Phosphate solubilising bacteria

Abstract

The present invention relates to bacteria with phosphate solubilising activity. The present invention also relates to the use of these bacteria for solubilising phosphate and for growing plants.

Description

[0001] PHOSPHATE SOLUBILISING BACTERIA

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to bacteria with phosphate solubilising activity. The present invention also relates to the use of these bacteria for solubilising phosphate and for growing plants.

[0004] BACKGROUND OF THE INVENTION

[0005] Fertilizers provide essential nutrients for plants to grow and the most significant nutrients required are nitrogen, phosphorus and potassium. Phosphorus is a nutrient that is required in a number of plant cell processes including photosynthesis, cell division, respiration and energy storage and transfer. Phosphorus is critical to root development and flower and seed development and improves plant resilience to disease.

[0006] While soil often contains suitable amounts of phosphorus, it is often in a form that is inaccessible to plants. Adding phosphorus in the form of fertilizer is one solution, however, excess phosphorus is detrimental to the environment. Excess or unused fertilizer on farm, particularly in pasture, may be leached from the soil and provide a number of environmental hazards such as groundwater pollution, eutrophication, the increase in algal blooms in waterways and soil acidity.

[0007] Providing sufficient nutrients to crops and pastures to obtain good yield at an acceptable price is essential for cost effective and productive farming.

[0008] Biofertilizers have recently emerged as an ecofriendly and cost-effective alternative to chemical fertilizers. Plant-associated microbes with growth promoting properties can reduce environmental hazards and cost of chemicals on farm while enhancing plant biomass and crop yield. There is a need for alternative biofertilizers that contribute to cost effective and productive farming.

[0009] SUMMARY OF THE INVENTION

[0010] The present inventors have determined that at least some strains of Pantoea sp. having phosphate solubilising activity. Thus, in an aspect the present invention provides an isolated strain of Pantoea sp. having phosphate solubilising activity.

[0011] In an embodiment, the Pantoea sp. is P. rara.

[0012] In an embodiment, the strain was deposited with the National Measurement Institute under accession number V24 / 010669 or accession number V24 / 010670 on 18 June 2024, or an active variant thereof having phosphate solubilising activity. In an embodiment, the Pantoea sp., such as P. rara, has a mutant HisG protein when compared to a wild type Pantoea sp. protein, such as P. rara protein. In an embodiment, the HisG protein is C-terminally truncated. In an embodiment, the C- terminally truncated HisG consists of the amino acid sequence provided as SEQ ID NO: 46. In an embodiment, the strain lacks a wild-type HisG protein such as a HisG protein having the amino acid sequence provided as SEQ ID NO: 44.

[0013] In an embodiment, the strain has higher levels of one or more or all of hisl (EC:3.5.4.19), hisA (EG5.3.1.16), hisF (EC:4.3.2.10), hisB (LFC=EC:4.2.1.19), hisC (EC:2.6.1.9) and hisD (EC: 1.1.1.23) when compared to a wild type strain such as Lu_Sq_004_WT.

[0014] In an embodiment, the strain produces higher levels of amino-imidazole carboxamide riboside 5 '-phosphate (AICAR) when compared to a wild type strain such as Lu_Sq_004_WT.

[0015] In an embodiment, the strain comprises active phosphate transporter genes pstS, pstC, pstB and pslA. and active phosphonate solubilisation genes phoR, phoB, phnD, phnEl, phnE2, phnC, phnA and phnN, and active phosphate solubilisation genes pqqB, pqqC, pqqD, pqqE, gcd and gad.

[0016] In an embodiment, one or more or all of the following apply, i) the pstS gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 9, ii) the pstC gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 11, iii) the pstB gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 13, iv) the pstA gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 15, v) the phoR gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 17, vi) the phoB gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 19, vii) the phnD gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:21, viii) the phnEl gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:23, ix) the phnE2 gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:25, x) the phnC gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:27, xi) the phnA gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:29, xii) the phnN gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 31, xiii) the pqqB gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:33, xiv) the pqqC gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:35, xv) the pqqD gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 37, xvi) the pqqE gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 39, xvii) the gcd gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:41, and xviii) the gad gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:43.

[0017] In an embodiment, the strain comprises a polynucleotide encoding a 16S RNA molecule which is at least 95% identical, at least 97% identical, at least 99% identical or is identical, to a nucleotide sequence set forth in any one or more of all of SEQ ID NO’s 1 to 7.

[0018] In an aspect, the present invention provides a composition comprising the strain of the invention and a suitable carrier.

[0019] In an embodiment, the carrier is an agriculturally acceptable carrier.

[0020] In an embodiment, the composition is a biofertilizer.

[0021] In an embodiment, the composition is for applying to a seed.

[0022] In an embodiment, the composition is suitable for mixing with, or spraying on, soil. In an embodiment, the composition improves phosphorus solubilisation in the soil.

[0023] Examples of suitable carriers include, but are not limited to, one or more of peat, clay, soil material, mineral, vermiculite, polymer, fertilizer and compatible oil.

[0024] In an embodiment, the composition is in the form of powder, granules, liquid, semi-solid, a dust, suspension, emulsion or spray.

[0025] In an embodiment, the strain is encapsulated in alginate.

[0026] In an embodiment, a seed dressed with the strain is encapsulated in alginate. In an embodiment, a seed is encapsulated with an alginate composition comprising the strain.

[0027] In an embodiment, the composition comprises a polymer selected from the group consisting of alginate, polyco 2133, carrageenan, Gelrite™, guar gum, gellan gum, pectin, sodium pectate, carboxymethyl cellulose (CMC), agar, tragacanth gum and xanthan gum.

[0028] In an embodiment, the composition comprises about 102CFU / mL to about IO10CFU / mL (colony forming unit) of the strain.

[0029] In an embodiment, the composition is a culture of the strain.

[0030] In an aspect, the present invention provides a method of producing a composition of the invention, the method comprising culturing the strain and formulating the cultured strain into the composition.

[0031] In a further aspect, the present invention provides a method of improving phosphorus solubilisation in soil by applying to the soil a strain of the invention.

[0032] In another aspect, the present invention provides a method of increasing the level of bioavailable phosphorus, the method comprising contacting insoluble inorganic phosphorus and / or insoluble organic phosphate with a strain of the invention.

[0033] In an embodiment, the insoluble inorganic phosphorus and / or insoluble organic phosphate is in soil.

[0034] In an embodiment, the soil comprises a seed and / or roots of a plant.

[0035] In a further aspect, the present invention provides a method of producing a plant, the method comprising growing a plant in the presence of a strain of the invention.

[0036] In an embodiment, the method comprises contacting a seed with the strain and growing a plant from the seed.

[0037] In an embodiment, the plant is grown in soil comprising the strain.

[0038] In an embodiment, the method comprises spraying the soil with the strain.

[0039] In an embodiment, the soil comprises a pellet or power comprising the strain.

[0040] In an embodiment, the method improves root growth of the plant.

[0041] Also provided is a plant produced by a method of the invention.

[0042] Furthermore, provided is a seed coated with a strain of the invention.

[0043] The present inventors have also determined that strains of Pantoea sp., such as P. r r C can be used to inhibit fungal growth. Thus, another aspect the present invention provides a method of inhibiting the growth of, and / or reducing damage caused by, one or more fungal pathogen(s), the method comprising delivering to the one or more fungal pathogen(s), or contacting the one or more fungal pathogen(s) with, a strain of the invention. In an embodiment, the method comprises applying a composition of the invention to an area which comprises, or may comprise, one or more fungal pathogen(s).

[0044] In an embodiment, the area comprises a plant.

[0045] Also provided is the use of a strain of the invention to inhibit the growth of, and / or reduce damage caused by, one or more fungal pathogen(s).

[0046] In a further aspect, the present invention provides a kit comprising

[0047] (i) a strain of the invention,

[0048] (ii) optionally a delivery system for applying the strain to a plant or a part thereof, soil or an area; and

[0049] (iii) optionally instructions for using the strain.

[0050] In another aspect, the present invention provides a method of modifying a parental strain of Pantoea sp., such as Pantoea rar a, to produce a mutant strain with improved production of bioavailable phosphorus, the method comprising, a) mutating the HisG gene of the parental strain, b) testing the ability of the mutant strain with the mutated HisG gene to produce bioavailable phosphorus, and c) selecting the mutant strain if is produces more bioavailable phosphorus than the parent strain.

[0051] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0052] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0053] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0054] The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

[0055] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0056] Figure 1 - Provides a graphical representation of microbial strains isolated from commercial cultivar lucerne seeds when screened for phosphate solubilising activity on Pikovskaya agar; dot hatching - negative for phosphate solubilising activity (Bottom part of bars); diagonal hatching - low efficiency phosphate solubilising activity (phosphate solubilising index (PSI) 0 < 1.5) (middle portion of bars where appropriate); cross hatching - high efficiency phosphate solubilising activity (PSI 1.5 <) (top portion of bars where appropriate).

[0057] Figure 2 - Shows examples of microbes grown on Pikovskaya agar that have A: high efficiency PSI; B: low efficiency PSI; C: negative for phosphate solubilising activity.

[0058] Figure 3 - Shows the bacteria Lu_Sq_004 wild type when grown on Pikovskaya agar producing an indigo dye at Day 7 (A), which is lost by Day 10 (B).

[0059] Figure 4 - Shows the growth of UV mutated bacteria Lu_Sq_004_l_2 where a mutant was identified as having phosphate solubilising activity (A), and was isolated as a single colony and grown again on Pikovskaya agar showing significant phosphate solubilising activity (PSI = 4.13). (B).

[0060] Figure 5 - Provides a table showing the phosphate solubilising genes for high efficiency phosphate solubilising microbes wild type microbe (Lu_Sq_004_WT) and mutant Lu_Sq_004_5722_l_2 (Lu_Sq_004_l_2); low efficiency mutants

[0061] (Lu_Sq_004_5722_l_l and Lu_Sq_004_5722_4_2) and mutants with no phosphate solubilising activity (Lu_Sq_004_21722_l_l and Lu_Sq_004_21722_4_2). Blank cells indicate the absence of a gene in the genome of the microbe. The control strain is a Pantoea allii type strain.

[0062] Figure 6 - Graphical representation of mean root length (Y axis) and the inoculant and dilution level (X axis) for lucerne seeds inoculated with no microbe (control) or wild type microbe (Lu_Sq_004) at different dilutions 10°, 10’1, 10'2, 10'3and 10'4) grown on Pikovskaya agar. Mean root length increases with decreasing concentration.

