics and for comparing the de novo assembly against Pseudomonas protegens reference genomes (Table 1).
  • Short (609-1,266 nucleotides) and long (16,813 nucleotides) DNA sequences extracted from the genome assembly are annotated as SEQ ID NOs: 1-8.
  • ANI Average nucleotide identity
  • the DNA sequence of the 16S ribosomal RNA (rRNA) gene from the VIP strain was extracted from the assembled genome with ContEstl6S (available at www.ezbiocloud.net/tools/contestl6s) and compared to bacterial type strains using the Basic Local Alignment Search Tool (BLAST) (v.2.12.0).
  • BLAST Basic Local Alignment Search Tool
  • a pre-built database of 16S ribosomal RNA sequences from bacteria and archaea type strains was downloaded from NCBI and used as the reference database for BLAST.
  • the identified 16S ribosomal RNA sequence is encoded by SEQ ID NO: 9 (1,539 nucleotides).
  • Protein function of the translated sequence of SEQ ID NOs: 1-7 was predicted using BLAST protein alignment.
  • the UniProtKB reference proteomes and Swiss- Prot databases were used as reference databases.
  • the reference genome of Pseudomonas protegens belongs to the type strain CHAO (Table 1) for which a full-genome sequence is available. Comparing VIP sequencing reads to the whole genome of the reference genome as well as to fullgenomes of two closely related Pseudomonas protegens strains, namely Cab57 and Pf-5 (Table 1), revealed ⁇ 95% sequence similarity (Table 2). This suggests that VIP might be a novel Pseudomonas protegens strain.
  • Table 2 Percent sequence identity between VIP sequencing reads and Pseudomonas protegens reference genomes.
  • VIP sequencing reads were assembled into a de novo genome. Sequencing reads were assembled into larger fragments of DNA sequences (contigs) that were subsequently assembled and ordered into longer scaffolds. A total length of 7,078,551 nucleotides were obtained for the assembled genome of VIP with the longest scaffold representing 1,072,950 nucleotides of the genome ( Figure 2). 99.6% of the genome assembly is based on scaffolds of size 500 nucleotides, suggesting that the genome of VIP is bigger than the genome size of the type strain (Table 1).
  • Table 3 Percent sequence identity between VIP genome assembly and Pseudomonas protegens reference genomes.
  • 16S ribosomal RNA sequences (16S rRNA, ⁇ l,500 bp) 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. Extracting the DNA sequence encoding the 16S rRNA gene of the assembled genome of VIP (1,539 nucleotides, SEQ ID NO: 9) and comparing that to the NCBI 16S ribosomal database revealed 99% similarity to the type strain of Pseudomonas protegens (CHAO). Although the 16S rRNA region contains hypervariable regions that can be used for taxonomic discrimination, the analysis of 16S rRNA alone ignores the genome-wide variability.
  • ANI Average Nucleotide Identity
  • Table 1 Average Nucleotide Identity (ANI) scores of VIP genome assembly compared to Pseudomonas protegens reference genomes.
  • SEQ ID NO's: 1--5 Five random regions containing such open reading frames (SEQ ID NO's: 1-5) were extracted. Two additional and randomly selected genomic regions located outside of SEQ ID NO: 8 containing open reading frames were further included (SEQ ID NO's: 6-7). Comparing each of these sequences to the nucleotide database at NCBI using BLAST revealed sequence identities ⁇ 95.09% (Table 5), suggesting that SEQ ID NOs: 1-7 are highly selective for VIP. Insight into protein function may be obtained through protein sequence alignment. Subjecting SEQ ID NOs: 1-7 to a protein similarity search using the protein databases of UniProtKB and Swiss-Prot indicated that SEQ ID NOs: 1-7 may encode DNA-binding proteins, e.g., transcription factors. Although prediction scores were low ( ⁇ 50% similarity), some of the identified hypothetical proteins could be of regulatory importance for VIP.
  • Table 5 Maximum percent sequence coverage and sequence identity of SEQ ID NOs: 1-7 compared to the nucleotide database of NCBI using BLAST.
  • VIP Whole genome sequencing and analysis of the bacterial component of the VIP seed coat (here referred to as VIP) revealed firstly that VIP belongs to the plantprotecting Pseudomonas protegens species.
  • DNA sequences containing open reading frames and that have high specificity for VIP were identified; one long genomic region (16,813 nucleotides, SEQ ID NO: 8) wherein short DNA sequences (609-1,266 nucleotides) were extracted (SEQ ID NOs: 1-5) as well as two short DNA sequences identified outside of the long genomic region (SEQ ID NOs: 6-7). Combined or alone, these DNA sequences may be used to identify VIP at the DNA sequence level.
  • the aim is to demonstrate the ability of VIP to increase the availability of common plant nutrients otherwise supplemented as chemical fertilizer (nitrogen, phosphorous, and potassium).
  • a fluid culture of each bacterial strain was prepared in a concentration of 0.5 McFarland. The solution was then spotted onto plates containing the different media in repetitions of three. The plates were incubated at 8°C. This temperature was chosen to mimic the conditions during spring seeding in northern Europe. Growth and halo zone area were measured each day using imageJ.
  • the ability of VIP to increase the availability of nitrogen, phosphorous, and potassium was evaluated using plate assay and measuring growth and/or halo zone.
  • the results of the plate assay on media free of nitrogen or with an insoluble phosphate source are shown in Figure 3 and 4. Growth on nitrogen-free medium at day 4 was only observed with VIP and FloraGro, where the colony area was 28% larger with VIP ( Figure 3 (top) and 4A). As this medium is free of nitrogen, increased growth indicates greater nitrogen fixing. Nitrogen is essential to plant growth and present in limited amount in soil. Therefore, nitrogen fixing bacteria play and important role in promoting plant growth and development.
  • biofertilizer can increase the availability of essential nutrients to plants otherwise administered as chemical fertilizer. These results indicate that the novel biofertilizer VIP can fix nitrogen and solubilize insoluble phosphorous and potassium. These beneficial properties are evident even at low temperatures (8°C), ensuring functionality also during spring seeding in northern Europe unlike most competitors.
  • Example 4 Stimulatory traits of VIP seed coat on early plant growth
  • the scope of this example is to demonstrate the stimulatory effects of a VIP seed coat on early plant growth in vitro in two typical agricultural crops, namely winter wheat and oilseed rape.
  • Oilseed rape 2 McFarland standards of VIP were diluted in lx PBS + pectin (final concentration of 1.5% (by volume)). 1 g of oilseed rape seeds were coated with 3 mL of VIP-pectin solution.
  • the capacity of VIP seed coat to solubilize inorganic phosphate was determined by plating coated seeds from oilseed rape and winter wheat crops on Pikowskaya medium. Oilseed rape and winter wheat seeds were coated with VIP seed coat, as previously described, and immediately plated on Pikowskaya medium in 9.5 cm petri dishes (2 replicates, each containing 5 seeds). Petri dishes were kept in incubators at 24°C. The formation of clearing zones around plated seeds was examined at two days following seed plating. Fungal contamination was further evaluated at 4-5 days after plating of seeds on Pikowskaya medium.
  • Figure 8 shows the potential of VIP seed coat to solubilize inorganic phosphate. Significant clearing zones were observed around VIP-coated oilseed rape and winter wheat seeds at two days following coating and plating on Pikowskaya medium ( Figure 8, white arrowheads). Minor clearing zones were also observed around control winter wheat seeds, indicating that phosphate-solubilizing microbes are naturally present on winter wheat seeds. Clearing zones do however increase significantly in size around seeds that have been coated with VIP seed coat, suggesting that VIP seed coat has potential to improve phosphate availability.
  • FIG. 9 shows a significant growth of fungi in control (uncoated) seeds, especially in winter wheat seeds ( Figure 9, black arrowheads). Coating seeds with VIP seed coat resulted in an apparent decrease in fungal contamination. This suggests that VIP seed coat does not only have phosphate-solubilizing properties, but may also act as an antifungal.
  • VIP seed coat has multiple beneficial effects on early plant growth of agricultural crops in vitro. Coating seeds with VIP seed coat stimulates early germination processes by increasing the emergence of radicle and plumule, increases phosphate solubility in the vicinity of seeds, and results in less fungal contamination. Collectively, these data demonstrate the plant growth-promoting properties of VIP seed coat.
  • strain VIP may alter plant properties in the field.
  • FloraGro was bought from BioPlant whereas Serenade ASO was bought from Brdr Ewers. Both FloraGro and Serenade ASO contain pure cultures of Bacillus bacteria. FloraGro is a biostimulant.
  • Serenade ASO is a fungicide i tool designed to protect against the effects of soil and foliar bacterial and fungal diseases.
  • Seed treatments were carried out by applying lOOmL VIP or FloraGro onto 100kg seeds. The seeds where then airdried and sown on the 27 April 2021 on a field in Denmark with 4 replicates of each treatment. The daily mean temperature at sowing was 8°C.
  • the seed treatment fields were fertilized with NPK at a rate of 120kg N/ha and the fields were grown conventionally. The fields were harvested on the 15 august 2021.
  • Spray treatments were on fields that were sown on the 27 April 2021 on a field in Denmark with 4 replicates of each treatment.
  • the daily mean temperature at sowing was 8°C.
  • the fields were fertilized with NPK at a rate of 120kg N/ha and the fields were grown conventionally.
  • the products were used at a rate of 4L/ha and the products were diluted 1: 50 in water before application.
  • the sprayings were carried out on 10, 14 and 24 May with daily mean temperatures of 15°C, 14°C and 20°C, respectively. Spraying was carried out with a spray height of 50cm, a pressure of 2.4 bar, a nozzle size of 110 micrometers and a nozzle spacing of 50cm.
  • the fields were harvested on the 15 august 2021.
