Classifications

• • 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
  • A01P21/00—Plant growth regulators
• • 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
• • 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; 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
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; 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
• • 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

Definitions

• the present invention relates to a novel isolated plant microbiome strain, in particular an endophyte, plants infected with such strains and related methods.
• the isolated endophyte may be used in a composition for use as a biofertilizer and/or biostimulant or for bioprotection in plants.
• the composition may further comprise an adjuvant.
• the growth and/or yield of agricultural crops is limited by the amount of nitrogen that can be used or taken up by the plant.
• exogenous nitrogen is added to the soil before or after sowing.
• the nitrogen fertilizer can be of organic (e.g., urea, amino acids, manure, horn shavings) or mineral (e.g. ammonium nitrate, ammonium sulphate, potassium nitrate) origin in nature. What all these anthropogenic nitrogen fertilizers have in common is that their production, transport and/or application is energy-intensive and thus leads to a positive CO2 balance.
• ammonium nitrate and urea which are by far the most widely used nitrogen fertilizers worldwide, have a large CO2 footprint because their common precursor ammonia (NH3), is derived from the Haber-Bosch process.
• This process uses atmospheric nitrogen (N2) and hydrogen (H2) to synthesize ammonia (NH3) under high temperatures and pressures. This is process thus adds to climate change and is not good for the environment.
• Biofertilizers are made up of living microorganisms that are able to reduce atmospheric nitrogen and thus make it available to the plant in form of ammonium/ ammonia or organically bound nitrogen. These organisms have to be applied in a metabolically active form to the seed, root or in rarer cases to the leaf.
• biofertilizers apply the nitrogen-fixing microorganisms either to the surface of the seed, to the leaf or the soil. Due to this fact, the organisms are exposed to high stress from abiotic factors such as temperature, drought, pH, and wash-off from rain and high moisture. The effectiveness and efficiency of the biofertilizers is reduced. A large amount of the biofertilizers is thus needed to enable them to be effective in providing bioprotection to the plants and/or increasing yield of the crops. This increases the costs of farming.
• biotic factors such as high competition with the already predominant plant or soil microbiome or the presence of antibiotically active substances also reduce the survival rate of the exogenously added nitrogen-fixing microorganisms on the respective surfaces. This also reduces the efficiency of the currently available biofertilizers.
• FIGURES Figure 1 is a picture of maize growth (without treatment) after 6 eight weeks under different nitrogen fertilization (0 kgN/ha, 21 kgN/ha, 42 kgN/ha). Nutrient-poor Oxisol soil was used as test soil (substrate).
• Figure 2 is a picture of maize growth at 21 kgN/ha, once without (left) and once with application of Paenibacillus xylanexedens DSM 34353, which was applied in a mixture with S301® to the leaf only.
• Figure 3 is a graph showing shoot dry matter of maize at different nitrogen fertilizer levels, with and without application of Paenibacillus xylanexedens DSM 34353, which was applied in a mixture with S301®.
• the present invention attempts to solve the problems above by providing a novel isolated endophyte from Paenibacillus xylanexedens that is capable of being an effective and efficient biostimulant and/or biofertilizer.
• These newly isolated endophytes which due to their unique genetic equipment and/ or in the presence of specific adjuvants, are able to enable and especially enforce the penetration of these endophytes into the plant or part thereof to which the endophyte is brought into contact with.
• the newly isolated Paenibacillus xylanexedens strain is able to penetrate and proliferate in the plant tissue or seed.
• the newly isolated endophyte, Paenibacillus xylanexedens with Accession Number DSM 34353, solves the problem of low stability of microbial nitrogen fixers as it is an endophytic organism that has both an unprecedented high nitrogen fixation capacity and the ability to penetrate into the endosphere of the plant that most other existing endophytes used in agriculture do not have.
• the endophyte according to any aspect of the present invention was isolated from the inner plant tissue of a copper flower (Minuartia verna subsp. hercynica) and typed and sequenced as Paenibacillus xylanexedens sp. Greenhouse experiments demonstrated that Paenibacillus xylanexedens is capable of providing significant amounts of enzymatically fixed nitrogen to the plant in both seed and foliar applications to maize (Zea mays, variant LG 31 .224).