[0063] Figure 7 - Graphical representation of mean shoot length (Y axis) and the inoculant and dilution level (X axis) for lucerne plants inoculated with no microbe (control), wild type microbe (Lu_Sq_004_WT), high efficiency PSI mutant (Lu_Sq_004_l_2) and negative PSI mutant (Lu_Sq_004_6_5) at dilutions 10° and 10'4grown in potting mix for four weeks. The undiluted high efficiency solubilising mutant Lu_Sq_004_l_2 had statistically longer shoots after four weeks growth in potting mix in seedling trays. Figure 8 - Provides a photographic representation of inhibition of fungal growth by Lu_Sq_004_ WT when inoculated on a nutrient agar plate with a central inoculation of Didymella pinoides (a pathogenic fungi that causes Ascochyta blight in peas) (right hand plate) compared with a control sample that includes the fungal pathogen only (Control, left hand plate). Lu_Sq_004_l_2 showed similar effect (data not shown).

[0064] Figure 9 - Provides a table of genes that are up- or down-regulated in the test microbes Lu_Sq_004_ WT and Lu_Sq_004_l _2 when grown in the presence of pathogenic fungus Didymella pinoides.

[0065] KEY TO THE SEQUENCE LISTING

[0066] SEQ ID NO: 1 - First 16S rRNA encoding sequence of Pantoea rara strain Lu_Sq_004. SEQ ID NO:2 - Second 16S rRNA encoding sequence of Pantoea rara strain Lu_Sq_004.

[0067] SEQ ID NO:3 - Third 16S rRNA encoding sequence of Pantoea rara strain Lu_Sq_004. SEQ ID NO:4 - Fourth 16S rRNA encoding sequence of Pantoea rara strain Lu_Sq_004. SEQ ID NO: 5 - Fifth 16S rRNA encoding sequence of Pantoea rara strain Lu_Sq_004. SEQ ID NO:6 - Sixth 16S rRNA encoding sequence of Pantoea rara strain Lu_Sq_004. SEQ ID NO:7 - Seventh 16S rRNA encoding sequence of Pantoea rara strain Lu_Sq_004.

[0068] SEQ ID NO:8 - P. rara Lu_Sq_004_WT strain pstS gene encoding a Pi-binding periplasmic protein (identical in Lu_Sq_004_l_2 mutant).

[0069] SEQ ID NO:9 -P. rara Lu_Sq_004_WT strain pstS Pi-binding periplasmic protein.

[0070] SEQ ID NO: 10 -P. rara Lu_Sq_004_WT strain pstC gene encoding a membrane protein which forms part of a phosphate-specific transporter (identical in Lu_Sq_004_l_2 mutant).

[0071] SEQ ID NO: 11 - P. rara Lu_Sq_004_WT strain pstC membrane protein which forms part of a phosphate-specific transporter.

[0072] SEQ ID NO: 12 - P. rara Lu_Sq_004_WT strain pstB gene encoding an ATPase (identical in Lu_Sq_004_l_2 mutant).

[0073] SEQ ID NO: 13 -P. rara Lu_Sq_004_WT strain pstB ATPase protein.

[0074] SEQ ID NO: 14 - P. rara Lu_Sq_004_WT strain pstA gene encoding a transmembrane protein which forms part of a phosphate-specific transporter (identical in Lu_Sq_004_l_2 mutant).

[0075] SEQ ID NO: 15 - P. rara Lu_Sq_004_WT strain pstA membrane protein which forms part of a phosphate-specific transporter. SEQ ID NO : 16 - P. rar a Lu_Sq_004_WT strain phoR gene encoding a membrane-bound histidine kinase (identical in Lu_Sq_004_l_2 mutant).

[0076] SEQ ID NO: 17 -P. rara Lu_Sq_004_WT strain phoR membrane-bound histidine kinase which forms part of a two-component signal transduction system with phoB.

[0077] SEQ ID NO: 18 - P. rara Lu_Sq_004_WT strain phoB gene encoding a cytoplasmic response regulator that can be phosphorylated by PhoR (identical in Lu_Sq_004_l_2 mutant).

[0078] SEQ ID NO: 19 - P. rara Lu_Sq_004_WT strain phoB cytoplasmic response regulator that can be phosphorylated by PhoR and which forms part of a two-component signal transduction system with phoR.

[0079] SEQ ID NO:20 - P. rara Lu_Sq_004_WT strain phnD gene encoding a periplasmic binding protein (identical in Lu_Sq_004_l_2 mutant).

[0080] SEQ ID NO:21 -P. rara Lu_Sq_004_WT strain phnD periplasmic binding protein.

[0081] SEQ ID NO:22 - P. rara Lu_Sq_004_WT strain phnEl gene encoding a periplasmic binding protein which, along with phnE2, forms the transmembrane channel of the PhnCDE transporter (identical in Lu_Sq_004_l_2 mutant).

[0082] SEQ ID NO:23 - P. rara Lu_Sq_004_WT strain phnEl periplasmic binding protein which, along with phnE2, forms the transmembrane channel of the PhnCDE transporter. SEQ ID NO:24 - P. rara Lu_Sq_004_WT strain phnE2 gene encoding a periplasmic binding protein which, along with phnEl, forms the transmembrane channel of the PhnCDE transporter (identical in Lu_Sq_004_l_2 mutant).

[0083] SEQ ID NO:25 - P. rara Lu_Sq_004_WT strain phnE2 periplasmic binding protein which, along with phnEl, forms the transmembrane channel of the PhnCDE transporter. SEQ ID NO:26 - P. rara Lu_Sq_004_WT strain phnC gene encoding an ATP-binding subunit (identical in Lu_Sq_004_l_2 mutant).

[0084] SEQ ID NO:27 -P. rara Lu_Sq_004_WT strain phnC ATP-binding subunit.

[0085] SEQ ID NO:28 - P. rara Lu_Sq_004_WT strain phnA gene encoding a phosphonoacetate hydrolase protein (identical in Lu_Sq_004_l_2 mutant).

[0086] SEQ ID NO:29 -P. rara Lu_Sq_004_WT strain phnA phosphonoacetate hydrolase.

[0087] SEQ ID NO:30 - P. rara Lu_Sq_004_WT strain phnN gene encoding ribose 1,5- bisphosphokinase (identical in Lu_Sq_004_l_2 mutant).

[0088] SEQ ID NO:31 - P. rara Lu_Sq_004_WT strain phnN ribose 1,5 -bisphosphokinase protein.

[0089] SEQ ID NO:32 - P. rara Lu_Sq_004_WT strain pqqB gene (identical in Lu_Sq_004_l_2 mutant). SEQ ID NO:33 - P. rar a Lu_Sq_004_WT strain pqqB protein involved with biosynthesis of pyrroloquinoline quinone.

[0090] SEQ ID NO:34 - P. rara Lu_Sq_004_WT strain pqqC gene (identical in Lu_Sq_004_l_2 mutant).

[0091] SEQ ID NO:35 - P. rara Lu_Sq_004_WT strain pqqC protein involved with biosynthesis of pyrroloquinoline quinone.

[0092] SEQ ID NO:36 - P. rara Lu_Sq_004_WT strain pqqD gene (identical in Lu_Sq_004_l_2 mutant) .

[0093] SEQ ID NO:37 - P. rara Lu_Sq_004_WT strain pqqD protein involved with biosynthesis of pyrroloquinoline quinone.

[0094] SEQ ID NO:38 - P. rara Lu_Sq_004_WT strain pqqE gene (identical in Lu_Sq_004_l_2 mutant).

[0095] SEQ ID NO:39 -P. rara Lu_Sq_004_WT strain pqqE protein involved with biosynthesis of pyrroloquinoline quinone.

[0096] SEQ ID NO:40 - P. rara Lu_Sq_004_WT strain gcd gene encoding a glucose dehydrogenase protein (identical in Lu_Sq_004_l_2 mutant).

[0097] SEQ ID NO:41 -P. rara Lu_Sq_004_WT strain glucose dehydrogenase protein (gcd). SEQ ID NO:42 - P. rara Lu_Sq_004_WT strain gad gene encoding a glutamate decarboxylase protein (identical in Lu_Sq_004_l_2 mutant).

[0098] SEQ ID NO:43 -P. rara Lu_Sq_004_WT strain glutamate decarboxylase protein (gad). SEQ ID NO:44 - Wild type P. rara HisG protein sequence (Accession WP_179895341). SEQ ID NO:45 - Wild type P. rara HisG gene sequence.

[0099] SEQ ID NO:46 - Mutant P. rara HisG protein sequence.

[0100] SEQ ID NO:47 - Mutant P. rara HisG gene sequence.

[0101] DETAILED DESCRIPTION OF THE INVENTION

[0102] General Techniques and Definitions

[0103] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in biofertilizers, bacteria with phosphate solubilising activity, compositions comprising bacteria with phosphate solubilising activity and uses thereof etc).

[0104] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning. As used herein, the term about, unless stated to the contrary, refers to + / - 10%, or more preferably + / - 5%, more preferably + / - 1%, of the designated value.

[0105] As used herein, the phrase “CFUs” or “Colony Forming Units” refers to the number of bacterial cells in a defined sample (e.g., millilitre of liquid, square centimetre of surface, one seed of grain, etc.) that form colonies and thereafter numbered, on a semisolid bacteriological growth medium.

[0106] The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. In the context of the invention, the GAP analysis aligns two sequences over their entire length.

[0107] The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. In the context of the invention, the GAP analysis aligns two sequences over their entire length.

[0108] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0109] Bacteria with phosphate solubilising activity

[0110] In an aspect, the present invention provides an isolated strain of Pantoea sp. having phosphate solubilising activity.

[0111] In an embodiment, the Pantoea sp. is selected from, but not limited to, Pantoea alvi, Pantoea agglomerans, Pantoea vagans, Pantoea brenneri Pantoea astica, Pantoea bathycoeliae, Pantoea bituminis, Pantoea borealis, Pantoea carbekii, Pantoea communis, Pantoea deserta, Pantoea edessiphila, Pantoea floridensis, Pantoea formicae, Pantoea gossypiicola, Pantoea haifensis, Pantoea multigeneris, Pantoea persica, Pantoea rara, Pantoea soli, Pantoea superficialis, Pantoea symbiotica and Pantoea varia.

[0112] In an aspect, the present invention provides an isolated strain of Pantoea rara having phosphate solubilising activity.