  • Average harvest parameters for the VIP treated plots were (yield 10.25 tons/ha, hectoliter weight 74.10 kg/hl, protein content 10.55%, total protein yield 1.08 tons/ha).
  • the non-treated control group resulted in the following average harvest parameters (yield 9.66 tons/ha, hectoliter weight 73.88 kg/hl, protein content 10.43%, total protein yield 1.01 tons/ha), while the FloraGro treated benchmark group gave the following parameters (yield 9.91 tons/ha, hectoliter weight 73.44 kg/hl, protein content 10.38%, total protein yield 1.03 tons/ha).
  • Hectoliterweight is also known as the test weight.
  • VIP as a seed treatment and/or a spray treatment may increase emergence, harvest yield, hectoliter weight, protein content and protein yield.
  • Example 6 Example 6 - VIP and potential applications in the inhibition of bacteria in human, animal and plant health
  • AMR Antimicrobial resistance
  • VRE Gram-positive vancomycin resistant enterococci
  • MRSA Gram-positive methicillin resistant staphylococcus aureus
  • ESBL Gram-negative extended spectrum betalactamase producing Escherichia coli
  • VRE VRE
  • ESBL Fig 1A
  • MRSA Fig 1C
  • VIP inhibits a diverse selection of multi-resistant bacteria that cause diseases.
  • VIP may therefore find use as a probiotic, a cosmetic ingredient, a food additive, a feed additive, a veterinary medicine, a biocide, an antibiotic, a pharmaceutical, or a biotechnological product.
  • Example 7 Field applications in winter crops
  • a bacterial solution containing 10 McF VIP bacteria in 2% pecton was added to winter wheat and rapeseed seeds in a rotating seed coating machine at a ratio of 8mL/kg (winter wheat) and 32mL/kg (rapeseed). The seeds were then air dried and stored in a bag until sowing. The rape seed was sown in August 2022 and the winter wheat was sown in October 2022, both on fields on Zealand, Denmark. Emergence was counted 21 days after sowing (rapeseed) and 33 days after sowing (winter wheat) and was quantified as plants/m 2 . Above ground biomass of rapeseed was measured by cutting, drying and weighing plants from 2 x 0.5m 2 at 2 months after sowing.
  • VIP can enhance the emergence of crops as diverse as rapeseed (belonging to the plant family Brassicaceae, a dicot) and winter wheat (belonging to the plant family Poaceae, a monocot). VIP is also capable of increasing the biomass of a crop and therefore the rate of photosynthesis, carbon capture and carbon storage in biomass.
  • the aim of this study was to test the ability of VIP to affect the growth of various spring crops when applied as a seed coat before sowing.
  • Seeds from spring crops listed in Table 6 were used for field trials (performed by an independent third-party contract research organization (VKST, Denmark)). Seed treatments were carried out by applying a bacterial seed coat solution containing 4xl0 9 CFU/mL VIP to seeds at a rate of 2.5 mL/kg. Seeds were stirred during the seed coat application to ensure an even distribution of the seed coat. Untreated seeds were used as controls for each respective crop (Table 6). Following seed coat application, seeds were dried over night at 8C° and sown on fields April 2023 (between April 10 and 22, mean temperature April 2023 was 7C°).
  • Field areas were fertilized with either a combination of nitrogen-sulfur (NS; 30% nitrogen, 10% sulfur) + phosphate-potassium-sulfur (PKS; 10% phosphate, 20% potassium, 3% nitrogen) fertilizers or a recycled organic fertilizer (0GRO;
  • Table 6 Spring crops used for field trials. Trials were carried out by an independent third-party contract research organization (VKST, Denmark). A combination of nitrogen-sulfur (NS) + phosphate-potassium-sulfur (PKS) fertilizers or a recycled organic fertilizer (0GRO) were applied to fields where oat, spring barley and spring wheat seeds were sowed.
  • NS nitrogen-sulfur
  • PES phosphate-potassium-sulfur
  • (0GRO) a recycled organic fertilizer
  • Treating seeds from some of the most common grown spring crops in Denmark, including grain and legume crops, with a bacterial solution containing VIP before sowing demonstrates that VIP can enhance the emergence of crops in the field and lead to a higher harvest yield.
  • Example 9 - VIP increases the yield of vegetables
  • the aim of this study was to test the ability of VIP to affect the yield of vegetables.
  • Treating onion sets, seed potatoes and maize seeds with a bacterial solution containing VIP before sowing in fields demonstrates that VIP can affect the harvest yield of vegetables as diverse as onions, potatoes and maize by a minimum of 18%.
  • Example 10 - VIP has mineral-dissolving properties at low, medium and high temperatures
  • VIP was cultivated on different solid media: nitrogen- free agar; Jensen medium, insoluble phosphate agar; Pikowskaya agar, and insoluble potassium agar; Aleksandrow agar. These three solid media are recognized for being specific for detection and cultivation of nitrogen-fixing, phosphate-solubilizing, and potassium-solubilizing soil microorganisms. Growth on Jensen agar indicates nitrogen-fixating abilities, as this medium is free of nitrogen. A halo zone on Pikowskaya and Aleksandrow agar indicates solubilization of calcium phosphate and potassium alumino-silicate, respectively.
  • SI solubilization index
  • experiments were performed at low (5°C), medium (15°C), and high (25°C) temperatures. Typically, spring seeding will occur at soil temperatures around 6- 8°C in Northern Europe, and activity at lower temperatures is therefore of great importance at this latitude.
  • a commercial microbial biostimulant and biofungicide, biol and bio2 respectively, were included in the experiment as benchmarking controls.
  • the active microorganisms in these products were isolated and included in the test on equal footing with VIP: A fluid culture of each bacterial strain was prepared in a concentration of 0.5 McFarland. The solution was then spotted onto plates containing the different media in repetitions of three. The plates were incubated at either 5°C, 15°C, or 25°C. Colony and halo zone areas were measured each day using imageJ.
  • VIP The ability of VIP 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 were quantified and furthermore compared to bacterial strains isolated from commercial biostimulant products (Figure 12).
  • VIP 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.
  • VIP exerted mineral-dissolving properties at all temperatures (Figure 12B and C) while the bacterial control strains isolated from products only demonstrated little (Figure 12B) or no (Figure 12C) capacity towards dissolving phosphate or potassium, respectively. This suggests that VIP has a better capacity towards dissolving minerals which are otherwise unavailable for plants.

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Abstract

The present invention relates in particular to an identified strain of bacteria, namely the Pseudomonas protegens strain V1P deposited with deposit number DSM 34378 and uses thereof, such as a plant growth enhancer.

Description

NOVEL PSEUDOMONAS STRAIN
Technical field of the invention
The present invention relates to isolated bacteria or biologically pure bacterial culture of the species Pseudomonas protegens. 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.
Background of the invention
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 about 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.
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 consequences for our environment and climate. 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.
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.
There are many mechanisms by which beneficial microorganisms may promote plant growth or survival. Beneficial bacteria may help the plant access strongly bound nutrients in the soil that would otherwise not be plant-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 or interior of plants and seeds such as their leaves, stem 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, green houses, gardens, landscaping, reforestation, carbon capture and storage and in the restoration of degraded areas. In addition, bacteria that are capable of solubilizing nutrient containing minerals or of inhibiting pathogenic microorganisms 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.
WO 2020/214843 Al discloses a method of incorporating 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. Hence, provision of bacteria capable of improving plant growth would be advantageous, and in particular, more efficient and/or reliable compositions comprising such bacteria would be advantageous.
Summary of the invention
The inventing team has isolated, identified, characterized and propagated a newly identified strain of bacteria, namely the Pseudomonas protegens strain VIP deposited with deposit number DSM 34378 (otherwise indicated as VIP). 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.
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, animals and humans. This may be done by placing the strain on the surface of the body, by placing it in a cavity in the body or by injecting it into the body of the recipient plant, animal or human. The strain may also be used as a probiotic for ingestion. Specifically, the strain may be used to treat extended spectrum beta-lactamase (ESBL)-producing bacteria, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin- resistant enterococci (VRE) infections in animals and humans.
The strain may also be used to dissolve otherwise poorly soluble substances such as minerals and salts. These minerals and salts may be present naturally in soil or other plant growth media, examples are calcium phosphate, iron phosphate, aluminium phosphate and potassium alumino silicate. They may also form when macronutrients or micronutrients, possibly in the form of fertilizer, are added to the soil or other plant growth media. The minerals and salts may also be components of nutrient bearing materials destined for agricultural use such as wastewater, sludge, biochar, manure, green manures, compost, biogas and pyrolysis residues, biowaste, household waste and other biomasses. The purpose of adding the strain would in these cases be to dissolve the minerals or salts and thereby release nutrients such as phosphate, potassium, calcium, magnesium, sulfate, manganese and iron that can enhance plant growth.
The strain may also be used to dissolve otherwise poorly soluble substances such as minerals and salts for applications outside plants. It may be used to bioleach ores or waste materials. It may therefore by used in mining, recycling, or urban mining.
In summary:
Example 1 shows how the VIP strain has been identified and isolated.
Example 2 shows, using bioinformatic tools, that the VIP strain has a unique genomic signature and therefore is considered a newly identified strain.
Example 3 shows that the VIP strain can increase the availability of common plants nutrients.
Example 4 demonstrates the stimulatory effects of a VIP seed coat on early plant growth in vitro in two typical agricultural crops, namely winter wheat and oilseed rape.
Example 5 shows how the VIP strain positively alters plant properties in the field. Example 6 shows that the VIP strain may also be relevant for medical purposes. Example 7 demonstrates that VIP can enhance the emergence of crops as diverse as rapeseed (belonging to the plant family Brassicaceae, a dicot) and winter wheat (belonging to the plant family Poaceae, a monocot). VIP is also capable of increasing the biomass of a crop and therefore the rate of photosynthesis, carbon capture and carbon storage in biomass. Example 8 shows the ability of VIP to affect the growth of various spring crops. Treating seeds, including grain and legume crops, with a bacterial solution containing VIP before sowing demonstrates that VIP can enhance the emergence of crops in the field and lead to a higher harvest yield.
