WO2018197433A1 - Method and composition for improving nutrient acquisition of plants - Google Patents

Method and composition for improving nutrient acquisition of plants Download PDF

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Publication number
WO2018197433A1
WO2018197433A1 PCT/EP2018/060378 EP2018060378W WO2018197433A1 WO 2018197433 A1 WO2018197433 A1 WO 2018197433A1 EP 2018060378 W EP2018060378 W EP 2018060378W WO 2018197433 A1 WO2018197433 A1 WO 2018197433A1
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WO
WIPO (PCT)
Prior art keywords
alkyl
independently
ammonium
plants
aryl
Prior art date
Application number
PCT/EP2018/060378
Other languages
French (fr)
Inventor
Günter Neumann
Original Assignee
Eurochem Agro Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eurochem Agro Gmbh filed Critical Eurochem Agro Gmbh
Priority to AU2018258983A priority Critical patent/AU2018258983A1/en
Priority to RU2019137648A priority patent/RU2019137648A/en
Priority to EP18719854.4A priority patent/EP3615495A1/en
Priority to CN201880042434.4A priority patent/CN110831914A/en
Priority to BR112019022106A priority patent/BR112019022106A2/en
Publication of WO2018197433A1 publication Critical patent/WO2018197433A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05C—NITROGENOUS FERTILISERS
    • C05C3/00—Fertilisers containing other salts of ammonia or ammonia itself, e.g. gas liquor
    • C05C3/005—Post-treatment
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05C—NITROGENOUS FERTILISERS
    • C05C3/00—Fertilisers containing other salts of ammonia or ammonia itself, e.g. gas liquor
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
    • C05D9/00—Other inorganic fertilisers
    • C05D9/02—Other inorganic fertilisers containing trace elements
    • 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
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F3/00—Fertilisers from human or animal excrements, e.g. manure
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C05G3/90—Mixtures of one or more fertilisers with additives not having a specially fertilising activity for affecting the nitrification of ammonium compounds or urea in the soil
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
    • Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Definitions

  • the present invention relates to a method for improving nutrition of plants with one or more minerals, comprising the administration of at least one ammonium source A, at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties, and at least one nitrification inhibitor C to a substrate S on which the plants are cultivated or intended to be cultivated. Further, the present invention relates to a composition and a kit usable for this purpose.
  • nitrogen and phosphate salts are among the most limiting factors in most soils. Therefore, nitrogen-based fertilizers are widely used which can be based on synthetic fertilizers such as ammonium or nitrate salts, or organic wastes such as manure, compost, clearing sludge, moist rest residue of a biogas plant, or the like.
  • At least one other mineral becomes the limiting factor such as often phosphor (P, typically phosphate), iron (Fe), zinc (Zn) and/or manganese (Mn).
  • P typically phosphate
  • Fe iron
  • Zn zinc
  • Mn manganese
  • soil microbial communities comprise populations with both, beneficial and inhibitory effects on plant growth.
  • Many beneficial microorganisms are able to increase the plant availability of mineral nutrients by chemical mobilization or by stimulation of root growth, while others are effective in the suppression of pathogens.
  • Attempts to supplement soils with additional amounts of plant growth-promoting microorganisms showed some beneficial activity.
  • the success was limited since the conditions promoting a successful establishment of these populations are not well understood and obviously affected by many external factors.
  • a first aspect of the invention relates to a method for improving nutrition of plants with one or more sparingly soluble minerals selected from the group consisting of phosphor, iron, zinc and manganese, said method comprising the following steps:
  • At least one ammonium source A component A
  • at least 50 mol% of the total nitrogen content of A is present as ammonium and/or organically bound nitrogen
  • B at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties (component B), wherein B does not form part of A, and
  • step (iii) enabling growth of the plants in the substrate S obtained from step (ii).
  • the method according to the present invention enables improving nutrition of plants without the need of applying the substrate S with soluble salts of the (sparingly soluble) minerals, such as minerals selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese. Accordingly, the method does preferably not comprise a step of adding such soluble salts to the substrate S.
  • the substrate S is not supplemented with soluble salts of the (sparingly soluble) minerals, such as minerals selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese.
  • the substrate S is not supplemented with (considerable amounts of, i.e., more than traces of) soluble salts of phosphor (typically phosphate).
  • the components typically may comprise traces of phosphor (typically phosphate).
  • the components A, B and C as described herein are essentially not supplied in combination with a mineral selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese, in particular are essentially not supplied in combination with phosphor (typically phosphate).
  • the components A, B and C are (essentially) not supplied in combination with such salts in solid form and/or of synthetic origin.
  • the amount of phosphor (e.g., present as phosphate) supplied to the substrate S is below 10% by weight (w/w), based on the sum of the weight of the components A, B and C supplied on a substrate S within one month, one week or one month from the point of time of supplying the components A, B and C to the substrate A.
  • the amount of phosphor supplied to the substrate S is below 5% (w/w), preferably below 1 % (w/w) or below 0.1 % (w/w), based on the sum of the weight of the components A, B and C supplied on a substrate S within one week from the point of time of supplying the components A, B and C to the substrate A.
  • sparingly soluble may be understood in the broadest sense as not being well water soluble.
  • a sparingly soluble mineral will be understood as a salt or complex having a water solubility of ⁇ 0.1 g per liter, preferably not more than 100 mg per liter, more preferably not more than 10 mg per liter, in particular not more than 10 mg per liter (at ambient temperature of 20°C).
  • sparingly soluble usually means a solubility of not more than 1 mg per liter, even in well fertilized soils.
  • the method according to the present invention also achieves improving the effectivity of the bacteria or fungal species B.
  • the bacteria or fungal species B also designated as biostimulants
  • the bacteria or fungal species B are boosted (i.e., promoted) by means of applying these in combination with at least one ammonium source A and at least one nitrification inhibitor C.
  • essentially inaccessible mineral salts such as poorly accessible phosphor (typically phosphate), iron, zinc and/or manganese salts
  • the bacteria were surprisingly found to be particularly effective when working in combination with components A and C.
  • the method according to the present invention also achieves improving the effectivity of the fertilization with an ammonium source A and a nitrification inhibitor C.
  • the combination of components A and C is boosted by means of applying it in combination with at bacteria or fungal species B.
  • the at least one bacteria or fungal species B according to the present invention has phosphate-solubilizing and/or root growth-promoting properties.
  • component B may comprise (or consist of) at least one bacteria or fungal species B having phosphate-solubilizing and/or root growth-promoting properties; and/or at least one fungal species B having phosphate-solubilizing and/or root growth-promoting properties.
  • the "at least one bacteria or fungal species B" according to the invention may be selected from the list consisting of: - a bacteria species having phosphate-solubilizing properties;
  • root growth-promoting may be understood in the broadest sense as any method that stimulates root (rhizome) growth. Moreover, it is considered that ammonium nutrition may promote the microbial production of auxins as a major hormonal factor for induction of root growth. A variety of bacteria and fungal species promoting root growth are well known in the art.
  • P-solubilizing may be understood in the broadest sense as making phosphate (anions) plant available, in other words, enable plants to take up the phosphate of the substrate S and improving the usability of the phosphate sources of the substrate S for plant growth.
  • rock phosphate present in many soils is not water soluble and also not or sparingly available for plants.
  • the rock phosphate contains phosphate anions present in the substrate S, but is still (essentially) not accessible and usably by the plants.
  • the bacteria or fungal species B according to the present invention having phosphate-solubilizing properties render the phosphate available for the plants.
  • solubilization i.e., mobilization
  • solubilization of phosphate occurs directly in proximity to the rhizosphere of the plants. Therefore, typically, the phosphate solubilized by the bacteria and/or fungi B is rapidly taken up by the roots of the plants.
  • the at least one bacteria or fungal species B according to the present invention has phosphate-solubilizing properties. In a particularly preferred embodiment, the at least one bacteria or fungal species B according to the present invention has phosphate-solubilizing and root growth- promoting properties.
  • the method of the invention is for improving nutrition of plants with phosphor (typically phosphate). Alternatively or additionally, the method of the invention is for improving nutrition of plants with iron. Alternatively or additionally, the method of the invention is for improving nutrition of plants with zinc. Alternatively or additionally, the method of the invention is for improving nutrition of plants with manganese. In another preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate) and iron.
  • the method of the invention is for improving nutrition of plants with phosphor (typically phosphate) and zinc. In another preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate) and manganese. In another preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate), iron and zinc. In another preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate), iron and manganese. In another preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate), zinc and manganese.
  • the method of the invention is for improving nutrition of plants with phosphor (typically phosphate), iron, zinc and manganese.
  • the at least one bacteria or fungal species B does not form part of ammonium source A, preferably does not form part of A or substrate S (i.e., does neither form part of A or S).
  • the optionally inherently present bacterial and/or fungal content of component A is enriched/supplemented by the at least one bacteria or fungal species B.
  • the component B is not inherently comprised in the component A, but is added additionally. Therefore, component A may or may not comprise one or more bacteria and/or fungal species optionally having phosphate-solubilizing and/or root growth-promoting properties.
  • the at least one (additional) bacteria or fungal species B is supplied to the substrate S.
  • the components A and B and optionally C and optionally further components are premixed prior to being supplied to the substrate S.
  • this component A is for instance, clearing sludge, moist rest residue of a biogas plant, or a mixture of two or more thereof, this component A will typically comprise bacteria and, optionally, fungal species. Nevertheless, according to the present invention, at least one additional bacteria or fungal species B is added.
  • an amount of species B not forming part of A or S is employed in the method of the invention.
  • the terms “mineral”, “mineral salt”, “nutrient”, “trace mineral”, “plant nutrient” and the like should be understood interchangeably in the broadest sense as any inorganic entity (i.e., atom, molecule or, in particular, salt or ion) comprising at least one element as referred to (i.e., phosphor (typically phosphate), iron, zinc and/or manganese).
  • phosphor typically phosphate
  • iron, zinc and/or manganese refers to any kind of mineral salt comprising one or more of the aforementioned atoms. Typically, these atoms are not found as elements in nature, but rather as molecular bound ions.
  • a phosphor mineral in the context of the present invention may comprise anions like phosphate (PO 4 3- , HPO 4 2- (also designated as hydrogen phosphate), or H 2 PO 4 - (also designated as dihydrogen phosphate)).
  • hydrogen phosphate and dihydrogen phosphate are also considered as phosphate(s).
  • the phosphate is PO 4 3- .
  • the counter ion of such anion may be any cation.
  • the phosphor mineral may also form part of a complex.
  • an iron mineral in the context of the present invention may comprise cations like Fe 2+ or Fe 3+ .
  • the counter ion of such cation may be any anion.
  • the iron mineral may also form part of a complex.
  • the iron is present as the hydrated form of iron oxide.
  • a zinc mineral in the context of the present invention may comprise a cation like Zn 2+ .
  • the counter ion of such cation may be any anion.
  • the zinc mineral may also form part of a complex.
  • a manganese mineral in the context of the present invention may bear an oxidation state of 0, +1 , +2, +3, +4, +5 +6, or +7.
  • the binding partner, complexing partner or counter ion of such atom or cation may be any atom, molecule or cation sufficient for this purpose.
  • the manganese mineral may also form part of a complex.
  • the term "improving nutrition of plants” may be understood in the broadest sense as ameliorating the uptake of the respective mineral. This can be also designated as fertilizing the plants. Typically, improving nutrition leads to better growth and/or health of the plant. Further, the content of the mineral in the plant is preferably increased.
  • the person skilled in the art will directly and unambiguously understand what is meant by (relative) terms like “improving”, “better” or “increasing” in the context of the present invention.
  • Plant growth may be understood in the broadest sense as plant height (e.g., in centimeters (cm)) obtained after a given time and/or biomass weight (e.g., in grams (g) or kilograms (kg)) after a given time and/or stem diameter (e.g., in millimeters (mm)) after a given time.
  • biomass weight e.g., in grams (g) or kilograms (kg)
  • stem diameter e.g., in millimeters (mm)
  • Content of mineral may optionally be provided in milligram of the mineral per kilogram of total plant mass or plant dry mass (provided in mineral mass [mg] / total plant dry mass [kg]).
  • An ammonium source A may be any molecule or molecular composition either comprising ammonium ions (NH + ) or comprising (or consisting of) compounds serving as precursors for ammonium ions such as compounds that are typically metabolized (e.g., by bacteria) or chemically transformed to form ammonium ions, in particular organically bound nitrogen (in particular, primary amino groups, secondary amino groups and tertiary amino groups).
  • ammonium ions NH +
  • compounds serving as precursors for ammonium ions
  • such precursors may be selected from the group consisting of urea, uric acid and ammoniac.
  • At least 50 mol% i.e., >50 % of the total amount of the nitrogen atoms present in the ammonium source A
  • at least 55 mol% more preferably at least 60 mol%, at least 65 mol%, at least 70 mol%, or more than 75 mol%, of the total amount of the nitrogen of A is present as ammonium and organically bound nitrogen.
  • not more than 50 mol% i.e, ⁇ 50 % of the total amount of the nitrogen atoms present in the ammonium source A
  • preferably not more than 45 mol% preferably not more than 40 mol%, in particular not more than 40 mol% or less, is present as the sum of nitrate and nitrite.
  • the at least one ammonium source A is a chemical fertilizer comprising (or consisting of) at least one ammonium salt, manure (liquid or solid manure, in particular liquid), clearing sludge, moist rest residue of a biogas plant, or a mixture of two or more thereof.
  • the at least one ammonium source A is a chemical fertilizer comprising (or consisting of) at least one ammonium salt.
  • the at least one ammonium source A is a chemical fertilizer comprising (or consisting of) ammonium sulfate.
  • components A, B, and C may be each applied separately from another, or A and B may be premixed and C may be applied separately, or A and C may be premixed and B may be applied separately, or B and C may be premixed and A may be applied separately.
  • the components A, B, and C will inherently form an aqueous solution in the substrate S.
  • a aqueous solution i.e., adding the components A, B, and C to water (which is initially neutral, i.e., has a pH of approximately 7)
  • the obtained aqueous solution may have specific characteristics, including any pH.
  • the at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties may be any bacteria or fungal species suitible for this purpose.
  • the at least one bacteria and fungi species B is selected from the group consisting of Trichoderma species, Pseudomonas species, Bacillus species, and combinations thereof.
  • the at least one bacteria or fungal species B comprises bacteria selected from the group consisting of Paenibacillus mucilaginosus, Trichoderma harzianum, Pseudomonas sp. DMSZ 13134, Pseudo- monas fluorescens, Bacillus subtilis, Bacillus amyloliquefaciens, and combinations thereof.
  • the at least one bacteria or fungal species B comprises bacteria selected from the group consisting of Paenibacillus mucilaginosus, Trichoderma harzianum, Pseudomonas sp. DMSZ 13134, Pseudo- monas fluorescens, Bacillus subtilis, and combinations thereof.
  • such bacteria or fungal species B may be such exemplified in the Example section below and/or may be obtained from the suppliers exemplified in the Example section below.
  • the component B comprises at least one Bacillus species (e.g. Bacillus subtilis and/or Bacillus amyloliquefaciens) and is more preferably (essentially) free of other bacteria.
  • Bacillus species e.g. Bacillus subtilis and/or Bacillus amyloliquefaciens
  • the at least one bacteria or fungal species B is not Bacillus amyloliquefaciens.
  • the at least one bacteria or fungal species B is not a Bacillus species.
  • the component B comprises at least one Bacillus species (e.g. Bacillus subtilis and/or Bacillus amyloliquefaciens) and at least one Trichoderma species (e.g. Trichoderma harzianum) and is more preferably (essentially) free of other bacteria.
  • Bacillus species e.g. Bacillus subtilis and/or Bacillus amyloliquefaciens
  • Trichoderma species e.g. Trichoderma harzianum
  • a combination product of Bacillus/Trichoderma e.g., Trichoderma harzianum (OMG16) and Bacillus subtilis
  • a Trichoderma! Pseudomonasl- Bacillus e.g., Trichoderma harzianum (OMG16), Pseudomonas fluorescens, and Bacillus subtilis
  • Zn/Mn zinc and/or manganese
  • the bacteria and/or fungi may also be commercially available as mixtures such as, e.g., Proradix (Soucon Padena, Tubingen Germany; Pseudomonas sp.
  • the at least one bacteria and/or fungal species B is used in the composition as a spore formulation.
  • the bacteria and/or fungal species are stored in dry state, in particular as dried or freeze-dried spores. Such formulation may bear higher stress resistance and has a longer shelf-life.
  • the nitrification inhibitor C may be any nitrification inhibitor known in the art.
  • a nitrification inhibitor C may be selected and prepared as disclosed in WO 201 1/032904, WO2014/053401 , WO 2013/121384, EP-B 0 808 297, EP-B 1 021 416, EP-B 2 748 148, EP-B 1 120 388, WO 1996/024566, WO 2015/- 086823, EP-B 0 974 585, WO 2015/158853 and literature cited therein.
  • the nitrification inhibitor C may be selected from the group consisting of 3,4-di- methylpyrazol phosphate (DMPP), nitrapyrine, etridiazole, and 2-cyanoguanidine.
  • DMPP 3,4-di- methylpyrazol phosphate
  • the nitrification inhibitor C does (essentially) not bear anti-bacterial or anti-fungal properties, i.e., is preferably not bacteriozide or bacteriostatic, fungicide or arresting fungal proliferation, in order to avoid harming the bacteria and/or fungi of component B.
  • the at least one nitrification inhibitor C is a compound selected from the group consisting of compounds containing a pyrazole residue which can be substituted in their structure, 1 H-1 ,2,4-triazole, 2-chloro-6- (trichloromethyl)-pyridine, 5-ethoxy-3-trichloromethyl-1 ,2,4-thiadiazol, 2-amino-4- chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, 4-amino-1 ,2,4-triazole, 3-mercapto-1 ,2,4-triazole, 2,4-diamino-6-trichloro- methyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methyl carbamate and N-(2,6-di
  • the at least one nitrification inhibitor C may be selected from the group consisting of 3,4-dimethylpyrazolephosphate (DMPP), 2-(3,4- dimethyl-pyrazol-1 -yl)-succinic acid, 3,4-dimethylpyrazole (DMP), 1 H-1 ,2,4-triazo- le, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine, 5-ethoxy-3-tri- chloromethyl-1 ,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto- benzothiazole, 2-sulfanilamidothiazole, thiourea, 1 -hydroxypyrazole, 2-methyl- pyrazole-1 -carboxamide, 4-amino-1 ,2,4-triazole, 3-mercapto-1 ,2,4-triazole, 2,4- diannino-6-trichloronnethyl-5-tria
  • DMPP
  • A is aryl or hetaryl, wherein the aromatic ring may in each case be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from R 1A and R 2A are independently of each other selected from H and C 1 -C 2 -alkyl; and
  • R 3A is H, CrC 4 -haloalkyl, C 1 -C 4 -hydroxyalkyl, ethynylhydroxymethyl, phenyl- hydroxymethyl, or aryl, wherein the aromatic ring may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from R B ;
  • a 3- to 14-membered saturated or unsaturated carbocycle or heterocycle which may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR 1 b , O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R 1 b is H, C 1 -C 4 -alkyl, C 2 -C 4 -alkenyl, C 3 -C 6 -cycloalkyl, C 3 -C 6 - cycloalkylmethyl, or OR 9 ; or
  • B is aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially orfully substituted by substituents, which are independently of each other selected from R h ; or
  • a 3- to 14-membered saturated or unsaturated carbocycle or heterocycle which may contain 1 , 2, or 3 heteroatoms which, independently of each other, are selected from NR 1 b , O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R 1 b is H, C 1 -C 4 -alkyl, C 2 -C 4 -alkenyl, C 3 -C 6 -cycloalkyl, C 3 -C 6 - cycloalkylmethyl, or OR 9 ; or
  • two substituents R A together represent a carbocyclic or heterocyclic Ring, which is fused to A and may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR 1c , O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially orfully substituted by substituents which, independently of each other, are selected from R'; and wherein R 1c is H, C 1 -C 4 -alkyl, C2-C 4 -alkenyl, C3-C6-cycloalkyl, C3-C6- cycloalkylmethyl, C3-C6-heterocyclyl, C3-C6-heterocyclylmethyl or OR 9 ;
  • Y 1 , Y 2 and Y 3 are independently of each other selected from O, S and NR 1a , wherein R 1a is in each case independently H, CrC 4 -alkyl, C 2 -C 4 -alkenyl, C3-C6- cycloalkyl, C 3 -C 6 -cycloalkylmethyl, OR 9 , SR g or NR m R n ;
  • R a and R b are independently of each other selected from
  • aryl or hetaryl wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from R b ; or
  • a 3- to 10-membered, saturated or unsaturated heterocycle which may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR 1 b , O, and S, wherein S may be oxidized and/or wherein the heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R 1b is H, C 1 -C 4 - alkyl, C 2 -C 4 -alkenyl, C3-C6-cycloalkyl, C3-C6-cycloalkylmethyl, or OR 9 ;
  • R f is C 1 -C 4 -alkyl, C 1 -C 4 -haloalkyl, NR j R k , OR 1 , SR 1 , aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from R h ;
  • R g is H or C 1 -C 4 -alkyl
  • (ii) NR 1d , wherein R 1d is H, C 1 -C 4 -alkyl, C 2 -C 4 -alkenyl, C 3 -C 6 -cycloalkyl, C 3 -C 6 - cycloalkylmethyl, or OR 9 ;
  • A is phenyl or a 6- membered hetaryl, preferably phenyl, wherein the aromatic ring may in each case be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from R A .
  • R 1 and R 2 both represent hydrogen.
  • R 3 is hydrogen, C 1 -C 4 -haloalkyl or ethinylhydroxymethyl, and preferably R 3 is hydrogen.
  • R A in said compound of formula (I), is
  • Ci- C 4 -alkylene or C 2 -C 4 -alkenylene chain may in each case be unsubstituted or may be partially or fully substituted by CN or halogen;
  • a 3- to 14-membered saturated or unsaturated heterocycle which may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR 1 b , O, and S, wherein S may be oxidized and/or wherein the heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R 1 b is H, C 1 -C 4 - alkyl, C2-C 4 -alkenyl, C3-C6-cycloalkyl, C3-C6-cycloalkylmethyl, or OR 9 , wherein preferably
  • Y 1 , Y 2 and Y 3 are independently of each other selected from O, S and NR 1 a , wherein R 1a is in each case independently H, C 1 -C 4 -alkyl, OH, or NH 2 .
  • R a and R b are independently of each other selected from
  • R f is C 1 -C 4 -alkyl
  • R g is H
  • R h is halogen or C 1 -C 4 -alkoxy
  • R 1 and R 2 both represent hydrogen
  • R a and R b are in each case independently of each other selected from H, C 1 -C 2 -alkyl, NH 2 , CrC 2 -hydroxyalkyl, or wherein R a and R b may together with the nitrogen atom to which they are bonded form a morpholine ring.
  • the at least one nitrification inhibitor C is selected from the group consisting of 3,4-dimethylpyrazolephosphate (DMPP), 2-(3,4-di- methyl-pyrazol-1 -yl)-succinic acid, 3,4-dimethylpyrazole (DMP), 1 H-1 ,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine and 5-ethoxy-3- trichloromethyl-1 ,2,4-thiadiazol.