[0113] In an embodiment, the present invention provides an isolated strain of Pantoea rara deposited with the National Measurement Institute under accession number V24 / 010669 on 18 June 2024, or an active variant thereof having phosphate solubilising activity (also referred to herein as Lu_Sq_004 or Lu_Sq_004_WT). In an embodiment, the present invention provides an isolated strain of Pantoea rara deposited with the National Measurement Institute under accession number V24 / 010670 on 18 June 2024, or an active variant thereof having phosphate solubilising activity (also referred to herein as Lu_Sq_004_l_2).

[0114] The term “isolated”, as used herein, is intended to mean that a strain of the disclosure has been isolated from its natural source, substrate, habitat or environment and transferred to a culture media e.g., using methods known in the field bacteriology. For example, an “isolated” strain of the invention may encompass bacteria which has been grown or produced in culture. An “isolated” strain of the invention also encompasses bacteria which have been purified.

[0115] In one embodiment, a strain of the invention is an active variant of Pantoea rara deposited with the National Measurement Institute under accession number V24 / 010669 on 18 June 2024, and / or Pantoea rara deposited with the National Measurement Institute under accession number V24 / 010670 on 18 June 2024.

[0116] As used herein, the term “active variant” refers to the P. rara having phosphate solubilising activity. In an embodiment, the active variant has phosphate solubilising activity which is at about least 50%, at least about 75%, at least about 90%, or at about least about 95% that of a deposited strain outlined above, when tested in a suitable assay such as described in the Examples. Such variants, which may be identified using appropriate screening techniques, are a part of the present invention. In an embodiment, the active variant has a mutant HisG protein as described herein.

[0117] In an embodiment, the active variant is a mutant. In the present context, the term "mutant" or "mutant strain" refers to a strain derived, or a strain which can be derived, from a strain of the invention (or the parental strain) by means of e.g. genetic engineering, mutagenesis, radiation and / or chemical treatment. In an embodiment, the mutant comprises a transgene expressing a polynucleotide and / or protein of interest.

[0118] In an embodiment, the Pantoea sp., such as P. rara has a mutant HisG protein when compared to a wild type Pantoea sp. protein, such as a P. rara HisG protein. As used herein, wild type HisG gene encodes a HisG protein which is an ATP phosphoribosyltransferase. HisG plays a crucial role in histidine biosynthesis, catalysing the first step in this pathway which is the condensation of ATP and phosphoribosyl pyrophosphate (PRPP) to form N'-5'-phosphoribosyl-ATP (PR-ATP) and inorganic pyrophosphate. This reaction is essential for histidine production, and the enzyme's activity is often a point of regulation for the entire pathway.

[0119] In an embodiment, the strain comprises active phosphate transporter genes pstS, pstC, pstB and pstA, and active phosphonate solubilisation genes ^hoR, phoB, phnD, phnEl, phnE2, phnC, phnA and phnN, and active phosphate solubilisation genes pqqB, pqqC, pqqD, pqqE, gcd and gad.

[0120] The pstS, pstC, pstA and pstB genes are members of the bacterial phosphatespecific transporter (pstSCAB) gene cluster which encodes for an ATP -binding cassette (ABC) permease, the primary mechanism for inorganic phosphate transport. PstC works in conjunction with pstA, another integral membrane protein, to form the transmembrane domain of the pst system. Together, they create a pathway for phosphate to move from the periplasmic space into the cytoplasm.

[0121] As used herein, the pstS gene encodes a Pi-binding periplasmic protein. In an embodiment, the pstS gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:8. In an embodiment, the pstS protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:9.

[0122] As used herein, the pstC gene encodes a membrane protein which forms part of a phosphate-specific transporter. In an embodiment, the pstC gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO: 10. In an embodiment, the pstC protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO: 11.

[0123] As used herein, the pstB gene encodes an ATPase. In an embodiment, the pstB gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO: 12. In an embodiment, the pstB protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO: 13.

[0124] As used herein, the pstA gene encodes a transmembrane protein which forms part of a phosphate-specific transporter. In an embodiment, the pstA gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO: 14. In an embodiment, the pstA protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO: 15.

[0125] As used herein, the phoR gene encodes a membrane-bound histidine kinase that acts as a sensor for external phosphate levels. It monitors the extracellular phosphate concentration. When phosphate levels are low, PhoR protein undergoes autophosphorylation at a conserved histidine residue. This transfers the phosphoryl group is then transferred from PhoR to PhoB. In an embodiment, the phoR gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO: 16. In an embodiment, the phoR protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO: 17.

[0126] As used herein, the phoB gene encodes a cytoplasmic response regulator that, when phosphorylated by PhoR protein, becomes activated. Phosphorylated PhoB binds to specific DNA sequences called Pho boxes in the promoter regions of target genes, activating transcription in a wide range of phosphate solubilisation genes. In an embodiment, the phoB gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO: 18. In an embodiment, the phoB protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO: 19.

[0127] As used herein, the phnD gene encodes a substrate-binding protein of the PhnCDE transport system, which is specifically designed to bind and transport phosphonates (such as methylphosphonate, ethylphosphonate, aminomethylphosphonate and 2- aminnoethylphosphonate). In an embodiment, the phnD gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:20. In an embodiment, the phnD protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:21.

[0128] As used herein, the phoEl and phoE2 genes encode integral membrane proteins. PhnEl / 2 protein, along with another copy of itself, forms the transmembrane channel of the PhnCDE transporter. This channel allows phosphonates to pass through the cell membrane. PhnEl / 2 protein helps couple the energy from ATP hydrolysis (performed by PhnC) to the actual transport of phosphonates across the membrane. In an embodiment, the phnEl gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:22. In an embodiment, the phnEl protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:23. In an embodiment, the phnE2 gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:24. In an embodiment, the phnE2 protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:25.

[0129] As used herein, the phnC gene encodes an ATP -binding subunit. In an embodiment, the phnC gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:26. In an embodiment, the phnC protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:27.

[0130] As used herein, the phnA gene encodes a phosphonoacetate hydrolase protein. In an embodiment, the phnA gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:28. In an embodiment, the phnA protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:29.

[0131] As used herein, the phnN gene encodes a ribose 1,5 -bisphosphokinase that produces 5-phospho-d-ribosyl a- 1 -diphosphate (PRPP) which is a key intermediate in the C-P lyase pathway for phosphonate degradation. In an embodiment, the phnN gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:30. In an embodiment, the phnN protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO: 31.

[0132] As used herein, the pqqB gene is a member of the pqq operon responsible for the biosynthesis of pyrroloquinoline quinone (PQQ). In an embodiment, the pqqB gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:32. In an embodiment, the pqqB protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:33. As used herein, the pqqC gene is a member of the pqq operon responsible for the biosynthesis of pyrroloquinoline quinone (PQQ). In an embodiment, the pqqC gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:34. In an embodiment, the pqqC protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:35.

[0133] As used herein, the pqqD gene is a member of the pqq operon responsible for the biosynthesis of pyrroloquinoline quinone (PQQ). In an embodiment, the pqqD gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:36. In an embodiment, the pqqD protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:37.

[0134] As used herein, the pqqE gene is a member of the pqq operon responsible for the biosynthesis of pyrroloquinoline quinone (PQQ). In an embodiment, the pqqE gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:38. In an embodiment, the pqqE protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:39.

[0135] As used herein, the gcd gene encodes a glucose dehydrogenase protein. In an embodiment, the gcd gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:40. In an embodiment, the gcd protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:41.

[0136] As used herein, the gad gene encodes a glutamate decarboxylase protein. In an embodiment, the gad gene comprises a nucleotide sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of nucleotides set forth in SEQ ID NO:42. In an embodiment, the gad protein comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of amino acids set forth in SEQ ID NO:43.

[0137] In an embodiment, a strain of the invention comprises a polynucleotide encoding a 16S RNA molecule which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% identical to a nucleotide sequence set forth in any one or more of all of SEQ ID NO’s 1 to 7. In an embodiment, a strain of the invention comprises a polynucleotide encoding a 16S RNA molecule which is at least 99%, at least 99.5%, at least 99.9% or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 1. In an embodiment, a strain of the invention comprises a polynucleotide encoding a 16S RNA molecule which is at least 99%, at least 99.5%, at least 99.9% or 100% identical to a nucleotide sequence set forth in SEQ ID NO:2. In an embodiment, a strain of the invention comprises a polynucleotide encoding a 16S RNA molecule which is at least 99%, at least 99.5%, at least 99.9% or 100% identical to a nucleotide sequence set forth in SEQ ID NO:3. In an embodiment, a strain of the invention comprises a polynucleotide encoding a 16S RNA molecule which is at least 99%, at least 99.5%, at least 99.9% or 100% identical to a nucleotide sequence set forth in SEQ ID NO:4. In an embodiment, a strain of the invention comprises a polynucleotide encoding a 16S RNA molecule which is at least 99%, at least 99.5%, at least 99.9% or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 5. In an embodiment, a strain of the invention comprises a polynucleotide encoding a 16S RNA molecule which is at least 99%, at least 99.5%, at least 99.9% or 100% identical to a nucleotide sequence set forth in SEQ ID NO:6. In an embodiment, a strain of the invention comprises a polynucleotide encoding a 16S RNA molecule which is at least 99%, at least 99.5%, at least 99.9% or 100% identical to a nucleotide sequence set forth in SEQ ID NO:7.

[0138] Mutant HisG

[0139] Amino acid sequence mutants (modifications) of a HisG polypeptide / protein can be prepared by introducing appropriate nucleotide changes into a nucleic acid encoding the protein. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final protein product possesses the desired characteristics, namely improved bioavailable phosphorus when produced in a Pantoea sp. such as P. rar a .

[0140] The mutation of the HisG gene may be achieved by various means known in the art. In some embodiments, the mutation is introduced into the live bacteria strain by genetical engineering. In some embodiments, the mutation may be achieved by homologous recombination, e.g., double homologous recombination. In some embodiments, the mutation is carried out by targeted mutagenesis, such as via CRISPR (see, for example Benz et al., 2025), TALEN or ZFN technologies. In another embodiment, the mutant is produced by chemical mutagenesis. A mutant strain of Pantoea sp., such as P. rar a, comprising a mutated HisG gene can be tested for improved production of bioavailable phosphorus using a method described herein, such as an assay provided in Examples 3 or 4.

[0141] In an embodiment, the mutant HisG protein is C-terminally truncated.

[0142] In an embodiment, the mutant HisG has a frameshift mutation which encodes a C-terminally truncated HisG protein.

[0143] In an embodiment, the mutant is null for HisG. In an embodiment, the mutant does not have a HisG gene encoding a polypeptide with ATP phosphoribosyltransferase activity.