Example 9 shows the ability of VIP to affect the yield of vegetables such as onions, potatoes and maize. Treating onion sets, seed potatoes and maize seeds with a bacterial solution containing VIP before sowing in fields demonstrates that VIP can affect the harvest yield of vegetables as diverse as onions, potatoes and maize by a minimum of 18%.
Example 10 shows the ability of VIP to increase the availability of common plant nutrients, such as nitrogen, phosphate and potassium, specifically under low, medium, and high temperature conditions. This demonstrates that VIP 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.
The aforementioned deposit were made by University of Southern Denmark (SDU) on September 15, 2022. The deposit was given the following reference number: DSM 34378.
Thus, an object of the present invention relates to the provision of novel bacterial strains/biostimulants for improving plant growth.
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.
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: 1-7; b) a genomic sequence having at least 84% sequence identity to any of SEQ ID NOs: 1-7, preferably such as at least 90%, more preferably 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: 1-7; and/or c) a fragment of one or more of the sequences of a) or b), having a length of at least 500 nucleotides.
Another aspect of the present invention relates to a fertilizer and/or inoculant and/or biostimulant composition comprising the bacteria or biologically pure bacterial culture according to the invention.
Yet another aspect of the present invention is to provide 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.
Still another aspect of the present invention is to provide a plant seed coated with the composition according to the invention or coated with a coating composition according to the invention.
An aspect of the invention relates to the use of 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, as a plant growth promoting agent, such as a fertilizer or inoculum or biostimulant.
In a further aspect, the 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 forrow, soil and/or plant growth medium.
In yet a further aspect, the 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
• 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.
Thus an aspect of the invention relates to 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 for use as a medicament. As shown in the example section the bacteria according to the invention may have anti-bacterial properties. It may also be used to promote animal or human health, such as by dissolving or freeing nutrients.
A further aspect of the invention relates to the use of the bacteria or biologically pure bacterial culture according to the invention or the composition according to the invention for solubilizing minerals in ores.
Brief description of the figures
Figure 1
Figure 1 shows initial screening of retrieved capture pills from the microbeTRAP on agar plated with VRE. B) Shows a clearing zone surrounding one capture pill. Isolated bacteria from this capture pill was spotted onto ESBL expressing E. coli (B), methicillin resistant staphylococcus aureus (MRSA) (C) and VRE (D).
Figure 2
Figure 2 shows de novo genome assembly of VIP. Scaffolds of VIP genome assembly (arrow bars) depicted along with BLAST results against the type strain of Pseudomonas protegens CHAO (top bars). White areas in the bars at the top indicate no sequence overlap between VIP and the type strain. A gradient from grey to black indicates percent identity, where black represents 100% sequence similarity. Note: The assembly has been divided into two overlapping sections.
Figure 3
Figure 3 shows bacterial growth and solubilization halo of VIP, Serenade, and FloraGro on solid nitrogen-free medium (top row) or medium with insoluble phosphate source (calcium phosphate) (bottom row) at day 4, 8°C. Figure 4
Figure 4 shows A) Colony area of VIP, Serenade, and FloraGro when grown on nitrogen-free medium; and B) Colony area and C) halo area of VIP, Serenade, and FloraGro when grown on medium with insoluble phosphate source (calcium phosphate) at day 4, 8°C.
Figure 5
Figure 5 shows bacterial growth and solubilization halo of VIP and FloraGro on solid medium with insoluble potassium source (potassium alumino silicate) at day 15, 8°C.
Figure 6
Figure 6 shows A) colony area and B) halo area of VIP and FloraGro when grown on a medium containing insoluble potassium (potassium alumino silicate) at day 15, 8°C.
Figure 7
Figure 7 shows early germination performance of VIP seed coat in crops in vitro. A) Emergence of radicle from uncoated (control) or VIP-coated (VIP) seeds from oilseed rape and winter wheat crops at one day following seed plating in petri dishes. B) Emergence of plumule from uncoated (control) or VIP-coated (VIP) seeds from oilseed rape and winter wheat crops at two days following seed plating in petri dishes. Bar plots are depicted as mean percentage emergence across seeds from 4 individual replicates. Error bars represent standard error of the mean (SEM) across replicates. * indicates statistical significance (p<0.05) of VIP seed coat vs control using a one-sided Wilcoxon test.
Figure 8
Figure 8 shows in vitro solubilization of inorganic phosphate using VIP seed coat. Uncoated (control) and VIP-coated (VIP seed coat) seeds from oilseed rape and winter wheat crops, respectively, on top of Pikowskaya medium. Pictures of petri dishes were taken two days following coating and plating and show significant clearing zones formed around VIP-coated seeds (exemplified by white arrow heads). Figure 9
Figure 9 shows anti-fungi properties of VIP seed coat in vitro. Uncoated (control) and VIP-coated (VIP seed coat) seeds from oilseed rape and winter wheat crops, respectively, on top of Pikowskaya medium. Pictures of petri dishes were taken four (oilseed rape) and five (winter wheat) days following plating. Black arrowheads indicate fungal contamination. White arrowheads indicate clearing zones formed around seeds as a result of phosphate solubilization.
Figure 10
Figure 10 shows the effect of VIP on early and late emergence of spring crop plants in the field. Early (14 days after sowing) and late emergence (42 days after sowing) of faba bean plants (A), pea plants (B), barley 'Evergreen' plants (C), barley 'KWS Irina' plants (D), oat plants (E) and spring wheat plants (F) per m2 from control or VIP-treated seeds. Seeds were sown in a Danish field (using an independent third-party contract research organization (VKST, Denmark)) April 2023. Field areas were fertilized with either a combination of nitrogen-sulfur (NS; 30% nitrogen, 10% sulfur) + phosphate-potassium-sulfur (PKS; 10% phosphate, 20% potassium, 3% nitrogen) fertilizers (C and F) or a recycled organic fertilizer (0GRO; 10% nitrogen 3% phosphate, 1% potassium) (D and E) at the day before sowing (faba bean and pea fields were not fertilized (A and B)). Fertilizers were applied at two rates; 100% and 75%. Asterisks indicate statistical significance (p<0.05) of VIP seed coat vs control within early emergence, late emergence and fertilizer groups using a one-sided t-test.
Figure 11
Figure 11 shows the effect of VIP on onion, potato and maize yields from fields.
A) Yield of onions per plant from control or VIP-treated onion sets (50 sets planted in total per treatment). B) Yield of potatoes per plant from control or VIP- treated seed potatoes (50 seed potatoes planted in total per treatment). C) Yield of maize cobs per plant from control or VIP-treated maize seeds (10 maize seeds planted in total per treatment). Bars represent the mean yield per vegetable across treatments. Error bars represent the standard error of the mean (SEM) across yields. Asterisks indicate statistical significance (p<0.03) of VIP seed coat vs control using a one-sided t-test.
Figure 12
Figure 12 shows in vitro mineral-dissolving properties of VIP and commercially available biostimulants at low, medium and high temperatures. A) Colony size of VIP and two bacterial strains (Biol and Bio2) isolated from two commercially available microbial plant products (a biofungicide and a biostimulant) 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 VIP, Biol and Bio2 grown on Pikowskaya medium (insoluble phosphate agar) for 7 days (B) or Aleksandrow medium (insoluble potassium agar) for 10 days (C) at low, medium or high temperatures. Each experiment was carried out in three replicates and bars in each plot represent the mean across replicates. Error bars represent standard deviations across replicates in each experimental group.
The present invention will now be described in more detail in the following.
Detailed description of the invention
Definitions
Prior to discussing the present invention in further details, the following terms and conventions will first be defined: Effective amount
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.
Inoculant
The term "inoculant" as described in this invention is defined in several Federal, or State regulations as:
(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);
(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
(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).
Biostimulant
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.
In here the terms "fertilizer", "inoculant" and "biostimulant" may be used interchangeably.
Isolated bacteria and biologically pure bacteria! culture
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.
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 number of bacteria in the culture.
Preferably the bacteria according to the invention is an isolated bacteria, such as forming part of an isolated composition.
Plant growth promoting agent
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.
Seguence identity
In the present context, the term "sequence identity" indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length or between two nucleic acid sequences of substantially equal length. The two sequences to be compared must be aligned to best possible fit with the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as
Figure imgf000015_0001
, wherein
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 . Lipman (1988)).
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.
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%.
Hectoliter weight
Hectoliter weight relates to the harvest yield, which is also provided as kilogram (kg) per 100 liter (L) or kg per hectoliter (kg/hL).
Bacteria or biologically pure bacterial culture
As outlined above, the inventing team has identified, isolated and propagated a newly identified strain of bacteria, namely the Pseudomonas protegens strain VIP deposited with deposit number DSM 34378 (otherwise indicated as VIP). The strain may be used to promote a wide variety of plant health parameters. 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: 1-7; b) a genomic sequence having at least 84% sequence identity to any of SEQ ID NOs: 1-7, preferably at least 90%, such as at least 95%, more preferably 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: 1-7; and/or c) a fragment of one or more of the sequences of a) or b), having a length of at least 500 nucleotides.
Preferably the bacteria is an isolated bacteria.
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: 1, 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: 1.
In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 97% sequence identity to SEQ ID NO: 2, 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: 2.
In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 85% sequence identity to SEQ ID NO: 3, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 3.
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: 4, 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: 4. In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 90% sequence identity to SEQ ID NO: 5, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 5.
In an embodiment under b), the bacteria or biologically pure bacterial culture comprises a genomic sequence having at least 90% sequence identity to SEQ ID NO: 6, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 6.
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.