  • DMPP 3,4-dimethylpyrazolephosphate
  • DMP 3,4-di-methyl-pyrazol-1 -yl)-succinic acid
  • DMP 3,4-dimethylpyrazole
  • 1 H-1 ,2,4-triazole 3-methylpyrazole (3-MP)
  • 2-chloro-6-(trichloromethyl)-pyridine 2-chloro-6-(trichloromethyl)-pyridine and 5-ethoxy-3-
  • the at least one nitrification inhibitor C is selected from the group consisting of 3,4-dimethylpyrazolephosphate (DMPP) and 2-(3,4-dimethyl-pyrazol-1 -yl)-succinic acid or a salt thereof, in particular an alkali salt thereof.
  • DMPP 3,4-dimethylpyrazolephosphate
  • 2-(3,4-dimethyl-pyrazol-1 -yl)-succinic acid or a salt thereof, in particular an alkali salt thereof is selected from the group consisting of 3,4-dimethylpyrazolephosphate (DMPP) and 2-(3,4-dimethyl-pyrazol-1 -yl)-succinic acid or a salt thereof, in particular an alkali salt thereof.
  • the at least one nitrification inhibitor C is a compound of general formula (II)
  • radicals R 1 , R 2 , R 3 and R 4 independently from another have the following meanings:
  • R 1 , R 2 , R 3 and R 4 can be hydrogen, C 1 - to C 2 o-alkyl, C 3 - to C 8 -cycloalkyl, C 5 - to C 2 o-aryl or alkylaryl, it being possible for these 4 radicals to be monosubstituted or disubstituted by halogen and/or hydroxyl,
  • R 1 , R 2 , R 3 can also be halogen or nitro; R 4 can also be a radical having the formula (III)
  • R 5 and R 6 independently from another are hydrogen, C 1 - to C 20 -alkyl which can be monosubstituted or disubstituted by halogen and/or hydroxyl, a carboxyl group, a carboxymethyl group or a functional derivative of the two last-mentioned groups, and
  • R 7 is a carboxyl radical or a carboxy-(C 1 - to C 3 -alkyl) radical or a functional derivative of these groups,
  • the at least one nitrification inhibitor C is 3,4-dimethylpyrazol phosphate (DMPP).
  • the plants may be further supplied with one or more additional salts further improving nutrition of plants as component E.
  • additional salts E may be chosen to reduce stress of the plants.
  • additional salts E may exemplarily be selected from the list consisting of one or more zink salts, one or more magnesium salts, one or more seaweed extracts, and combinations thereof.
  • the plant may, in general, be any plant that is to be grown.
  • the plant is a plant that is used in agriculture, including farming and horticulture.
  • the plants are crop plants, in particular crop plants selected from the group consisting of group consisting of maize, wheat, and tomato.
  • the plant is maize.
  • the substrate S of agricultural use on which the plants are cultivated or intended to be cultivated may be any kind of substrate including any kind of soil and any kind of artificial plant substrate (e.g., expanded clay aggregate, mineral wool, seet gel, perlit, polymers (e.g., styromull, polystyrene, polyurethane, etc.) or combinations of two or more thereof, optionally further comprising additives such as e.g., superabsorber).
  • it may have any pH.
  • the pH may be in the range of from 4 to 10.
  • the pH of the substrate S, before conducting the method according to the present invention is in the range of from 5 to 9, more preferably in the range of from 5.0 to 8.5, even more preferably in the range of from 5 to 8, in particular in the range of from 5.5 to 7.5, such as, e.g., in the range of from 5.5 to 6.0, from 6.0 to 6.5, from 6.5 to 7.0, or from 7.0 to 7.5.
  • the pH of the substrate S after conducting the method according to the present invention, is decreased (i.e., the soil is acidified), remained unchanged, or merely slightly increased by not more than 1 pH unit, more preferably decreased, remained unchanged, or increased by not more than 0.5 pH units, in particular decreased, remained unchanged, or increased by not more than 0.1 pH units.
  • the proton (H + ) concentration in the substrate S is preferably either increased upon conducting the method according to the present invention, is remained unchanged, or is decreased by not more than a factor of 10, 3 or 1 .3.
  • the pH of the substrate S, after conducting the method according to the present invention is decreased (i.e., the soil is acidified), wherein the decrease may exemplarily be a decrease up to 4 pH units, up to 3 pH units, up to 2 pH units, up to 1 pH unit, 0.1 to 3 pH units, 0.2 to 2 pH units, or 0.5 to 1 pH units.
  • the proton (H + ) concentration in the substrate S is preferably increased upon conducting the method according to the present invention, wherein the increase is an increase of the proton concentration in the substrate S up to 10000fold, up to 100Ofold, up to 100fold, up to 10fold, by 1 .3 to 100Ofold, by 1 .6 to 100 fold, or 3 to 10fold.
  • the weight ratio between the nitrogen in the at least one ammonium source A and the at least one nitrification inhibitor C (A : C) is in the range of between 20:1 and 10000 : 1 , preferably in the range of between 20:1 and 5000 : 1 , in the range of between 50:1 and 1000 : 1 , or in the range of between 75:1 and 200 : 1 .
  • the amounts of nitrification inhibitor C will be adapted to the individual chemical compounds. For example, it may be adapted to the molecular weight. The above amounts may be particularly beneficial for a nitrification inhibitor like DMPP.
  • the one or more bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties may be used in any amount.
  • between 10 7 and 10 11 colony forming units (cfu) kg -1 substrate S are used, preferably between 10 8 and 10 8 cfu kg -1 substrate S are used, exemplarily, 10 9 cfu kg -1 substrate S are used.
  • a decrease of the pH of the soil may optionally also be linked with a larger spatial extension of root-induced rhizosphere acidification as a consequence of a larger root system.
  • the substrate S is a soil containing sparingly soluble salts of at least one mineral which can preferably be mobilized by the bacteria or fungal species B.
  • the substrate S may or may not comprise at least one phosphate salt or inorganic recycling fertilizer product (ashes, slugs).
  • Insufficient nutrition of the plants with one or more sparingly soluble minerals may have different reasons. Exemplarily, it may be due to poor plant availability of the minerals from the substrate S and/or may be due to insufficient development of the root systems of the plants.
  • the substrate S before conducting step (ii), comprises less than 50 mg, more preferably not more than 30 mg, in particular not more than 20 mg, plant available phosphate per kg of the substrate S.
  • the plant available phosphate is determined according to the (German) calcium acetate lactate (CAL) extraction method conducted at pH 6.5-8.5) (with or without amendments of sparingly soluble P fertilisers such as rock-P or sewage sludge ash).
  • the CAL determination of sparingly soluble minerals, in particular phosphate (PCAL, CAL P), as used herein means determination according to data sheet VDLUFA I, P und K, CAL-loslich, A 6.2.1
  • plant available phosphate may be understood in the broadest sense as the usability of the phosphate for plant growth.
  • rock phosphate present in many soils is not water soluble and also not or sparingly available for plants.
  • the rock phosphate contains phosphate anions present in the substrate S, but is still (essentially) not accessible and usably by the plants.
  • phosphate is, for many plants, typically no longer a growth limiting factor under field conditions.
  • the plants bear root systems insufficient for efficient nutrient uptake, in particular wherein the plants show early root growth and/or have been subjected to stress conditions such as low root zone temperature.
  • a low temperature may exemplarily be a temperature in the range of below 15°C, exemplarily in the range of from 0 to 20 °C, preferably in the range of 5 to 15°C, in particular in the range of 10 to 15°C, exemplarily in the range of 12 to 14°C.
  • the method of the present invention is also suitible for phytosanitary uses such as increasing the plant's resistance against pathogens such as, e.g., fungi, bacteria and(or viruses, in particular in the rhizosphere, but also all over the plant as a whole.
  • pathogens such as, e.g., fungi, bacteria and(or viruses, in particular in the rhizosphere, but also all over the plant as a whole.
  • the method is particularly useful for substrate S bearing a shortage of at least one mineral.
  • the substrate S prior to conducting the method of the present invention, preferably comprises merely low amounts of plant available soluble salts of the mineral.
  • Step (ii) of the method i.e., supplying the components A, B and C to the substrate S
  • a liquid composition may be prepared and applied to the plants and/or the seeds of the plants by drenching.
  • seed row starter application by fertigation or drenching of the substrate S in nursery pots may be used.
  • seed placement of granulated formulations showing superior root colonization efficiency may be used.
  • Seed dressing may be used.
  • fertigation of liquid formulations is used.
  • the one two or all of the components A, B and C may be applied to the plants as one or more powders or pellets/granules.
  • pellet and “granule” may be understood interchangeably as any solid particle in the milli- or micrometer range (e.g., in the range of from 0.01 to 1 mm, 0.1 to 2 mm or 0.5 to 5 mm). Such powders or granules may also be placed incorporated into the substrate S near the roots. Alternatively, the one two or all of the components A, B and C may be applied to the plants as a depot. Such depot may also be placed buried near the roots. As indicated above, the components A, B and C may be applied in combination with another or may be party combined with another or may be administered separately from another.
  • the method comprises step (i-l) of premixing the components A, B and C thereby forming mixture ABC, optionally in an aqueous suspension or in solid state (e.g., as a pellet/granule),
  • step (ii) is
  • the premixed composition obtained from step (i-l) may be a liquid, a powder or a syrup.
  • one, two or all of the components A, B and/or C may also be pelletized, either form pellets comprising one two, or all of the components A, B and/or C. This will depend on the components and optional further ingredients, in particular on the ammonium source A (which is typically the largest volume) and on the optional presence of a solvent such as water in which the components may be dissolved.
  • the components A, B and C form an aqueous suspension, wherein preferably components A and C are at least partly dissolved and the bacteria and/or fungal B are suspended.
  • pellets/granules comprising the components A and C are prepared and coated with component B, exemplarily, by means of soaking or spraying the component B on the pellets/granules comprising the components A and C.
  • the mixture ABC in particular when provided in solid state (e.g., as a pellet/granule), may be added to the rhizosphere, exempla- rily as a reservoir/depot.
  • the method comprises step
  • step (ii) comprises the steps
  • step (ii-b) applying the component B to substrate S of agricultural use on which the plants are cultivated or intended to be cultivated, wherein step (ii-a) can be conducted concomitant with, prior to or subsequent to step (ii-b).
  • steps (ii-a) and (ii-b) separately from another may minimize risks of toxicity to the microorganisms of component B and may provide higher flexibility for combinations.
  • the applying the component B to substrate S of agricultural use may be performed by soaking a seedling of the plant and then planting the plants. Then, the mixture AC may be supplied to the substrate S subsequently to this step.
  • the applying the component B to substrate S of agricultural use may be performed by supplying reservoirs( depots comprising the component B in the rhizisphere and the mixture AC may be additionally added to the substarte S by any means.
  • the seeds of the plants may be applied to the substrate S prior, concomitantly or subsequently to conducting step (ii).
  • applying at least one of the components A, B and/or C to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S.
  • steps (ii) and (ii) refer to:
  • step (iii) enabling (starting) growth of the plants in the substrate S obtained from step (ii).
  • applying component A to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S.
  • applying component B to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S.
  • applying component C to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S.
  • applying a combination of the components A and B (AB) to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S.
  • applying a combination of the components A and C (AC) to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S.
  • applying a combination of the components B and C (BC) to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S.
  • applying a combination of the components A, B and C (ABC) to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S.
  • the seeds and the one or more seeds can be applied independently from another or combined with another. The further components are added separately, which can be conducted concomitantly, precedingly or subsequently.
  • the seed is coated with a composition comprising one or more of the components A, B and/or C.
  • the seed may be coated with a composition comprising component A, or a composition comprising component B, or a composition comprising component C, or a composition comprising components A and B (AB), or a composition comprising components A and C (AC), or a composition comprising components C and B (CB), or a composition comprising components A, B and C (ABC).
  • a coating can optionally comprise further ingredients such as those selected from the group consisting of binders (e.g, one or more sugars), fillers, minerals promoting plant growth, bacteria and/or fungal nutrients, and combinations of two or more thereof. The person skilled in the art will know a number of ingredients usable in such coatings. The further components are added separately, which can be conducted concomitantly, precedingly or subsequently.
  • the seeds may be coated with a composition comprising at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth- promoting properties (component B) and a combination of an ammonium source (component A) comprising nitrification inhibitor (component C) may be added additionally.
  • steps (ii) and (ii) refer to: (ii) applying
  • composition comprising at least one ammonium source A and cat least one nitrification inhibitor (e.g., DMPP)
  • step (iii) enabling (starting) growth of the plants in the substrate S obtained from step (ii).
  • the substrate S is prepared by means of the method according to the present invention prior to sowing the plants. Then, steps (ii) and (ii) refer to:
  • step (ii*) applying seeds of the plants to substrate S obtained from step (ii); and (iii) enabling (starting) growth of the plants in the substrate S obtained from step
  • step (iii) means enabling further growths of the plants in the substrate S obtained from step (ii). Then, steps (ii) and (ii) refer to:
  • step (iii) enabling further growth of the plants in the substrate S obtained from step (ii).
  • the method is for improving root growth of the plants.
  • the invention bases on the interplay of components A, B and C. Accordingly, the intended application of said components, in particular the concomitant supplementation of said components to the substrate S is encom- passed by the present invention.
  • plant root may be interpreted in the broadest sense as growth of any part of the rhizosphere, i.e., root growth.
  • biomass of the roots is enhanced in comparison to the biomass obtained for the roots of a plant grown on comparable substrate S* under comparable conditions not subjected to the method according to the present invention.
  • the method is for improving plant root of the plants. Therefore, the growth of the roots is preferably stimulated.
  • the biomass of the roots (total and/or dry weight thereof) is enhanced in comparison to those of a comparable plant grown on comparable substrate S* under comparable conditions not subjected to the method according to the present invention. It will be understood that a promoted root growth may also promote nutrient uptake, water uptake and/or total plant growth, in particular root growth.
  • a further aspect of the present invention relates to the use of (a combination of) at least one ammonium source A, wherein at least 50 mol% of the total nitrogen content of A is present as ammonium and/or organically bound nitrogen, at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth- promoting properties, wherein B does not form part of A, and at least one nitrification inhibitor C for improving nutrition of plants with one or more sparingly soluble minerals selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese.
  • phosphor typically phosphate
  • iron zinc and manganese
  • the use is preferably further characterized by one or more features as laid out in the context of the method laid out herein.
  • the term "combination of may be understood in the broadest sense as using all of the components A, B and C. It does not necessarily mean that these ingredients are comprised in a single composition. The can also be applied separately from another.
  • a still further aspect of the present invention relates to a composition usable for a method according to the present invention, said composition comprising (in particular consisting of):
  • (F) optionally one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers,
  • composition wherein at least 50 mol% of the total nitrogen content of the composition is present as ammonium and/or organically bound nitrogen, and
  • composition does not comprise an ammonium source A comprising bacteria or fungal species with phosphate-solubilizing and/or root growth-promoting properties. It will be understood that all definitions and preferred embodiments laid out in the context of the method according to the present invention mutatis mutandis also apply to the composition of the present invention.
  • the composition does not comprise considerable amounts of soluble salts of (sparingly soluble) minerals, such as minerals selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese. It will be understood that the components may comprise traces of such salts.
  • the composition does (essentially) not comprise salts selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese salts, in particular does (essentially) not comprise such salts in solid form and/or of synthetic origin, phosphor (typically phosphate) salts in solid form and/or of synthetic origin.
  • the composition does (essentially) not comprise such salts in solid form and/or of synthetic origin, in particular does (essentially) not comprise phosphor (typically phosphate) salts in solid form and/or of synthetic origin.
  • the amount of phosphor in the composition is below 10% (w/w), preferably below 5% (w/w), more preferably below 1 % (w/w) or below 0.1 % (w/w), based on the sum of the weight of the components A, B and C.
  • the at least one bacteria or fungal species B is not Bacillus amyloliquefaciens and the amount of phosphor in the composition is below 10% (w/w), preferably below 5% (w/w), more preferably below 1 % (w/w) or below 0.1 % (w/w), based on the sum of the weight of the components A, B and C and the total amount of phosphor.
  • the composition is free of such ammonium sources A comprising bacteria or fungal species with phosphate-solubilizing and/or root growth-promoting properties. Therefore, the composition will be typically exempla- rily free of manure, clearing sludge, moist rest residue of a biogas plant, and soil.
  • the one or more ammonium sources A is/are one or more ammonium salts, more preferably a chemical fertilized comprising at least one ammonium salt, in particular ammonium sulfate.
  • the components A and B may or may not be spatially separated from another.
  • a and B are mixed powders.
  • a and B are provided in spatially separated containers or bags.
  • composition may also be used as a coating for seeds of the plants.
  • present invention also refers to seed coated with a composition according to the present invention (typically (essentially) without water).
  • composition may also be pelletized.
  • present invention also refers to pellets comprising (or consisting of) a composition according to the present invention.
  • the composition comprises one or more additional salts further improving nutrition of plants as component E.
  • additional salts E may be chosen to reduce stress of the plants.
  • additional salts E may exemplarily be selected from the list consisting of one or more zinc (Zn) salts, one or more magnesium (Mg) salts, one or more manganese (Mn) salts, one or more seaweed extracts, and combinations thereof.
  • the component E comprises (or consists of) a Zn/Mn mixture (i.e., a mixture of Zn and Mn salt(s)).
  • the composition may be a powder, a liquid or a syrup. Optionally, it may be a commercial product.
  • the user completes the composition just prior to its use, i.e., prior applying the composition to the substrate S of agricultural use on which the plants are cultivated or intended to be cultivated.
  • the user adds the components B, C and, optionally, D, E and/or F to one or more ammonium sources A prior use.
  • the present invention also refers to a packaging unit such as a tank or a sachet/bag comprising the composition according to the present invention.
  • the composition is a pellet/granule comprising the components A, B and C and optionally one or more of the components C, D and/or E.
  • the core of such pellet/granule may comprise the components A and C and the shell may comprise the component B. This may optionally be achieved by spraying or soaking of precursor pellets/- granules comprising the components A and C by a suspension comprising component B.
  • component B i.e., the one or more bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties
  • the optional premixing of components and/or the application formu- lation will be chosen.
  • separate use of liquid formulations comprising components A and C, liquid microbial inoculants for seed row application, soil incorporation or soil drenching of nursery pots may be used.
  • granulated formulations e.g mixed product with separate granules for fertilizers and microorganisms e.g. for under seed placement, may be used.
  • a still further aspect of the present invention refers to a kit for use in a method according to the present invention comprising at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties, wherein B does not form part of A, and at least one nitrification inhibitor C, and optionally one or more ammonium sources A.
  • B does not form part of A
  • C at least one nitrification inhibitor
  • ammonium sources A optionally one or more ammonium sources A.
  • the bacteria and/or fungal species are stored in dry state, in particular as dried or freeze-dried spores. Such formulation may bear higher stress resistance and has a longer shelf-life.
  • the bacteria and/or fungal species (spores) may be packed in any form, e.g., in a container or a sachet.
  • the kit may comprise one, two or all of the components A, B and/or C in pelletized (granulized) form, either separated or in combination with another as described above.
  • the kit comprises (or consists of), as functional components:
  • (F) optionally one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers, and
  • kit will typically further comprise means for packaging the functional components.
  • the kit further comprises user instructions for conducting a method according to the present invention.
  • the kit according to the present invention does not comprise (i.e., is (essentially) free of) other bacteria species X than bacteria or fungal species B.
  • the kit according to the present invention may comprise (or consist of), as functional components:
  • B a premixed composition comprising (or consisting of) components B and C and, optionally, A, D, E and/or F;
  • the kit comprises (or consists of):
  • composition optionally a composition comprising (or consisting of) any of components A and/or F.
  • the kit comprises (or consists of):
  • composition optionally a composition comprising (or consisting of) any of components A and/or F.
  • the kit comprises (or consists of):
  • composition optionally a composition comprising (or consisting of) any of components A and/or F.
  • the kit comprises (or consists of):
  • composition optionally a composition comprising (or consisting of) any of components A and/or F.
  • the kit comprises (or consists of):
  • composition optionally a composition comprising (or consisting of) any of components A and/or F.
  • the kit comprises (or consists of):
  • mixing of the components with another can be conducted by any means known in the art.
  • mixing can be conducted in undissolved/undiluted state (e.g., as mixing one or more dry powders and/or one or more syrups with another) or can be completely or partly dissolved and/or diluted before mixing, e.g., in an aqueous solvent such as water.
  • the kit preferably comprises (or consists of) components (I) and (II), (I*) and (II), (I) and (II*), (I*) and (II*), (l-ll) or ( ⁇ - ⁇ II*) only, and the user adds one or both of these components to the component A of interest just before supplying the substrate S therewith.
  • the kit comprises (or consists of) (l-ll-lll): a powder comprising (or consisting of) components A, B and C and, optionally, components E and/or F.
  • the kit comprises (or consists of) ( ⁇ - ⁇ - ⁇ II*): a liquid or syrup composition comprising (or consisting of) components A, B and C and, optionally, components E and/or F.
  • the kit according to the present invention comprises (or consists of) a composition according to the present invention.
  • the composition may be an aqueous suspension or may be a powder.
  • a further aspect of the present invention relates to a composition
  • a composition comprising or consisting of:
  • (F) optionally one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers,
  • composition wherein preferably at least 50 mol% of the total nitrogen content of the composition is present as ammonium and/or organically bound nitrogen.
  • the components are each defined as above.
  • the presence of the ammonium sources A as being of synthetic origin may be understood in the broadest sense in that the ammonium sources A forms part of or is a chemical fertilizer.
  • This composition may or may not comprise more than 20% (w/w), 10% (w/w), 5% (w/w), 1 % (w/w) or 0.1 % (w/w) of phosphate (typically in the form of phosphate), based on the total weight of the composition as a whole.
  • the composition is a solid composition comprising the components A, B and C and, optionally, E and, optionally F, as mixed powders. Then, preferably, water is preferably (essentially) absent in the storable composition. Water may optionally be added just before supplying a substrate S with the composition.
  • the terms are defined as laid out throughout the present invention.
  • composition of the present invention may comprise or consist of:
  • composition of the present invention may comprise or consist of:
  • composition of the present invention may comprise or consist of:
  • (F) 0-10% (w/w) of one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers.
  • the amounts, in particular the amount of nitrification inhibitor C, will be adapted to the individual chemical compounds.
  • Figure 1 demonstrates effects of soil-buffering capacity on growth stimulation of maize by microbial biofertilizer-induced Ca-P solubilisation on a substrate with Ca- P and Rock-P as exclusive P sources at six weeks after sowing (Nkebiwe, 2017).
  • Figure 2 demonstrates rhizosheath formation of maize under field conditions and along single roots in a rhizobox culture system with root observation window.
  • Figure 3 shows the synergistic effect of a nitrification inhibitor (DMPP), microbial biofertilizers and ammonium fertilization. This figure depicts the comparison between the synergistic effects of different microbial biofertilizers.