[0144] In an embodiment, the mutant HisG protein lacks at least the 50, 100 or 150 C- terminal amino acids of a wild type HisG protein.

[0145] Compositions

[0146] The present invention provides a composition (formulation) comprising a strain of the invention. Typically, the composition comprises a suitable carrier.

[0147] In an embodiment, the carrier is an agriculturally acceptable carrier. Any carrier suitable for agricultural use can form part of the compositions and / or formulations of the present invention. The carrier may be any one or more of a number of carriers that confer a variety of properties, including increased stability, wettability, dispersibility, etc. Any other suitable carrier material known in the art that is agriculturally compatible and that would not significantly reduce the viability of the cells can be used without limiting the scope of the present disclosure.

[0148] In an embodiment, the composition is a biofertilizer. As used herein, a biofertilizer is a composition which contains living micro-organisms (in this case a strain of the invention) which, when applied to seeds, plant surfaces, or soil, promotes growth by increasing the supply or availability of primary nutrients (in this case bioavailable phosphorus) to the host plant.

[0149] Suitable compositions that may be prepared include wettable powders, granules, gels, agar strips or pellets, and the like, microencapsulated particles, and the like, liquids such as aqueous flowables, aqueous suspensions, water-in-oil emulsions, etc. The formulation may include grain or legume products (e.g., ground grain or beans, broth or flour derived from grain or beans), starch, sugar, or oil. In certain embodiments, the carrier is a seed, and the composition may be applied or coated onto the seed or allowed to saturate the seed.

[0150] According to certain embodiments, the composition in a form selected from the group consisting of a powder such as a wettable powder (WP), freeze dried powder or water-soluble powder (SP), a liquid, a semi-solid, a suspension, composition an emulsion concentrate (EC), a microemulsion concentrate (MEC), a water-soluble concentrate (SL), a suspoemulsion (SE), an oil dispersion (OD), a concentrated emulsion (BW) such as oil-in-water and water-in-oil emulsion, sprayable solution or emulsion, a microemulsion (ME), a capsule suspension (CS), a suspension concentrate (SC), a dust (DP), an oil-miscible solution (OL), a seed-dressing product, granules (GR) such as in the form of micro granules, spray granules, coated granules, absorption granules, granules for soil application, granules for soil application broadcasting, water-soluble granules (SG) or water-dispersible granules (WDG), a ULV formulation, microcapsules, and waxes. Each possibility represents a separate embodiment of the present invention.

[0151] According to some embodiments, the agricultural carrier may be soil or other plant growth medium. Other agricultural carriers that may be used include water, plantbased oils, humectants, or combinations thereof. Alternatively, the agricultural carrier may be a solid, such as diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, seed cases, other plant and animal products, or combinations, including granules, pellets, or suspensions. Mixtures of any of the aforementioned ingredients are also contemplated as carriers, such as, but not limited to, pesta (flour and kaolin clay), agar or flour-based pellets in loam, sand, clay, etc. Formulations may include food sources for the cultured organisms, such as barley, rice, or other biological materials such as seed, plant parts, sugar cane bagasse, hulls or stalks from grain processing, ground plant material ("yard waste") or wood from building site refuse, sawdust, or small fibers from recycling of paper, fabric, or wood. Other suitable formulations will be known to those skilled in the art.

[0152] In the liquid form, e.g., solutions or suspensions, the strain may be mixed or suspended in water or in aqueous solutions. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum distillates, or other liquid carriers. In an embodiment, the formulation may be a fermentation broth.

[0153] Solid compositions can be prepared by dispersing the strain in and on an appropriately divided solid carrier, such as peat, wheat bran, vermiculite, clay, talc, bentonite, a polymer, diatomaceous earth, mineral, fuller's earth, soil such as pasteurized soil, and the like. When such formulations are used as wettable powders, biologically compatible dispersing agents such as non-ionic, anionic, amphoteric, or cationic dispersing and emulsifying agents can be used.

[0154] Wetting agents such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or a combination thereof, can be included in a composition of the invention. Water-in-oil emulsions can also be used to formulate a composition comprising a strain of the present invention (see, for example, U.S. Patent No. 7,485,451).

[0155] The composition / formulation as used herein can also refer to a customary formulation in an effective amount to be applied either to the soil (i.e., in-furrow), to a portion of the plant (i.e., drench) or on the seed before planting (i.e., seed coating or dressing). Customary formulations include solutions, emulsifiable concentrates, wettable powders, suspension concentrates, soluble powders, granules, suspensionemulsion concentrates, natural and synthetic materials impregnated with active compounds, and very fine controlled release capsules in polymeric substances. In certain embodiments of the present invention, the strain is formulated in a powder that is available in either a ready-to-use formulation or are otherwise mixed at the time of use. In either embodiment, the powder may be admixed with the soil prior to or at the time of planting.

[0156] Depending on the final formulation, one or more suitable additives can also be introduced to the compositions of the present invention. Adhesives such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or latexes, such as gum arabic, chitin, polyvinyl alcohol and polyvinyl acetate, as well as natural phospholipids, such as cephalins and lecithins, and synthetic phospholipids, can be added to the compositions / formulation of the present invention.

[0157] According to certain embodiments, the strain is formulated in a single, stable solution, or emulsion, or suspension. For solutions, the chemical compounds are typically dissolved in solvents before the strain is added. Suitable liquid solvents include petroleum-based aromatics, such as xylene, toluene or alkylnaphthalenes; aliphatic hydrocarbons, such as cyclohexane or paraffins, for example petroleum fractions; mineral and vegetable oils; alcohols, such as butanol or glycol as well as their ethers and esters; ketones, such as methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone; and strongly polar solvents, such as dimethylformamide and dimethyl sulphoxide.

[0158] In use, the liquid can be sprayed or can be applied to the plant foliage as an atomized spray or in-furrow at the time of planting the crop. The liquid composition can be introduced in an effective amount on the seed (i.e., seed coating or dressing) or to the soil (i.e., in-furrow) before germination of the seed or directly to the soil in contact with the roots by utilizing a variety of techniques known in the art including, but not limited to, drip irrigation, sprinklers, soil injection or soil drenching. Optionally, stabilizers and buffers can be added, including alkaline and alkaline earth metal salts and organic acids, such as citric acid and ascorbic acid, inorganic acids, such as hydrochloric acid or sulfuric acid. According to certain currently exemplary embodiments, the agricultural composition is in the form of a seed coating. In an embodiment, the seed is drenched with a bacterial composition comprising the strain of the invention and then is coated with a seed coating. In an embodiment, the seed coating composition comprises the strain of the invention. According to these embodiments, the seed coating formulation may further comprise at least one agent selected from the group consisting of a binding agent and a wetting agent.

[0159] In an embodiment, the seed coating formulation comprises a polymer selected from alginate, agar, carboxymethyl cellulose, poly co 2133, carrageenan, Gelrite™, guar gum, sodium pectate, pectin, tragacanth gum, xanthan gum and gellan gum. In an embodiment, the seed coating formulation comprises alginate, especially calcium alginate as described in WO 2013 / 177616.

[0160] In an embodiment, the strain is encapsulated in alginate, especially calcium alginate. In an embodiment, the seed is encapsulated in alginate, especially calcium alginate, where the strain is coated on the seed or wherein the strain is within the calcium alginate encapsulation material.

[0161] In an embodiment, the composition comprises macro and / or micronutrients. Examples of nutrients include, but are not limited to, the chemical salts of the ions of K+, NO3’, NH4+, PO43', SO42' , Mg2+, Ca2+, BO33', Fe2+, Mn2+, Zn2+, Cu2+, Mo2+and Co2+.

[0162] In an embodiment, the composition further comprises at least one additional active agent selected from the group consisting of a fertilizer, an acaricide, a fungicide, an insecticide, a nematicide, a pesticide, sun protecting agent, an additional plant growth regulator, a nutrient, and any combination thereof.

[0163] Any fertilizer as is known in the art can be added to the compositions / formulations of the present invention, as long as the fertilizer does not interfere with the bacterial growth and activity. According to certain embodiments, the fertilizer is selected from the group consisting of chemical or biological fertilizer. The amount of the at least one chemical or biological fertilizer employed can vary depending on the final formulation as well as the size of the plant and / or seed to be treated.

[0164] In an embodiment, the composition comprises a phosphate solubilising bacteria other than a strain of the invention. Examples include, but are not limited to, some strains of Rhizobium sp., Azospirillum sp., Bacillus sp. such as Bacillus coagulans o Bacillus megaterium, Curtobacterium sp., Erwinia sp., Pantoea sp., Pseudomonas sp., Stenotrophomonas sp., Azotobacter sp., Burkholderia sp., Herbaspirillum sp., Rhodococcus sp., Arthrobacter sp., Serratia sp., Chryseobacterium sp., Gordonia sp., Phyllobacterium sp., Delftia sp., Xanthomonas sp., Enterobacter sp. or Xanthobacter agilis.

[0165] In an embodiment, the composition is a culture of the strain such as a starter culture.

[0166] Examples of the types of compositions which may comprise a strain of the invention include those described in, but not limited to, W02024 / 006500, W02020 / 021549, WO 2024 / 100602, WO 2023 / 218262, WO 2021 / 021030, WO 2019 / 078806 and WO 2018 / 234996.

[0167] In an embodiment, the amount of the strain within the composition / formulation is sufficient to interact, colonize and / or localize in a cultivated plant, typically crop plant, treated with same.

[0168] According to certain embodiments, the strain is about 2% w / w to about 80% w / w of the entire formulation / composition. According to other embodiments, the strain is about 5% w / w to about 65% w / w, or about 10% w / w to about 60%, w / w by weight of the entire formulation / composition.

[0169] In an embodiment, the composition comprises about 102CFU / mL to about 1010CFU / mL (colony forming unit), or about 104CFU / mL to about 108CFU / mL, of the strain.

[0170] According to certain embodiments, the composition provided herein is formulated to provide stability for the strain. Optionally, a shelf-stable formulation is in a dry form, e.g., a powder formulation, or a lyophilized formulation. According to certain embodiments, the composition is substantially stable at temperatures between about 4°C and about 37°C for at least about 5, 10, 15, 20, 25, 30 or more days. According to certain exemplary embodiments, the strain may be shelf-stable, wherein at least 0.01% of the CFU or spores are viable after storage in desiccated form (i.e., moisture content of 30% or less) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more than 25 weeks at 4°C or at room temperature.