As outlined in example 2, no sequence which such sequence identities have been identified.
In an embodiment, the bacteria or biologically pure bacterial culture according to the invention comprises any of SEQ ID NO's: 1-7, such as one or more of SEQ ID NO's: 1-7. 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, or preferably such as all of SEQ ID NO: 1-7, or with a sequence identity for SEQ ID NO: 1-7 as defined above.
SEQ ID No's: 1-5 forms part of SEQ ID NO: 8, and have been identified to comprise reading frames with low sequence identity to other known proteincoding genes as also illustrated in example 2. SEQ ID NO's: 6 and 7 lies outside SEQ ID NO: 8 and have also been identified to comprise open reading frames.
Thus, in another embodiment, the bacteria or biologically pure bacterial culture according to the invention comprises a) a genomic sequence according to SEQ ID NO: 8; b) a genomic sequence having at least 90% sequence identity to SEQ ID NO: 6, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 8; and/or c) a fragment of the sequence of a) or b), having a length of at least 5000 nucleotides, such as at least 8000 nucleotides, such as at least 10000 nucleotides, such as at least 14000 nucleotides.
The VIP strain according to the invention has been annotated as being a Pseudomonas protegens species. Thus, in an embodiment, the bacteria or biologically pure bacterial culture is of the genus Pseudomonas, such as species Pseudomonas protegens.
In an embodiment the bacteria or biologically pure bacterial culture comprises a 16S rRNA gene encoded by SEQ ID NO: 9. As shown in example 2, the strain of the invention has been identified to comprise a 16S rRNA gene encoded by SEQ ID NO: 9.
In an embodiment, the isolated bacteria or biologically pure bacterial culture being Pseudomonas protegens, DSM 34378 deposited with the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) on 15 September 2022]. In here this strain is also named VIP.
In yet an aspect the invention relates to an (isolated) bacteria or biologically pure bacterial culture being Pseudomonas protegens, DSM 34378 deposited with the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) on 15 September 2022].
For colder regions it would be an advantage if the bacteria was active under colder conditions. Thus, in an embodiment, the bacteria or biologically pure bacterial culture according to the invention is
- capable of solubilizing calcium phosphate at 8°C and/or potassium aluminum silicate at 8°C; and/or
- capable of inhibiting MRSA, VRE or ESBL. In an embodiment, the bacteria or biologically pure bacterial culture is dehydrated, such as spray-dried.
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 involved in producing carcinogenic secondary metabolites.
Fertilizer or inoculant or biostimulant
The isolated bacteria or biologically pure bacterial culture according to the invention may form part of different compositions. Thus, an aspect of the invention relates to a fertilizer and/or inoculant and/or biostimulant and/or biofungicide and/or antimicrobial composition comprising the bacteria or biologically pure bacterial culture according to the invention.
The composition may comprise other microorganisms. Thus, in an embodiment, the fertilizer and/or inoculant and/or biostimulant composition further comprises other microorganisms, such as other microorganisms able to function as a biostimulant.
In an embodiment, the composition further comprises one or more agriculturally acceptable carriers.
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 cryoprotectant, a drying additive, a preservative, a polymer, biopolymer, a coating, or a combination thereof.
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 or a biopolymer derived from a natural source, possibly chemically modified afterwards.
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.
In yet another embodiment, the composition is formulated as a granular formulation or a powder formulation.
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, abacterial 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, or a combination thereof.
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.
Coating composition
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.
In an embodiment, the coating composition comprises a biopolymer promoting adherent to a plant seed. As shown in Example 3, pectin has been tested as biopolymers promoting adherence to plant seeds.
In an embodiment, the coating composition is formulated as an aqueous or oilbased solution for application to seeds, preferably aqueous.
In another embodiment, the coating composition is formulated as a powder or granular formulation for application to seeds.
Coated plant seed
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.
In an embodiment, the plant seed is a dicotyledon, monocotyledon or a gymnosperm seed.
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, wheat, such as winter or spring wheat. Oats, triticale, maize, rye, grass, clover, broad bean, lupines, strawberries, peas, potatoes, onions, carrots and sugar beets.
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.
In yet an embodiment, the plant seed being selected from the group of trees, bushes or grasses.
Uses and methods
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.
In an embodiment, the use as a plant growth-promoting agent is for:
• solubilizing inorganic minerals or salts, such as phosphate and/or potassium; 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.
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.
In an embodiment, the inorganic minerals or salts solubilized 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, 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. As shown in the example section, the isolated bacteria or biologically pure bacterial culture can grow under low temperature conditions. Thus, in an embodiment, the use takes place at field temperatures (measured as the soil surface temperature) in the range -10°C to 15°C, such as -5°C to 15°C, preferably 0 to 15°C, more preferably such as 2 to 10°C, such 2 to 8°C.
In an embodiment, the use takes place at field temperatures in the range 2 to 8°C. It is shown in the examples, such as examples 3, 5, and 10, the isolated bacteria or biologically pure bacterial culture are used and grown at temperatures at 8°C and below.
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.
In yet another embodiment, the use takes place in Scandinavia, such as Norway, Sweden, Finland or Denmark, such as in Jutland, Fyn, and/or Zealand.
In an embodiment, the plant is a dicotyledon, monocotyledon or a gymnosperm.
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, and vegetables, such as onion. As shown in the Examples 7-9, the (isolated) bacteria or biologically pure bacterial culture according to the invention have been used on the above-mentioned plant varieties and demonstrates increased growth and yield.
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.
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), Trema nd raceae, 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).
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.
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).
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.
Method for stimulating plant growth
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 forrow, soil and/or plant growth medium.
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:
- to a plant growth medium, such as sphagnum; and/or
- to a plant growth medium prior to, concurrently with, or after planting of seeds, seedlings, cuttings, bulbs, or plants in the plant growth medium; or
- to plant leaves, roots, or stems; and/or
- to plant seeds, and/or
- to a watering system for the plants, such as hydroponics, aeroponics and/or aquaponics. 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.
Kit
In yet a further aspect, the invention relates to a kit of parts for stimulating plant growth comprising
• 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
• 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.
In an embodiment, the kit of parts further comprises one or more containers comprising fertilizers, nutrients, and/or other microorganisms.
Medical uses
As outlined in example 6, the invention may also have medical uses. Thus, an aspect of the invention relates to 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 for use as a medicament. As shown in the example section (Example 6) the bacteria according to the invention may have anti-bacterial properties. It is shown in Example 6 that VIP inhibits a diverse selection of multi-resistant bacteria that cause diseases.
VIP may therefore find use as a probiotic, a cosmetic ingredient, a food additive, a feed additive, a veterinary medicine, a biocide, an antibiotic, a pharmaceutical, or a biotechnological product.
Thus, an aspect relates to the (isolated) bacteria or biologically pure bacterial culture according to the invention or composition according to the invention for use in the treatment, alleviation and/or prevention of bacterial infections, such as E. coli, spectrum beta-lactamase (ESBL)-producing bacteria, Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).
Ores
A further aspect of the invention relates to the 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. 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.
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.
All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.
The invention will now be described in further details in the following non-limiting examples.
Examples
Example 1 - Isolation of the strain and assessment of its anti-bacterial properties
Aim of study
Isolation of the strain deposited under DSM 34378 and testing of its antibacterial properties.
Materials and methods
Tricalcium phosphate (TCP), myristic acid (MA) and LB medium was from Sigma Aldrich. The microbeTRAP was 3D printed in polyamide (PA12) by Materialize. MRSA, ESBL and VRE were donated by the Department of Clinical Microbiology at Odense University Hospital. Strain VIP was isolated from the soil of an old beech wood in October 2019 (coordinates: 55°21'57.6"N 10°25'55.2"E) using a microbeTRAP [W02021/180941 Al]. The microbeTRAP was 3D printed from PA (SLS, Materialize) with the overall dimensions of a microtiterplate, it contained 16 central chambers that contained a nutrient pills, each of these chambers were connected to 4 separate outer chambers that contained a capture pills. Nutrient and capture pills were made as previously described. Nutrient pills were made by compressing LB medium into a pellet using a pill press. Capture pills were made by first suspending 25g TCP in 6g MA that had been melted to 70°C on a hot-plate, the suspension was then cast to pills using a silicone mold and the pills were then carbonized for 1 hours at 400°C and sintered for 2 hours at 1100°C resulting in porous ceramic cylinders. Nutrient pills and capture pills were placed in the microbeTRAP and it was sealed with microtiter plate seal. Forest soil was collected from an old beech forest in Odense, Denmark (coordinates 55°21'57.6"N 10°25'55.2"E) in October 2020. The soil was placed in a biosafety plastic box, the microbeTRAP was placed in the middle of the soil and the soil was added IL of sterile water to create a moist environment that would promote nutrient exchange and chemotaxis. The soil box was then incubated for 5 days at 37°C.
The microbeTRAP was excavated and the capture pills retrieved. These were placed on agar plates covered with either vancomycin resistant enterococci (VRE) or extended spectrum beta-lactamase (ESBL) expressing E. coli. After 24 hours of incubation at 37°C, the plates were placed at 4°C for 5 days. They were then investigated for clearing zones surrounding the capture pills after which they were incubated for 24 hours more at 37°C followed by 3 days at 4C before being investigated for clearing zones again.