  • DMPP nitrification inhibitor
  • microbial biofertilizers ammonium fertilization
  • NoP background control without phosphate source
  • NoBE rock phosphate without microbial biofertilizers (negative control)
  • Trianum P Trichoderma harzianum T22 (deposit No.: ATCC 20847);
  • Proradix Pseudomonas sp., (deposit No.: DMSZ 13134);
  • CombifectorA comprising: Trichoderma harzianum OMG16, Pseudomonas fluorescens, Bacillus subtilis, and micronutrients;
  • Rhizovital Rhizovital FZB42: Bacillus amyloliquefaciens subsp.
  • the star (*) indicates that the t-test is significant (0.05 alpha) compared to NoBE.
  • the lowercase letters indicate statistically distinguishable groups.
  • Figure 5 shows the synergistic synergistic effect of a nitrification inhibitor (DMPP), microbial biofertilizers and ammonium fertilization at different pH values on plant growth, (herein: 1 : rock phosphate without microbial biofertilizers (negative control); 2: combination of the nitrification inhibitor DMPP and ammonium salt; 3: combination of the nitrification inhibitor DMPP, ammonium salt and Rhizovital FZB42; 4: soluble phosphor source added and nitrate added).
  • DMPP nitrification inhibitor
  • 1 rock phosphate without microbial biofertilizers (negative control)
  • 2 combination of the nitrification inhibitor DMPP and ammonium salt
  • 3 combination of the nitrification inhibitor DMPP, ammonium salt and Rhizovital FZB42
  • 4 soluble phosphor source added and nitrate added.
  • the lowercase letters indicate statistically distinguishable groups.
  • Figure 6 shows synergistic effect of microbial biofertilizers and ammonium fertilization on root length of maize.
  • the lowercase letters indicate statistically distinguishable groups.
  • Figure 7 shows the effect of stabilized ammonium fertilization and microbial biostimulant inoculation (FZB42) on plant available P (CAL-extractable P) in the rhizosphere of a low-P soil with pH 5.6.
  • FZB42 stabilized ammonium fertilization and microbial biostimulant inoculation
  • the specific microbial inoculants are introduced into the rhizosphere of the target crop, where they can propagate by supply of plant root exudates as energy source and increase the nutrient availability for the host plant (Menzies et al. 201 1 ; Sharma et al., 2013).
  • a range of so-called microbial "biofertilizer” products is already commercially available. However, limited reproducibility of the desired fertilizer effects under practical conditions still represents a major undissolved problem (Menzies et al. 201 1 ).
  • Bacteria were chosen that enable soil acidification by plant roots, which is induced by ammonium-dominated fertilization which can increase the plant availability of phosphate (P) and micronutrients particularly on neutral and alkaline soils (Marschner, 1995; Neumann and Romheld 2002). These soil microorganisms were identified as also being able to use ammonium as cationic nitrogen source, associated with proton extrusion for charge-balance, which results in medium acidification, (Menzies et al., 201 1 ; Nkebiwe 2017).
  • Microbial inoculants based on strains of Bacillus, Pseudomonas and Trichoderma, representative for many commercial biofertilizers, have been characterized for Ca- P solubilisation on artificial growth media, and utilization of ammonium sulfate as nitrogen source.
  • DMPP dimethylpyrazolphosphate
  • the bacteria or fungal species B were obtained from the following suppliers:
  • Trianum P Trichoderma harzianum T22 (deposit No.: ATCC 20847; Koppert Biological Systems Nederland, Veilingweg 14, 2651 BE Berkel en Rodenrijs, The Netherlands)
  • Rhizovital FZB42 Bacillus amyloliquefaciens subsp. Plantarum, synonymous: Bacillus velezensis FZB42 (Taxonomy ID: 326423; deposit No.: DSM231 17; ABiTEP GmbH, Glienicker Weg 185, 12489 Berlin, Germany)
  • Bacillus atrophaeus (ABI02A1 , deposit No.: DSM 32019; ABiTEP GmbH, Glienicker Weg 185, 12489 Berlin, Germany) Paenibacillus mucilaginosus (ABiTEP GmbH, Glienicker Weg 185, 12489 Berlin, Germany)
  • Trichoderma harzianum OMG16 (Anhalt University of Applied Sciences Center of Life Sciences, Institute of Bioanalytical Sciences (IBAS) Strenzfelder Allee 28, 06406 Bernburg, Germany)
  • Table 1 Shoot biomass production (g plant -1 ) of maize (cv Colisee) on a sand-soil substrate (calcareous Loess subsoil pH 7.6; PCAL5 mg kg -1 soil) with different levels of liming (0 and 25% Ca(COs)2) and sparingly soluble Ca-Phosphates as exclusive P source (120 mg P kg -1 substrate as Rock-P). Effect of nitrate and DMPP-Ammonium fertilization (100 mg N kg -1 substrate as Ca(NO 3 )2 or DMPP- (NH ) 2 SO 4 Novatec Solub, Compo, Germany) and three weekly inoculations with Proradix (10 9 cfu kg -1 substrate).
  • Table 2 Shoot dry matter production (g plant -1 ) of maize (cv Colisee) on two different clay-loam field soils with low P availability ( ⁇ 20 mg P C AL kg -1 substrate) supplied with Rock-P (RP) (120 mg P kg -1 substrate) as exclusive P source.
  • the effect of nitrate and DMPP-Ammonium fertilization (100 mg N kg -1 substrate as Ca(NO 3 ) 2 or DMPP-(NH 4 ) 2 SO 4 Novatec Solub, Compo, Germany) and three weekly inoculations by fertigation with Proradix (10 9 cfu kg -1 substrate)
  • Proradix and DMPP-Ammonium fertilization on plant growth stimulation are not restricted to maize and could be similarly demonstrated in a pot experiment with spring wheat on a low-P silty loam organic farming soil (pH 6.4, PCAI_:7 mg kg -1 ) supplied with Rock-P (RP) as sparingly soluble P source.
  • Proradix inoculation in combination with DMPP-Ammonium significantly increased final grain yield by 34 % as compared with the non-inoculated control, while the Proradix effect in combination with nitrate fertilization was not significant (+ 8%).
  • Table 3 Biomass production and grain yield of spring wheat (cv Schirocco) on a low P silty loam organic farming soil (pH 7.6; 7 mg P C AL kg -1 soil) supplied with Rock-P (RP) (150 mg P kg -1 substrate ) as exclusive P source. Effect of nitrate and DMPP-Ammonium fertilization (100 mg N kg -1 substrate as Ca(NO 3 )2 or DMPP- (NH ) 2 SO 4 Novatec Solub, Compo, Germany) and three inoculations by fertigation with Proradix (Pro, 10 9 cfu kg -1 substrate) at 0, 24 and 34 days.
  • Plant growth promotion by combination of DMPP-ammonium with different microbial inoculants Plant growth promotion by combination of DMPP-ammonium with different microbial inoculants
  • a range of seven microbial biofertilizer products, based on strains of Bacillus, Pseudomonas Trichoderma, Penicillium and combinations thereof were tested for their plant growth-promoting potential with Maize cv Colisee in combination with DMPP-Ammonium fertilization on a low-P, clay-loam organic farming soil pH 6.8 (available P: 20 mg CAL-P kg -1 soil) supplied with Rock-P (100 mg P kg -1 soil) as sparingly-soluble P source. Variants without P fertilization (No P) and with soluble P supply (100 mg P kg -1 soil as Ca(H 2 PO 4 )2) were included as negative and positive controls, respectively.
  • DMPP-Ammonium with Rock-P fertilization but without biofertilizers induced approximately 60% of the shoot biomass production as compared with maize plants supplied with full soluble P fertilization (NO3_Soluble P).
  • Ammonium-DMPP in combination with all tested biofertilizer products based on strains of Pseudomonas, Bacillus and Trichoderma significantly increased the shoot biomass production even for those products, which have been previously proven to be ineffective in combination with nitrate fertilization (Fig .1 ; Table3).
  • the only exception was the Penicillium-based biofertilizer (BFOD).
  • Fungal biofertilizers Trianum.P (Trichoderma harzianum T22), BFOD (Penicillium bilaii); Bacterial biofertilizers: Proradix (Pseudomonas sp DMSZ13134), Rhizovital (Bacillus amyloliquefaciens FZB42), Paenibacillus mucilaginosus; Combination products: Vit SP1 1 (Bacillus subtilis, Pseudomonas sp., Streptomyces spp., humic acids, Ascophyllum nodosum extract), CombiFectA (Trichoderma harzianum OMG16, 3 Bacillus strains, Pseudomonas sp Zn and Mn).
  • the black frame exemplarily shows the synergistic effect of DMPP-Ammonium and Proradix as compared with DMPP-Ammonium or with Proradix combined with nitrate fertilization.
  • Pseudomonas-, Bacillus-, and Trichoderma-based biofertilizers used in the descry- bed maize experiments in combination with DMPP-Ammonium fertilization, exerted similar effects also in a pot experiment conducted with spring wheat, grown on a low-P Cambisol with 14 - 28 % increase in shoot biomass production and a 26 - 35 % increase in grain yield.
  • Table 6 Effect of microbial bioeffectors on biomass production and grain yield in a Ppot experiment with spring wheat (cv. Schirocco, KWS, Germany) on a low-P organic farming soil (PCAL 7 mg kg -1 soil) silty loam Cambisol, pH 6.4 with DMPP- stabilized ammonium fertilization (NH 150mg N kg -1 soil placed by point injection) and rock phosphate (RP 150 mg P kg -1 soil)) as P fertilizer.
  • Plant growth promotion in maize supplied with different P sources by combination of DMPP-ammonium with microbial inoculants based on Bacillus. Pseudomonas and Trichoderma strains.
  • Low soil temperature in spring is a major constraint for cultivation of tropical and sub-tropical crops in temperate climates and is associated with inhibition of root growth and activity.
  • Various strategies have been proposed as practical measures to counteract low temperature stress in crops including (i) fertilizer placement (P, micronutrients, such as Zn and Mn) close to the seedling roots, (ii) application of plant and seaweed extracts with antioxidative and membrane-protective properties, and (iii) improving root growth and plant nutrient acquisition by inoculation with plant growth-promoting microorganisms (Bradacova et al. 2016).
  • Leaf damage was generally more expressed under nitrate supply as compared with ammonium fertilization (Table 8). The highest shoot biomass production was achieved by CombifectorA (77g), followed by BFOD (70g) under ammonium fertilization and cold-stress, which was significantly higher than all other tested variants and untreated controls (Table 8)
  • Table 8 Effects of microbial inoculants (Abi02, cold-resistant Bacillus atrophaeus; BFOD Penicillium sp.; CombifectorA and Zn/Mn seed dressing (Lebosol GmbH, Germany) on shoot biomass production Fresh weight (FW g plant -1 ) and oxidative leaf damage (number of chlorlotic/necrotic) leaves plant -1 ) in maize, induced by two weeks exposure to root zone temperatures of 12-14°C on a clay loam field soil pH 6.8 with nitrate or DMPP stabilised ammonium sulfate fertilization.
  • microbial inoculants Abi02, cold-resistant Bacillus atrophaeus; BFOD Penicillium sp.; CombifectorA and Zn/Mn seed dressing (Lebosol GmbH, Germany
  • the cold stress-suppressive effect of CombifectorA was also reflected by significantly increased superoxide dismutase (SOD) activity and polyphenols content in maize shoot tissue, which was amore expressed in ammonium as compared to nitrate fertilization (not shown), reflecting a higher expression of defence mechanisms against oxidative stress depending on micronutrients (Zn, Mn, Cu, Fe) as enzymatic co-factors. Accordingly, a critical micronutrient status was identified as growth limiting factor in maize plants exposed to low root-zone temperature (Table. 9).
  • Micronutrient seed application the microbial inoculants and rhizosphere acidification induced by DMPP stabilized ammonium fertilization, obviously increased the plant availability of the critical micronutrients in the rhizosphere (Table 9), thereby stimulating the expression of the antioxidative stress defence. Since the same defence mechanisms are also involved in plant tolerance to other abiotic and biotic stress factors, comparable investigations are currently conducted also for plants exposed to water limitation.
  • Ammonium nutrition induces rhizosphere acidification due to root-induced proton extrusion for charge-balance of ammonium uptake (Table 14), with the well- documented effects on increased solubility of Ca phosphates, Rock P and micronutrients, with particular importance on slightly acidic to alkaline soils with limited solubility of the respective nutrients (Neumann and Romheld 2002).
  • microorganisms release protons in response to ammonium uptake (Menzies et al., 201 1 ).
  • Table 14 Changes in rhizosphere pH (rel. to bulk soil) along seminal roots of maize supplied with rock P and nitrate or DMPP-stabilized ammonium fertilization with or without inoculation with the Pseudomonas-based biofertilizer Proradix on a low P clay-loam organic farming soil, pH 6.8. Measurements conducted with antimony micro-electrodes, 1 mm in diameter (Haussling et al., 1985).
  • rhizoheaths improves the root soil contact (Fig.2) with positive effects on nutrient uptake and mobilization of sparingly available nutrients in the rhizosphere.
  • soil bound in rhizosheaths shows a higher water holding capacity as compared with the bulk soil as an important benefit under conditions of water limitation (Huang et al. 1993)
  • the size of the rhizosheaths and thus the extension of the rhizosphere is largely determined by the length of the root hairs (Fig. 2; Hailing et al., 2014).
  • Root growth responses including root hair formation are strongly determined by nutrient availability with particularly high variability in response to ammonium fertilization, depending on dosage, soil pH, placement of the fertilizer and genotype (Kania et al., 2007; Pan et al. 2016).
  • Root growth stimulation by microbial inoculants is thought to be mediated by microbial production of hormonal factors (mostly, auxins) or interference with the plant hormonal signalling systems (via quorum sensing signals or degradation of ethylene precursors).
  • hormonal factors mostly, auxins
  • interference with the plant hormonal signalling systems via quorum sensing signals or degradation of ethylene precursors.
  • the potential of the respective metabolic activities in microbial inoculants is most frequently demonstrated on artificial growth media on agar plates and investigations under rhizosphere conditions are rare.
  • Table 16 Auxin production g -1 microbial biomass (rel. values, Salkowski Assay) of bacterial populations re-isolated from rhitosphere soil attached to the roots of maize plants grown on three different field soils (clay loam-silty loam pH 5.8-7.5) and nitrate or DMPP-stabilized ammonium (DMPP NH ) fertilization, with and without microbial inoculants (Proradix; ECAG2895) and rock phosphate (RP) or soluble Ca(H 2 PO 4 ) 2 (Psol) as P fertilizers.
  • DMPP NH nitrate or DMPP-stabilized ammonium
  • root growth-stimulating and P-solubilizing BEs can synergistically support the root- mediated nutrient mobilization induced by ammonium-triggered proton extrusion (Table 14) as a general response in all crops, and also by the ammonium - induced rhizosphere extension demonstrated in Table 15.
  • DMPP nitrification inhibitor
  • DMPP is used as nitrification inhibitor in the samples.
  • microbial biofertilizers show statistically significant beneficial impact on plant growth in soil that contains sparingly soluble calcium phosphates as exclusive P source. Similar synergistic ammonium effects were also found after inoculation with other bacteria and fungi belonging to the genera Trichoderma, Penicillium, Pseudomonas, Bacillus, Paenibacillus, and Streptomyces.
  • This example shows the positive effect of a combination of microbial biofertilizers (biostimulants) and stabilized ammonium nitrogen on plant growth by induced increased P-uptake from rock phosphate.
  • Stabilized ammonium fertilization synergistically supports plant growth promotion in maize supplied with sparingly soluble Ca-P (Rock-P)
  • This example shows the positive effect of a combination of biostimulants and stabilized ammonium N on plant growth by induced increased P-uptake from rock phosphate on a silty loam soil pH 6.9. This effect was not found for soils where ammonium is replaced by nitrate, in other words, larger amounts of nitrate salts are added.
  • auxin indole acetic acid
  • cytokinins zeatin
  • giberrellic acid in the shoot tissue of maize plants in comparison with the nitrate-fertilized control (Table 17, determined by UHPLC-MS analysis according to Moradtalab et al . 2018).
  • Ammonium-induced increased internal levels of growth hormones may increase the responsiveness of the host plants to hormone production of microbial inoculants.
  • Table 17 Phytohormone levels in maize shoots (3 weeks after sowing) on a clay loam soil pH 6.9 as affected by the form of nitrogen fertilization (Ca-nitrate or
  • DMPP nitrification inhibitor
  • microbial biofertilizer microbial biofertilizer
  • ammonium fertilization on plant growth at different pH values

Abstract

The present invention relates to a method for improving nutrition of plants with one or more minerals, comprising the administration of at least one ammonium source A, at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties, and at least one nitrification inhibitor C to a substrate S on which the plants are cultivated or intended to be cultivated. Further, the present invention relates to a composition and a kit usable for this purpose.

Description

Method and Composition for Improving Nutrient Acquisition of Plants
The present invention relates to a method for improving nutrition of plants with one or more minerals, comprising the administration of at least one ammonium source A, at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties, and at least one nitrification inhibitor C to a substrate S on which the plants are cultivated or intended to be cultivated. Further, the present invention relates to a composition and a kit usable for this purpose.
Since far more than hundred years, it is commonly known that plants need certain amounts of minerals (also designated as trace minerals or plant nutrients) for their growth and maintaining their health. According to the "Law of the Minimum" established by Justus von Liebig, plant growth is limited by the scarcest mineral, rather than the total amount of mineral resources available. Accordingly, a soil of agricultural use on which plants of interest are cultivated or intended to be cultivated should bear sufficient amounts of all minerals required by the plants of interest. Cultivating crops for a longer time exhausts the soil for a number of minerals. Any shortage of a single mineral involved in plant growth in a plant- accessible form limits the plants' growth and thus diminishes the obtainable harvest. Accordingly, agriculture typically involves and often requires fertilizing. Traditionally, nitrogen and phosphate salts are among the most limiting factors in most soils. Therefore, nitrogen-based fertilizers are widely used which can be based on synthetic fertilizers such as ammonium or nitrate salts, or organic wastes such as manure, compost, clearing sludge, moist rest residue of a biogas plant, or the like.
When fertilizing plants with sufficient amounts of such nitrogen-based fertilizers, typically at least one other mineral becomes the limiting factor such as often phosphor (P, typically phosphate), iron (Fe), zinc (Zn) and/or manganese (Mn). The lack of such minerals in a plant available form is a widespread problem for many crops.
In contrast to nitrogen, the total content of these minerals in the soil is often theoretically sufficient. However, the vast majority of these minerals are present in the soil in a form not accessible by plants, such as in the form of poorly soluble or essentially insoluble rocks. Therefore, there is often a shortage of accessible phosphor (typically phosphate), iron, zinc and/or manganese minerals in soils of agricultural use. Traditionally, such shortage is compensated by supplementing the plants not only with nitrogen-based fertilizers but additionally also with fertilizers comprising such well-soluble salts of these minerals (e.g, by foliar or soil application).
However, particularly for phosphate fertilization, strong fixation in soils in combination with the low acquisition potential of many crops, limits the current use efficiency of phosphate fertilizers to approximately 20%, associated with a high risk of non-sustainable and ecologically problematic over-fertilization. Evidently, this bears several significant drawbacks. First, the production of such salts requires significant amounts of natural, energy and economic resources. Additio- nally, adding more well-soluble salts of these minerals to the soil often leads to either rapid leaching of these salts from the soil. Alternatively, depending on the counter-ions present in the soil, adding more well-soluble salts of such minerals can also lead to the rapid formation of poorly soluble or essentially insoluble salts. In both cases the long-term effectivity of the fertilizing is rather poor.
It is well-documented that soil microbial communities comprise populations with both, beneficial and inhibitory effects on plant growth. Many beneficial microorganisms are able to increase the plant availability of mineral nutrients by chemical mobilization or by stimulation of root growth, while others are effective in the suppression of pathogens. Attempts to supplement soils with additional amounts of plant growth-promoting microorganisms showed some beneficial activity. However, the success was limited since the conditions promoting a successful establishment of these populations are not well understood and obviously affected by many external factors. In view of the above, there is still a need for effective and sustainable means of improving nutrition of plants of interest with minerals of limited solubility, such as phosphor (typically phosphate), iron, zinc and/or manganese, but also to increase the general use efficiency of fertilizers, preferably by reducing the need of adding soluble salts of these minerals.
Surprisingly, it has been found that the combined supplementation of an ammonium source, a bacterial and/or fungal species promoting root growth and/or mobilizing phosphates, and a nitrification inhibitor frequently leads to the desired improvement in the mobilization of minerals with limited solubility and due to root growth promotion additionally increased the efficiency of nutrient acquisition in general.
Accordingly, a first aspect of the invention relates to a method for improving nutrition of plants with one or more sparingly soluble minerals selected from the group consisting of phosphor, iron, zinc and manganese, said method comprising the following steps:
(i) providing the following components:
(A) at least one ammonium source A (component A), wherein at least 50 mol% of the total nitrogen content of A is present as ammonium and/or organically bound nitrogen,
(B) at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties (component B), wherein B does not form part of A, and
(C) at least one nitrification inhibitor C (component C);
(ii) applying the components A, B and C to a substrate S of agricultural use on which the plants are cultivated or intended to be cultivated; and
(iii) enabling growth of the plants in the substrate S obtained from step (ii).
The method according to the present invention enables improving nutrition of plants without the need of applying the substrate S with soluble salts of the (sparingly soluble) minerals, such as minerals selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese. Accordingly, the method does preferably not comprise a step of adding such soluble salts to the substrate S. In a preferred embodiment, the substrate S is not supplemented with soluble salts of the (sparingly soluble) minerals, such as minerals selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese. Thus, in a preferred embodiment, the substrate S is not supplemented with (considerable amounts of, i.e., more than traces of) soluble salts of phosphor (typically phosphate). The person skilled in the art will notice that the components typically may comprise traces of phosphor (typically phosphate). In other words, preferably, the components A, B and C as described herein are essentially not supplied in combination with a mineral selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese, in particular are essentially not supplied in combination with phosphor (typically phosphate). In a preferred embodiment, the components A, B and C are (essentially) not supplied in combination with such salts in solid form and/or of synthetic origin.
In a preferred embodiment, the amount of phosphor (e.g., present as phosphate) supplied to the substrate S is below 10% by weight (w/w), based on the sum of the weight of the components A, B and C supplied on a substrate S within one month, one week or one month from the point of time of supplying the components A, B and C to the substrate A. In a preferred embodiment, the amount of phosphor supplied to the substrate S is below 5% (w/w), preferably below 1 % (w/w) or below 0.1 % (w/w), based on the sum of the weight of the components A, B and C supplied on a substrate S within one week from the point of time of supplying the components A, B and C to the substrate A.
As used herein, the term "sparingly soluble" may be understood in the broadest sense as not being well water soluble. Typically, a sparingly soluble mineral will be understood as a salt or complex having a water solubility of < 0.1 g per liter, preferably not more than 100 mg per liter, more preferably not more than 10 mg per liter, in particular not more than 10 mg per liter (at ambient temperature of 20°C). In soil solutions, sparingly soluble usually means a solubility of not more than 1 mg per liter, even in well fertilized soils.