• endophyte is an endosymbiont, which refers to a bacterial or fungal strain that lives within a plant for at least part of its life cycle without causing apparent disease.
• the bacteria or the fungus is closely associated with the plant where the term ‘closely associated’ refers to the bacteria or fungus living on, in or in close proximity to the plant.
• it may be endophytic, and living within the internal tissues of the plant, or epiphytic, and growing externally on the plant. There are many different endophytes that have been discovered.
• endophytic inoculants for agriculture such as arbuscular mycorrhizae, rhizobia, and Azospirillium.
• Clavicipitaceous fungi is also an endophyte that is used in agriculture.
• the endophyte according to any aspect of the present invention is a strain of Paenibacillus xylanexedens with Accession Number DSM 34353.
• the term “substantially purified” refers to an endophyte being free of other organisms.
• the term includes, for example, an endophyte in axenic culture.
• the endophyte is at least about 90% pure, more particularly at least about 95% pure, even more particularly at least about 98%, 99% or 99.5% pure.
• the term ‘isolated’ refers to an endophyte according to any aspect of the present invention that is removed from its original environment (e.g., the natural environment if it is naturally occurring).
• a naturally occurring endophyte present in a living plant is not isolated, but the same endophyte separated from some or all of the coexisting materials in the natural system, is isolated.
• the isolated endophyte according to any aspect of the present invention may be a pure culture of a single strain and this single strain was submitted to the German Collection of Microorganisms and Cell Cultures (DSMZ) located in InhoffenstraBe 7B, 38124 Braunschweig, Germany on 11 th August 2022 and has Accession Number DSM 34353. Particles for keeping and modifying cells are available from the prior art, for example Sambrook/Fritsch/Maniatis (1989).
• bioprotection and/or biostimulant may refer to the endophyte according to any aspect of the present invention possessing genetic and/or metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with, the endophyte.
• Such beneficial properties or phenotypes resulting from the endophyte being present in the plant include improved resistance to pests and/or diseases, improved tolerance to water and/or nutrient stress, enhanced biotic stress tolerance, enhanced drought tolerance, enhanced water use efficiency, reduced toxicity and enhanced vigour in the plant with which the endophyte is associated, in comparison to a plant which is not associated with the endophyte according to any aspect of the present invention or to a endophyte such as standard toxic (ST) endophyte.
• the bioprotection and/or biostimulant phenotype according to any aspect of the present invention includes nitrogen fixation in the plant into which the endophyte is introduced.
• the pests and/or diseases may include, but are not limited to, fungal and/or bacterial pathogens, particularly, fungal.
• the endophyte may result in the production of the bioprotectant compound in the plant with which it is associated.
• bioprotectant compound refers to a compound that provides or aids in providing bioprotection to the plant with which it is associated against pests and/or diseases, such as bacterial and/or fungal pathogens.
• a bioprotectant compound may also be known as a ‘biocidal compound’.
• biostimulant refers to 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.
• the endophyte according to any aspect of the present invention acts as a biostiumulant to the plant and/ or part thereof to which it comes in contact with.
• a more detailed definition of biostimulant is provided at least in Ricci, M., General Principles to Justify Plant Biostimulant Claims, Frontiers in Plant Science (2019), 10.
• the term ‘introduce’ refers to the contact and/or treatment of a plant or part thereof with an endophyte where the endophyte is delivered to the plant.
• the endophyte is introduced into the plant or part thereof to encourage the endophyte to grow there.
• Any method of introduction of the endophyte according to any aspect of the present invention to the plant or part thereof may be used.
• the endophyte may be sprayed on or inoculated in the plant or part thereof.
• the endophyte may be inoculated for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 days before they start to grow in the plant.
• active portion refers to an amino acid sequence or a nucleic acid sequence, which is less than the full-length amino acid sequence or codes for less than the full-length amino acid sequence, respectively, wherein the amino acid sequence or the amino acid sequence encoded, respectively retains at least some of its essential biological activity.