The capture pills displaying clearing zones (n=9) were sampled for microorganisms using an inoculation loop that were swept from the pill out to the edge of the clearing zone. The microorganisms were then spread on 5% blood agar and CSPA agar plates. These plates were incubated at 37°C and the colonies that developed were further isolated on subsequent plates. The anti-microbial properties of these colonies were then assessed using the "spot on lawn" technique. The colonies were spotted onto Mueller-Hinton or 5% blood agar plates containing a lawn of either VRE, MRSA, ESBL or C. albicans. The plates were incubated for 24 hours at 37°C. Colonies that successfully displayed an inhibition zone (n=3) were reisolated from the inhibition zone onto fresh 5% blood agar plates. There the microorganism responsible for the inhibition was identified using MALDI-TOF mass spectrometry (Biotyper, Bruker). Another round of inhibition zone assessment was conducted to confirm their inhibitory ability, in this round 2/3 strains failed to recreate the inhibition zone. The remaining inhibiting colony was then re-cultured on CSPA agar, its identity reconfirmed using mass spectrometry and finally DNA was extracted from the colonies using a kit (Qiagen). The DNA was sent to sequencing at Novogene and a partial genome assembly was carried out. The strain was tested for antibiotics susceptibility against a panel of compounds commonly used to treat infections by bacteria in the Pseudomonas genus at Odense University Hospital.
Results
Initial screening of retrieved capture pills from the microbeTRAP on agar plated with VRE showing a clearing zone surrounding one capture pill (Figure IB). Bacteria from this capture pill were isolated and led to the discovery of a bacterial isolate that produced a zone of clearance when spotted onto ESBL expressing E. coli (Figure 1A), methicillin resistant staphylococcus aureus (MRSA) (Figure 1C) and VRE (Figure ID). The bacteria was identified to be a Pseudomonas protegens strain and was dubbed VIP as it was isolated from "VRE plate number 1" and was a Pseudomonas. Antibiotics susceptibility testing revealed that the bacteria was inhibited by all antibiotics commonly used to treat infections by Pseudomonas bacteria.
Conclusion
The microbeTRAP technology enabled the capture of an anti-bacterial microorganism from Danish forest soil that was later identified to be a new Pseudomonas protegens strain (see Example 2). The strain proved capable of inhibiting ESBL E. coli, MRSA and VRE and was susceptible to many antibiotics.
Example 2 - VIP is a novel plant-protecting Pseudomonas protegens strain
Aim of study
The aim is to demonstrate that VIP is a novel bacterial strain belonging to the plant-protecting Pseudomonas protegens species. Materials and methods
DNA extraction, library preparation, and whole genome sequencing
DNA was extracted from a bacterial culture of VIP using the DNease UltraClean Microbial Kit from Qiagen following the instructions from the manufacturer. 50 ng of bacterial 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. DNA libraries were sequenced on an Illumina MiniSeq machine. A total of 4,681,350 reads were obtained, corresponding to a genome coverage of lOOx (compared to the reference genome size of Pseudomonas protegens CHAO (Table 1)).
Quality control of sequencing reads
Adapter contaminants from the library preparation step as well as nucleotides with a Phred score <20 were removed from raw sequencing reads using Trim Galore (vO.6.7). Only trimmed reads were used for downstream analyses.
Taxonomic classification of sequencing reads
The metagenomic classification tool KrakenUniq (vl.0.1) was used for taxonomic assignment of trimmed sequencing reads according to the National Center for Biotechnology Information (NCBI) Taxonomy. KrakenUniq utilizes an advanced k- mer-based approach where short DNA sequences of k length are matched to a nucleotide reference database. For the reference database, a pre-built database containing all current (as of 6/16/2022) and completely assembled bacterial, archaeal, viral and human RefSeq genomes as well as vector sequences (downloaded from https://benlangmead.github.io/aws-indexes/k2) was used.
Alignment-based analysis of sequencing reads
Trimmed sequencing reads were aligned against three Pseudomonas protegens genomes (Table 1) using the burrows-wheeler aligner (BWA) (vO.7.17) with the BWA-MEM algorithm. Pseudomonas protegens genomes were downloaded from NCBI (Table 1).
Table 1: Pseudomonas protegens reference genomes.
Figure imgf000035_0001
Figure imgf000036_0001
De novo genome assembly
SPAdes (v3.15.5) was used for assembling trimmed sequencing reads into de novo contigs that were subsequently joined into putative scaffolds. A read errorcorrection step (BayesHammer module) was performed prior to the genome assembly step to minimize the number of mismatches in the resulting contigs. Following the genome assembly step, QUAST (v5.0.2) was used for computing assembly quality metrics and for comparing the de novo assembly against Pseudomonas protegens reference genomes (Table 1).
Short (609-1,266 nucleotides) and long (16,813 nucleotides) DNA sequences extracted from the genome assembly are annotated as SEQ ID NOs: 1-8.
Average nucleotide identity (ANI) analysis
FastANI (vl.l) was used for computing average nucleotide identity (ANI) scores of the assembled genome compared to Pseudomonas protegens reference genomes (Table 1).
16S rRNA extraction and analysis
The DNA sequence of the 16S ribosomal RNA (rRNA) gene from the VIP strain was extracted from the assembled genome with ContEstl6S (available at www.ezbiocloud.net/tools/contestl6s) and compared to bacterial type strains using the Basic Local Alignment Search Tool (BLAST) (v.2.12.0). A pre-built database of 16S ribosomal RNA sequences from bacteria and archaea type strains was downloaded from NCBI and used as the reference database for BLAST.
The identified 16S ribosomal RNA sequence is encoded by SEQ ID NO: 9 (1,539 nucleotides).
Visualization of genome assembly and variable regions compared to type strain Proksee (available at https://proksee.ca/) was used for visualization of VIP genome assembly along with BLAST results where VIP was compared to the type strain of Pseudomonas protegens (CHAO). Protein analysis
Protein function of the translated sequence of SEQ ID NOs: 1-7 was predicted using BLAST protein alignment. The UniProtKB reference proteomes and Swiss- Prot databases were used as reference databases.
Results
Initial taxonomic classification of VIP sequencing reads using a reference database composed of bacterial, archaeal, viral and human genomes as well as vector sequences, revealed that 98.56% of all reads were assigned to the Pseudomonas genus. Analysis at species level revealed that 62.02% of all reads could further be assigned to Pseudomonas protegens; a known class of plantprotecting bacterial species.
The reference genome of Pseudomonas protegens belongs to the type strain CHAO (Table 1) for which a full-genome sequence is available. Comparing VIP sequencing reads to the whole genome of the reference genome as well as to fullgenomes of two closely related Pseudomonas protegens strains, namely Cab57 and Pf-5 (Table 1), revealed <95% sequence similarity (Table 2). This suggests that VIP might be a novel Pseudomonas protegens strain.
Table 2: Percent sequence identity between VIP sequencing reads and Pseudomonas protegens reference genomes.
Figure imgf000037_0001
To obtain further biological insight into VIP as a putative novel strain belonging to the Pseudomonas protegens taxon, VIP sequencing reads were assembled into a de novo genome. Sequencing reads were assembled into larger fragments of DNA sequences (contigs) that were subsequently assembled and ordered into longer scaffolds. A total length of 7,078,551 nucleotides were obtained for the assembled genome of VIP with the longest scaffold representing 1,072,950 nucleotides of the genome (Figure 2). 99.6% of the genome assembly is based on scaffolds of size 500 nucleotides, suggesting that the genome of VIP is bigger than the genome size of the type strain (Table 1). Evaluating the assembled genome against the reference genomes of Pseudomonas protegens revealed sequence similarities that were similar to the alignment-based analysis of the shorter sequencing reads alone (Table 3). The variability between VIP and the type strain of Pseudomonas protegens (CHAO) is further depicted in Figure 2. Genome annotation of the assembled genome importantly showed no presence of the rhizoxin-biosynthetic gene cluster (including rhi and rzx gene loci) which may be involved in producing carcinogenic secondary metabolites.
Table 3: Percent sequence identity between VIP genome assembly and Pseudomonas protegens reference genomes.
Figure imgf000038_0001
16S ribosomal RNA sequences (16S rRNA, ~l,500 bp) 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. Extracting the DNA sequence encoding the 16S rRNA gene of the assembled genome of VIP (1,539 nucleotides, SEQ ID NO: 9) and comparing that to the NCBI 16S ribosomal database revealed 99% similarity to the type strain of Pseudomonas protegens (CHAO). Although the 16S rRNA region contains hypervariable regions that can be used for taxonomic discrimination, the analysis of 16S rRNA alone ignores the genome-wide variability. Average Nucleotide Identity (ANI) has been proposed as a robust measure of whole genome similarity of prokaryotic strains and is commonly used for delineating species identity. Although consensus criteria for species boundaries are missing, organisms showing 95% ANI at the full-genome level are typically assigned to the same species. Defining thresholds based on whole genome comparisons at the strain level is an even bigger challenge due to the high genetic relatedness. Thus, ANI is not useful, at least at the moment, beyond species-level. Computing ANI values of VIP against the three Pseudomonas protegens reference genomes used here (Table 1) revealed ANI scores <99% (Table 4), suggesting that VIP is a novel strain within the Pseudomonas protegens family. Table 4: Average Nucleotide Identity (ANI) scores of VIP genome assembly compared to Pseudomonas protegens reference genomes.
Figure imgf000039_0001
To further signify VIP as a novel Pseudomonas protegens strain, genomic regions within the VIP genome assembly showing no or low concordance with Pseudomonas protegens references (Table 1) were extracted and subjected to sequence similarity analysis using the nucleotide database at NCBI. This identified a genomic region of 16,813 nucleotides (SEQ ID NO: 8) showing <50% coverage in various Pseudomonas and Azotobacter species and importantly <2% coverage in Pseudomonas protegens strains. Thus, this region seems specific for VIP. Interestingly, several long open reading frames were discovered within this genomic region, which may represent important functional elements, e.g., protein-coding genes. To investigate potential functions of such "hypothetical proteins", five random regions containing such open reading frames (SEQ ID NO's: 1-5) were extracted. Two additional and randomly selected genomic regions located outside of SEQ ID NO: 8 containing open reading frames were further included (SEQ ID NO's: 6-7). Comparing each of these sequences to the nucleotide database at NCBI using BLAST revealed sequence identities <95.09% (Table 5), suggesting that SEQ ID NOs: 1-7 are highly selective for VIP. Insight into protein function may be obtained through protein sequence alignment. Subjecting SEQ ID NOs: 1-7 to a protein similarity search using the protein databases of UniProtKB and Swiss-Prot indicated that SEQ ID NOs: 1-7 may encode DNA-binding proteins, e.g., transcription factors. Although prediction scores were low (<50% similarity), some of the identified hypothetical proteins could be of regulatory importance for VIP.