The method according to the present invention also achieves improving the effectivity of the bacteria or fungal species B. In other words, the bacteria or fungal species B (also designated as biostimulants) are boosted (i.e., promoted) by means of applying these in combination with at least one ammonium source A and at least one nitrification inhibitor C. It was found that such bacteria or fungal species B are able to convert essentially inaccessible mineral salts (such as poorly accessible phosphor (typically phosphate), iron, zinc and/or manganese salts) to their counterparts which are accessible by plants. Thereby the bacteria and/or fungal B are in principle able to mobilize the respective minerals. The bacteria were surprisingly found to be particularly effective when working in combination with components A and C.
The method according to the present invention also achieves improving the effectivity of the fertilization with an ammonium source A and a nitrification inhibitor C. In other words, the combination of components A and C is boosted by means of applying it in combination with at bacteria or fungal species B.
The at least one bacteria or fungal species B according to the present invention has phosphate-solubilizing and/or root growth-promoting properties. In other words, component B may comprise (or consist of) at least one bacteria or fungal species B having phosphate-solubilizing and/or root growth-promoting properties; and/or at least one fungal species B having phosphate-solubilizing and/or root growth-promoting properties. Exemplarily, the "at least one bacteria or fungal species B" according to the invention may be selected from the list consisting of: - a bacteria species having phosphate-solubilizing properties;
- a bacteria species having root growth-promoting properties;
- a bacteria species having phosphate-solubilizing and root growth-promoting properties;
- a mixture of two, three, four, five or more bacteria species each having phosphate-solubilizing and/or root growth-promoting properties;
- a mixture of two, three, four, five or more bacteria species each having phosphate-solubilizing properties;
- a mixture of two, three, four, five or more bacteria species each having root growth-promoting properties;
- a mixture of two, three, four, five or more bacteria species each having phosphate-solubilizing and root growth-promoting properties;
and root growth-promoting properties;
- one bacteria or fungal species B having root growth-promoting properties;
- one bacteria or fungal species B having phosphate-solubilizing and root growth- promoting properties;
- a fungal species having phosphate-solubilizing properties; - a fungal species having root growth-pronnoting properties;
- a fungal species having phosphate-solubilizing and root growth-pronnoting properties;
- a mixture of two, three, four, five or more fungal species each having phosphate- solubilizing and/or root growth-promoting properties;
- a mixture of two, three, four, five or more fungal species each having phosphate- solubilizing properties;
- a mixture of two, three, four, five or more fungal species each having root growth- promoting properties;
- a mixture of two, three, four, five or more fungal species each having phosphate- solubilizing and root growth-promoting properties; and
- a mixture of one or more bacteria species with one or more fungal species each as described above. As used herein, "root growth-promoting" may be understood in the broadest sense as any method that stimulates root (rhizome) growth. Moreover, it is considered that ammonium nutrition may promote the microbial production of auxins as a major hormonal factor for induction of root growth. A variety of bacteria and fungal species promoting root growth are well known in the art.
As used herein, "P-solubilizing" (" phosphate-solubilizing") may be understood in the broadest sense as making phosphate (anions) plant available, in other words, enable plants to take up the phosphate of the substrate S and improving the usability of the phosphate sources of the substrate S for plant growth. Typically, rock phosphate present in many soils is not water soluble and also not or sparingly available for plants. Thus, the rock phosphate contains phosphate anions present in the substrate S, but is still (essentially) not accessible and usably by the plants. The bacteria or fungal species B according to the present invention having phosphate-solubilizing properties render the phosphate available for the plants. Typically, solubilization (i.e., mobilization) of phosphate occurs directly in proximity to the rhizosphere of the plants. Therefore, typically, the phosphate solubilized by the bacteria and/or fungi B is rapidly taken up by the roots of the plants.
In a highly preferred embodiment, the at least one bacteria or fungal species B according to the present invention has phosphate-solubilizing properties. In a particularly preferred embodiment, the at least one bacteria or fungal species B according to the present invention has phosphate-solubilizing and root growth- promoting properties. In a preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate). Alternatively or additionally, the method of the invention is for improving nutrition of plants with iron. Alternatively or additionally, the method of the invention is for improving nutrition of plants with zinc. Alternatively or additionally, the method of the invention is for improving nutrition of plants with manganese. In another preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate) and iron. In another preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate) and zinc. In another preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate) and manganese. In another preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate), iron and zinc. In another preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate), iron and manganese. In another preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate), zinc and manganese. In a particularly preferred embodiment, the method of the invention is for improving nutrition of plants with phosphor (typically phosphate), iron, zinc and manganese. According to the present invention, the at least one bacteria or fungal species B does not form part of ammonium source A, preferably does not form part of A or substrate S (i.e., does neither form part of A or S). This means, the optionally inherently present bacterial and/or fungal content of component A is enriched/supplemented by the at least one bacteria or fungal species B. In other words, the component B is not inherently comprised in the component A, but is added additionally. Therefore, component A may or may not comprise one or more bacteria and/or fungal species optionally having phosphate-solubilizing and/or root growth-promoting properties. In any case, the at least one (additional) bacteria or fungal species B is supplied to the substrate S. This does not exclude that the components A and B and optionally C and optionally further components are premixed prior to being supplied to the substrate S. If the component A is for instance, clearing sludge, moist rest residue of a biogas plant, or a mixture of two or more thereof, this component A will typically comprise bacteria and, optionally, fungal species. Nevertheless, according to the present invention, at least one additional bacteria or fungal species B is added.
Preferably, an amount of species B not forming part of A or S is employed in the method of the invention.
As used herein, the terms "mineral", "mineral salt", "nutrient", "trace mineral", "plant nutrient" and the like should be understood interchangeably in the broadest sense as any inorganic entity (i.e., atom, molecule or, in particular, salt or ion) comprising at least one element as referred to (i.e., phosphor (typically phosphate), iron, zinc and/or manganese). It will be understood that the term "minerals" in the context of phosphor (typically phosphate), iron, zinc and manganese refers to any kind of mineral salt comprising one or more of the aforementioned atoms. Typically, these atoms are not found as elements in nature, but rather as molecular bound ions. Exemplarily, a phosphor mineral in the context of the present invention may comprise anions like phosphate (PO4 3-, HPO4 2- (also designated as hydrogen phosphate), or H2PO4- (also designated as dihydrogen phosphate)). As used herein, hydrogen phosphate and dihydrogen phosphate are also considered as phosphate(s). Most typically the phosphate is PO4 3-. The counter ion of such anion may be any cation. Optionally, the phosphor mineral may also form part of a complex. Exemplarily, an iron mineral in the context of the present invention may comprise cations like Fe2+ or Fe3+. The counter ion of such cation may be any anion. Optionally, the iron mineral may also form part of a complex. Most commonly, the iron is present as the hydrated form of iron oxide. Exemplarily, a zinc mineral in the context of the present invention may comprise a cation like Zn2+. The counter ion of such cation may be any anion. Optionally, the zinc mineral may also form part of a complex. Exemplarily, a manganese mineral in the context of the present invention may bear an oxidation state of 0, +1 , +2, +3, +4, +5 +6, or +7. The binding partner, complexing partner or counter ion of such atom or cation may be any atom, molecule or cation sufficient for this purpose. Optionally, the manganese mineral may also form part of a complex. The term "improving nutrition of plants" may be understood in the broadest sense as ameliorating the uptake of the respective mineral. This can be also designated as fertilizing the plants. Typically, improving nutrition leads to better growth and/or health of the plant. Further, the content of the mineral in the plant is preferably increased. The person skilled in the art will directly and unambiguously understand what is meant by (relative) terms like "improving", "better" or "increasing" in the context of the present invention. These terms refer to the comparison of a plant cultivated on a substrate S subjected to the method according to the present invention with a comparable (i.e., an (essentially) identical) substrate S* not subjected to the method according to the present invention, wherein the plants are cultivated on both soils S and S* under comparable (i.e., (essentially) the same) conditions obtained after a given (identical) time. Thus, stronger plant growth or higher contents of the mineral means an improvement. "Plant growth" may be understood in the broadest sense as plant height (e.g., in centimeters (cm)) obtained after a given time and/or biomass weight (e.g., in grams (g) or kilograms (kg)) after a given time and/or stem diameter (e.g., in millimeters (mm)) after a given time. Alternatively, also the amount of crops may be taken as a parameter for determining plant growth. "Content of mineral" may optionally be provided in milligram of the mineral per kilogram of total plant mass or plant dry mass (provided in mineral mass [mg] / total plant dry mass [kg]).
An ammonium source A may be any molecule or molecular composition either comprising ammonium ions (NH +) or comprising (or consisting of) compounds serving as precursors for ammonium ions such as compounds that are typically metabolized (e.g., by bacteria) or chemically transformed to form ammonium ions, in particular organically bound nitrogen (in particular, primary amino groups, secondary amino groups and tertiary amino groups). Exemplarily, such precursors may be selected from the group consisting of urea, uric acid and ammoniac.
As mentioned before, according to the present invention, at least 50 mol% (i.e., >50 % of the total amount of the nitrogen atoms present in the ammonium source A) is present as the sum of ammonium and/or organically bound nitrogen, In a preferred embodiment, at least 55 mol%, more preferably at least 60 mol%, at least 65 mol%, at least 70 mol%, or more than 75 mol%, of the total amount of the nitrogen of A is present as ammonium and organically bound nitrogen. Preferably, not more than 50 mol% (i.e, ≤50 % of the total amount of the nitrogen atoms present in the ammonium source A), preferably not more than 45 mol%, more preferably not more than 40 mol%, in particular not more than 40 mol% or less, is present as the sum of nitrate and nitrite.
In a preferred embodiment, the at least one ammonium source A is a chemical fertilizer comprising (or consisting of) at least one ammonium salt, manure (liquid or solid manure, in particular liquid), clearing sludge, moist rest residue of a biogas plant, or a mixture of two or more thereof.
In a more preferred embodiment, the at least one ammonium source A is a chemical fertilizer comprising (or consisting of) at least one ammonium salt.
In a particularly preferred embodiment, the at least one ammonium source A is a chemical fertilizer comprising (or consisting of) ammonium sulfate.
It will be understood that the components A, B, and C may be each applied separately from another, or A and B may be premixed and C may be applied separately, or A and C may be premixed and B may be applied separately, or B and C may be premixed and A may be applied separately.
At the latest when applied to the substrate S, the components A, B, and C will inherently form an aqueous solution in the substrate S. When mixing the components in a aqueous solution, i.e., adding the components A, B, and C to water (which is initially neutral, i.e., has a pH of approximately 7), the obtained aqueous solution may have specific characteristics, including any pH.
The at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties may be any bacteria or fungal species suitible for this purpose.
In a preferred embodiment, the at least one bacteria and fungi species B is selected from the group consisting of Trichoderma species, Pseudomonas species, Bacillus species, and combinations thereof. In a more preferred embodiment, the at least one bacteria or fungal species B comprises bacteria selected from the group consisting of Paenibacillus mucilaginosus, Trichoderma harzianum, Pseudomonas sp. DMSZ 13134, Pseudo- monas fluorescens, Bacillus subtilis, Bacillus amyloliquefaciens, and combinations thereof.
In a highly preferred embodiment, the at least one bacteria or fungal species B comprises bacteria selected from the group consisting of Paenibacillus mucilaginosus, Trichoderma harzianum, Pseudomonas sp. DMSZ 13134, Pseudo- monas fluorescens, Bacillus subtilis, and combinations thereof.
Exemplarily, such bacteria or fungal species B may be such exemplified in the Example section below and/or may be obtained from the suppliers exemplified in the Example section below.
In a highly preferred embodiment, the component B comprises at least one Bacillus species (e.g. Bacillus subtilis and/or Bacillus amyloliquefaciens) and is more preferably (essentially) free of other bacteria. In an alternative preferred embodiment, the at least one bacteria or fungal species B is not Bacillus amyloliquefaciens. In another preferred embodiment, the at least one bacteria or fungal species B is not a Bacillus species.
In another highly preferred embodiment, the component B comprises at least one Bacillus species (e.g. Bacillus subtilis and/or Bacillus amyloliquefaciens) and at least one Trichoderma species (e.g. Trichoderma harzianum) and is more preferably (essentially) free of other bacteria.
Exemplarily, a combination product of Bacillus/Trichoderma (e.g., Trichoderma harzianum (OMG16) and Bacillus subtilis) or a Trichoderma! Pseudomonasl- Bacillus (e.g., Trichoderma harzianum (OMG16), Pseudomonas fluorescens, and Bacillus subtilis), optionally further comprising zinc and/or manganese (Zn/Mn), may be used in the context of the present invention. The bacteria and/or fungi may also be commercially available as mixtures such as, e.g., Proradix (Soucon Padena, Tubingen Germany; Pseudomonas sp. DMSZ 13134), VitalinSPI (Vitalin Pflanzengesundheit, Oberamstadt, Germany); mixture of Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Ascophyllum nodosum extract and humic acids), Rhizovital (ABITEP GmbH, Berlin, Germany; Bacillus amyloliquefaciens FZB42), or CombifectorA (comprising: Trichoderma harzianum OMG16, Pseudomonas fluorescens, Bacillus subtilis). Further specific examples are provided in the example section below.
Particularly preferably, the at least one bacteria and/or fungal species B is used in the composition as a spore formulation. In a preferred embodiment, the bacteria and/or fungal species are stored in dry state, in particular as dried or freeze-dried spores. Such formulation may bear higher stress resistance and has a longer shelf-life.
The nitrification inhibitor C may be any nitrification inhibitor known in the art. Exemplarily, a nitrification inhibitor C may be selected and prepared as disclosed in WO 201 1/032904, WO2014/053401 , WO 2013/121384, EP-B 0 808 297, EP-B 1 021 416, EP-B 2 748 148, EP-B 1 120 388, WO 1996/024566, WO 2015/- 086823, EP-B 0 974 585, WO 2015/158853 and literature cited therein. Exemplarily, the nitrification inhibitor C may be selected from the group consisting of 3,4-di- methylpyrazol phosphate (DMPP), nitrapyrine, etridiazole, and 2-cyanoguanidine. Preferably, the nitrification inhibitor C does (essentially) not bear anti-bacterial or anti-fungal properties, i.e., is preferably not bacteriozide or bacteriostatic, fungicide or arresting fungal proliferation, in order to avoid harming the bacteria and/or fungi of component B. In a preferred embodiment, wherein the at least one nitrification inhibitor C is a compound selected from the group consisting of compounds containing a pyrazole residue which can be substituted in their structure, 1 H-1 ,2,4-triazole, 2-chloro-6- (trichloromethyl)-pyridine, 5-ethoxy-3-trichloromethyl-1 ,2,4-thiadiazol, 2-amino-4- chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, 4-amino-1 ,2,4-triazole, 3-mercapto-1 ,2,4-triazole, 2,4-diamino-6-trichloro- methyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methyl carbamate and N-(2,6-dimethyl- phenyl)-N-(methoxyacetyl)-alanine methyl ester. In a preferred embodiment, the at least one nitrification inhibitor C may be selected from the group consisting of 3,4-dimethylpyrazolephosphate (DMPP), 2-(3,4- dimethyl-pyrazol-1 -yl)-succinic acid, 3,4-dimethylpyrazole (DMP), 1 H-1 ,2,4-triazo- le, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine, 5-ethoxy-3-tri- chloromethyl-1 ,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto- benzothiazole, 2-sulfanilamidothiazole, thiourea, 1 -hydroxypyrazole, 2-methyl- pyrazole-1 -carboxamide, 4-amino-1 ,2,4-triazole, 3-mercapto-1 ,2,4-triazole, 2,4- diannino-6-trichloronnethyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methyl carbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester. In a preferred embodiment, the at least one nitrification inhibitor C is a compound of formula (I)
Figure imgf000014_0001
or a stereoisomer, salt, tautomer or N-oxide thereof, wherein
A is aryl or hetaryl, wherein the aromatic ring may in each case be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from R1A and R2A are independently of each other selected from H and C1-C2-alkyl; and
R3A is H, CrC4-haloalkyl, C1-C4-hydroxyalkyl, ethynylhydroxymethyl, phenyl- hydroxymethyl, or aryl, wherein the aromatic ring may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from RB;
and wherein
RA is
(i) halogen, CN, NRaRb, ORc, SRC, C(=Y1)RC, C(=Y1)ORc, C(=Y1)SRC, C(=Y1)NRaRb, Y2C(=Y1)RC, Y2C(=Y1)ORc, Y2C(=Y1)SRC, Y2C(=Y1)NRaRb, Y3Y2C(=Y1)RC, NRgN=C(Rd)(Re), C(=N-ORc)Rg, C(=N-ORc)Rg, C(=N-SRc)Rg, C(=N-NRaRb)Rg, S(=O)2Rf, NRgS(=O)2Rf, S(=O)2Y2C(=Y1)Rc, S(=O)2Y2C(=Y1)ORc, S(=O)2Y2C(=Y1)SRc, S(=O)2Y2C(=Y1)NRaRb, NO2, NON-CN, C1-C6-alkyl, C2-C6- alkenyl, C2-C6-alkynyl, C1-C4-haloalkyl, C1-C4-cyanoalkyl, C1-C4-hydroxyalkyl, C1- C4-alkoxy, C2-C4-alkynyl-C1-C2-hydroxyalkyl, C2-C4-alkynyloxy; (ii) C1-C4-alkylene-C(=Y1)Rc, C2-C4-alkenylene-C(=Y1)Rc, C1-C4- alkylene- C(=Y1)ORc, C2-C4-alkenylene-C(=Y1)OR°, C1-C4-alkylene-C(=Y1)SRc, C2-C4- alkenylene-C(=Y1)SRc, C1-C4-alkylene-C(=Y1)NRaNRb, C2-C4-alkenylene- C(=Y1)NRaNRb, C1-C4-alkylene-Y2-C(=Y1)Rc, C2-C4-alkenylene-Y2-C(=Y1)Rc, d- C4-alkylene-NRaRb, C2-C4-alkenylene-NRaRb, C1-C4-alkylene-ORc, C2-C4- alkenylene-ORc, C1-C -alkylene-SRc, C2-C -alkenylene-SRc, wherein the C1-C - alkylene or C2-C4-alkenylene chain may in each case be unsubstituted or may be partially or fully substituted by OR9, CN, halogen or phenyl;
(iii) aryl, aryl-C1-C2-alkyl, hetaryl or hetaryl-C1-C2-alkyl, wherein the aromatic Ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rh;
(iv) a 3- to 14-membered saturated or unsaturated carbocycle or heterocycle, which may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR1 b, O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R1 b is H, C1-C4-alkyl, C2-C4-alkenyl, C3-C6-cycloalkyl, C3-C6- cycloalkylmethyl, or OR9; or
(v) L-B, wherein
L is -CH2-, -CH=CH-, -CEC-, -C(=O)- or -CH=, and
B is aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially orfully substituted by substituents, which are independently of each other selected from Rh; or
a 3- to 14-membered saturated or unsaturated carbocycle or heterocycle, which may contain 1 , 2, or 3 heteroatoms which, independently of each other, are selected from NR1 b, O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R1 b is H, C1-C4-alkyl, C2-C4-alkenyl, C3-C6-cycloalkyl, C3-C6- cycloalkylmethyl, or OR9; or
(vi) two substituents RA together represent a carbocyclic or heterocyclic Ring, which is fused to A and may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR1c, O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially orfully substituted by substituents which, independently of each other, are selected from R'; and wherein R1c is H, C1-C4-alkyl, C2-C4-alkenyl, C3-C6-cycloalkyl, C3-C6- cycloalkylmethyl, C3-C6-heterocyclyl, C3-C6-heterocyclylmethyl or OR9;
and wherein
RB is NH-C(=O)-(C1-C4-alkyl), NH-C(=O)-(C2-C4-alkenyl), NH-C(=0)-(C1-C2- alkoxy-C1-C2- alkyl), NH-C(=O)-(C3-C6-cycloalkyl), NH-S(=O)2-(C1 -C4-alkyl), or NO2; and wherein
Y1, Y2 and Y3 are independently of each other selected from O, S and NR1a, wherein R1a is in each case independently H, CrC4-alkyl, C2-C4-alkenyl, C3-C6- cycloalkyl, C3-C6-cycloalkylmethyl, OR9, SRg or NRmRn;
Ra and Rb are independently of each other selected from
(i) H, NRRk, OR1, SR1, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyCl,1-C4- hydroxyalkyl, C1-C4-alkoxy, C(=Y1)R', C(=Y1)OR', C(=Y1)SR', C(=Y1)NRjRk, C(=Y1)C(=Y2) R', S(=O)2Rf;
(ii) aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rb; or
Ra and Rb together with the nitrogen atom to which they are bound form
(iii) a hetaryl group which may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rb; or
(iv) a 3- to 10-membered, saturated or unsaturated heterocycle, which may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR1 b, O, and S, wherein S may be oxidized and/or wherein the heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R1b is H, C1-C4- alkyl, C2-C4-alkenyl, C3-C6-cycloalkyl, C3-C6-cycloalkylmethyl, or OR9;
Rc is
(i) H, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C(=O)OR', C(=O)SR', C(=O)NRjRk;
(ii) C1-C4-alkylene-C(=O)R', C1-C4-alkylene-C(=O)OR', wherein the C1-C4- alkylene chain may in each case be unsubstituted or may be partially orfully substituted by OR9, CN, halogen, or phenyl;
(iii) aryl, aryl-C1-C2-alkyl, hetaryl, or hetaryl-C1-C2-alkyl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rh; or (iv) a 3- to 10-membered saturated or unsaturated carbocycle or heterocycle, which may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR1 b, O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from Rl; and wherein R1 b is H, C1-C4-alkyl, C2-C4-alkenyl, C3-C6-cycloalkyl, C3-C6- cycloalkylmethyl, or OR9;
Rd and Re are independently selected from C1-C4-alkyl, C1-C4-haloalkyl, NRjRk, OR', SR', CN, C(=Y1)R', C(=Y1)OR', C(=Y1)SR', or C(=Y1)NRjRk;
Rf is C1-C4-alkyl, C1-C4-haloalkyl, NRjRk, OR1, SR1, aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rh;
Rg is H or C1-C4-alkyl;
Rh is halogen, CN, NO2, NRjRk, OR1, SR1, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-haloalkyl, C1-C4-alkoxy, C2-C4-alkynyloxy, C(=Y1)RI, C(=Y1)OR', C(=Y1)SR', C(=Y1)NRjRk, aryl, aryloxy, hetaryl and hetaryloxy;
R' is
(i) halogen, CN, C1 -C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-haloalkyl, C2- C4-haloalkenyl;
(ii) =NR1d, wherein R1d is H, C1-C4-alkyl, C2-C4-alkenyl, C3-C6-cycloalkyl, C3-C6- cycloalkylmethyl, or OR9;
(iii) =0, =S, NRjRk, OR1, SR1, C(=Y1)R', C(=Y1)OR', C(=Y1)SR', C(=Y1)NRjRk;
(iv) aryl, aryl-C1-C2-alkyl, hetaryl, or hetaryl-C1-C2-alkyl, wherein the aromatic Ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from halogen, CN, C1-C4-alkyl, C1 -C4-haloalkyl, C1-C4-alkoxy, C2-C4-alkynyloxy, OR9, and SR9; or
(vi) C3-C6-cycloalkyl, or 3- to 6-membered heterocyclyl, wherein the cycloalkyl Ring or the heterocyclyl ring may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from halogen, CN, C1-C4-alkyl, OR9, and SR9;
Rj and Rk are independently selected from H, OR9, SR9, C(=Y1)R9, C(=Y1)OR9, C(=Y1)SR9, C(=Y1)NRmRn, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4- haloalkyl, aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently selected from halogen, CN, C1-C4-alkyl, C1-C4-haloalkyl, C1-C4- alkoxy, C2-C4-alkynyloxy, OR9, and SRg;
R1 is H, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-haloalkyl, C(=Y1)Rg, C(=Y1)ORg, C(=Y1)SRg, C(=Y1)NRmRn, aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently selected from halogen, CN, C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C2-C4-alkynyloxy, OR9, and SRg; and Rm and Rn are independently selected from H and C1-C4-alkyl. In a preferred embodiment, in said compound of formula (I), A is phenyl or a 6- membered hetaryl, preferably phenyl, wherein the aromatic ring may in each case be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from RA. In a more preferred embodiment, in said compound of formula (I), R1 and R2 both represent hydrogen.