[0171] The present invention further provides a method of producing a composition of the invention, the method comprising culturing the strain and formulating the cultured strain into the composition. Methods of culturing Enterobacterales, such as Pantoea sp. including P. rara. are well known in the art. One example is the use of R2A media which is described in the Examples herein. In an embodiment, the strain is cultured overnight in liquid culture medium to an OD 600 of between about 0.5 to 0.7, especially about 0.6.

[0172] In an embodiment, the cultured strain is freeze dried as a powder before mixing with a carrier, wherein the powder has viable cells. Bioavailable Phosphorous

[0173] A strain of the invention can be used to improve phosphorus solubilisation in soil by applying the strain to soil.

[0174] A strain of the invention can also be used to increase the level of bioavailable phosphorus in, for example, soil by contacting insoluble inorganic phosphorus and / or insoluble organic phosphate with the strain.

[0175] In an embodiment, the solubilised phosphorus or the bioavailable phosphorus is inorganic phosphate (Pi) in the soil.

[0176] The insoluble inorganic phosphorus and / or insoluble organic phosphate includes, but is not limited to, one or more or all of rock phosphate, hydroxyapatite, phosphonate, phytate and metal phosphates.

[0177] Furthermore, a strain of the invention can be used to produce a plant by growing the plant in the presence of the strain.

[0178] In an embodiment, the seed is coated with the strain, placed in soil and grown. In an embodiment, the plant is grown in soil comprising the strain.

[0179] According to some embodiments, the seed is in soil, and administration is via infurrow application, seed treatment, broadcast spray pre-planting incorporation, broadcast spraying on the soil surface, and / or drip irrigation. In an embodiment, a method of the invention comprises spraying soil with the strain. In an embodiment, soil comprising a pellet or power comprising the strain is used in a method of the invention.

[0180] In an embodiment, a method of the invention improves root growth of the plant. For example, the method improves one or more or all of root biomass, root coverage, root density or root length.

[0181] As used herein, the term “root coverage” refers to the total area or volume of soil or of any plant-growing medium encompassed by the roots of a plant. According to some embodiments of the invention, the root coverage is the minimal convex volume encompassed by the roots of the plant.

[0182] It should be noted that since each plant has a characteristic root system, e.g., some plants exhibit a shallow root system (e.g., only a few centimetres below ground level), while others have a deep in soil root system (e.g., a few tens of centimetres or a few meters deep in soil below ground level), measuring the root coverage of a plant can be performed in any depth of the soil or of the plant-growing medium, and comparison of root coverage between plants of the same species (e.g., plant inoculated with bacteria of some embodiments of the invention and a control plant) should be performed by measuring the root coverage in the same depth. As used herein, the term “root length” refers to the total length of the longest root of a single plant.

[0183] As used herein, the term "increasing" or “improving” refers to at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%, increase when compared to a suitable control. For example, increase in a trait (e.g., root growth, yield, seed yield, biomass, growth rate, vigor, photosynthetic capacity, early flowering, grain filling period, harvest index or plant height) of a plant as compared to a control plant (e.g., isogenic plant), i.e., a plant not inoculated with the strain under the same (e.g., identical) growth conditions.

[0184] The present invention can be used on a wide variety of plants including a monocot plant or a dicot plant. In an embodiment, the plant is an angiosperm.

[0185] In an embodiment, the plant is selected from the group consisting of a grain crop plant, an oilseed crop plant, a vegetable crop plant, a forage crop plant, an industrial crop plant, or a woody crop plant. The plant may be produced, typically as a crop, for food, feed, fibre, construction, fuel purposes and more.

[0186] The term “plant” encompasses a whole plant, a grafted plant, ancestor(s) and progeny of the plants and plant parts (also referred to herein as “a portion”), including seeds, shoots, stems, roots (including tubers), rootstock, scion, and plant cells, tissues, and organs. The plant may be in any form, including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. Plants that are particularly useful in the methods of the invention include those selected from the group comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Cannabaceae, Cannabis, Cannabis sativa, Hemp, industrial Hemp, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Chlor is spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia villosa, Pagopyrum spp., Feijoa sellowlana, Festuca spp., Fragaria spp., Flemingia spp, Freycinetia banks li, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgar e, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Lolium spp. such as Lolium perenne and Lolium multiflorum, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago spp. such as Medicago sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Paspalum spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize, wheat, barley, rye, oat, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. Each possibility represents a separate embodiment of the present invention.

[0187] According to some embodiments of the invention, the plant is selected from the group consisting of rice, maize, wheat, barley, peanut, potato, sesame, olive tree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa, millet, Leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, poplar, or cotton.

[0188] According to some embodiments of the invention, the plant is selected from a forage grass such as perennial ryegrass, tall festuca, paspalum or crown grass, lucerne and Rhodes grass. According to certain exemplary embodiments, the monocotyledonous plant includes a monocotyledonous species such as: maize (Zea mays), common wheat (Triticum aestivum), spelt (Triticum spelta), einkorn wheat (Triticum monococcum), emmer wheat (Triticum dicoccum), durum wheat (Triticum durum), Asian rice (Oryza sativa), African rice (Oryza glabaerreima). wild rice (Zizania aquatica, Zizania latifolia, Zizania palustris, Zizania texana), barley (Hordeum vulgare). Sorghum (Sorghum bicolor), Finger millet (Eleusine coracana), Proso millet (Panicum miliaceum), Pearl millet (Pennisetum glaucum), Foxtail millet (Setaria italica), Oat (Avena sativa), Triticale (Triticosecale), rye (Secale cereale), Russian wild rye (Psathyrostachys juncea), bamboo (Bambuseae), or sugarcane (e.g., Saccharum arundinaceum, Saccharum barberi, Saccharum bengalense, Saccharum edule, Saccharum munja, Saccharum officinarum, Saccharum procerum, Saccharum ravennae, Saccharum robustum, Saccharum sinense, or Saccharum spontaneum).

[0189] According to further certain exemplary embodiments, the dicotyledonous plant includes a dicotyledonous species such as: soybean (Glycine max), canola and rapeseed cultivars (Brassica napus), cotton (genus Gossypium), alfalfa (Medicago sativa), cassava (genus Manihof), potato (Solanum tuberosum), tomato (Solanum lycopersicum), pea (Pisum sativum), chickpea (Cicer arietinum), lentil (Lens culinaris), flax (Linum usitatissimum), or many varieties of vegetables.

[0190] In an embodiment, the plant to which the strain is inoculated is free of that microbe before inoculation.

[0191] Controlling Fungi

[0192] The present invention also provides a method of inhibiting the growth of, and / or reducing damage caused by, one or more fungal pathogen(s), the method comprising delivering to the one or more fungal pathogen(s), or contacting the one or more fungal pathogen(s) with, a strain of the invention.

[0193] In an embodiment, the method comprises applying a composition of the invention to an area which comprises, or may comprise, one or more fungal pathogen(s). In an embodiment, the area comprises one or more plants, such as a crop.

[0194] In an embodiment the one or more fungal pathogen(s) causes a disease in the plant such as, but not limited to, stem rust, leaf rust, stripe rust, powdery mildew, head blight, crown rot, foot rot, pink snow mold, spot blotch, common root rot, blast, leaf blight, anthracnose and southern corn blight.

[0195] In an embodiment, the one or more fungal pathogen(s) at least infect one or more of the followings plants; wheat, barley, oats, rye, rice, maize, peas, lentils or sorghum. In an embodiment, the one or more fungal pathogen(s) is a Didymella sp., Puccinia sp., Blumeria sp., Fusarium sp., Magnoporthe sp., Bipolar is sp., Oidium sp., Gibber ella sp., Cochliobolus sp., Exser ohilum sp., Uredo sp. Microdochium sp., Helminthosporium sp., Monographella sp., Colletotrichum sp., Uromyces sp. or Erysiphe sp..

[0196] In an embodiment, the one or more fungal pathogen(s) is a Didymella sp., Puccinia sp., Blumeria sp., Fusarium sp., Magnoporthe sp., Bipolaris sp., Cochliobolus sp., Exserohilum sp. o Erysiphe sp..

[0197] In an embodiment, the Didymella sp. is Didymella pinoides, Didymella pinodella. Didymella ly roper sici. Didymella fabae. Didymella arachidicola or Didymella aliena: especially Didymella pinoides.

[0198] In an embodiment, the Puccinia sp. is Puccinia graminis, Puccinia triticina, Puccinia tritici-duri, Puccinia recondita or Puccinia striiformis.

[0199] In an embodiment, the Puccinia graminis is Puccinia graminis f. sp. tritici (Ug99).

[0200] In an embodiment, the Puccinia recondita is Puccinia recondita f. sp. tritici.

[0201] In an embodiment, the Fusarium sp. is Fusarium oxysporum, Fusarium proliferation, Fusarium pseudograminearum. Fusarium graminearum Group II, Fusarium avenaceum. Fusarium culmorum and Fusarium nivale.

[0202] In an embodiment, the Blumeria sp. is Blumeria graminis. In an embodiment, the Blumeria sp. is Blumeria graminis f. sp. tritici.

[0203] In an embodiment, the Bipolaris sp. is Bipolaris sorokiniana.

[0204] In an embodiment, the Gibberella sp. is Gibberella avenacea or Gibberella zeae .

[0205] In an embodiment, the Erysiphe sp. is Erysiphe graminis. In an embodiment, the Erysiphe graminis is Erysiphe graminis f. sp. tritici.

[0206] In an embodiment, the Exserohilum sp. is Exserohilum turcicum.

[0207] In an embodiment, the Magnoporthe sp. is Magnaporthe grisea.

[0208] In an embodiment, the Uredo sp. is Uredo glumarum.

[0209] In an embodiment, the Microdochium sp. ox Microdochium nivale.

[0210] In an embodiment, the Monographella sp. is Monographella nivalis.

[0211] In an embodiment, the Cochliobolus sp. is Cochliobolus sativus.

[0212] In an embodiment, the Helminthosporium sp. is Helminthosporium sativum.

[0213] In an embodiment, the Oidium sp. is Oidium monilioides.

[0214] In an embodiment, the Colletotrichum sp. is Colletotrichum sublineolum.

[0215] In an embodiment, the Uromyces sp. is Uromyces eragrostidis . Kits

[0216] According to certain aspects, the present invention provides a kit comprising a strain of the invention. In an embodiment, the strain is present in a composition of the invention.

[0217] In an embodiment, the kit further comprises a delivery system for applying the strain to a plant or a part thereof, soil or an area.

[0218] In an embodiment, the kit further comprises instructions for using the strain. According to certain embodiments, the instructions comprise instructions for the amounts and frequency of applying a composition of the invention so as to, for example, enhance the bioavailable phosphorus levels in soil.