Table 5: Maximum percent sequence coverage and sequence identity of SEQ ID NOs: 1-7 compared to the nucleotide database of NCBI using BLAST.
Figure imgf000039_0002
Figure imgf000040_0001
Conclusion
Whole genome sequencing and analysis of the bacterial component of the VIP seed coat (here referred to as VIP) revealed firstly that VIP belongs to the plantprotecting Pseudomonas protegens species.
Secondly, thorough sequence comparisons to the reference genome of Pseudomonas protegens (type strain CHAO) and to the genomes of two closely related Pseudomonas protegens strains (Cab57 and Pf-5) importantly suggested that VIP indeed is a novel Pseudomonas protegens strain.
Thirdly, several DNA sequences containing open reading frames and that have high specificity for VIP were identified; one long genomic region (16,813 nucleotides, SEQ ID NO: 8) wherein short DNA sequences (609-1,266 nucleotides) were extracted (SEQ ID NOs: 1-5) as well as two short DNA sequences identified outside of the long genomic region (SEQ ID NOs: 6-7). Combined or alone, these DNA sequences may be used to identify VIP at the DNA sequence level.
Example 3 - Assessment of mineral dissolving properties
Aim of study
The aim is to demonstrate the ability of VIP to increase the availability of common plant nutrients otherwise supplemented as chemical fertilizer (nitrogen, phosphorous, and potassium).
Materials and methods
To demonstrate the ability of VIP to increase the availability of nitrogen, phosphorous, and potassium it was cultivated on different solid media by plate assay: 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-fixing, as this medium is nitrogen free. A halo zone on Pikovskaya and Aleksandrow agar indicates solubilization of calcium phosphate and potassium alumino-silicate respectively. Two known bio stimulants were included in the experiment as benchmarking controls: Serenade (Bayer CropScience Ltd, UK) and FloraGro (RhizoVital, Andermatt Biocontrol AG, Switzerland).
A fluid culture of each bacterial strain was prepared in a concentration of 0.5 McFarland. The solution was then spotted onto plates containing the different media in repetitions of three. The plates were incubated at 8°C. This temperature was chosen to mimic the conditions during spring seeding in northern Europe. Growth and halo zone area were measured each day using imageJ.
Results
The ability of VIP to increase the availability of nitrogen, phosphorous, and potassium was evaluated using plate assay and measuring growth and/or halo zone. The results of the plate assay on media free of nitrogen or with an insoluble phosphate source are shown in Figure 3 and 4. Growth on nitrogen-free medium at day 4 was only observed with VIP and FloraGro, where the colony area was 28% larger with VIP (Figure 3 (top) and 4A). As this medium is free of nitrogen, increased growth indicates greater nitrogen fixing. Nitrogen is essential to plant growth and present in limited amount in soil. Therefore, nitrogen fixing bacteria play and important role in promoting plant growth and development. Growth on medium with an insoluble phosphate source When grown on medium with an insoluble phosphate source the area of VIP was 33 mm2 and the solubilization index was 8 at day 4 (i.e. ability to create a halo zone 8 times greater than the colony area) (Figure 3 (bottom) and 4B/C). This indicates that VIP is able to solubilize phosphorous from an insoluble inorganic source. In soil phosphorous exist to a large extent bound to insoluble inorganic minerals, which are unavailable for plants. Thus, solubilization of phosphorous by VIP may improve plant availability and promote growth. No growth or halo zone was observed with Serenade and FloraGro when grown on medium with an insoluble phosphate source (Figure 3 (bottom) and 4B/C). The results of the plate assay on medium with an insoluble potassium source are shown in Figure 5 and 6. The colony area of VIP was 29 mm2 after 15 days at 8°C, and the solubilization index 1.6. Like phosphorous, potassium exists in insoluble complexes in soil. Therefore, increasing the availability of potassium may similarly promote plant growth. No growth or halo zone was observed with the controls.
Conclusion
Use of biofertilizer can increase the availability of essential nutrients to plants otherwise administered as chemical fertilizer. These results indicate that the novel biofertilizer VIP can fix nitrogen and solubilize insoluble phosphorous and potassium. These beneficial properties are evident even at low temperatures (8°C), ensuring functionality also during spring seeding in northern Europe unlike most competitors.
Example 4 - Stimulatory traits of VIP seed coat on early plant growth
Aim of study
The scope of this example is to demonstrate the stimulatory effects of a VIP seed coat on early plant growth in vitro in two typical agricultural crops, namely winter wheat and oilseed rape.
Materials and methods
Winter wheat seeds (RGT Saki seeds from Danish Agro) and oilseed rape seeds (organic seeds from Danish Agro) were coated with VIP along with pectin, acting as a biopolymer, using the following preparations:
• Oilseed rape: 2 McFarland standards of VIP were diluted in lx PBS + pectin (final concentration of 1.5% (by volume)). 1 g of oilseed rape seeds were coated with 3 mL of VIP-pectin solution.
• Winter wheat: 10 McFarland standards of VIP were diluted in lx PBS + pectin (final concentration of 1.5% (by volume?)). 5 g of winter wheat seeds were coated with 6 mL of VIP-pectin solution.
To obtain a homogenous seed coating, seeds were mixed with a VIP-pectin solution (named VIP seed coat from hereafter), as previously described, and stirred at 300 rpm for 30 min. Following stirring, seeds were sieved to remove excess coating solution. For early germination performance experiments, coated seeds and control seeds (uncoated) were placed on top of presoaked sterile blue germination paper within 9.5 cm petri dishes (4 replicates, each containing 25 seeds). Petri dishes were kept at room temperature (23±2) with a 16/8 h lig ht/dark regime. Early germination processes, here evaluated as the emergence of the primary root (radicle) and the young shoot (plumule), respectively, were recorded from the day after seeds were plated. Radicle and plumule emergence were averaged across replicates (n=4) and plotted as mean emergence for each crop and treatment in bar plots with standard error of the mean (SEM) as error bars. Statistical testing of VIP effects on radicle and plumule emergence were performed using a onesided Wilcoxon test where * denotes a p-value <0.05.
The capacity of VIP seed coat to solubilize inorganic phosphate was determined by plating coated seeds from oilseed rape and winter wheat crops on Pikowskaya medium. Oilseed rape and winter wheat seeds were coated with VIP seed coat, as previously described, and immediately plated on Pikowskaya medium in 9.5 cm petri dishes (2 replicates, each containing 5 seeds). Petri dishes were kept in incubators at 24°C. The formation of clearing zones around plated seeds was examined at two days following seed plating. Fungal contamination was further evaluated at 4-5 days after plating of seeds on Pikowskaya medium.
Results
Germination:
Early germination was evaluated by the emergence of the primary root (radicle) followed by the emergence of the young shoot (plumule) at one and two days following plating of oilseed rape and winter wheat seeds in petri dishes, respectively. Figure 7 shows the emergence of the radicle (Figure 7A) and the plumule (Figure 7B) in oilseed rape and winter wheat seeds, respectively. Coating winter wheat seeds with VIP seed coat resulted in a significant (p<0.05) increase of both radicle (6-fold) and plumule (1.5-fold) emergence, respectively, compared to control winter wheat seeds that have not received coating (Figure 7A and 7B, bars to the right). A similar tendency was also observed for radicle and plumule emergence in oilseed rape seeds that were coated with VIP seed coat (Figure 7A and 7B, bars to the left). These findings suggest that VIP seed coat has a stimulatory effect on early plant growth. Phosphate availability:
Plant growth is often limited by the availability of phosphate in soil. Figure 8 shows the potential of VIP seed coat to solubilize inorganic phosphate. Significant clearing zones were observed around VIP-coated oilseed rape and winter wheat seeds at two days following coating and plating on Pikowskaya medium (Figure 8, white arrowheads). Minor clearing zones were also observed around control winter wheat seeds, indicating that phosphate-solubilizing microbes are naturally present on winter wheat seeds. Clearing zones do however increase significantly in size around seeds that have been coated with VIP seed coat, suggesting that VIP seed coat has potential to improve phosphate availability.
Anti-fungal effect:
Keeping seeds on Pikowskaya medium for an additional 4-5 days further demonstrated potential antifungal effects of VIP seed coat. Figure 9 shows a significant growth of fungi in control (uncoated) seeds, especially in winter wheat seeds (Figure 9, black arrowheads). Coating seeds with VIP seed coat resulted in an apparent decrease in fungal contamination. This suggests that VIP seed coat does not only have phosphate-solubilizing properties, but may also act as an antifungal.
Conclusion
Administration of beneficial microbes to crop seeds before sowing can benefit plant growth by for example increasing the availability of phosphate in soil. Here, we show that VIP seed coat has multiple beneficial effects on early plant growth of agricultural crops in vitro. Coating seeds with VIP seed coat stimulates early germination processes by increasing the emergence of radicle and plumule, increases phosphate solubility in the vicinity of seeds, and results in less fungal contamination. Collectively, these data demonstrate the plant growth-promoting properties of VIP seed coat.
The compatibility of the VIP bacteria with other biopolymers has been tested and it is compatible with biopolymers as different as starch, carboxymethylcellulose and chitosan. It may therefore likely work with other biopolymers such as agar, agarose, guar gum, xanthan gum and alginate. Example 5 - Field testing of VIP
Aim of Study
To test whether strain VIP may alter plant properties in the field.