In another preferred embodiment, in said compound of formula (I), R3 is hydrogen, C1-C4-haloalkyl or ethinylhydroxymethyl, and preferably R3is hydrogen.
In another preferred embodiment, in said compound of formula (I), RA, if present, is
(i) halogen, CN, NRaRb, ORc, C(=Y1)RC, C(=Y1)ORc, C(=Y1)SRC, C(=Y1)NRaRb, Y2C(=Y1)RC, Y2C(=Y1)NRaRb, NRgN=C(Rd)(Re), S(=O)2Rf, NO2, Ci -C6-alkyl, C2-C6- or C1-C4-haloalkyl, C1-C4- alkoxy, C2-C4-alkynyl-C1-C2-hydroxyalkyl, C2-C4- alkynyloxy;
(ii) C2-C4-alkenylene-C(=Y1)Rc, C2-C4-alkenylene-Y2-C(=Y1)Rc, wherein the Ci- C4-alkylene or C2-C4-alkenylene chain may in each case be unsubstituted or may be partially or fully substituted by CN or halogen;
aryl, wherein the aromatic ring of the aryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rh; or
(iii) a 3- to 14-membered saturated or unsaturated heterocycle, which may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR1 b, O, and S, wherein S may be oxidized and/or wherein the heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R1 b is H, C1-C4- alkyl, C2-C4-alkenyl, C3-C6-cycloalkyl, C3-C6-cycloalkylmethyl, or OR9, wherein preferably
Y1, Y2 and Y3 are independently of each other selected from O, S and NR1 a, wherein R1a is in each case independently H, C1-C4-alkyl, OH, or NH2.
Ra and Rb are independently of each other selected from
(i) H, NH2, C1-C4-alkyl, C1-C4-hydroxyalkyl, C(=O)H, C(=S)H, C(=N-H)H, C(=N-(C C4)alkyl))H, C(=N-OH)H, C(=N-NH2)H, or Ra and Rb together with the nitrogen atom to which they are bound form
(iv) a 3- to 10-membered, saturated or unsaturated heterocycle, which may contain 1 , 2, or 3 heteroatoms which, independently of each other, are selected from NR1 b, O, and S, wherein S may be oxidized and/or wherein the heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected ' from C1-C4-alkyl, C1-C4-haloalkyl, C2- C4-haloalkenyl, and =O; and wherein R1 b is H, C1-C4-alkyl, or OH;
Rc is
(i) H, C1-C4-alkyl; or
(iv) a 3- to 10-membered saturated or unsaturated carbocycle or heterocycle, which may contain 1 , 2, or 3 heteroatoms which, independently of each other, are selected from NR1 b, O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from C1-C4-alkyl, C1-C4-haloalkyl, C2-C4-haloalkenyl, and =O; and wherein R1 b is preferably H, C1-C4-alkyl, or OH;
Rd and Re are independently selected from NH2 and C(=O)0H;
Rf is C1-C4-alkyl;
Rg is H;
Rh is halogen or C1-C4-alkoxy; and
R' is
(i) C1-C4-alkyl, C1-C4-haloalkyl, C2-C4-haloalkenyl; or (iii) =O.
In a preferred embodiment, in said compound of formula (I),
R1 and R2 both represent hydrogen,
R3 is hydrogen, and A is phenyl, wherein the aromatic ring is substituted by 1 ,2, or 3 substituent(s) RA, wherein the substituent(s) RA are independently of each other selected from halogen, CN, NH2, C(=O)NRaRb, NHC(=0)NRaRb, NHC(=S)NRaRb, NHC(=O)H, C1-C4-alkoxy, C2-C4-alkynyl-Ci-C2-hydroxyalkyl, and C2-C4-alkynyloxy,
wherein Ra and Rb are in each case independently of each other selected from H, C1-C2-alkyl, NH2, CrC2-hydroxyalkyl, or wherein Ra and Rb may together with the nitrogen atom to which they are bonded form a morpholine ring.
In a more preferred embodiment, the at least one nitrification inhibitor C is selected from the group consisting of 3,4-dimethylpyrazolephosphate (DMPP), 2-(3,4-di- methyl-pyrazol-1 -yl)-succinic acid, 3,4-dimethylpyrazole (DMP), 1 H-1 ,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine and 5-ethoxy-3- trichloromethyl-1 ,2,4-thiadiazol. It will be understood that the formulae shown herein also comprise the acid form as well as the neutralized form, in particular alkali salts thereof (e.g., the respective sodium, potassium or lithium salt).
In a highly preferred embodiment, the at least one nitrification inhibitor C is selected from the group consisting of 3,4-dimethylpyrazolephosphate (DMPP) and 2-(3,4-dimethyl-pyrazol-1 -yl)-succinic acid or a salt thereof, in particular an alkali salt thereof.
In a highly preferred embodiment, the at least one nitrification inhibitor C is a compound of general formula (II)
Figure imgf000020_0001
or acid addition salts thereof, where the radicals R1, R2, R3 and R4 independently from another have the following meanings:
R1, R2, R3 and R4 can be hydrogen, C1- to C2o-alkyl, C3- to C8-cycloalkyl, C5- to C2o-aryl or alkylaryl, it being possible for these 4 radicals to be monosubstituted or disubstituted by halogen and/or hydroxyl,
R1, R2, R3 can also be halogen or nitro; R4 can also be a radical having the formula (III)
Figure imgf000021_0001
where
- R5 and R6 independently from another are hydrogen, C1- to C20-alkyl which can be monosubstituted or disubstituted by halogen and/or hydroxyl, a carboxyl group, a carboxymethyl group or a functional derivative of the two last-mentioned groups, and
- R7 is a carboxyl radical or a carboxy-(C1- to C3-alkyl) radical or a functional derivative of these groups,
or is selected from the group consisting of 1 H-1 ,2,4-triazole, 2-chloro-6-(trichloro- methyl)-pyridine and 5-ethoxy-3-trichloromethyl-1 ,2,4-thiadiazol. In a particularly preferred embodiment, the at least one nitrification inhibitor C is 3,4-dimethylpyrazol phosphate (DMPP).
Optionally, the plants may be further supplied with one or more additional salts further improving nutrition of plants as component E. Such additional salts E may be chosen to reduce stress of the plants. Such additional salts E may exemplarily be selected from the list consisting of one or more zink salts, one or more magnesium salts, one or more seaweed extracts, and combinations thereof.
The plant may, in general, be any plant that is to be grown. Preferably, the plant is a plant that is used in agriculture, including farming and horticulture.
Accordingly, in a preferred embodiment, the plants are crop plants, in particular crop plants selected from the group consisting of group consisting of maize, wheat, and tomato.
In a particularly preferred embodiment, the plant is maize. The substrate S of agricultural use on which the plants are cultivated or intended to be cultivated may be any kind of substrate including any kind of soil and any kind of artificial plant substrate (e.g., expanded clay aggregate, mineral wool, seet gel, perlit, polymers (e.g., styromull, polystyrene, polyurethane, etc.) or combinations of two or more thereof, optionally further comprising additives such as e.g., superabsorber). Before conducting the method according to the present invention, it may have any pH. Exemplarily, the pH may be in the range of from 4 to 10. Preferably, the pH of the substrate S, before conducting the method according to the present invention, is in the range of from 5 to 9, more preferably in the range of from 5.0 to 8.5, even more preferably in the range of from 5 to 8, in particular in the range of from 5.5 to 7.5, such as, e.g., in the range of from 5.5 to 6.0, from 6.0 to 6.5, from 6.5 to 7.0, or from 7.0 to 7.5.
Preferably, the pH of the substrate S, after conducting the method according to the present invention, is decreased (i.e., the soil is acidified), remained unchanged, or merely slightly increased by not more than 1 pH unit, more preferably decreased, remained unchanged, or increased by not more than 0.5 pH units, in particular decreased, remained unchanged, or increased by not more than 0.1 pH units. In other words, the proton (H+) concentration in the substrate S is preferably either increased upon conducting the method according to the present invention, is remained unchanged, or is decreased by not more than a factor of 10, 3 or 1 .3.
More preferably, the pH of the substrate S, after conducting the method according to the present invention, is decreased (i.e., the soil is acidified), wherein the decrease may exemplarily be a decrease up to 4 pH units, up to 3 pH units, up to 2 pH units, up to 1 pH unit, 0.1 to 3 pH units, 0.2 to 2 pH units, or 0.5 to 1 pH units. In other words, the proton (H+) concentration in the substrate S is preferably increased upon conducting the method according to the present invention, wherein the increase is an increase of the proton concentration in the substrate S up to 10000fold, up to 100Ofold, up to 100fold, up to 10fold, by 1 .3 to 100Ofold, by 1 .6 to 100 fold, or 3 to 10fold.
In a preferred embodiment, the weight ratio between the nitrogen in the at least one ammonium source A and the at least one nitrification inhibitor C (A : C) is in the range of between 20:1 and 10000 : 1 , preferably in the range of between 20:1 and 5000 : 1 , in the range of between 50:1 and 1000 : 1 , or in the range of between 75:1 and 200 : 1 . For example, the used amount of the nitrification inhibitor C is between 0.1 and 20% (w/w), preferably between 0.2 and 12% (w/w) or between 0.5 and 1 % (w/w), referred to the total content of nitrogen contained in the one or more ammonium sources A (total N set = 100%). For example, the used amount of the nitrification inhibitor C may be 0.6% (w/w), 0.7% (w/w), 0.8% (w/w), 0.9% (w/w), 1 .0% (w/w), 1 .1 % (w/w), 1 .2% (w/w), 1 .3% (w/w), 1 .4% (w/w), 1 .5% (w/w), 1 .6% (w/w), 1 .7% (w/w), 1 .8% (w/w), 1 .9% (w/w), 2% (w/w), 3% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 1 1 % (w/w), or 12% (w/w), referred to the total content of nitrogen contained in the one or more ammonium sources A (total N set = 100%). In the exemplified Novatec Solub (Compo) compositions, the amount of the nitrification inhibitor DMPP is typically in the range of 0.8% (w/w), referred to the total content of nitrogen (set = 100%). The amounts of nitrification inhibitor C, will be adapted to the individual chemical compounds. For example, it may be adapted to the molecular weight. The above amounts may be particularly beneficial for a nitrification inhibitor like DMPP.
The one or more bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties may be used in any amount. In a preferred embodiment, between 107 and 1011 colony forming units (cfu) kg-1 substrate S are used, preferably between 108 and 108 cfu kg-1 substrate S are used, exemplarily, 109 cfu kg-1 substrate S are used.
A decrease of the pH of the soil may optionally also be linked with a larger spatial extension of root-induced rhizosphere acidification as a consequence of a larger root system.
Preferably, the substrate S is a soil containing sparingly soluble salts of at least one mineral which can preferably be mobilized by the bacteria or fungal species B.
The substrate S may or may not comprise at least one phosphate salt or inorganic recycling fertilizer product (ashes, slugs).
Insufficient nutrition of the plants with one or more sparingly soluble minerals (e.g, phosphor (typically phosphate), iron, zinc and/or manganese) may have different reasons. Exemplarily, it may be due to poor plant availability of the minerals from the substrate S and/or may be due to insufficient development of the root systems of the plants.
In a preferred embodiment, before conducting step (ii), the substrate S comprises less than 50 mg, more preferably not more than 30 mg, in particular not more than 20 mg, plant available phosphate per kg of the substrate S.
The plant available phosphate is determined according to the (German) calcium acetate lactate (CAL) extraction method conducted at pH 6.5-8.5) (with or without amendments of sparingly soluble P fertilisers such as rock-P or sewage sludge ash). The CAL determination of sparingly soluble minerals, in particular phosphate (PCAL, CAL P), as used herein means determination according to data sheet VDLUFA I, P und K, CAL-loslich, A 6.2.1 .1 in Handbuch der Bodenuntersuchung: Terminologie, Verfahrensvorschriften und Datenblatter; physikalische, chemische, biologische Untersuchungsverfahren: gesetzliche Regelwerke. Hrsg. DIN, Deutsches Institut fur Normung e. V., Beuth Verlag, 2000, ISBN 3-410-14590-7. This is further exemplified in the example section.
The term "plant available phosphate" as used herein may be understood in the broadest sense as the usability of the phosphate for plant growth. Typically, rock phosphate present in many soils is not water soluble and also not or sparingly available for plants. Thus, the rock phosphate contains phosphate anions present in the substrate S, but is still (essentially) not accessible and usably by the plants. Typically, at concentrations of approximately 50 mg or above of plant available phosphate per kg of the substrate S (CAL P/kg soil), phosphate is, for many plants, typically no longer a growth limiting factor under field conditions.
In a preferred embodiment, the plants bear root systems insufficient for efficient nutrient uptake, in particular wherein the plants show early root growth and/or have been subjected to stress conditions such as low root zone temperature.
Then, the method of the present invention may promote acquisition of critical nutrients under conditions when soil contents are sufficient but the ability of the root system for nutrient acquisition is limited. As used herein, a low temperature may exemplarily be a temperature in the range of below 15°C, exemplarily in the range of from 0 to 20 °C, preferably in the range of 5 to 15°C, in particular in the range of 10 to 15°C, exemplarily in the range of 12 to 14°C.
The method of the present invention is also suitible for phytosanitary uses such as increasing the plant's resistance against pathogens such as, e.g., fungi, bacteria and(or viruses, in particular in the rhizosphere, but also all over the plant as a whole.
As indicated above, the method is particularly useful for substrate S bearing a shortage of at least one mineral. Thus, prior to conducting the method of the present invention, the substrate S preferably comprises merely low amounts of plant available soluble salts of the mineral.
Step (ii) of the method, i.e., supplying the components A, B and C to the substrate S, may be conducted by any means. In a preferred embodiment, a liquid composition may be prepared and applied to the plants and/or the seeds of the plants by drenching. Exemplarily, seed row starter application by fertigation or drenching of the substrate S in nursery pots may be used. Also seed placement of granulated formulations showing superior root colonization efficiency may be used. Seed dressing may be used. Particularly preferably, fertigation of liquid formulations is used. Alternatively, the one two or all of the components A, B and C may be applied to the plants as one or more powders or pellets/granules. As used herein, the terms "pellet" and "granule" may be understood interchangeably as any solid particle in the milli- or micrometer range (e.g., in the range of from 0.01 to 1 mm, 0.1 to 2 mm or 0.5 to 5 mm). Such powders or granules may also be placed incorporated into the substrate S near the roots. Alternatively, the one two or all of the components A, B and C may be applied to the plants as a depot. Such depot may also be placed buried near the roots. As indicated above, the components A, B and C may be applied in combination with another or may be party combined with another or may be administered separately from another.
In a preferred embodiment, the method comprises step (i-l) of premixing the components A, B and C thereby forming mixture ABC, optionally in an aqueous suspension or in solid state (e.g., as a pellet/granule),
subsequent to step (i) and prior to conducting step (ii), wherein step (ii) is
(ii) applying the mixture ABC to substrate S of agricultural use on which the plants are cultivated or intended to be cultivated.
The premixed composition obtained from step (i-l) may be a liquid, a powder or a syrup. Optionally, one, two or all of the components A, B and/or C may also be pelletized, either form pellets comprising one two, or all of the components A, B and/or C. This will depend on the components and optional further ingredients, in particular on the ammonium source A (which is typically the largest volume) and on the optional presence of a solvent such as water in which the components may be dissolved. In a preferred embodiment, the components A, B and C form an aqueous suspension, wherein preferably components A and C are at least partly dissolved and the bacteria and/or fungal B are suspended.
In a particularly preferred embodiment, pellets/granules comprising the components A and C are prepared and coated with component B, exemplarily, by means of soaking or spraying the component B on the pellets/granules comprising the components A and C.
In a highly preferred embodiment, the mixture ABC, in particular when provided in solid state (e.g., as a pellet/granule), may be added to the rhizosphere, exempla- rily as a reservoir/depot.
In an alternative preferred embodiment, the method comprises step
(i-ll) of premixing the components A and C thereby forming mixture AC, optionally in an aqueous solution or suspension,
subsequent to step (i) and prior to conducting step (ii), wherein step (ii) comprises the steps
(ii-a) applying the mixture AC to substrate S of agricultural use on which the plants are cultivated or intended to be cultivated, and
(ii-b) applying the component B to substrate S of agricultural use on which the plants are cultivated or intended to be cultivated, wherein step (ii-a) can be conducted concomitant with, prior to or subsequent to step (ii-b).
Depending of the choice of organisms, applying steps (ii-a) and (ii-b) separately from another (i.e., not concomitantly) may minimize risks of toxicity to the microorganisms of component B and may provide higher flexibility for combinations.
In a particularly preferred embodiment, the applying the component B to substrate S of agricultural use may be performed by soaking a seedling of the plant and then planting the plants. Then, the mixture AC may be supplied to the substrate S subsequently to this step.
In another particularly preferred embodiment, the applying the component B to substrate S of agricultural use may be performed by supplying reservoirs( depots comprising the component B in the rhizisphere and the mixture AC may be additionally added to the substarte S by any means.
The seeds of the plants may be applied to the substrate S prior, concomitantly or subsequently to conducting step (ii).
In a preferred embodiment, applying at least one of the components A, B and/or C to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S.
Then, steps (ii) and (ii) refer to:
(ii) applying the components A, B and C and seeds of the plants (concomitantly) to substrate S of agricultural use on which the plants are intended to be cultivated; and
(iii) enabling (starting) growth of the plants in the substrate S obtained from step (ii).
Exemplarily, applying component A to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S. Alternatively, applying component B to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S. Alternatively, applying component C to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S. Alternatively, applying a combination of the components A and B (AB) to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S. Alternatively, applying a combination of the components A and C (AC) to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S. Alternatively, applying a combination of the components B and C (BC) to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S. Alternatively, applying a combination of the components A, B and C (ABC) to the substrate S according to step (ii) is conducted concomitant with applying seeds of the plant to the substrate S. In each of these cases of applying one or more of the components to the substrate S according to step (ii) concomitant with applying the seeds, the seeds and the one or more seeds can be applied independently from another or combined with another. The further components are added separately, which can be conducted concomitantly, precedingly or subsequently.
In a preferred embodiment, the seed is coated with a composition comprising one or more of the components A, B and/or C. Exemplarily, the seed may be coated with a composition comprising component A, or a composition comprising component B, or a composition comprising component C, or a composition comprising components A and B (AB), or a composition comprising components A and C (AC), or a composition comprising components C and B (CB), or a composition comprising components A, B and C (ABC). It will be well understood that a coating can optionally comprise further ingredients such as those selected from the group consisting of binders (e.g, one or more sugars), fillers, minerals promoting plant growth, bacteria and/or fungal nutrients, and combinations of two or more thereof. The person skilled in the art will know a number of ingredients usable in such coatings. The further components are added separately, which can be conducted concomitantly, precedingly or subsequently.
Exemplarily, the seeds may be coated with a composition comprising at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth- promoting properties (component B) and a combination of an ammonium source (component A) comprising nitrification inhibitor (component C) may be added additionally. In this example, steps (ii) and (ii) refer to: (ii) applying
(a) seeds coated with a composition comprising at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth- promoting properties, and
(b) a composition comprising at least one ammonium source A and cat least one nitrification inhibitor (e.g., DMPP)
to substrate S of agricultural use on which the plants are intended to be cultivated; and
(iii) enabling (starting) growth of the plants in the substrate S obtained from step (ii).
In an alternative preferred embodiment, the substrate S is prepared by means of the method according to the present invention prior to sowing the plants. Then, steps (ii) and (ii) refer to:
(ii) applying the components A, B and C to substrate S of agricultural use on which the plants are intended to be cultivated;
(ii*) applying seeds of the plants to substrate S obtained from step (ii); and (iii) enabling (starting) growth of the plants in the substrate S obtained from step
(ii).
In an alternative preferred embodiment, the plants are already grown before conducting the method according to the present invention, and step (iii) means enabling further growths of the plants in the substrate S obtained from step (ii). Then, steps (ii) and (ii) refer to:
(ii) applying the components A, B and C to substrate S of agricultural use on which the plants are (already) cultivated; and
(iii) enabling further growth of the plants in the substrate S obtained from step (ii).
As indicated above, in a preferred embodiment, the method is for improving root growth of the plants.
As indicated above, the invention bases on the interplay of components A, B and C. Accordingly, the intended application of said components, in particular the concomitant supplementation of said components to the substrate S is encom- passed by the present invention. As used herein, the term "plant root" may be interpreted in the broadest sense as growth of any part of the rhizosphere, i.e., root growth. Particularly preferably, the biomass of the roots (total and/or dry weight thereof) is enhanced in comparison to the biomass obtained for the roots of a plant grown on comparable substrate S* under comparable conditions not subjected to the method according to the present invention.
In a particularly preferred embodiment, the method is for improving plant root of the plants. Therefore, the growth of the roots is preferably stimulated. The biomass of the roots (total and/or dry weight thereof) is enhanced in comparison to those of a comparable plant grown on comparable substrate S* under comparable conditions not subjected to the method according to the present invention. It will be understood that a promoted root growth may also promote nutrient uptake, water uptake and/or total plant growth, in particular root growth.
A further aspect of the present invention relates to the use of (a combination of) at least one ammonium source A, wherein at least 50 mol% of the total nitrogen content of A is present as ammonium and/or organically bound nitrogen, at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth- promoting properties, wherein B does not form part of A, and at least one nitrification inhibitor C for improving nutrition of plants with one or more sparingly soluble minerals selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese. It will be understood that all definitions and preferred embodiments laid out in the context of the method according to the present invention mutatis mutandis also apply to the use. Thus, the use is preferably further characterized by one or more features as laid out in the context of the method laid out herein. As used in the context of the use, the term "combination of may be understood in the broadest sense as using all of the components A, B and C. It does not necessarily mean that these ingredients are comprised in a single composition. The can also be applied separately from another. A still further aspect of the present invention relates to a composition usable for a method according to the present invention, said composition comprising (in particular consisting of):
(A) one or more ammonium sources A,
(B) one or more bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties,
(C) one or more nitrification inhibitors C,
(D) optionally water,
(E) optionally one or more additional salts further improving nutrition of plants, and
(F) optionally one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers,
wherein at least 50 mol% of the total nitrogen content of the composition is present as ammonium and/or organically bound nitrogen, and
wherein the composition does not comprise an ammonium source A comprising bacteria or fungal species with phosphate-solubilizing and/or root growth-promoting properties. It will be understood that all definitions and preferred embodiments laid out in the context of the method according to the present invention mutatis mutandis also apply to the composition of the present invention.