[0219] EXAMPLES

[0220] Example 1 : Isolation of phosphate solubilising microbes

[0221] A library of microbes isolated from commercial cultivars of Medicago sativa (Lucerne) were screened for phosphate solubilising activity using Pikovskaya agar (PKA).

[0222] Bacterial isolates from the library were taken from -80°C glycerol stock and plated onto R2A media and grown for 5 days (21.5°C, 30% RH). Single bacterial colonies were taken from the isolate plates and transferred to Pikovskaya agar plates (21.5°C, 30% RH). After 10 days the plates were reviewed and assessed for phosphate solubilisation efficiency as shown in Figures 1 and 2. A PSI of over 1.5 indicated efficient phosphate solubilisation, between 0 and 1.5 indicated low efficiency phosphate solubilisation and 0 indicated no phosphate solubilisation.

[0223] A total of 15 isolates with efficient phosphate solubilisation were identified. Two were isolated from the Aurora cultivar, one from Force-5 cultivar, one from Hunter River cultivar, 3 from Ryno06 cultivar, two from Sequel cultivar and 6 from SiRiver cultivar.

[0224] The genomes of candidate phosphate solubilising microbes were sequenced. These novel strains were retrieved from -80°C glycerol storage, inoculated onto R2A plates and grown at room temperature for five days. A single colony was taken from each plate and grown in 30mL nutrient broth (NB) and incubated at 25°C for 24 hours at 170 cycles a minute. DNA extraction was performed using the Wizard® Genomic DNA Purification Kit (Al 120, Promega). The sequencing library was prepared using an inhouse protocol modified from the official protocols library preparation kits (SQK- LSK112.24, ONT, Oxford, UK). All libraries were sequenced on a MinlON MklB platform (MIN- 10 IB) with R10.4 flow cells (FLO-MINI 06) and under the control of MinKNOW software. After the sequencing run finished, the fast5 files that contain raw read signals were transferred to a separate, high performance computing Linux server for basecalling and demultiplexing using ONT’s Albacore software (Version 2.3.1) with default parameters.

[0225] For libraries prepared with the barcoding kit (SQK-RBK004), barcode demultiplexing was achieved during basecalling. The sequencing summary file produced by Albacore was processed by the R script minion qc (https: / / github.com / roblanf / minion_qc) and NanoPlot to assess the quality of each sequencing run, while Porechop (Version 0.2.3, https: / / github.com / rrwick / Porechop) was used to remove adapter sequences from the reads (De Coster et al., 2018). Reads which were shorter than 300 bp were removed and the worst 5% of reads (based on quality) were discarded by using Filtlong (Version 0.2.0, https: / / github.com / rrwick / Filtlong). Output reads from Oxford Nanopore Technologies (ONT) MinlON were assembled using Try cycler, a high-quality long-read-only bacterial genomic assembly tool (Wick et al., 2021). The full suite of phosphate solubilisation genes were only identified in two of the isolates Lu_Sq_004 and Lu_R6_006. The 16S rRNA encoding sequences of Lu_Sq_004 (SEQ ID NO’s 1 to 7) was compared to reference samples and the inventors classified the bacteria as a strain of Pantoea rara.

[0226] One of these microbes Lu_Sq_004 included a pigment when being grown on PKA providing a purple colour between days 4 and 8 (Figure 3). The indigo pigment was only present when grown on PKA not when grown on R2A agar. The production of pigment occurred only in colonies that were at least 18 mm apart and the pigment was not light labile. Lu_Sq_004 was used in further experiments as the colour acted as a marker for the presence or absence of the microbe.

[0227] Example 2: Mutation of Phosphate solubilising microbe Lu Sq 004

[0228] UV mutagenesis of commercial lucerne isolates was performed using a modified version of the Barrick lab’s E. coli mutagenesis protocol (https: / / barricklab.org / twiki / bin / view / Lab / ProtocolsUVLibrary). In a preliminary experiment, Lu_Sq_004 was retrieved from -80°C glycerol storage, inoculated onto R2A plates and grown at room temperature for five days. A single colony was taken from the plate was and grown in 30mL nutrient broth (NB) and incubated at 25°C for 24 hours at 170 cycles a minute. Using an Eppendorf tube, ImL of overnight culture was pelleted at 3,000 ref for 5 minutes. The supernatant was removed, and the cells were resuspended in ImL of saline solution. Two 1 in 10 serial dilutions were performed to achieve a workable quantity of cells. Using thin-walled PCR tubes, 120 pL of dilute cells were exposed to the UV Crosslinker. Preliminary experiments were conducted to determine the ideal UV exposure time (s) to generate a 90% cell death for Lu_Sq_004. Tubes were exposed to the UV source for between 0 and 300 seconds plated on R2A and growth for two days. Cell counts were then performed to determine the rate of cell death. It was determined that 60 seconds of exposure to the UV source reduced the cell count by 88%, while 90 seconds of exposure to the UV source reduced the cell count by 93%.

[0229] The tubes were exposed to either 60 or 90 seconds, depending on treatment. A series of 10 pL aliquots were plated on PKA. Control, 60 seconds and 90 seconds treatments were also plated on R2A to ensure that 90% cell death was maintained across round of mutation. Surviving colonies were then screened for altered phosphate solubilisation activity. The colonies were plated at 25°C for ten days and examined for altered pigment production or increase, reduction or absence of clear halos. The phosphate solubilising index (PSI) of potential increased-function, reduced-function and null mutants were calculated using the method used to assess the lucerne seed isolate library.

[0230] One mutant, designated Lu_Sq_004_5722_l_2 was found to have significant phosphate solubilisation efficiency with a PSI of 4.13 compared to the wild type microbe which has a PSI of 1.74 (Figure 4).

[0231] In silico screening was performed on the wild type microbe Lu_Sq_004_WT, the high efficiency mutant Lu_Sq_004_5722_l_2 as well as two selected low efficiency mutants Lu_Sq_004_5622_l_l and Lu_Sq_004_5722_4_2 and two negative mutants Lu_Sq_004_21722_6_3 and Lu_Sq_004_21722_6_4. Figure 5 indicates that the low efficiency and negative mutants all lacked the gcd gene involved in phosphate solubilisation whereas the wild type and high efficiency mutant had a full suite of phosphate solubilisation genes.

[0232] Example 3 : Seedling plate assay

[0233] Pantoea rara strain Lu_Sq_004_WT was cultured in 30mL nutrient broth (NB) and incubated at 25°C for 24 hours at 170 cycles a minute. The following day, lucerne seeds were sterilised by soaking in 80% ethanol for 5 minutes and then washing 5 times in sterile distilled water. An OD reading of the culture was taken to determine the CFU / mL of the microbe. Lucerne seeds (cv Siriver) were inoculated with the wild type microbe Lu_Sq_004_WT at different concentrations by soaking the seed in a solution of microbe at concentrations of 10°, 10-1, 1 O'2, 1 O'3and 10'4for 4 hours at 26°C with shaking. Single seeds that germinated on the same day were placed on solid Pikovskaya’s agar and the seed were allowed to grow for 7 days at 21.5°C. A control that was not exposed to microbe but soaked in PBS without bacteria for 4 hours at 26°C was also included.

[0234] After a total of 10 days growth, the plates were photographed and the mean root length and shoot length for the seedlings was then measured. Data was statistically analysed using a one-way ANOVA and Tukey test to detect the presence of any significant difference (p < 0.05) between the treatments using OriginPro 2023 (Version bl0.0.0.154). The results in Figure 6 indicate that the microbial effect is concentration dependent with lower concentrations of microbe resulting in longer roots.

[0235] Example 4: Seedling pot assay

[0236] Sterile lucerne seeds (cultivar Siriver) were prepared as in Example 3. Overnight cultures of Lu_Sq_004_WT, Lu_Sq_004_5722_l_2 (high efficiency mutant) and Lu_Sq_004_6_5 (no activity mutant) were prepared. A single colony was taken from the plate was and grown in 30mL nutrient broth (NB) and incubated at 25°C for 24 hours at 170 cycles a minute and an OD reading was taken to determine the CFU / mL.

[0237] A neat solution (10°) and a PBS diluted solution (10'4) were prepared and the sterile seeds were soaked in the bacterial solutions for 4 hours at 26°C in a shaking incubator, 30 seeds per treatment. Control seeds were inoculated with media containing no microbe. The seeds were then planted in a seedling tray containing potting mix with one seed per cell. Germination rates were calculated at 5 days after planting.

[0238] After 4 weeks of growth, the seedlings were photographed and the root and shoot lengths of the seedlings were measured. Data was statistically analysed using a one-way ANOVA and Tukey test to detect the presence of any significant difference (p < 0.05) between the treatments using OriginPro 2023 (Version bl0.0.0.154). The results are shown in Figure 7. The plants inoculated with undiluted high-phosphate solubilising mutant had statistically longer shoots after four weeks growth in potting mix in seedling trays.

[0239] Example 5: Bioprotectant properties

[0240] Lu_Sq_004_WT and Lu_Sq_004_l_2 mutant were inoculated onto a nutrient agar plate with a central inoculation of Didymella pinoides, a pathogenic fungi that causes Ascochyta blight in peas and incubated (21 °C, 30% RH) for 7 days. The Lu_Sq_004_WT and Lu_Sq_004_l_2 mutant inhibited the growth of the pathogenic fungi. The results for Lu_Sq_004_WT are shown in Figure 8, the same result was observed for the mutant Lu_Sq_004_l_2 (not shown).

[0241] RNA was extracted using the ZymBiomics RNA Miniprep Kit and rRNA depletion achieved using the NEBNext rRNA Depletion Kit. RNA libraires were prepared with the NEXTFLEX Rapid Directional RNA-Seq Kit 2.0. Transcript-level quantification estimates for RNA-seq were calculated using Salmon (version: 1.10.1, https: / / github.com / COMBINE-lab / salmon). Wasabi converted Salmon output files into a Sleuth-compatible format (version: 1.0.1, https: / / github.com / COMBINE-lab / wasabi). Using Sleuth, transcripts with less than 3 reads that appeared in less than 47% of samples per treatment were removed and differential analysis was performed using likelihood ratio tests (LRT) and Wald tests (version: 0.30.1, h tips : / / gi dmb.com / pach ted ab / sleu th) . The transcriptomic profile of Lu_Sq_004_WT and Lu_Sq_004_l_2 mutant exposed to the fungi was obtained and compared to Lu_Sq_004_WT and Lu_Sq_004_l_2 mutant control samples not exposed to fungi and demonstrated significant fold changes in expression in the presence of pathogenic fungi (Figures 8 and 9).