Materials and methods
Spring Barley seeds (Hordeum vulgare, variety "Fantex"), were used in both trials. FloraGro was bought from BioPlant whereas Serenade ASO was bought from Brdr Ewers. Both FloraGro and Serenade ASO contain pure cultures of Bacillus bacteria. FloraGro is a biostimulant.
Serenade ASO is a fungicide i tool designed to protect against the effects of soil and foliar bacterial and fungal diseases.
The trials were carried out by an independent third-party contract research organization (Agrolab).
Seed treatments were carried out by applying lOOmL VIP or FloraGro onto 100kg seeds. The seeds where then airdried and sown on the 27 April 2021 on a field in Denmark with 4 replicates of each treatment. The daily mean temperature at sowing was 8°C. The seed treatment fields were fertilized with NPK at a rate of 120kg N/ha and the fields were grown conventionally. The fields were harvested on the 15 august 2021.
Spray treatments were on fields that were sown on the 27 April 2021 on a field in Denmark with 4 replicates of each treatment. The daily mean temperature at sowing was 8°C. The fields were fertilized with NPK at a rate of 120kg N/ha and the fields were grown conventionally. The products were used at a rate of 4L/ha and the products were diluted 1: 50 in water before application. The sprayings were carried out on 10, 14 and 24 May with daily mean temperatures of 15°C, 14°C and 20°C, respectively. Spraying was carried out with a spray height of 50cm, a pressure of 2.4 bar, a nozzle size of 110 micrometers and a nozzle spacing of 50cm. The fields were harvested on the 15 august 2021.
Results The use of VIP as a seed treatment for spring barley resulted in the emergence of 282 plants on average per m2 at 4 weeks after sowing. Whereas, on average 261.3 and 269.3 seeds emerged per m2 for the non-treated groups and FloraGro treated groups, respectively.
Average harvest parameters for the VIP treated plots were (yield 10.25 tons/ha, hectoliter weight 74.10 kg/hl, protein content 10.55%, total protein yield 1.08 tons/ha). The non-treated control group resulted in the following average harvest parameters (yield 9.66 tons/ha, hectoliter weight 73.88 kg/hl, protein content 10.43%, total protein yield 1.01 tons/ha), while the FloraGro treated benchmark group gave the following parameters (yield 9.91 tons/ha, hectoliter weight 73.44 kg/hl, protein content 10.38%, total protein yield 1.03 tons/ha).
The use of VIP as a spray treatment for spring barley resulted in the following average harvest parameters (yield 7.28 tons/ha, hectoliter weight 70.63 kg/hl, protein content 9.48%, total protein yield 0.69 tons/ha). The non-treated control group resulted in the following average harvest parameters (yield 7.00 tons/ha, hectoliter weight 70.20 kg/hl, protein content 9.18%, total protein yield 0.64 tons/ha), while the Serenade treated benchmark group gave the following parameters (yield 7.26 tons/ha, hectoliter weight 70.35 kg/hl, protein content 8.93%, total protein yield 0.65 tons/ha).
Hectoliterweight is also known as the test weight.
Conclusion
The use of VIP as a seed treatment and/or a spray treatment may increase emergence, harvest yield, hectoliter weight, protein content and protein yield.
Example 6 - VIP and potential applications in the inhibition of bacteria in human, animal and plant health
Aim of study
Antimicrobial resistance (AMR) in pathogens of plants, animals and humans is becoming a major problem for food production and human health. The aim of this example is to demonstrate that the VIP can inhibit multi-resistant bacteria. Broad classes of multi-resistant bacteria were exemplified with the Gram-positive vancomycin resistant enterococci (VRE), the Gram-positive methicillin resistant staphylococcus aureus (MRSA) and the Gram-negative extended spectrum betalactamase producing Escherichia coli (ESBL), which are all major pathogens of humans and animals.
Materials and methods
Mueller-Hinton or 5% blood agar plates containing a lawn of either VRE, MRSA, ESBL, were created by plating a 0.5 McFarland solution of these pathogens onto the agar plates using a cotton swab and a petri dish rotator. VIP were spotted from pure cultures onto these lawns and were incubated 24h at 37°C. Inhibition zones were then visualized and photographed.
Results
VIP produced a clear inhibition zone on VRE (Fig ID), ESBL (Fig 1A) and MRSA (Fig 1C) showing that it inhibits these pathogens.
Conclusion
VIP inhibits a diverse selection of multi-resistant bacteria that cause diseases.
VIP may therefore find use as a probiotic, a cosmetic ingredient, a food additive, a feed additive, a veterinary medicine, a biocide, an antibiotic, a pharmaceutical, or a biotechnological product.
Example 7 - Field applications in winter crops
Aim of study
To test the ability of the bacteria to affect emergence and growth of winter crops.
Materials and methods
A bacterial solution containing 10 McF VIP bacteria in 2% pecton was added to winter wheat and rapeseed seeds in a rotating seed coating machine at a ratio of 8mL/kg (winter wheat) and 32mL/kg (rapeseed). The seeds were then air dried and stored in a bag until sowing. The rape seed was sown in August 2022 and the winter wheat was sown in October 2022, both on fields on Zealand, Denmark. Emergence was counted 21 days after sowing (rapeseed) and 33 days after sowing (winter wheat) and was quantified as plants/m2. Above ground biomass of rapeseed was measured by cutting, drying and weighing plants from 2 x 0.5m2 at 2 months after sowing.
Results
Emergence in winter wheat treated with VIP was 327 plants/m2 whereas it was 311 plants/m2 in the non-treated control group giving an increase in emergence of 5.3% (paired t-test p = 0.00041). Emergence in rapeseed treated with VIP was 46 plants/m2 whereas it was 31 plants/m2 in the non-treated control group giving an increase in emergence of 37.4% (paired t-test p = 0.0014). Biomass in rapeseed treated with VIP was 154 g/m2 whereas it was 113 g/m2 in the nontreated control group giving an increase in biomass of 41 g/m2 or 36% (paired t- test p = 0.011).
Conclusion
VIP can enhance the emergence of crops as diverse as rapeseed (belonging to the plant family Brassicaceae, a dicot) and winter wheat (belonging to the plant family Poaceae, a monocot). VIP is also capable of increasing the biomass of a crop and therefore the rate of photosynthesis, carbon capture and carbon storage in biomass.
Example 8 - Field applications in spring crops
Aim of study
The aim of this study was to test the ability of VIP to affect the growth of various spring crops when applied as a seed coat before sowing.
Materials and methods
Seeds from spring crops listed in Table 6 were used for field trials (performed by an independent third-party contract research organization (VKST, Denmark)). Seed treatments were carried out by applying a bacterial seed coat solution containing 4xl09 CFU/mL VIP to seeds at a rate of 2.5 mL/kg. Seeds were stirred during the seed coat application to ensure an even distribution of the seed coat. Untreated seeds were used as controls for each respective crop (Table 6). Following seed coat application, seeds were dried over night at 8C° and sown on fields April 2023 (between April 10 and 22, mean temperature April 2023 was 7C°). Field areas were fertilized with either a combination of nitrogen-sulfur (NS; 30% nitrogen, 10% sulfur) + phosphate-potassium-sulfur (PKS; 10% phosphate, 20% potassium, 3% nitrogen) fertilizers or a recycled organic fertilizer (0GRO;
10% nitrogen 3% phosphate, 1% potassium) (Table 6) at the day before sowing (faba bean and pea fields were not fertilized). Fertilizers were applied at two rates: 75% NS+PKS; 287 kg/ha NS + 188 kg/ha PKS, 100% NS+PKS; 382 kg/ha NS + 250 kg/ha PKS, 75% 0GRO; 667 kg/ha, 100% 0GRO; 889 kg/ha. Early and late emergence of plants were quantified at 14 and 42 days following sowing as plants per m2. Fields were harvested in the period August-September 2023.
Table 6: Spring crops used for field trials. Trials were carried out by an independent third-party contract research organization (VKST, Denmark). A combination of nitrogen-sulfur (NS) + phosphate-potassium-sulfur (PKS) fertilizers or a recycled organic fertilizer (0GRO) were applied to fields where oat, spring barley and spring wheat seeds were sowed.
Figure imgf000049_0001
Results
Treating seeds from oat, spring barley, pea, spring wheat and faba bean crops (Table 6) with a seed coat containing a bacterial solution of VIP before sowing affected the emergence in all spring crops tested in fields (Figure 10). The early emergence (14 days after sowing) was significantly (p<0.05, one-sided t-test) enhanced in five crops (faba bean, Pea, barley (variety 'KWS Irina'), oat and wheat) (Figure 10A, B, D, E and F) and this was interestingly observed on both non-fertilized (Figure 10A and B) and fertilized fields (Figure 10D, E and F). Moreover, faba bean plants developing from VIP-treated seeds produced a significant (p=0.03032, one-sided t-test) higher yield of faba beans (482 kg more per ha) compared to control plants. Even though the early emergence seemed to be most improved in crops grown on non-fertilized fields (between 18 and 19%), VIP was still able to enhance the early emergence significantly (p<0.05, onesided t-test) in crops grown on fertilized fields (between 4% and 11%), indicating that VIP can improve crop emergence even in the presence of a fertilizer. A similar tendency was also observed for the late emergence time point (42 days after sowing) where the emergence was significantly (p<0.05, one-sided t-test) increased in four crops (pea, barley (varieties 'Evergreen' and 'KWS Irina') and wheat) (Figure 1OB, C, D and F) and this was also observed on both nonfertilized (18%) (Figure 1OB) and fertilized fields (between 5% and 9%) (Figure IOC, D and F).
Conclusion
Treating seeds from some of the most common grown spring crops in Denmark, including grain and legume crops, with a bacterial solution containing VIP before sowing demonstrates that VIP can enhance the emergence of crops in the field and lead to a higher harvest yield.