As indicated above, in a preferred embodiment, the composition does not comprise considerable amounts of soluble salts of (sparingly soluble) minerals, such as minerals selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese. It will be understood that the components may comprise traces of such salts. In a preferred embodiment, the composition does (essentially) not comprise salts selected from the group consisting of phosphor (typically phosphate), iron, zinc and manganese salts, in particular does (essentially) not comprise such salts in solid form and/or of synthetic origin, phosphor (typically phosphate) salts in solid form and/or of synthetic origin. In a preferred embodiment, the composition does (essentially) not comprise such salts in solid form and/or of synthetic origin, in particular does (essentially) not comprise phosphor (typically phosphate) salts in solid form and/or of synthetic origin. In a preferred embodiment, the amount of phosphor in the composition is below 10% (w/w), preferably below 5% (w/w), more preferably below 1 % (w/w) or below 0.1 % (w/w), based on the sum of the weight of the components A, B and C. In a preferred embodiment, the at least one bacteria or fungal species B is not Bacillus amyloliquefaciens and the amount of phosphor in the composition is below 10% (w/w), preferably below 5% (w/w), more preferably below 1 % (w/w) or below 0.1 % (w/w), based on the sum of the weight of the components A, B and C and the total amount of phosphor.
It will be understood that the composition is free of such ammonium sources A comprising bacteria or fungal species with phosphate-solubilizing and/or root growth-promoting properties. Therefore, the composition will be typically exempla- rily free of manure, clearing sludge, moist rest residue of a biogas plant, and soil. In a preferred embodiment, the one or more ammonium sources A is/are one or more ammonium salts, more preferably a chemical fertilized comprising at least one ammonium salt, in particular ammonium sulfate.
In a preferred embodiment, the components A and B may or may not be spatially separated from another. In a preferred embodiment, A and B are mixed powders. In another preferred embodiment, A and B are provided in spatially separated containers or bags.
Optionally, such composition may also be used as a coating for seeds of the plants. Accordingly, the present invention also refers to seed coated with a composition according to the present invention (typically (essentially) without water).
Optionally, such composition may also be pelletized. Accordingly, the present invention also refers to pellets comprising (or consisting of) a composition according to the present invention.
In a preferred embodiment, the composition comprises one or more additional salts further improving nutrition of plants as component E. Such additional salts E may be chosen to reduce stress of the plants. Such additional salts E may exemplarily be selected from the list consisting of one or more zinc (Zn) salts, one or more magnesium (Mg) salts, one or more manganese (Mn) salts, one or more seaweed extracts, and combinations thereof. In a preferred embodiment, the component E comprises (or consists of) a Zn/Mn mixture (i.e., a mixture of Zn and Mn salt(s)). The composition may be a powder, a liquid or a syrup. Optionally, it may be a commercial product. Preferably however, the user completes the composition just prior to its use, i.e., prior applying the composition to the substrate S of agricultural use on which the plants are cultivated or intended to be cultivated. Particularly preferable, the user adds the components B, C and, optionally, D, E and/or F to one or more ammonium sources A prior use. The present invention also refers to a packaging unit such as a tank or a sachet/bag comprising the composition according to the present invention.
In a particularly preferred embodiment, the composition is a pellet/granule comprising the components A, B and C and optionally one or more of the components C, D and/or E. Exemplarily, the core of such pellet/granule may comprise the components A and C and the shell may comprise the component B. This may optionally be achieved by spraying or soaking of precursor pellets/- granules comprising the components A and C by a suspension comprising component B.
Depending on the microorganisms of component B (i.e., the one or more bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties), the optional premixing of components and/or the application formu- lation will be chosen. Exemplarily, separate use of liquid formulations comprising components A and C, liquid microbial inoculants for seed row application, soil incorporation or soil drenching of nursery pots may be used. Exemplarily, granulated formulations (e.g mixed product with separate granules for fertilizers and microorganisms e.g. for under seed placement, may be used.
A still further aspect of the present invention refers to a kit for use in a method according to the present invention comprising at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties, wherein B does not form part of A, and at least one nitrification inhibitor C, and optionally one or more ammonium sources A. It will be understood that all definitions and preferred embodiments laid out above in the context of the method and the composition according to the present invention mutatis mutandis also apply to the kit of the present invention. In a preferred embodiment, the at least one bacteria and/or fungal species B is present in the kit as a spore formulation. In a particularly preferred embodiment, the bacteria and/or fungal species are stored in dry state, in particular as dried or freeze-dried spores. Such formulation may bear higher stress resistance and has a longer shelf-life. The bacteria and/or fungal species (spores) may be packed in any form, e.g., in a container or a sachet.
Optionally, the kit may comprise one, two or all of the components A, B and/or C in pelletized (granulized) form, either separated or in combination with another as described above.
In a preferred embodiment, the kit comprises (or consists of), as functional components:
(B) one or more bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties,
(C) one or more nitrification inhibitors C,
(A) optionally one or more ammonium sources A,
(D) optionally water,
(E) optionally one or more additional salts further improving nutrition of plants,
(F) optionally one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers, and
It will be understood that the kit will typically further comprise means for packaging the functional components.
In a preferred embodiment, the kit further comprises user instructions for conducting a method according to the present invention.
In a preferred embodiment, the kit according to the present invention does not comprise (i.e., is (essentially) free of) other bacteria species X than bacteria or fungal species B. The kit according to the present invention may comprise (or consist of), as functional components:
A two or more components premixed with another and one or more other components provided separately;
B a premixed composition comprising (or consisting of) components B and C and, optionally, A, D, E and/or F;
C all components B and C and, optionally, A D, E and/or F provided separately.
In a particularly preferred embodiment, the kit comprises (or consists of):
(I) a powder comprising (or consisting of) component B and, optionally, components E and/or F;
(II) a powder comprising (or consisting of) component C and, optionally, component F; and
(III) optionally a composition comprising (or consisting of) any of components A and/or F.
In an alternative preferred embodiment, the kit comprises (or consists of):
(l-ll) a powder comprising (or consisting of) components B and C and, optionally, components E and/or F; and
(III) optionally a composition comprising (or consisting of) any of components A and/or F.
In an alternative preferred embodiment, the kit comprises (or consists of):
( ) a liquid or syrup composition comprising (or consisting of) component B and, optionally, components E and/or F;
(II) a powder comprising (or consisting of) component C and, optionally, component F; and
(III) optionally a composition comprising (or consisting of) any of components A and/or F.
In an alternative preferred embodiment, the kit comprises (or consists of):
(I) a powder composition comprising (or consisting of) component B and, optionally, components E and/or F; (II*) a liquid or syrup composition comprising (or consisting of) component C and, optionally, component F; and
(III) optionally a composition comprising (or consisting of) any of components A and/or F.
In an alternative preferred embodiment, the kit comprises (or consists of):
(II*) a liquid or syrup composition comprising (or consisting of) component B and, optionally, components E and/or F;
(II*) a liquid or syrup composition comprising (or consisting of) component C and, optionally, component F; and
(III) optionally a composition comprising (or consisting of) any of components A and/or F.
In an alternative preferred embodiment, the kit comprises (or consists of):
(Ι- II*) a liquid or syrup composition comprising (or consisting of) components B and C and, optionally, components E and/or F; and
(III) optionally a composition comprising (or consisting of) any of components A and/or F. As far as appropriate and desired, mixing of the components with another can be conducted by any means known in the art. Exemplarily, mixing can be conducted in undissolved/undiluted state (e.g., as mixing one or more dry powders and/or one or more syrups with another) or can be completely or partly dissolved and/or diluted before mixing, e.g., in an aqueous solvent such as water.
When it is intended to use liquid chemical fertilizer comprising at least one ammonium salt, manure, clearing sludge, moist rest residue of a biogas plant, or a mixture of two or more thereof, in particular ammonium sulfate, as ammonium source (component A), the kit preferably comprises (or consists of) components (I) and (II), (I*) and (II), (I) and (II*), (I*) and (II*), (l-ll) or (Ι-ΙII*) only, and the user adds one or both of these components to the component A of interest just before supplying the substrate S therewith.
In an alternative preferred embodiment, the kit comprises (or consists of) (l-ll-lll): a powder comprising (or consisting of) components A, B and C and, optionally, components E and/or F. In an alternative preferred embodiment, the kit comprises (or consists of) (Ι-ΙΙ-ΙII*): a liquid or syrup composition comprising (or consisting of) components A, B and C and, optionally, components E and/or F.
In a further preferred embodiment, the kit according to the present invention comprises (or consists of) a composition according to the present invention. Herein, as described above, the composition may be an aqueous suspension or may be a powder.
A further aspect of the present invention relates to a composition comprising or consisting of:
(A) one or more ammonium sources A of synthetic origin,
(B) one or more bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties,
(C) one or more nitrification inhibitors C,
(D) optionally water,
(E) optionally one or more additional salts further improving nutrition of plants, and
(F) optionally one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers,
wherein preferably at least 50 mol% of the total nitrogen content of the composition is present as ammonium and/or organically bound nitrogen.
The components are each defined as above. The presence of the ammonium sources A as being of synthetic origin may be understood in the broadest sense in that the ammonium sources A forms part of or is a chemical fertilizer. This composition may or may not comprise more than 20% (w/w), 10% (w/w), 5% (w/w), 1 % (w/w) or 0.1 % (w/w) of phosphate (typically in the form of phosphate), based on the total weight of the composition as a whole.
In a preferred embodiment, the composition is a solid composition comprising the components A, B and C and, optionally, E and, optionally F, as mixed powders. Then, preferably, water is preferably (essentially) absent in the storable composition. Water may optionally be added just before supplying a substrate S with the composition. The terms are defined as laid out throughout the present invention.
In a preferred embodiment, a composition of the present invention may comprise or consist of:
(A) 5-99.9% (w/w), preferably 5-50% (w/w) of one or more ammonium sources A,
(B) 0.05-1 % (w/w) of one or more bacteria or fungal species B,
(C) 0.05-20% (w/w) of one or more nitrification inhibitors C, referred to the total composition,
preferably between 0.1 and 10% (w/w), more between 0.2 and 12% (w/w), referred to the total content of nitrogen in the one or more ammonium sources A,
(D) 0-80% (w/w) of water,
(E) 0-80% (w/w) of one or more additional salts further improving nutrition of plants, and
(F) 0-80% (w/w) of one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers.
In a preferred embodiment, a composition of the present invention may comprise or consist of:
(A) 5-99.85% (w/w), preferably 5-20% (w/w) of nitrogen comprised in one or more ammonium sources A;
(B) 0.05-1 % (w/w) of one or more bacteria or fungal species B,
(C) 0.1 -2% (w/w) of one or more nitrification inhibitors C, , referred to the total composition,
preferably between 0.1 and 10% (w/w), more between 0.2 and 12% (w/w), referred to the total content of nitrogen in the one or more ammonium sources A,
(D) 0-80% (w/w) of water,
(Ε') 0-50% (w/w) of phosphate in phosphate-containing salts,
(E") 0-50% (w/w) of one or more other additional salts, which are not phosphate- containing, for further improving nutrition of plants, and 0-80% (w/w) of one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers. In a preferred embodiment, a composition of the present invention may comprise or consist of:
(A) 5-99.83% (w/w), preferably 7-15% (w/w) of nitrogen comprised in one or more ammonium sources A;
(B) 0.07-0.5% (w/w) of one or more bacteria or fungal species B,
(C) 0.1 -5% (w/w) of one or more nitrification inhibitors C, referred to the total composition,
preferably between 0.1 and 20% (w/w), more between 0.2 and 12% (w/w), referred to the total content of nitrogen in the one or more ammonium sources A,
(D) 0-1 % (w/w) of water,
(Ε') 0-40% (w/w) of phosphate in phosphate-containing salts,
(E") 0-50% (w/w) of one or more other additional salts, which are not phosphate- containing, for further improving nutrition of plants, and
(F) 0-10% (w/w) of one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers.
The amounts, in particular the amount of nitrification inhibitor C, will be adapted to the individual chemical compounds.
It will be understood that the following examples are not intended to limit the scope of the claims. The following examples are provided to exemplify the invention further and to provide specific embodiments thereof. Brief description of the Figures
Figure 1 demonstrates effects of soil-buffering capacity on growth stimulation of maize by microbial biofertilizer-induced Ca-P solubilisation on a substrate with Ca- P and Rock-P as exclusive P sources at six weeks after sowing (Nkebiwe, 2017). (No. BEs = rock phosphate without microbial biofertilizers (negative control); +P = soluble P 150 mg P kg-1 soil as Ca(H2PO4)2, (positive control); Pseu = Pseudomonas sp. DMSZ 13134 = Proradix® (Soucon Padena, Tubingen Germany); SP1 1 = VitalinSPI® (Vitalin Pflanzengesundheit, Oberamstadt, Germany): Mixture of Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Ascophyllum nodosum extract, humic acids; Penic = Penicillium sp.; Paenibac = Paenibacillus mucilagenosus, Three weekly inoculations wit)
Figure 2 demonstrates rhizosheath formation of maize under field conditions and along single roots in a rhizobox culture system with root observation window. Figure 3 shows the synergistic effect of a nitrification inhibitor (DMPP), microbial biofertilizers and ammonium fertilization. This figure depicts the comparison between the synergistic effects of different microbial biofertilizers. (Herein: NoP = background control without phosphate source; NoBE = rock phosphate without microbial biofertilizers (negative control); Trianum P = Trichoderma harzianum T22 (deposit No.: ATCC 20847); Proradix = Pseudomonas sp., (deposit No.: DMSZ 13134); CombifectorA comprising: Trichoderma harzianum OMG16, Pseudomonas fluorescens, Bacillus subtilis, and micronutrients; Rhizovital = Rhizovital FZB42: Bacillus amyloliquefaciens subsp. plantarum, synonymous: Bacillus velezensis FZB42 (Taxonomy ID: 326423; deposit No.: DSM231 17); Paenibacillus = Paenibacillus mucilaginosus); BFOD = Penicillium bilaii; VitSP1 1 = Vitalin SP1 1 ; NO3- = nitrate salt added; NH + = ammonium salt added; Rock-P = sparingly soluble calcium phosphates as exclusive P source; Solub-P = soluble phosphor source added). The star (*) indicates that the t-test is significant (0.05 alpha) compared to NoBE.
Figure 4 shows the synergistic synergistic effect of a nitrification inhibitor (DMPP), microbial biofertilizers and ammonium fertilization on the auxin generation in maize (herein: NoBE = rock phosphate without microbial biofertilizers (negative control); Rhizovital = Rhizovital FZB42: Bacillus amyloliquefaciens subsp. plantarum, synonymous: Bacillus velezensis FZB42 (Taxonomy ID: 326423; deposit No.: DSM231 17); NO3- = nitrate salt added; NH4 + = ammonium salt added). The lowercase letters indicate statistically distinguishable groups.
Figure 5 shows the synergistic synergistic effect of a nitrification inhibitor (DMPP), microbial biofertilizers and ammonium fertilization at different pH values on plant growth, (herein: 1 : rock phosphate without microbial biofertilizers (negative control); 2: combination of the nitrification inhibitor DMPP and ammonium salt; 3: combination of the nitrification inhibitor DMPP, ammonium salt and Rhizovital FZB42; 4: soluble phosphor source added and nitrate added). The lowercase letters indicate statistically distinguishable groups.
Figure 6 shows synergistic effect of microbial biofertilizers and ammonium fertilization on root length of maize. The lowercase letters indicate statistically distinguishable groups. Figure 7 shows the effect of stabilized ammonium fertilization and microbial biostimulant inoculation (FZB42) on plant available P (CAL-extractable P) in the rhizosphere of a low-P soil with pH 5.6. (1 : unfertilized; 2: rock-P, NH +, 3: rock-P, NH +, FZB42 (Bacillus amyloliquefaciens); 4: soluble P, NO3-). The lowercase letters indicate statistically distinguishable groups.
Examples
Background
The specific microbial inoculants are introduced into the rhizosphere of the target crop, where they can propagate by supply of plant root exudates as energy source and increase the nutrient availability for the host plant (Menzies et al. 201 1 ; Sharma et al., 2013). A range of so-called microbial "biofertilizer" products is already commercially available. However, limited reproducibility of the desired fertilizer effects under practical conditions still represents a major undissolved problem (Menzies et al. 201 1 ).
Accordingly, in a recent research project on P-solubilizing microorganisms, more than 10 experiments conducted in eight countries with four crops (maize, wheat, barley, tomato) and 13 commercial or newly developed bacterial and fungal biofertilizer products, revealed no indications for mobilization of sparingly-soluble P forms in the rhizosphere, although all microbial strains showed P-solubilizing potential in lab-screening assays on artificial growth media (Sanchez-Esteva et al. 2016; Lekfeldt et al. 2016, Nkebiwe, 2017; Thonar et al. 2017). Further investigations suggested that a high buffering capacity of many field soils is an important factor, which limits the expression of microbial-induced modifications in soil chemistry supporting P solubilisation (Fig. 1 ).
On the highly buffered calcareous soil (B) only soluble P (+P) stimulates maize growth, while P-solubilizing microorganisms (Pseu, SP1 1 , Penic, Paenibac) were ineffective (Fig. 1 B). However, lowering the pH buffering capacity of the substrate by addition of quartz sand (70% w/w) stimulates plant growth promotion, in particular root growth promotion, by the microbial inoculants (A) The potential of the fertilizer-based strategy to increase the efficiency of microbial biofertilizers as plant inoculants was investigated.
Bacteria were chosen that enable soil acidification by plant roots, which is induced by ammonium-dominated fertilization which can increase the plant availability of phosphate (P) and micronutrients particularly on neutral and alkaline soils (Marschner, 1995; Neumann and Romheld 2002). These soil microorganisms were identified as also being able to use ammonium as cationic nitrogen source, associated with proton extrusion for charge-balance, which results in medium acidification, (Menzies et al., 201 1 ; Nkebiwe 2017).
In the present examples, effects of ammonium fertilization combined with nitrification inhibition on root morphology were documented, including elongation of root hairs, which is associated with a higher binding capacity of soil particles for the formation of so-called rhizosheaths, improving the root soil contact and increasing the radial extension of the rhizosphere for nutrient acquisition and plant- microbial interactions.
Microbial inoculants based on strains of Bacillus, Pseudomonas and Trichoderma, representative for many commercial biofertilizers, have been characterized for Ca- P solubilisation on artificial growth media, and utilization of ammonium sulfate as nitrogen source. DMPP (dimethylpyrazolphosphate) was identified as one of the suitable examples for nitrification inhibitors without negative effects on growth of the respective inoculants (Nkebiwe 2017). Summarized, it was surprisingly found that the plant/root growth promoting potential of microbial inoculants based on strains of Pseudomonas, Bacillus and Trichoderma can be induced and synergistically increased by combination with DMPP-Ammonium fertilization. The effects have been demonstrated for biofertilizer products containing three different strains of Pseudomonas, three strains of Trichoderma and five strains of Bacillus and combinations thereof, so far with three different crops (maize, wheat, tomato).
The bacteria or fungal species B were obtained from the following suppliers:
Trianum P; Trichoderma harzianum T22 (deposit No.: ATCC 20847; Koppert Biological Systems Nederland, Veilingweg 14, 2651 BE Berkel en Rodenrijs, The Netherlands)
Rhizovital FZB42: Bacillus amyloliquefaciens subsp. Plantarum, synonymous: Bacillus velezensis FZB42 (Taxonomy ID: 326423; deposit No.: DSM231 17; ABiTEP GmbH, Glienicker Weg 185, 12489 Berlin, Germany)
Bacillus atrophaeus (ABI02A1 , deposit No.: DSM 32019; ABiTEP GmbH, Glienicker Weg 185, 12489 Berlin, Germany) Paenibacillus mucilaginosus (ABiTEP GmbH, Glienicker Weg 185, 12489 Berlin, Germany)
Proradix; Pseudomonas sp., (deposit No.: DMSZ 13134; Sourcon Padena GmbH & Co. KG, Hechinger Str. 262, 72072 Tubingen, Germany)
Biological Fertilizer OD, Penicillium sp. (Bayer CropScience Biologies GmbH, Inselstr. 12, 23999 Malchow/Poel, Germany)
Trichoderma harzianum OMG16 (Anhalt University of Applied Sciences Center of Life Sciences, Institute of Bioanalytical Sciences (IBAS) Strenzfelder Allee 28, 06406 Bernburg, Germany)
Vitalin SP1 1 (Vitalin Pflanzengesundheit GmbH, Pragelatostraβe 113, 64372 Ober-Ramstadt, Germany) Statistics: if not indicated separately statistical analysis was performed by one-way ANNOVA with Tukey test, p=0.05
Synergistic interaction of DMPP-ammonium fertilization and Pseudomonas sp. on plant growth promotion
Using the Pseudomonas-based biofertilizer Proradix® (Sourcon Padena, Tubingen, Germany) as model inoculant, a synergistic effect with DMPP-stabilized ammonium sulfate fertilization was demonstrated on biomass production of maize, grown on a sand-soil substrate pH 7.5 with sparingly soluble calcium phosphates as exclusive P source (Rock-P) with and without increased pH buffering by liming (Ca(CO3)2 20% (w/w) (Table 1 ).
Table 1 : Shoot biomass production (g plant-1) of maize (cv Colisee) on a sand-soil substrate (calcareous Loess subsoil pH 7.6; PCAL5 mg kg-1 soil) with different levels of liming (0 and 25% Ca(COs)2) and sparingly soluble Ca-Phosphates as exclusive P source (120 mg P kg-1 substrate as Rock-P). Effect of nitrate and DMPP-Ammonium fertilization (100 mg N kg-1 substrate as Ca(NO3)2 or DMPP- (NH )2SO4 Novatec Solub, Compo, Germany) and three weekly inoculations with Proradix (109 cfu kg-1 substrate).
Figure imgf000044_0001
The same synergistic effect was detected also in four greenhouse experiments with maize on real field soils (pH 6.8-7.5) with low P availability (< 20 mg PCAL kg-1 substrate) and Rock-P as sparingly soluble P fertilizer (Table 2). On average, the combination of DMPP-Ammonium with Proradix increased maize shoot biomass production by approx. 53% as compared with nitrate fertilization, and an increase of 21 % could be attributed to the Proradix effect.
Table 2: Shoot dry matter production (g plant-1) of maize (cv Colisee) on two different clay-loam field soils with low P availability (< 20 mg PCAL kg-1 substrate) supplied with Rock-P (RP) (120 mg P kg-1 substrate) as exclusive P source. The effect of nitrate and DMPP-Ammonium fertilization (100 mg N kg-1 substrate as Ca(NO3)2 or DMPP-(NH4)2SO4 Novatec Solub, Compo, Germany) and three weekly inoculations by fertigation with Proradix (109 cfu kg-1 substrate)
Figure imgf000045_0001
n.d. = not determined
Synergistic effects between Proradix and DMPP-Ammonium fertilization on plant growth stimulation are not restricted to maize and could be similarly demonstrated in a pot experiment with spring wheat on a low-P silty loam organic farming soil (pH 6.4, PCAI_:7 mg kg-1) supplied with Rock-P (RP) as sparingly soluble P source. Proradix inoculation in combination with DMPP-Ammonium significantly increased final grain yield by 34 % as compared with the non-inoculated control, while the Proradix effect in combination with nitrate fertilization was not significant (+ 8%).