[0242] Example 6: Transcriptome Analysis

[0243] Materials and Methods

[0244] Transcript abundance was estimated using Salmon (Patro et al., 2017). A transcriptome index was first generated from the annotation genome of Lu_Sq_004_WT, generated by Prokka, using the salmon index function. Quantification was performed on rRNA-depleted, paired-end RNA-seq reads using the salmon quant function. The following parameters were applied: -1 A, — numBootstraps 1000, — seqBias and - numBiasSampels 5000000. Differential gene expression (DGE) analysis was conducted using R package sleuth64 with kallisto-generated transcript abundances. First, transcriptlevel pairwise analysis of differential expression analysis was performed using the Sleuth R package (Pimentel et al., 2017). Low-abundance transcripts were filtered using a custom function requiring transcripts to have >3 estimated counts in at least 47% of samples.

[0245] A full model (-condition) and a reduced model (-1) were fitted using bootstrapped transcript abundances. Likelihood ratio tests (LRT) and Wald tests were performed to identify differentially expressed transcripts. Transcripts were considered significant at a false discovery rate (FDR) threshold of q < 0.05. Overlapping sets of significant transcripts from both tests were extracted to identify robust DEG candidates. Heatmaps of log2 fold changes were generated using the ComplexHeatmap R package (Gu et al., 2016). Transcript counts normalised prior to model fitting were exported for downstream interpretation. To perform KEGG pathway enrichment analysis, differentially expressed genes (DEGs) identified using Sleuth were processed using clusterProfiler’s enricher function. Annotated genes sets were then mapped to KEGG pathways using local EggNOG-derived functional annotations. Adjusted p-values (FDR- corrected) were used to identify the pathways that were significantly enriched when the bacterial transcriptome profiles on PVK and R2A were compared (Wu et al., 2021). Results were visualized using ggplot2 (Wickham and Sievert, 2009).

[0246] Soluble Phosphate Limitation Drives Transcriptional Changes in Lu Sq 004 WT and Mutants

[0247] Pathway enrichment across the five time points was visualized and sorted based on their temporal enrichment profiles (Table 1). Overall, 80 pathways were significantly enriched in at least one condition at least one recorded time point. The number of core pathways, here defined as pathways enriched by all three microbes (namely Lu_Sq_004_WT, Lu_Sq_004_l_2 and null mutant Lu_Sq_004_6_5) at the same timepoint, highlighted the transcriptomic shifts the bacteria with and without phosphate- solubilising capacity, exposed to varying levels of soluble phosphate, had in common. The number of pathways increased steadily from Day 2 (1 pathway), Day 4 (8 pathways), Day 6 (37 pathways), Day 8 (50 pathways) until Day 10, when the number of core pathways decreased to six. There were five temporal enrichment profiles.

[0248] The first (Group A) contained five pathways enriched in phosphate solubilizers (WT and 1 2), but never in null phosphate solubilization mutant.

[0249] Three Group A pathways, ascorbate and aldarate metabolism, histidine metabolism, inositol phosphate metabolism, have been previously associated with either phosphate starvation or phosphate solubilisation. Given the potential mechanistic importance of phosphate solubilizer-exclusive enrichment, the expression differences within these KEGG pathway were visualised.

[0250] Group B contained 21 pathways which were more consistently enriched in enhanced function mutant than in either the wild-type or null phosphate solubilisation mutant 6 5.

[0251] Group C contained two pathways, amino sugar and nucleotide sugar metabolism, and microbial metabolism in diverse environments, which had uniform enrichment in the wildtype and null phosphate solubilisation mutant, but unique temporal enrichment in the enhanced function mutant. Group D contained 23 pathways, which showed, simultaneously, earlier enrichment in the null phosphate solubilisation mutant and delayed enrichment in the enhanced mutant, compared to the wildtype.

[0252] Group E contained six pathways, glycerine, serine and threonine, methane metabolism, 2-oxocarboxylic acid metabolism, biosynthesis of secondary metabolites, biosynthesis of amino acids and carbon metabolism, which had more consistent enrichment in the null phosphate solubilisation mutant compared to both the phosphate solubilizers.

[0253] Example 7: Mutational impact on ‘canonical’ phosphate solubilization gene expression

[0254] In silico functional analysis of P. rara Lu_Sq_004_WT, based on the presence / absence of members of the phosphate solubilisation ‘canon’ in the genome, suggested that the isolate had the capacity to secrete both gluconic acid and a fleet of enzymes (phytase, phosphatase and C-P lyase) to solubilise recalcitrant forms of phosphate from its environment (Hone et al., 2025). The genes governing these mechanisms (inorganic (gcd, / x / z / BCDE, ppk, ppx. / zs / SCAB, / ?z7AB), organic (ctpp . phnACDE) and regulatory (phoR, phoB, phoU) phosphate solubilisation genes) were therefore examined to elucidate both the wild-type baseline and the expression differences in the phosphate solubilisation mutant.

[0255] As Day 8 had simultaneously the highest number of ‘core’ enriched pathways, 58, (Table 1), and the highest RichFactor for phosphate solubilizers, this timepoint was selected for DEG comparison. Differential gene expression was assessed using a Wald test, and log2 fold changes (LFC) were reported (e.g., LFC = 1.5 corresponds to ~2.8- fold upregulation). All differentially expressed genes (DEGs) discussed met a significance threshold of q < 0.05.

[0256] In both phosphate solubilizers, there was significant upregulation of the high- affinity inorganic phosphate transporter operon, / zs / SCAB. In the wildtype, there was an upregulation of phn. (LFC=2.67) but a downregulation of phnW (LFC=-1.48) and acpD (LFC=-0.42).

[0257] Contrary to expectations, in the wildtype, there was also a downregulation of low- affinity inorganic phosphate transporter pitB, organic phosphate transporter phnP), and regulator phoB

[0258] Conversely, ppk. / zs / SCAB, pilMP phoBB and phoU were exclusively upregulated in the enhanced phosphate solubilisation mutant.

[0259] The only phosphate solubilisation gene downregulated in the enhanced mutant was enzymolysis-associated phnW. Table 1 - Differential expression in point mutation affected genes in enhanced phosphate solubilisation mutant (Lu_Sq_004_l_2) under Soluble phosphate limitation at Day 8, compared to Lu_Sq_004_WT under the same conditions

[0260] None of the key phosphate solubilisation genes were differentially expressed under limited soluble phosphate conditions in the null phosphate solubilisation mutant.

[0261] Notably, gcd, responsible for the production of glucose hydrogenase was not upregulated in either of the phosphate solubilizers at any of the five time points. In fact, gcd was downregulated in the wildtype on Day 8 (LFC=-0.42).

[0262] The lack of differential expression in gcd suggests that may not be the key organic acid responsible for mediating the zone of clearing observed on PVK (limited soluble phosphate conditions). As a result, genes associated with the production of alternative organic acids, such as citric acid, acetic acid, lactic acid, and oxalic acid, which although frequently observed in phosphate solubilising microbes are rarely used as markers for phosphate solubilisation, were also considered.

[0263] Only three of the organic acid genes, ackA (LFC=0.26) and poxB (LFC=0.77), which are associated with acetic acid production and pflB (LFC=1.3), associated with formic acid, were upregulated in the wildtype.

[0264] The upregulation of mqo could point to the accumulation of oxaloacetate, which has, in the past, been linked to the production of oxalic and acetic acid by the action of oxaloacetate hydrolase (oah) (Kobayashi et al., 2014). However, as the oah gene was not identified in the genome of P. rara Lu_Sq_004_WT this seems unlikely.

[0265] However, in the enhanced function mutant, there was significant upregulation of acetic acid genes poxB (LFC=0.79), / ?to (LFC=1.67 ) and ackA (LFC=1.41), formic acid genes pflB (LFC=2.23), and citric acid genes gltA (LFC=0.93).

[0266] Example 8: Evaluating the impact of UV-induced point mutations on gene transcription soluble phosphate limitation responses

[0267] Genome sequencing identified 24 unique point mutations between Lu_Sq_004_WT and Lu_Sq_004_l_2 (Hone et al., 2025). Twenty of these mutations fell within annotated genes, and four fell within the inter-genic space between annotated genes (Hone et al., 2025).

[0268] Fourteen mutated genes (tsgA, bfr, GMKMPLAH 00426, hflX, cpdB, GMKMPLAH 00626, recX, cysM, hisG, GMKMPLAH 01529, htpX l, rluB, cntO l, and GMKMPLAH 03887) and four of the genes (hslR, yrfG, GMKMPLAH 02763, yigB) located on either side of the inter-genic mutations showed differential expression when the transcriptome of the WT or mutants were grown under conditions with limited soluble phosphate.

[0269] Notably, none of these genes showed differential expression under soluble phosphate limitation (SPL) in the null phosphate solubilisation mutant. A pairwise comparison of Lu_Sq_004_WT and enhanced mutant Lu_Sq_004_l_2 under soluble phosphate limitation on Day 8 was performed to allow direct comparison of the transcriptomic impact of the UV-induced point mutations.

[0270] Genes GMKMPLAH 00426, cpdB, cysM, rluB, hslR, GMKMPAH 02763, although differentially expressed when the bacterial transcriptomes on R2A and PVK at the timepoint were compared, showed no significant differences in expression when the wildtype and enhanced mutant were compared on PVK (soluble phosphate limitation).

[0271] The / r / A 1, hisG, cntO 1, recX, GMKMPLAH 00626, yigB, and hflX genes were upregulated in the enhanced mutant, compared to the wildtype. Notably, the hisG codes for a ATP phosphoribosyltransferase that acts as the first step of histidine metabolism, one of the five Group A KEGG pathways that were exclusively expressed in phosphate solubilizers when exposed to soluble phosphate limitation (Malykh et al., 2018).

[0272] The remaining genes were responsible for a variety of functions.

[0273] Both htpX 1 and hflX code for heat shock proteins, the former a zinc-dependent membrane endoprotease and the latter an ATP-dependent RNA helicase. yigB codes for a flavin mononucleotide (FMN) phosphatase, a crucial step in the biosynthesis of riboflavin.

[0274] Riboflavin metabolism demonstrated a ‘B-type’ temporal enrichment pattern, more consistently expressed in the enhanced function mutant than in either the wild-type or null phosphate solubilisation mutant 6 5 (Table 2).

[0275] The cnto 1 gene codes for a TonB-dependent receptor associated with the uptake of siderophores, vitamin B12, and saccharides. Finally, recX is an important negative regulator of RecA, a key actor in SOS response activation, DNA repair, and recombination.