Example 9 - VIP increases the yield of vegetables
Aim of study
The aim of this study was to test the ability of VIP to affect the yield of vegetables.
Materials and methods
Onion
Onion sets (Allium cepa 'Sturon', 10-21 mm) were dipped in a bacterial solution containing 1 McF VIP (n=50) or water (control, n = 50). Onions were air dried for 2 hours and planted in a field on Funen, Denmark, on April 21 2023 in 5 rows of 10 plants at a depth leaving only the top of the onions exposed. Onions were planted with 10 cm between each set and each row was placed 20 cm apart. Onions were harvested on July 19-20 2023.
Potato
Seed potatoes (Solanum tuberosum 'Bintje') were dipped in a bacterial solution containing 1 McF VIP (n=50) or water (control, n = 50). Seed potatoes were air dried for 2 hours and planted in a field on Funen, Denmark, on April 21 2023 in 5 rows of 10 plants at a depth of 2.5 cm with 30 cm between each seed potato. Rows were placed 50 cm apart. Potatoes were harvested on August 12-13 2023. Maize
Maize seeds (Zea mays, silage hybrid) were dipped in a bacterial solution containing 1 McF VIP (n= 10) or water (control, n = 10). Immediately following dipping, maize seeds were sown in a field on Funen, Denmark, on April 21 2023 in rows at a depth of 2 cm with 2 cm between each seed. Rows were placed 50 cm apart. Maize plants were harvested on August 25 2023.
Results
Treating onion sets, seed potatoes and maize seeds with a bacterial solution of VIP before sowing led to significant increases in harvest yields across all tested vegetables (Figure 11). The fresh weight of onions harvested from VIP treated onion sets was on average 122 grams per plant compared to 101 grams per control plant (Figure 11A), resulting in a significant increase in onion yield of 21% (p-value = 0.02759, one-sided t-test). The fresh weight of potatoes harvested from VIP treated seed potatoes was on average 693 grams per plant compared to 586 grams per control plant (Figure 11B), resulting in a significant increase in potato yield of 18% (p-value = 0.02292, one-sided t-test). The fresh weight of maize cobs harvested from VIP treated seeds was on average 512 grams per plant compared to 363 grams per control plant (Figure 11C), resulting in a significant increase in maize yield of 41% (p-value = 0.004187, one-sided t- test).
Conclusion
Treating onion sets, seed potatoes and maize seeds with a bacterial solution containing VIP before sowing in fields demonstrates that VIP can affect the harvest yield of vegetables as diverse as onions, potatoes and maize by a minimum of 18%.
Example 10 - VIP has mineral-dissolving properties at low, medium and high temperatures
Aim of study
The aim of this study is to demonstrate the ability of VIP to increase the availability of common plant nutrients specifically under low, medium, and high temperature conditions. Materials and methods
To demonstrate the ability of VIP to increase the availability of nitrogen, phosphate, and potassium, VIP was cultivated on different solid media: nitrogen- free agar; Jensen medium, insoluble phosphate agar; Pikowskaya agar, and insoluble potassium agar; Aleksandrow agar. These three solid media are recognized for being specific for detection and cultivation of nitrogen-fixing, phosphate-solubilizing, and potassium-solubilizing soil microorganisms. Growth on Jensen agar indicates nitrogen-fixating abilities, as this medium is free of nitrogen. A halo zone on Pikowskaya and Aleksandrow agar indicates solubilization of calcium phosphate and potassium alumino-silicate, respectively. The solubilization capacity was evaluated by calculating a solubilization index (SI, SI = Areanaio/Areacoiony). To demonstrate temperature-dependent activities, experiments were performed at low (5°C), medium (15°C), and high (25°C) temperatures. Typically, spring seeding will occur at soil temperatures around 6- 8°C in Northern Europe, and activity at lower temperatures is therefore of great importance at this latitude. A commercial microbial biostimulant and biofungicide, biol and bio2 respectively, were included in the experiment as benchmarking controls. The active microorganisms in these products were isolated and included in the test on equal footing with VIP: A fluid culture of each bacterial strain was prepared in a concentration of 0.5 McFarland. The solution was then spotted onto plates containing the different media in repetitions of three. The plates were incubated at either 5°C, 15°C, or 25°C. Colony and halo zone areas were measured each day using imageJ.
Results
The ability of VIP 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 were quantified and furthermore compared to bacterial strains isolated from commercial biostimulant products (Figure 12). VIP 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, VIP exerted mineral-dissolving properties at all temperatures (Figure 12B and C) while the bacterial control strains isolated from products only demonstrated little (Figure 12B) or no (Figure 12C) capacity towards dissolving phosphate or potassium, respectively. This suggests that VIP has a better capacity towards dissolving minerals which are otherwise unavailable for plants.
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 VIP was able to grow and display bacterial activity at 5°C, demonstrating that VIP 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.
Figure imgf000054_0001
Figure imgf000054_0002
FOR RECEIVING OFFICE USE ONLY
Figure imgf000054_0003
FOR INTERNATIONAL BUREAU USE ONLY
Figure imgf000054_0004

Claims

Claims
1. A bacteria, preferably isolated, or biologically pure bacterial culture comprising a) a genomic sequence according to any of SEQ ID NOs: 1-7; b) a genomic sequence having 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: 1-7; and/or c) a fragment of one or more of the sequences of a) or b), having a length of at least 500 nucleotides.
2. The bacteria or biologically pure bacterial culture according to claim 1 comprising any of SEQ ID NO: 1-7.
3. The bacteria or biologically pure bacterial culture according to claims 1 or 2, comprising a) a genomic sequence according to SEQ ID NO: 8; b) a genomic sequence having at least 90% sequence identity to SEQ ID NO: 8, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.9 % sequence identity to SEQ ID NO: 8; and/or c) a fragment of the sequence of a) or b), having a length of at least 5000 nucleotides, such as at least 8000 nucleotides, such as at least 10000 nucleotides, such as at least 14000 nucleotides.
4. The bacteria or biologically pure bacterial culture according to any of claims 1-
3, being of the genus Pseudomonas, such as species Pseudomonas protegens.
5. The bacteria or biologically pure bacterial culture according to any of claims 1-
4, comprising a 16S rRNA gene encoded by SEQ ID NO: 9.
6. A bacteria or biologically pure bacterial culture being Pseudomonas protegens, DSM 34378 deposited with the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany] on 15 September 2022.
7. The bacteria or biologically pure bacterial culture according to any of the preceding claims, being
- capable of solubilizing calcium phosphate at 8°C and/or potassium aluminum silicate at 8°C; and/or
- capable of inhibiting MRSA, VRE or ESBL.
8. 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 the preceding claims.
9. The fertilizer and/or inoculant and/or biostimulant and/or biofungicide and/or antimicrobial composition according to claim 8 further comprising other microorganisms, such as other microorganisms able to function as a biostimulant.
10. A coating composition, preferably a seed coating composition, comprising the bacteria or biologically pure bacterial culture according to any of the claims 1-7 and/or the composition according to claims 8 or 9.
11. The coating composition according to claim 10 comprising a biopolymer promoting adherence to a plant seed.
12. A plant seed coated with the composition according to claims 8 or 9 or coated with a coating composition according to claims 10 or 11.
13. Use of an bacteria or biologically pure bacterial culture according to any of claims 1-7, the composition according to claims 8 or 9 or the coating composition according to claims 10 or 11, as a plant growth promoting agent, such as a fertilizer or inoculum or biostimulant.
14. The use according to claim 13, wherein the use as a plant growth-promoting agent is for:
• solubilizing inorganic minerals or salts, such as phosphate and/or potassium; 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.
15. The use according to claim 14, 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, 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.
16. The use according to any of claims 13-15, 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.
17. The use according to any of claims 13-16, wherein the use takes place at field temperatures in the range 2°C to 8°C.
18. A method for stimulating plant growth comprising applying the bacteria or biologically pure bacterial culture according to any of claims 1-7, the composition according to claims 8 or 9 or the coating composition according to claims 10 or 11 to a plant, plant seed, a sowing forrow, soil and/or plant growth medium.
19. The method for stimulating plant growth according to claim 18, wherein 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, and vegetables, such as onion.
20. 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 1-7, the composition according and/or the coating composition according to claims 10 or 11; and
• instructions for applying the inoculum to plants, plant seeds, or a plant growth medium.
21. The bacteria or biologically pure bacterial culture according to any of claims 1- 7, the composition according to claims 8 or 9 and/or the coating composition according to claims 10 or 11 for use as a medicament.
22. The bacteria or biologically pure bacterial culture according to any of claims 1- 7, the composition according to claims 8 or 9 for use in the treatment, alleviation and/or prevention of bacterial infections, such as E. coll, spectrum beta-lactamase (ESBL)-producing bacteria, Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).
23. Use of the bacteria or biologically pure bacterial culture according to any of claims 1-7, the composition according to claims 8 or 9 for solubilizing minerals in ores.
PCT/EP2023/081608 2022-11-18 2023-11-13 Novel pseudomonas strain WO2024104961A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020214843A1 (en) 2019-04-17 2020-10-22 Andes Ag, Inc. Novel seed treatment methods and compositions for improving plant traits and yield
WO2021119867A1 (en) * 2019-12-20 2021-06-24 Universidad de Concepción Strain of pseudomonas protegens rgm 2331 and use thereof to produce a biostimulant with antifungal properties to promote growth in plants
WO2021180941A1 (en) 2020-03-13 2021-09-16 Syddansk Universitet Device for capturing microorganisms from the environment
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WO2020214843A1 (en) 2019-04-17 2020-10-22 Andes Ag, Inc. Novel seed treatment methods and compositions for improving plant traits and yield
WO2021119867A1 (en) * 2019-12-20 2021-06-24 Universidad de Concepción Strain of pseudomonas protegens rgm 2331 and use thereof to produce a biostimulant with antifungal properties to promote growth in plants
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