Table 3: Biomass production and grain yield of spring wheat (cv Schirocco) on a low P silty loam organic farming soil (pH 7.6; 7 mg PCAL kg-1 soil) supplied with Rock-P (RP) (150 mg P kg-1 substrate ) as exclusive P source. Effect of nitrate and DMPP-Ammonium fertilization (100 mg N kg-1 substrate as Ca(NO3)2 or DMPP- (NH )2SO4 Novatec Solub, Compo, Germany) and three inoculations by fertigation with Proradix (Pro, 109 cfu kg-1 substrate) at 0, 24 and 34 days.
Figure imgf000046_0001
Plant growth promotion by combination of DMPP-ammonium with different microbial inoculants
A range of seven microbial biofertilizer products, based on strains of Bacillus, Pseudomonas Trichoderma, Penicillium and combinations thereof were tested for their plant growth-promoting potential with Maize cv Colisee in combination with DMPP-Ammonium fertilization on a low-P, clay-loam organic farming soil pH 6.8 (available P: 20 mg CAL-P kg-1 soil) supplied with Rock-P (100 mg P kg-1 soil) as sparingly-soluble P source. Variants without P fertilization (No P) and with soluble P supply (100 mg P kg-1 soil as Ca(H2PO4)2) were included as negative and positive controls, respectively. Table 3 shows the effects of the microbial inoculants in combination with DMPP-Ammonium as relative changes (%) compared with full soluble P fertilization and nitrate supply (NO3 Soluble P = 100%, positive control) and the unfertilized negative control (No P = 0%).
DMPP-Ammonium with Rock-P fertilization but without biofertilizers (NH _RP) induced approximately 60% of the shoot biomass production as compared with maize plants supplied with full soluble P fertilization (NO3_Soluble P). However, Ammonium-DMPP in combination with all tested biofertilizer products based on strains of Pseudomonas, Bacillus and Trichoderma (Table 4,) significantly increased the shoot biomass production even for those products, which have been previously proven to be ineffective in combination with nitrate fertilization (Fig .1 ; Table3). The only exception was the Penicillium-based biofertilizer (BFOD). In case of the combination products Vit SP1 1 and CombifectorA, the obtained biomass increase was not significantly lower than the positive control with soluble P fertilization (Table 3). With exception of the Trichoderma-based biofertilizer Trianum-P, shoot growth stimulation was associated with a promotion of root growth and the P shoot content reached 80-90% compared with the positive control with soluble P supply (Table 4).
Table 4: Synergistic effects of stabilized ammonium (NH ) fertilization and inoculation with fungal and bacterial biofertilizers on growth and P acquisition from sparingly soluble Rock-P (RP) in maize (cv Colisee) on a low P organic farming soil. Relative values (%) compared with conventional nitrate-based fertilization with full soluble P supply (NO3_soluble P = 100%).
Figure imgf000047_0001
Fungal biofertilizers: Trianum.P (Trichoderma harzianum T22), BFOD (Penicillium bilaii); Bacterial biofertilizers: Proradix (Pseudomonas sp DMSZ13134), Rhizovital (Bacillus amyloliquefaciens FZB42), Paenibacillus mucilaginosus; Combination products: Vit SP1 1 (Bacillus subtilis, Pseudomonas sp., Streptomyces spp., humic acids, Ascophyllum nodosum extract), CombiFectA (Trichoderma harzianum OMG16, 3 Bacillus strains, Pseudomonas sp Zn and Mn).
The black frame exemplarily shows the synergistic effect of DMPP-Ammonium and Proradix as compared with DMPP-Ammonium or with Proradix combined with nitrate fertilization.
Apart from P, also the acquisition of other mineral nutrients, such as N, K and Mn was improved by combination of DMPP-Ammonium with microbial biofertilizers as compared with sole DMPP-Ammoniunn/Rock-P fertilization (Table 5). These findings demonstrate that not only P nutrition but the plant-nutritional status in general can be improved by the synergistic effects of Ammonium-DMPP fertilization and microbial inoculants on root growth and nutrient solubilisation.
Table 5: Effects of DMPP-Ammonium fertilization and DMPP-Ammonium combined with microbial biofertilizers on shoot nutrient contents of maize (cv Colisee) on a low P organic farming soil supplied with Rock-P as sparingly-soluble P source. Relative changes (%) compared with conventional nitrate-based fertilization with full soluble P supply (NO3_soluble P = 100%, positive control). For more details see Table 3.
Figure imgf000048_0001
a = not significantly different from soluble P; b = significantly different from NH4 RP A selection of Pseudomonas-, Bacillus-, and Trichoderma-based biofertilizers (Proradix, Rhizovital, Paenibacillus mucilaginosus, Trianum-P) used in the descry- bed maize experiments in combination with DMPP-Ammonium fertilization, exerted similar effects also in a pot experiment conducted with spring wheat, grown on a low-P Cambisol with 14 - 28 % increase in shoot biomass production and a 26 - 35 % increase in grain yield.
Table 6: Effect of microbial bioeffectors on biomass production and grain yield in a Ppot experiment with spring wheat (cv. Schirocco, KWS, Germany) on a low-P organic farming soil (PCAL 7 mg kg-1 soil) silty loam Cambisol, pH 6.4 with DMPP- stabilized ammonium fertilization (NH 150mg N kg-1 soil placed by point injection) and rock phosphate (RP 150 mg P kg-1 soil)) as P fertilizer.
Figure imgf000049_0001
Plant growth promotion in maize supplied with different P sources by combination of DMPP-ammonium with microbial inoculants based on Bacillus. Pseudomonas and Trichoderma strains.
To test the potential of the investigated microbial inoculants in combination with DMPP-ammonium fertilization to promote plant P acquisition from different sparingly-soluble P sources (native soil P, Rock-P, inorganic recycling fertilizes, such as sewage sludge ash, and struvite derived from processed biogas digestates), maize (cv Colisee) was grown on a low-P, clay-loam organic farming soil pH 6.8 (available P: 20 mg CAL-P kg-1 soil) supplied with the various sparingly-soluble P sources (100 mg P kg-1 soil). A variant with soluble P supply (100 mg P kg-1 soil as Ca(H2PO4)2 ) was included as positive control. Proradix and CombifectorA were used as microbial inoculants, due to their superior plant growth-promoting potential demonstrated in Table 4.
The results demonstrated that all fertilizer or biofertilizer treatments could increase plant biomass production to a level not significantly different from positive control plants supplied with full soluble P and nitrate fertilization (Table 5). Proradix combined with ammonium had significant growth-promoting effects on plants supplied with Rock-P and without P fertilization as compared with sole DMPP- Ammonium nutrition, while CombifectorA improved plant growth on sewage sludge ash (SSA). Shoot P accumulation was significantly improved on Rock-P by Proradix and CombifectorA and on Struvite by CombifectorA, associated with particular intense stimulation of root growth in case of CombifectorA (Table 5). The data demonstrate that the combination of DMPP-ammonium fertilization with microbial biofertilizers based on strains of Pseudomonas, Bacillus and Trichoderma not only promotes plant growth and P acquisition from rock phosphate but also from other sparingly soluble soil P forms and inorganic recycling fertilizer products.
Table 7: Effects of inorganic P fertilizers (Rock-P, sewage sludge ash SSA, struvite, soluble P) and inoculation with microbial biofertilizers (Proradix; CombifectorA) on growth and shoot P accumulation of maize (cv Colisee), grown on a low-P, clay-loam organic farming soil pH 6.8 with DMPP-stabilized ammonium as nitrogen source. Results are expressed as relative changes (%) compared to full soluble P fertilization (= 100%) with nitrate supply.
Figure imgf000051_0001
b significantly different from the positive control supplied with soluble P and nitrate fertilization, a significantly different from the respective controls without biofertilizer application DMPP-ammonium to increase the performance of microbial inoculants as cold stress protectants in maize.
Low soil temperature in spring is a major constraint for cultivation of tropical and sub-tropical crops in temperate climates and is associated with inhibition of root growth and activity. Various strategies have been proposed as practical measures to counteract low temperature stress in crops including (i) fertilizer placement (P, micronutrients, such as Zn and Mn) close to the seedling roots, (ii) application of plant and seaweed extracts with antioxidative and membrane-protective properties, and (iii) improving root growth and plant nutrient acquisition by inoculation with plant growth-promoting microorganisms (Bradacova et al. 2016). In an experimental setup with controlled root-zone temperature the Zn/Mn- Bacillus/Trichoderma-based combination product CombifectorA was tested in comparison with other microbial inoculants (BCSB = Penicillium-based inoculant; ABI02 = cold-tolerant Bacillus strain) with maize under nitrate-, and DMPP stabilised ammonium-fertilization exposed to cold-stress (12-14°C controlled root- zone temperature). Oxidative leaf damage (necrosis, chlorosis and anthocyanin formation) compared to non-inoculated controls declined in the order: No biofertilizers > ABI02+ZnMn > ZnMn alone = BFOD+ZnMn > CombifectorA> uncooled control. Leaf damage was generally more expressed under nitrate supply as compared with ammonium fertilization (Table 8). The highest shoot biomass production was achieved by CombifectorA (77g), followed by BFOD (70g) under ammonium fertilization and cold-stress, which was significantly higher than all other tested variants and untreated controls (Table 8)
Table 8: Effects of microbial inoculants (Abi02, cold-resistant Bacillus atrophaeus; BFOD Penicillium sp.; CombifectorA and Zn/Mn seed dressing (Lebosol GmbH, Germany) on shoot biomass production Fresh weight (FW g plant-1) and oxidative leaf damage (number of chlorlotic/necrotic) leaves plant-1) in maize, induced by two weeks exposure to root zone temperatures of 12-14°C on a clay loam field soil pH 6.8 with nitrate or DMPP stabilised ammonium sulfate fertilization.
Figure imgf000052_0001
The cold stress-suppressive effect of CombifectorA was also reflected by significantly increased superoxide dismutase (SOD) activity and polyphenols content in maize shoot tissue, which was amore expressed in ammonium as compared to nitrate fertilization (not shown), reflecting a higher expression of defence mechanisms against oxidative stress depending on micronutrients (Zn, Mn, Cu, Fe) as enzymatic co-factors. Accordingly, a critical micronutrient status was identified as growth limiting factor in maize plants exposed to low root-zone temperature (Table. 9). Micronutrient seed application, the microbial inoculants and rhizosphere acidification induced by DMPP stabilized ammonium fertilization, obviously increased the plant availability of the critical micronutrients in the rhizosphere (Table 9), thereby stimulating the expression of the antioxidative stress defence. Since the same defence mechanisms are also involved in plant tolerance to other abiotic and biotic stress factors, comparable investigations are currently conducted also for plants exposed to water limitation. Table 9: Effects of microbial inoculants (Abi02, cold-resistant Bacillus atrophaeus; BFOD Penicillium sp.; CombifectorA and Zn/Mn seed dressing (Zn/MnS, Lebosol GmbH, Germany) on Zn and Mn nutritional status and changes in rhizosphere soil pH (relative to the bulk soil pH) in maize, induced by two-weeks exposure to root zone temperatures of 12-14°C on a clay-loam field soil pH 6.8 with nitrate or DMPP-stabilised ammonium sulfate fertilization. NoBE = untreated control.
Figure imgf000053_0001
Field Performance
To investigate the potential of DMPP-Ammonium in combination with Bacillus, Trichoderma, and Pseudomonas-based biofertilizers to improve P acquisition of crops under real practice conditions, field experiments were conducted on soils with low to moderate P availability for production of silage maize (Germany, Czech Republic, Southern Italy) and winter wheat (Germany).
Silage maize production
Field sites (Table 10) in Germany and in the Czech Republic were characterized by silty loam to sandy loam soils with medium P availability (-50 mg PCAL kg-1 soil) and moderately acidic pH (5.2-5.9), while the experiment in Italy was conducted on a clay loam soil with high pH (8.6) and moderately low P availability (1 1 mg Poisen kg-1 soil). Biomass production of local maize varieties was determined for DMPP- Ammonium/Rock-P fertilization with and without inoculation of Bacillus/Tricho- derma and Pseudomonas-based biofertilizers in comparison local farming practices and a full soluble P fertilization with Superphosphate or Triple- Superphosphate.
Figure imgf000054_0001
On the low-P alkaline soil in Italy, Bacillus-based biofertilizers (FZB42) in combination with humic acids and seaweed extracts, as well as Bacillus- Trichoderma combination products enriched with Zn/Mn (CobifectorA B) were tested. In combination with DMPP-Ammonium, all biofertilizers stimulated maize growth to a similar extent in comparison with non-inoculated controls. In the variants without P fertilization depending on native soil P, biomass production was increased by the inoculants on average by 34.5 %. Even for Rock-P, which exhibits particularly low solubility on alkaline soils, the inoculants increased biomass production by 1 1 .5% and compared with farmers practice based on non- stabilized urea/di-ammonium phosphate fertilization (50 kg P ha-1), biomass production was increased by 22.4% (Table 1 1 ). Table 11 : Shoot dry matter [g plant-1] of maize cv Limagrain (LG) 30.600 at the field site Castel Volturno, Italy as determined by different forms of N and P fertilization and inoculation with Bacillus (FZB42) and Bacillus+Trichoderma (CombifectA/B)-based microbial biofertilizers
Figure imgf000055_0001
By contrast, on the more acidic soils with moderate P availability in Germany and the Czech Republic, comparison with non-fertilized variants revealed that only N but not P was a growth limiting factor. Under these conditions, microbial inoculants based on Paenibacillus mucilaginosus, Pseudomonas sp and the Bacillus/Trichoderma combination product CombifectorA exerted no significant growth promoting effects in combination with DMPP-Ammonium.
Winter wheat production
A field experiment with winter wheat (cv. Kometus) to test the combination of the Pseudomonas-based biofertilizer product Proradix with different nitrogen and phosphate fertilizers was conducted 2015/2016 at a grassland conversion field site near Horb am Neckar, South-West Germany, with slightly acidic soil pH of 6.6 (CaCl2) and low phosphorus availability (21 mg CAL P kg-1 soil). Proradix was applied as aqueous suspension with top-soil incorporation before sowing and on top of the wheat plants at vegetation start after winter at rates of 23 kg ha-1. The different fertilization variants are summarized in Table 12. Table 12: Fertilizer application in the winter-wheat field trial 2015/16 Horb, Southwest Germany - (ASN = Ammonium sulfate/ nitrate; CAN =Calcium-ammonium nitrate, AS = ammonium sulfate; MP = chicken manure pellets, Agriges, Italy)
Figure imgf000056_0001
Proradix inoculation showed a trend for increased grain yield (+ 9-1 1 %) both, in combination with rock phosphate and DMPP-ammonium fertilization and with DMPP-stabilized organic chicken manure fertilizer, but not with standard and non- stabilized DAP fertilization (Table 13), confirming the results of the related pot experiments. However, comparing all tested fertilization strategies, only Proradix+Rock P with DMPP stabilized ammonium could compete with the standard fertilization practice (Table 13). Table 13: Grain yield (1000 kg ha-1) of a winter wheat field trial 2015/16 in Horb, South-West Germany, as determined by different forms of N and P fertilization and inoculation with the Pseudomonas-based microbial biofertilizer Proradix. Data represent mean values ± standard error of the mean (SEM; n = 5). Significant differences are indicated by different characters (Tukey test, p < 0.05).
Figure imgf000057_0001
Mode of action
The reported effects are most probably a consequence of complex, multiple plant- fertilizer-microbe interactions at the level of rhizosphere chemistry, hormonal effects and alterations in root morphology.
Ammonium-induced rhizosphere acidification
Ammonium nutrition induces rhizosphere acidification due to root-induced proton extrusion for charge-balance of ammonium uptake (Table 14), with the well- documented effects on increased solubility of Ca phosphates, Rock P and micronutrients, with particular importance on slightly acidic to alkaline soils with limited solubility of the respective nutrients (Neumann and Romheld 2002). In principle, also microorganisms release protons in response to ammonium uptake (Menzies et al., 201 1 ). However, in the example shown in Table 14, inoculation of maize plants, grown on a low-P organic farming soil (pH 6.8), with the Pseudomonas-based biofertilizer Proradix obviously did not significantly contribute to the intensity of rhizosphere acidification, although ammonium is an efficient nitrogen source for the inoculated bacterium (Nkebiwe, 2017). However, Proradix may induce longer-lasting rhizosphere acidification (Table 14). Together with stimulation of root growth, this leads to a larger acidifying root system and a temporal extension of ammonium-induced rhizosphere acidification. However, the results presented in Table 9 suggest, that other microbial inoculants may be also able to intensify locally the ammonium-induced acidification of the rhizosphere soil. Table 14: Changes in rhizosphere pH (rel. to bulk soil) along seminal roots of maize supplied with rock P and nitrate or DMPP-stabilized ammonium fertilization with or without inoculation with the Pseudomonas-based biofertilizer Proradix on a low P clay-loam organic farming soil, pH 6.8. Measurements conducted with antimony micro-electrodes, 1 mm in diameter (Haussling et al., 1985).
Figure imgf000058_0001
Spatial expansion of the rhizosphere
The formation of so-called rhizosheaths, formed by soil particles bound in a matrix of exopolysaccharides released from plant roots and rhizoshere microorganisms, which can cover large parts of the root system (Figure 2) is a typical feature of many grasses but also of drought-adapted plant species (McCully, 1999).
Formation of rhizoheaths improves the root soil contact (Fig.2) with positive effects on nutrient uptake and mobilization of sparingly available nutrients in the rhizosphere. Moreover soil bound in rhizosheaths shows a higher water holding capacity as compared with the bulk soil as an important benefit under conditions of water limitation (Huang et al. 1993) The size of the rhizosheaths and thus the extension of the rhizosphere is largely determined by the length of the root hairs (Fig. 2; Hailing et al., 2014). Root growth responses including root hair formation are strongly determined by nutrient availability with particularly high variability in response to ammonium fertilization, depending on dosage, soil pH, placement of the fertilizer and genotype (Kania et al., 2007; Pan et al. 2016). Investigating the root growth responses of maize to stabilized DMPP-ammonium vs nitrate fertilization, (both 100 mg N kg-1 soil) with and without Proradix (Pseudomonas sp). inoculation on a low P organic farming soil (pH 6.8) supplied with rock-phosphate as P fertilizer, revealed a stimulation of root hair elongation and a corresponding extension of the rhizosheath diameter induced by DMPP- ammonium fertilization (Table 15). Although, Proradix inoculation had no significant effects on root hair length and extension of the rhizosheaths (Table 15), the total number of root hairs was increased by stimulation of root proliferation. Table 15: Effects of N form (nitrate vs DMPP stabilized ammonium nutrition) and Proradix inoculation (Pseudomonas sp. DMSZ13134) on root hair development and rhizosheath diameter of maize, grown on a low P clay-loam organic farming soil pH 6.8 with Rock-P fertilisation.
Figure imgf000059_0001
Root growth stimulation by microbial inoculants is thought to be mediated by microbial production of hormonal factors (mostly, auxins) or interference with the plant hormonal signalling systems (via quorum sensing signals or degradation of ethylene precursors). However, the potential of the respective metabolic activities in microbial inoculants is most frequently demonstrated on artificial growth media on agar plates and investigations under rhizosphere conditions are rare.
As an alternative screening approach, in the rhizosphere studies conducted with maize as described above, bacterial populations were re-isolated from the rhizosphere of maize plants, supplied with nitrate or DMPP-stabilized ammonium fertilization, with or without pre-inoculation of different microbial biofertilizers, and subsequently the auxin production potential of the re-isolated populations was tested with a standard assay (Bharucha et al., 2013). Table 16 shows the results of three experiments conducted on three different field soils with two different microbial biofertilizer products (Proradix; ECAG2895) as inoculants. In all cases the auxin production of bacterial populations re-isolated from pre-inoculated plants supplied with ammonium fertilization was higher than without pre-inoculation with significant effects particularly for the microbial combination product ECAG2895) and this was associated with root growth-promoting effects in the respective treatments. The findings further promote a role of microbial auxin production as putative factor for root growth stimulation. The synergistic effect of stabilized ammonium fertilization is in line with literature reports on increased auxin production by Pseudomonades supplied with ammonium as nitrogen source on artificial growth media (Bharucha et al. 2013)
Table 16: Auxin production g-1 microbial biomass (rel. values, Salkowski Assay) of bacterial populations re-isolated from rhitosphere soil attached to the roots of maize plants grown on three different field soils (clay loam-silty loam pH 5.8-7.5) and nitrate or DMPP-stabilized ammonium (DMPP NH ) fertilization, with and without microbial inoculants (Proradix; ECAG2895) and rock phosphate (RP) or soluble Ca(H2PO4)2 (Psol) as P fertilizers.
Figure imgf000060_0001
This is however, not a general principle. The synergistic effects of DMPP- stabilized ammonium fertilization and different microbial inoculants can rely on different modes of action. As shown in the experiment described in Table 4, the fungal Trichoderma-based biofertilizer (Trianum P) improved plant growth and P acquisition from Rock P in maize in combination with DMPP-ammonium. In this case, improved P acquisition was not associated with any promotion of root growth, suggesting a direct effect on P solubilisation via chemical changes in the rhizosphere. By contrast, plant growth promotion induced by the combined effect of DMPP-ammonium and the Bacillus/Trichodermal/Zn/Mn- combination product CombifectorA was associated with a massive promotion of root growth (Table 4) with significant positive effects on acquisition of different nutrients, such as N,K and Mn (Table 5). This may also offer the explanation for the broad expression of synergistic effects between ammonium fertilization and a wide range of different microbial inoculants documented so far. Independent of their mode of action, both, root growth-stimulating and P-solubilizing BEs can synergistically support the root- mediated nutrient mobilization induced by ammonium-triggered proton extrusion (Table 14) as a general response in all crops, and also by the ammonium - induced rhizosphere extension demonstrated in Table 15.
Synergistic effect of a nitrification inhibitor (DMPP), microbial biofertilizers and ammonium fertilization on plant growth - comparison between different microbial biofertilizers
This example has been conducted as laid out above. DMPP is used as nitrification inhibitor in the samples. As shown in Figure 3, microbial biofertilizers show statistically significant beneficial impact on plant growth in soil that contains sparingly soluble calcium phosphates as exclusive P source. Similar synergistic ammonium effects were also found after inoculation with other bacteria and fungi belonging to the genera Trichoderma, Penicillium, Pseudomonas, Bacillus, Paenibacillus, and Streptomyces. This example shows the positive effect of a combination of microbial biofertilizers (biostimulants) and stabilized ammonium nitrogen on plant growth by induced increased P-uptake from rock phosphate. Stabilized ammonium fertilization synergistically supports plant growth promotion in maize supplied with sparingly soluble Ca-P (Rock-P) This example shows the positive effect of a combination of biostimulants and stabilized ammonium N on plant growth by induced increased P-uptake from rock phosphate on a silty loam soil pH 6.9. This effect was not found for soils where ammonium is replaced by nitrate, in other words, larger amounts of nitrate salts are added. Synergistic effect of a nitrification inhibitor (DMPP), microbial biofertilizer and ammonium fertilization on auxin production and root growth
This example has been conducted as laid out above. DMPP is used as nitrification inhibitor in the samples. The rhizosphere-bacterial production potential of the growth factor auxin (determined after Glickmann & Dessaux, 1995) is significantly increased by a combination of a biostimulant (FZB42) with stabilized ammonium fertilization (cf. Figure 4), resulting in an increased plant- and root growth of the respective plants (cf. Figure 6). It was found that the production of growth factor auxin in maize is significantly increased by a combination with a microbial biofertilizer (biostimulant) (FZB42) with stabilized ammonium fertilization, resulting in an increased plant- and root growth. The results are depicted in Figure 4. Ammonium as N source increased the auxin production potential of bacterial strains, in vitro (A) and after re-isolation from the maize rhizosphere.