[0276] Conversely, GMKMPLAH 03887, tsgA, yrfG, GMKMPLAH 01529, and bfr were significantly downregulated under soluble phosphate limitation compared to the wildtype.

[0277] Bacterioferritin, encoded by bfr and part of porphyrin metabolism, regulates the storage and utilization of iron, which is essential for the growth and metabolism of microorganisms.

[0278] Porphyrin metabolism demonstrated a ‘B-type’ temporal enrichment pattern (Table 2). The yrfG gene codes for a GMP / IMP nucleotidase, specifically cytosolic 5'- nucleotidase II, which plays a crucial role in purine metabolism by catalyzing the hydrolysis of purine monophosphates.

[0279] The purine metabolism KEGG pathway displayed a ‘type D’ temporal expression pattern, early enrichment from the null phosphate solubilisation mutant, followed by staggered enrichment from the two phosphate solubilizers, in order of efficiency (Table 2).

[0280] Lastly, the tsgA gene codes for an MFS transporter, specifically belonging to the Fucose:H+ symporter family, involved in transporting substrates across cell membranes. Neither gene names nor KO numbers were successfully assigned to genes GMKMPLAH 00626, GMKMPLAH 03887, or GMKMPLAH 01529, confounding functional interpretation of these DEGs.

[0281] Differential gene expression of wildtype, gain of function, and loss of function mutants under PVK (limited soluble phosphate conditions), compared to R2A, was visualised to allow the identification of downstream mutational impacts. The downstream effects of the mutant-bfr gene and the mutant-yrfG gene on the purine metabolism pathways and porphyrin metabolism, respectively, were unclear.

[0282] However, the upregulation of mutant-hisG appears in concert with the downstream upregulation of:

[0283] • hisl (EC:3.5.4.19+3.6.1.31, LFC=1.36) • hisA (EC:5.3.1.16, LFC=0.87)

[0284] • hisF (EC:4.3.2.10, LFC=1.11)

[0285] • hisB (LFC=EC:4.2.1.19+3.1.3.15, LFC=1.05)

[0286] • hisC (EC:2.6.1.9, LFC=1.24)

[0287] • hisD (EC: 1.1.1.23, LFC=1.30) in enhanced phosphate solubilisation mutant Lu_Sq_004_l_2.

[0288] There was no differential expression in the his operon in either the wild-type or null phosphate solubilisation mutant in response to limited soluble phosphate. Excessive accumulation of amino-imidazole carboxamide riboside 5 '-phosphate (AICAR), a key by-product of histidine biosynthesis, has been associated with the regulation of the two- component PhoBR system, which in turn, regulates phosphate transport systems and phosphatases (Malykh et al., 2018, Kim et al., 2023).

[0289] The combination of phosphate solubilizer exclusive pathway enrichment in phosphate solubilizers, mutant-hisG led pathway upregulation, as well as the link between AICAR and the PhoBR regulon, indicates that the hisG mutation and histidine biosynthesis is the reason for the enhanced phosphate solubilization phenotype.

[0290] The present application claims priority from AU 2024902184 filed 15 July 2024, the entire contents of which are incorporated herein by reference.

[0291] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0292] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0293] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. REFERENCES

[0294] Benz et al. (2025) Nature Reviews Biotechnology, doi.org / 10.1038 / s44222-025-00311- 8. De Coster et al. (2018) Bioinformatics 34:2666-9.

[0295] Gu et al. (2016) Bioinformatics 32:2847-2849.

[0296] Hone et al. (2025) Front Microbiol DOI 10.3389 / fmicb.2025.1568162.

[0297] Kim et al. (2023) mBio 14:e0010223.

[0298] Malykh et al. (2018) Microbiol Cell Factories 17:42. Needleman and Wunsch (1970) J. Mol Biol. 45:443-53.

[0299] Patro et al. (2017) Nature Methods 14:417-419.

[0300] Pimentel et al. (2017) Nature Methods 14:687-690.

[0301] Wick et al. (2021) Genome Biology 22: 1-17.

[0302] Wickham and Sievert (2009) ggplot2: elegant graphics for data analysis, springer New York.

[0303] Wu et al. (2021) The Innovation 2: 100141.

Claims

1. CLAIMS1. An isolated strain of Pantoea sp. having phosphate solubilising activity.

2. The strain of claim 1 which is a strain of Pantoea rara.

3. The strain of claim 2 which was deposited with the National Measurement Institute under accession number V24 / 010670 or accession number V24 / 010669 on 18 June 2024, or an active variant thereof having phosphate solubilising activity.

4. The strain of claim 2 or claim 3 which has a mutant HisG protein when compared to a wild type Pantoea sp. protein.

5. The strain of claim 4, wherein the mutant HisG protein is C-terminally truncated.

6. The strain according to any one of claims 1 to 5 which comprises active phosphate transporter genes pstS, pstC, pstB and pstA, and active phosphonate solubilisation genes phoR, phoB, phnD, phnEl, phnE2, phnC, phnA and phnN, and active phosphate solubilisation genes pqqB, pqqC, pqqD, pqqE, gcd and gad.

7. The strain of claim 6, wherein one or more or all of the following apply, i) the pstS gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 9, ii) the pstC gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 11, iii) the pstB gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 13, iv) the pstA gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 15, v) the phoR gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 17, vi) the phoB gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 19, vii) the phnD gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:21,viii) the phnEl gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:23, ix) the phnE2 gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:25, x) the phnC gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:27, xi) the phnA gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:29, xii) the phnN gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 31, xiii) the pqqB gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:33, xiv) the pqqC gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:35, xv) the pqqD gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 37, xvi) the pqqE gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO: 39, xvii) the gcd gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:41, and xviii) the gad gene encodes a protein having an amino acid sequence which is at least 95% identical that set forth in SEQ ID NO:43.

8. The strain according to any one claims 1 to 7 which comprises a polynucleotide encoding a 16S RNA molecule which is at least 95% identical, at least 97% identical, at least 99% identical or is identical, to a nucleotide sequence set forth in any one or more of all of SEQ ID NO’s 1 to 7.

9. A composition comprising the strain according to any one of claims 1 to 8 and a suitable carrier.

10. The composition of claim 9, wherein the carrier is an agriculturally acceptable carrier.

11. The composition of claim 9 or claim 10 which is a biofertilizer.

12. The composition according to any one of claims 9 to 11 for applying to a seed.

13. The composition according to any one of claims 9 to 11 for mixing with, or spraying on, soil.

14. The composition of claim 13 which improves phosphorus solubilisation in the soil.

15. The composition according to any one of claims 9 to 14, wherein the carrier is at least one of peat, clay, soil material, mineral, vermiculite, polymer, fertilizer and compatible oil.

16. The composition according to any one of claims 9 to 15 which is in the form of powder, granules, liquid, semi-solid, a dust, suspension, emulsion or spray.

17. The composition according to any one of claims 9 to 16 which further comprises at least one additional active agent selected from the group consisting of a fertilizer, an acaricide, a fungicide, an insecticide, a nematicide, a pesticide, sun protecting agent, an additional plant growth regulator, a nutrient, and any combination thereof.

18. The composition according to any one of claims 9 to 17, wherein the strain is encapsulated in alginate.

19. The composition according to any one of claims 9 to 18 comprising about 102CFU / mL to about IO10CFU / mL (colony forming unit) of the strain.

20. The composition of claim 9 which is a culture of the strain.

21. A method of producing a composition according to any one of claims 9 to 20, the method comprising culturing the strain and formulating the cultured strain into the composition.

22. A method of improving phosphorus solubilisation in soil by applying to the soil a strain according to any one of claims 1 to 8.

23. A method of increasing the level of bioavailable phosphorus, the method comprising contacting insoluble inorganic phosphorus and / or insoluble organic phosphate with a strain according to any one of claims 1 to 8.

24. The method of claim 23, wherein the insoluble inorganic phosphorus and / or insoluble organic phosphate is in soil.

25. The method of claim 23 or claim 24, wherein the soil comprises a seed and / or roots of a plant.

26. A method of producing a plant, the method comprising growing a plant in the presence of a strain according to any one of claims 1 to 8.

27. The method of claim 26 which comprises contacting a seed with the strain and growing a plant from the seed.

28. The method of claim 26 or claim 27, wherein the plant is grown in soil comprising the strain.

29. The method of claim 28 which comprises spraying the soil with the strain.

30. The method of claim 28, wherein the soil comprises a pellet or power comprising the strain.

31. The method according to any one of claims 26 to 30 which improves root growth of the plant.

32. The method according to any one of claims 26 to 31, wherein the plant is selected from the group consisting of a monocot plant and a dicot plant.

33. The method according to any one of claims 26 to 32, wherein the plant is selected from the group consisting of a grain crop plant, an oilseed crop plant, a vegetable crop plant, a forage crop plant, an industrial crop plant, or a woody crop plant.

34. A plant produced by the method according to any one of claims 26 to 33.

35. A seed coated with a strain according to any one of claims 1 to 8.

36. A method of inhibiting the growth of, and / or reducing damage caused by, one or more fungal pathogen(s), the method comprising delivering to the one or more fungal pathogen(s), or contacting the one or more fungal pathogen(s) with, a strain according to any one of claims 1 to 8.

37. The method of claim 36 which comprises applying a composition according to any one of claims 9 to 20 to an area which comprises, or may comprise, one or more fungal pathogen(s).

38. The method of claim 37, wherein the area comprises a plant.

39. The method according to any one of claims 36 to 38, wherein the one or more fungal pathogen(s) is a Didymella sp. Puccinia sp., Blumeria sp., Fusarium sp., Magnoporthe sp., Bipolaris sp., Oidium sp., Gibberella sp., Cochliobolus sp., Exser ohilum sp., Uredo sp. Microdochium sp., Helminthosporium sp., Monographella sp., Colletotrichum sp., Uromyces sp. or Erysiphe sp..

40. Use of a strain according to any one of claims 1 to 8 to inhibit the growth of, and / or reduce damage caused by, one or more fungal pathogen(s).

41. A kit compri sing(i) a strain according to any one of claims 1 to 8;(ii) optionally a delivery system for applying the strain to a plant or a part thereof, soil or an area; and(iii) optionally instructions for using the strain.

42. A method of modifying a parental strain of Pantoea sp. to produce a mutant strain with improved production of bioavailable phosphorus, the method comprising, a) mutating the HisG gene of the parental strain, b) testing the ability of the mutant strain with the mutated HisG gene to produce bioavailable phosphorus, and c) selecting the mutant strain if is produces more bioavailable phosphorus than the parent strain.

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