• D 1 is P 1 2SiO2/2, D' is P 1 P 2 SiO2/2, o is 2, p is between 0 and 0.1 , q is between 1 .0 and 1.15,
• P 1 are independently hydrocarbyl having 1 to 8 carbon atoms
• P 2 are independently a polyether radical of the formula (III)
• P 3 are independently divalent hydrocarbyl radicals having 2 to 8 carbon atoms, P 5 is hydrogen; and/or
• R radicals are each independently identical or different, aliphatic or aromatic hydrocarbyl radicals having 1 to 10 carbon atoms
• the R4 radicals are each independently identical or different R
• the R1 , R2 and R3 radicals are each independently different polyether radicals of general formula (V)
• R5 are independently the same or different and are each a methyl, acetyl or hydrogen radical. More in particular, the adjuvants may be selected from the group consisting of BREAK-THRU® S 301 , BREAK-THRU® SP 133, BREAK-THRU® S 255.
• the adjuvants used according to any aspect of the present invention leads to a reduction of the surface tension at the stomata or where there are injuries and thus to a lower rejection or an improved flow of the particles (microorganisms) through the orifices into the plant or part thereof where the microorganisms particularly Paenibacillus xylanexedens is inoculated.
• the use of adjuvants enables/ accelerates the endophytic process of bacteria uptake.
• enhanced uptake also allows translocation of bacteria across the phloem from the leaf to the root, from where nitrogen fixation can also be enhanced. This results in the locally applied biostimulant acquiring a systemic character. Based on its systemic character, there is the advantage that in shoot-forming plants also the shoot is already inoculated with the biostimulant and thus passes the active ingredient on to the new generation.
• adjuvants also allows a uniform distribution of the Paenibacillus xylanexedens from the upper leaf surface to the lower leaf surface where most (opened) stomata are present. Thus, faster and more widespread penetration of endophytes into plant tissue is enabled. Without the use of biocompatible adjuvants, especially in the case of foliar application, reaching the stomata on the underside of the leaf would be particularly hard if not impossible.
• adjuvants with “anti- rinse-off’ properties also increases the residence time on the upper surface of the leaf and thus promotes endophytic uptake into the plant tissue. The presence of the adjuvant reduces early wash-off of the endophytic nitrogen fixating organisms.
• the adjuvant is (A):
• At least one radical R” corresponds to a radical of the formula R' — C(O) — . More in particular, M, D and T may be:
• polyglycerol esters of the mixture according to any aspect of the present invention is of the Formula (1(a)):
• the radicals R” of the formula R' — C(O) — may be independent of each another identical or different acyl radicals of saturated or unsaturated fatty acids, where the fatty acids include 4 up to 40 carbon atoms, particularly, the fatty acids are selected from the group consisting of butyric acid (butanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), capric acid (decanoic acid), lauric acid (dodecanoic acid), myristic acid (tetradecanoic acid), palmitic acid (hexadecanoic acid), stearic acid (octadecanoic acid), arachidic acid (eicosanoic acid), behenic acid (docosanoic acid), lig
• the fatty acid may be a mixture of rapeseed oil acids, soya fatty acids, sunflower fatty acids, peanut fatty acids and tall oil fatty acids.
• the fatty acids may be radicals of oleic acid.
• the molar mass of the lipophilic molecule moiety is the arithmetic mean of the total of the molar masses of all of the radicals R' which are present in the molecule.
• Sources of suitable fatty acids or fatty acid esters, especially glycerides can be vegetable or animal fats, oils or waxes.
• vegetable or animal fats, oils or waxes can be vegetable or animal fats, oils or waxes.
• the adjuvant is (B):
• D 1 is P 1 2SiO2/2, D' is P 1 P 2 SiO2/2, o is 2, p is between 0 and 0.1 , particularly 0 q is between 1.0 and 1.15, particularly between 1.0 and 1.10, especially particularly between 1 .00 and 1 .05,
• P 1 are independently hydrocarbyl having 1 to 8 carbon atoms, particularly methyl, ethyl, propyl or phenyl radicals, especially particularly methyl radicals
• P 2 are independently a polyether radical of the formula (III)

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• 2023
  • 2023-08-24 EP EP23764247.5A patent/EP4580408A1/de active Pending
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