Apart of stimulating effects on the auxin production potential of rhizosphere bacteria (cf. Figure 4), stabilized ammonium fertilization also increased the internal levels of plant growth-hormones, such as auxin (indole acetic acid), cytokinins (zeatin) and giberrellic acid in the shoot tissue of maize plants in comparison with the nitrate-fertilized control (Table 17, determined by UHPLC-MS analysis according to Moradtalab et al . 2018). Ammonium-induced increased internal levels of growth hormones may increase the responsiveness of the host plants to hormone production of microbial inoculants.
Table 17: Phytohormone levels in maize shoots (3 weeks after sowing) on a clay loam soil pH 6.9 as affected by the form of nitrogen fertilization (Ca-nitrate or
Figure imgf000062_0001
Synergistic effect of a nitrification inhibitor (DMPP), microbial biofertilizer and ammonium fertilization on plant growth at different pH values
This example has been conducted as laid out above. DMPP is used as nitrification inhibitor in the samples. It was found that the synergistic effect is increased at elevated soil pH compared to lower pH values, where overall maize plant growth is, however, lower. Without being bound to this theory, it is assumed that the solubility of the rock phosphate is higher at a lower pH value. This example demonstrates that present invention has a particularly high effect at neutral and slightly basic pH values. Maize growth on two African soils with contrasting pH supplied with stabilized ammonium fertilization and inoculated with Bacillus amyloliquefaciens FZB42.
The synergistic effect of a combination of rock phosphate - biostimulant - nitification inhibitor (Nl) stabilized ammonium is very clear at neutral to alkaline soil pH compared to lower pH environments (cf. Figure 5), because in latter case the solubility of rock phosphates is better anyway. However, the beneficial effects on P availability are still detectable even on more acidic soils, although they do not necessarily translate into better plant growth due to sufficient P supply already in the treatments without biostimulants (cf. Figure 7).
References Data sheet VDLUFA I, P und K, CAL-loslich, A 6.2.1 .1 , in: Handbuch der Bodenuntersuchung: Terminologie, Verfahrensvorschriften und Datenblatter; physikalische, chemische, biologische Untersuchungsverfahren: gesetzliche Regelwerke. Hrsg. DIN, Deutsches Institut fur Normung e. V., Beuth Verlag, 2000, ISBN 3-410-14590-7.
Bharucha U, Patel K, Trivedi UB (2013), Optimization of Indole Acetic Acid Production by Pseudomonas putida UB1 and its Effect as Plant Growth-Promoting Rhizobacteria on Mustard (Brassica nigra) Agric Res 2(3):215-221 . Glickmann, E., & Dessaux, Y. (1995). A Critical Examination of the Specificity of the Salkowski Reagent for Indolic Compounds Produced by Phytopathogenic Bacteria, 61 (2):793-796.
Huang B, North G, Nobel PS (1993) Soil sheaths, photosynthate distribution to roots and rhizosphere water relations for Opuntia ficus-indica. Int J Plant Sci 154:425-431 .
Kania, A., Guldner, M., Szabo, B., Kazem, S., Romheld, V., Neumann, G., Morhard, J., Evers, M., and Terlouw, T. (2007), Functional characterization of the stabilized organic turf grass fertilizer "Marathon". Rasen -Turf - Gazon 1/2007: 192-195.
Lekfeldt JDS, Rex M, Mercl F, Kulhanek M, Tlustos , Magid, de Neergaard (2016), Effect of bioeffectors and recycled P-fertiliser products on the growth of spring wheat, hem. Biol. Technol. Agric. 3:22. Marschner H. (1995), Rhizosphere pH effects on phosphorus nutrition. In: Johansen C, Lee KK, Sharma KK, Subbaro GV, Kueneman EA, eds. Genetic Manipulation of Crop Plants to Enhance Integrated Nutrient Management in Cropping Systems. 1 . Phosphorus. Proceedings of an FAO/ICRISAT Expert Consultary Workshop. ICRISAT Asia Center, India. Patancheru, Andhra Pradesh, India: International Crops Research Institute for the Semi-Arid Tropics, pp 107- 1 15.
McCully ME (1999), Roots in soil: unearthing the complexities of roots and their rhizospheres. Annu Rev Plant Physiol Plant Mol Biol 50:695-718.
Menzies N, Harbison D, Dart P (201 1 ), Soil chemistry - facts and fiction and their influence on the fertiliser decision making process. Proceedings of the 26th Annual Conference of The Grassland Society of NSW. 49-63.
Moradtalab N, Weinmann M, Walker F, Hoglinger B, Ludewig U and Neumann G (2018) Silicon Improves Chilling Tolerance During Early Growth of Maize by Effects on Micronutrient Homeostasis and Hormonal Balances. Front. Plant Sci. 9:420. doi: 10.3389/fpls.2018.0042.
Neumann G. Romheld V. (2002), Root-induced changes in the availability of nutrients in the rhizosphere. In: Plant Roots The Hidden Half, 3rd ed. Waisel Y., Eshel, A., Kafkafi U. eds. pp.617-649, Marcel Dekker, New York. Nkebiwe PM (2017), Fertilizer placement and the potential for its combination with bio-effectors to improve crop nutrient acquisition and yield. Dissertation, Universitat Hohenheim, Germany, submitted.
Pan WL, Madsen IJ, Bolton RP, Graves L, Sistrunk T (2016), Ammonia/Ammonium toxicity root symptoms induced by inorganic and organic fertilizers and placement. Agron. J. 108: 2485-2491 .
Sanchez-Esteva S, Gomez-Muhoz B Jensen LS, de Neergaard A, Magid J (2016), The effect of Penicillium bilaii on wheat growth and phosphorus uptake as affected by soil pH, soil P and application of sewage sludge. Chemical and Biological Technologies in Agriculture 3:21 . Sharma BS, Sayed RZ, Trivedi MH, Thivakaran AG. (2013), Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. Springer Plus 2: 587.
Thonar C, Lekfeldt JDS, Cozzolino V, Kundel D, Kulhanek M, Mosimann C, Neumann G, Piccolo A, Rex M, Symanczik S, Walder F, Weinmann M, de Neergaard A, Mader P (2017) Potential of three microbial bio-effectors to promote maize growth and nutrient acquisition from alternative phosphorous fertilizers in contrasting soils. Chem. Biol. Technol. Agric. 4:7.

Claims

Claims 1 . A method for improving nutrition of plants with one or more sparingly soluble elected from the group consisting of phosphor, iron, zinc and ma said method comprising the following steps:
(i) providing the following components:
(A) at least one ammonium source A, wherein at least 50 mol% of the total nitrogen content of A is present as ammonium and/or organically bound nitrogen,
(B) at least one bacteria or fungal species B with phosphate- solubilizing and/or root growth-promoting properties, wherein B does not form part of A, and
(C) at least one nitrification inhibitor C;
(ii) supplying the components A, B and C to a substrate S of agricultural use on which the plants are cultivated or intended to be cultivated; and
(iii) enabling growth of the plants in the substrate S obtained from step (ii). 2. The method of claim 1 , wherein the at least one ammonium source A is a chemical fertilizer comprising at least one ammonium salt, manure, clearing sludge, moist rest residue of a biogas plant, or a mixture of two or more thereof, more preferably a chemical fertilizer comprising at least one ammonium salt, in particular ammonium sulfate. 3. The method of any of claims 1 or 2, wherein the at least one bacteria and fungi species B is selected from the group consisting of Trichoderma species, Pseudomonas species, Bacillus species, and combinations thereof. 4. The method of any of claims 1 to 3, wherein the at least one bacteria or
Figure imgf000067_0001
fungal species B comprising bacteria selected from the group consisting of
Paenibacillus mucilaginosus, Trichoderma harzianum, Pseudomonas sp. DMSZ 13134, Pseudomonas fluorescens, Bacillus subtilis, Bacillus amyloliquefaciens, and combinations thereof. 5. The method of any of claims 1 to 4, wherein the at least one nitrification inhibitor C is not toxic for the at least one bacteria or fungal species B, in particular is inhibiting the ammonia monooxigenase of bacteria oxidizing ammonium. 6. The method of any of claims 1 to 5, wherein the at least one nitrification inhibitor C is a compound selected from the group consisting of compounds containing a pyrazole residue which can be substituted in their structure, 1 H-1 ,2,4-triazole, 2-chloro-6-(trichloromethyl)-pyridine, 5-ethoxy-3- trichloromethyl-1 ,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2- mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, 4-amino-1 ,2,4- triazole, 3-mercapto-1 ,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3- dihydro-2,2-dimethyl-7-benzofuranol methyl carbamate and N-(2,6- dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, or a compound of formula (I)
Figure imgf000067_0002
or a stereoisomer, salt, tautomer or N-oxide thereof, wherein
A is aryl or hetaryl, wherein the aromatic ring may in each case be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from R1A and R2A are independently of each other selected from H and C1-C2-alkyl; and R is H, C1-C4-haloalkyl, C1-C4-hydroxyalkyl, ethynylhydroxymethyl, phenylhydroxymethyl, or aryl, wherein the aromatic ring may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from RB;
and wherein
RA is
(i) halogen, CN, NRaRb, ORc, SRC, C(=Y1)RC, C(=Y1)ORc, C(=Y1)SRC, C(=Y1)NRaRb, Y2C(=Y1)RC, Y2C(=Y1)ORc, Y2C(=Y1)SRC, Y2C(=Y1)NRaRb, Y3Y2C(=Y1)RC, NRgN=C(Rd)(Re), C(=N-ORc)Rg, C(=N-ORc)Rg, C(=N- SRc)Rg, C(=N-NRaRb)Rg, S(=O)2Rf, NRgS(=O)2Rf, S(=O)2Y2C(=Y1)Rc, S(=O)2Y2C(=Y1)ORc, S(=O)2Y2C(=Y1)SRc, S(=O)2Y2C(=Y1)NRaRb, NO2, NON-CN, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-haloalkyl, C1-C4- cyanoalkyl, C1-C4-hydroxyalkyl, C1-C4-alkoxy, C2-C4-alkynyl-C1-C2- hydroxyalkyl, C2-C4-alkynyloxy;
(ii) C1-C4-alkylene-C(=Y1)Rc, C2-C4-alkenylene-C(=Y1)Rc, C1-C4- alkylene-C(=Y1)ORc, C2-C4-alkenylene-C(=Y1)OR°, C1-C4- alkylene- C(=Y1)SRC, C2-C4-alkenylene-C(=Y1)SRc, C1-C4-alkylene-C(=Y1)NRaNRb, C2-C4-alkenylene-C(=Y1)NRaNRb, C1-C4-alkylene-Y2-C(=Y1)Rc, C2-C4- alkenylene-Y2-C(=Y1)Rc, C1-C4-alkylene-NRaRb, C2-C4-alkenylene-NRaRb, C1-C4-alkylene-ORc, C2-C4-alkenylene-ORc, C1-C4-alkylene-SRc, C2-C4- alkenylene-SRc, wherein the C1-C4-alkylene or C2-C4-alkenylene chain may in each case be unsubstituted or may be partially or fully substituted by OR9, CN, halogen or phenyl;
(iii) aryl, aryl-C1-C2-alkyl, hetaryl or hetaryl-C1-C2-alkyl, wherein the aromatic Ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rh;
(iv) a 3- to 14-membered saturated or unsaturated carbocycle or heterocycle, which may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR1 b, O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R1 b is H, C1-C4-alkyl, C2-C4- alkenyl, C3-C6-cycloalkyl, C3-C6-cycloalkylmethyl, or OR9; or
(v) L-B, wherein
L is -CH2-, -CH=CH-, -CEC-, -C(=O)- or -CH=, and B is aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rh; or a 3- to 14-membered saturated or unsaturated carbocycle or heterocycle, which may contain 1 , 2, or 3 heteroatoms which, independently of each other, are selected from NR1 b, O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R1 b is H, C1-C4-alkyl, C2-C4- alkenyl, C3-C6-cycloalkyl, C3-C6-cycloalkylmethyl, or OR9; or
(vi) two substituents RA together represent a carbocyclic or heterocyclic ring, which is fused to A and may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR1c, O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R1c is H, C1- C4-alkyl, C2-C4-alkenyl, C3-C6-cycloalkyl, C3-C6-cycloalkylmethyl, C3-C6- heterocyclyl, C3-C6-heterocyclylmethyl or OR9;
and wherein
RB is NH-C(=O)-(C1-C4-alkyl), NH-C(=O)-(C2-C4-alkenyl), NH-C(=O)-(C1-C2- alkoxy-C1-C2- alkyl), NH-C(=O)-(C3-C6-cycloalkyl), NH-S(=O)2-(C1-C4-alkyl), or NO2; and wherein
Y1, Y2 and Y3 are independently of each other selected from O, S and NR1a, wherein R1 a is in each case independently H, C1-C4-alkyl, C2-C4-alkenyl, C3- C6-cycloalkyl, C3-C6-cycloalkylmethyl, OR9, SR9 or NRmRn;
Ra and Rb are independently of each other selected from
(i) H, NRRk, OR1, SR1, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4- hydroxyalkylC, 1-C4- alkoxy, C(=Y1)R', C(=Y1)OR', C(=Y1)SR', C(=Y1)NRjRk, C(=Y1)C(=Y2) R', S(=O)2Rf;
(ii) aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rb; or
Ra and Rb together with the nitrogen atom to which they are bound form
(iii) a hetaryl group which may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rb; or (iv) a 3- to 10-membered, saturated or unsaturated heterocycle, which may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR1 b, O, and S, wherein S may be oxidized and/or wherein the heterocycle may be unsubstituted or may be partially or fully substituted by substituents which, independently of each other, are selected from R'; and wherein R1 b is H, C1-C4-alkyl, C2-C4-alkenyl, C3-C6-cycloalkyl, C3-C6- cycloalkylmethyl, or OR9;
Rc is
(i) H, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C(=O)OR', C(=O)SR', C(=O)NRjRk;
(ii) C1-C4-alkylene-C(=O)R', C1-C4-alkylene-C(=O)OR', wherein the d- C4-alkylene chain may in each case be unsubstituted or may be partially orfully substituted by OR9, CN, halogen, or phenyl;
(iii) aryl, aryl-C1-C2-alkyl, hetaryl, or hetaryl-C1-C2-alkyl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rh; or
(iv) a 3- to 10-membered saturated or unsaturated carbocycle or heterocycle, which may contain 1 ,2, or 3 heteroatoms which, independently of each other, are selected from NR1 b, O, and S, wherein S may be oxidized and/or wherein the carbocycle or heterocycle may be unsubstituted or may be partially orfully substituted by substituents which, independently of each other, are selected from R1; and wherein R1 b is H, C1-C4-alkyl, C2-C4- alkenyl, C3-C6-cycloalkyl, C3-C6-cycloalkylmethyl, or OR9;
Rd and Re are independently selected from C1-C4-alkyl, C1-C4-haloalkyl,
NRjRk, OR', SR', CN, C(=Y1)R', C(=Y1)OR', C(=Y1)SR', or C(=Y1)NRjRk; Rf is C1-C4-alkyl, C1-C4-haloalkyl, NRjRk, OR1, SR1, aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from Rh;
R9 is H or C1-C4-alkyl;
Rh is halogen, CN, NO2, NRjRk, OR1, SR1, C1-Ca4l-kyl, C2-C4-alkenyl, C2-C4- alkynyl, C1-C4-haloalkyl, C1-C4-alkoxy, C2-C4-alkynyloxy, C(=Y1)RI, C(=Y1)OR', C(=Y1)SR', C(=Y1)NRjRk, aryl, aryloxy, hetaryl and hetaryloxy; R' is (i) halogen, CN, C1 -C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4- haloalkyl, C2-C4-haloalkenyl;
(ii) =NR1d, wherein R1d is H, C1-C4-alkyl, C2-C4-alkenyl, C3-C6-cycloalkyl, C3-C6-cycloalkyl methyl, or OR9;
(iii) =0, =S, NRjRk, OR1, SR1, C(=Y1)R', C(=Y1)OR', C(=Y1)SR', C(=Y1)NRjRk;
(iv) aryl, aryl-C1-C2-alkyl, hetaryl, or hetaryl-C1-C2-alkyl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from halogen, CN, C1-C4-alkyl, C1 -C4-haloalkyl, C1-C4- alkoxy, C2-C4-alkynyloxy, OR9, and SR9; or
(vi) C3-C6-cycloalkyl, or 3- to 6-membered heterocyclyl, wherein the cycloalkyi ring or the heterocyclyl ring may be unsubstituted or may be partially or fully substituted by substituents, which are independently of each other selected from halogen, CN, C1-C4-alkyl, OR9, and SR9;
Rj and Rk are independently selected from H, OR9, SR9, C(=Y1)R9, C(=Y1)OR9, C(=Y1)SR9, C(=Y1)NRmRn, C1-C4a- lkyl, C2-C4-alkenyl, C2-C4- alkynyl, C1-C4-haloalkyl, aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently selected from halogen, CN, C1-C4- alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C2-C4-alkynyloxy, OR9, and SR9;
R1 is H, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-haloalkyl, C(=Y1)R9, C(=Y1)OR9, C(=Y1)SR9, C(=Y1)NRmRn, aryl or hetaryl, wherein the aromatic ring of the aryl or hetaryl group may be unsubstituted or may be partially or fully substituted by substituents, which are independently selected from halogen, CN, C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C2-C4-alkynyloxy, OR9, and SR9; and
Rm and Rn are independently selected from H and C1-C4-alkyl.
7. The method of any of claims 1 to 6, wherein the at least one nitrification inhibitor C is a compound of general formula (II)
Figure imgf000071_0001
or acid addition salts thereof, where the radicals R1, R2, R3 and R4 independently from another have the following meanings:
R1, R2, R3 and R4 can be C1- to C2o-alkyl, hydrogen, C3- to Cs-cycloalkyl, C5- to C2o-aryl or alkylaryl, it being possible for these 4 radicals to be monosubstituted or disubstituted by halogen and/or hydroxyl,
R1, R2, R3 can also be halogen or nitro;
R4 can also be a radical having the formula (III)
Figure imgf000072_0001
where
- R5 and R6 independently from another are hydrogen, C1- to C2o-alkyl which can be monosubstituted or disubstituted by halogen and/or hydroxyl, a carboxyl group, a carboxymethyl group or a functional derivative of the two last-mentioned groups, and
- R7 is a carboxyl radical or a carboxy-(C1- to C3-alkyl) radical or a functional derivative of these groups, or is selected from the group consisting of 1 H-1 ,2,4-triazole, 2-chloro-6- (trichloromethyl)-pyridine and 5-ethoxy-3-trichloromethyl-1 ,2,4-thiadiazol, in particular is 3,4-dimethylpyrazol phosphate (DMPP) or 2-(3,4-dimethyl- pyrazol-1 -yl)-succinic acid or a salt thereof. 8. The method of any of claims 1 to 7, wherein, before conducting step (ii), the substrate S comprises less than 50 mg, preferably not more than 30 mg, in particular not more than 20 mg, plant available phosphate per kg of the substrate S.
Figure imgf000072_0002
The method of any of claims 1 to 8, wherein the plants bear root systems insufficient for efficient nutrient uptake, in particular wherein the plants show early root growth and/or have been subjected to stress conditions such as low root zone temperature.
10. The method of any of claims 1 to 9, wherein said method comprises step
(i-l) of premixing the components A, B and C thereby forming mixture ABC, optionally in an aqueous suspension or in solid state, subsequent to step (i) and prior to conducting step (ii), wherein step (ii) is
(ii) supplying the mixture ABC to substrate S of agricultural use on which the plants are cultivated or intended to be cultivated.
1 1 . The method of any of claims 1 to 9, wherein said method comprises step
(i-ll) of premixing the components A and C thereby forming mixture AC, optionally in an aqueous solution or suspension, subsequent to step (i) and prior to conducting step (ii), wherein step (ii) comprises the steps
(ii-a) supplying the mixture AC to substrate S of agricultural use on which the plants are cultivated or intended to be cultivated, and
(ii-b) supplying the component B to substrate S of agricultural use on which the plants are cultivated or intended to be cultivated, wherein step (ii-a) can be conducted concomitant with, prior to or subsequent to step (ii-b).
12. The method of any of claims 1 to 1 1 , wherein the method is for improving root growth of the plants.
13. The use of at least one ammonium source A, wherein at least 50 mol% of the total nitrogen content of A is present as ammonium and/or organically bound nitrogen, at least one bacteria or fungal species B with phosphate- solubilizing and/or root growth-promoting properties, wherein B does not form part of A, and at least one nitrification inhibitor C for improving nutrition of plants with one or more sparingly soluble minerals selected from the group consisting of phosphor, iron, zinc and manganese, in particular wherein the use comprises the characteristics of any of claims 1 to 12.
14. A composition usable for a method of any of claims 1 to 12 or for the use according to claim 13, said composition consisting of
(A) one or more ammonium sources A,
(B) one or more bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties,
(C) one or more nitrification inhibitors C,
(D) optionally water,
(E) optionally one or more additional salts further improving nutrition of plants, and
(F) optionally one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers,
wherein at least 50 mol% of the total nitrogen content of the composition is present as ammonium and/or organically bound nitrogen, and
wherein the composition does not comprise an ammonium source A comprising bacteria or fungal species with phosphate-solubilizing and/or root growth-promoting properties.
15. A kit for use in a method of any of claims 1 to 12 or for the use according to claim 13, comprising at least one bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties, wherein B does not form part of A, and at least one nitrification inhibitor C, and optionally one or more ammonium sources A.
16. A composition comprising:
(A) one or more ammonium sources A of synthetic origin,
(B) one or more bacteria or fungal species B with phosphate-solubilizing and/or root growth-promoting properties,
(C) one or more nitrification inhibitors C,
(D) optionally water, (E) optionally one or more additional salts further improving nutrition of plants, and
(F) optionally one or more additives selected from the group consisting of preservatives, colors, bacteria and/or fungal nutrients, organic solvents and fillers,
wherein preferably at least 50 mol% of the total nitrogen content of the composition is present as ammonium and/or organically bound nitrogen.
17. The composition of any of claims 14 or 16, wherein the composition is a solid composition comprising the components A, B and C and, optionally, E and, optionally, as mixed powders.
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CN116590170A (en) * 2022-12-26 2023-08-15 安徽科技学院 Preparation and application of microorganism strain, compound strain and growth-promoting disease-preventing organic fertilizer
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