Microbial inoculants, fertiliser compositions, growth mediums and methods for enhancing plant growth

Abstract

The present invention relates to microbial inoculants, fertiliser compositions, growth mediums and methods for enhancing plant growth. In one aspect, the invention relates to a method of increasing plant growth, plant productivity, seed germination or soil quality for a dicotyledonous plant or a monocotyledonous plant, the method comprising the step of: applying to the plant a treatment agent comprising at least one plant-beneficial Burkholderia-like species.

Claims

1. A method of increasing plant growth, plant productivity, seed germination or soil quality for: (i) a dicotyledonous plant, or (ii) a monocotyledonous plant selected from wheat, rice, and corn; the method comprising the step of: applying to the plant a treatment agent comprising at least one plant-beneficial Burkholderia-like species, wherein the at least one plant-beneficial Burkholderia-like species has at least 99% 16S rRNA sequence identity to Bacterium SOS3 in SEQ ID No: 3 (deposited on 2 Jun. 2016 under Accession No. V16/013910 at the National Measurement Institute, 1/153 Bertie Street Port Melbourne, Victoria, Australia 3207), and wherein the at least one plant-beneficial Burkholderia-like species is configured to form biofilms on the roots of the plant and wherein the at least one plant-beneficial Burkholderia-like species is not Burkholderia strain Q208.

2. The method of claim 1, wherein the method is a method of promoting plant growth; wherein the step of applying to the plant the treatment agent is a step of treating seeds or seedlings of the dicotyledonous plant, or the monocotyledonous plant with an effective amount of the treatment agent, wherein the at least one plant-beneficial Burkholderia-like species is configured to form biofilms on the roots of the seedlings or seedlings grown from the seeds; wherein the method further comprises the steps of: growing the seeds or seedlings in a soil-free growth medium for an initial time period to grow into plantlets; and transferring the plantlets to field or greenhouse conditions.

3. The method of claim 2, wherein at least a part of the soil-free growth medium comprises the treatment agent.

4. The method of claim 2, wherein the plantlets when transferred are cultivated in soil comprising fertiliser or manure treated with an additional amount of the treatment agent.

5. The method of claim 2, wherein the step of treating the seeds or seedlings comprises co-treatment with at least one of the group consisting of: a phospholipid, a phosphoprotein, a phosphoester, a sugar phosphate and a phytate.

6. The method of claim 2, wherein the at least one plant-beneficial Burkholderia-like species is Bacterium SOS3 (deposited on 2 Jun. 2016 under Accession No. V16/013910 at the National Measurement Institute, 1/153 Bertie Street Port Melbourne, Victoria, Australia 3207).

7. The method of claim 1, wherein the treatment agent is a fertiliser composition comprising the at least one plant-beneficial Burkholderia-like species.

8. The method of claim 7, wherein the at least one plant-beneficial Burkholderia-like species is Bacterium SOS3 (deposited on 2 Jun. 2016 under Accession No. V16/013910 at the National Measurement Institute, 1/153 Bertie Street Port Melbourne, Victoria, Australia 3207).

9. The method of claim 7, wherein the fertiliser composition further comprises at least one plant nutrient.

10. The method of claim 9, wherein the at least one plant nutrient comprises manure or compost.

11. The method of claim 9, wherein the at least one plant nutrient comprises one or more of the group consisting of: a phospholipid, a phosphoprotein, a phosphoester, a sugar phosphate and a phytate.

12. The method of claim 1, wherein the method is a method of growing seedlings, wherein the step of applying to the plant the treatment agent is a step of growing seedlings of the dicotyledonous plant, or the monocotyledonous plant in a soil-free growth medium comprising the at least one plant-beneficial Burkholderia-like species.

13. The method of claim 12, wherein the at least one plant-beneficial Burkholderia-like species is Bacterium SOS3 (deposited on 2 Jun. 2016 under Accession No. V16/013910 at the National Measurement Institute, 1/153 Bertie Street Port Melbourne, Victoria, Australia 3207).

14. The method of claim 12, wherein the soil-free growth medium further comprises at least one plant nutrient.

15. The method of claim 14, wherein the at least one plant nutrient comprises one or more of the group consisting of: a phospholipid, a phosphoprotein, a phosphoester, a sugar phosphate and a phytate.

16. The method of claim 1, wherein the at least one plant-beneficial Burkholderia-like species has 100% sequence identity to Bacterium SOS3 in SEQ ID No. 3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Various embodiments of the invention will be described with reference to the following drawings, in which:

(2) FIG. 1 illustrates a phylogenetic tree of 16S rRNA gene sequences showing the position of strains Bacterium SOS1, Bacterium SOS2, and Bacterium SO3 within the PBE clade of Burkholderia;

(3) FIG. 2 illustrates a phylogenetic tree with 10 bacterial clones isolated from the tomato rhizosphere and identified as Burkholderia using 16S rRNA primers OTU11burkhold_R: GTTGGCAACCCTCTGTTC) and 926F: AAACTYAAAKGAATTGACGG; and

(4) FIG. 3 illustrates the effect of plant-growth-promoting bacteria on plant growth and seed germination.

(5) Preferred features, embodiments and variations of the invention may be discerned from the following Description of Embodiments and Examples which provides sufficient information for those skilled in the art to perform the invention. The following Description of Embodiments and Examples are not to be regarded as limiting the scope of the preceding Summary of the Invention in any way.

DESCRIPTION OF EMBODIMENTS

(6) The invention in at least some embodiments relates to isolating a microorganism such as bacterium B. australis, Bacterium SOS1, Bacterium SOS2 or Bacterium SOS3 from the rhizosphere of plants and the disclosure encompasses methods for isolating microorganisms as previously discussed. Plant-beneficial Burkholderia-like species may have beneficial physiological characteristics including and ability for: (i) nitrogen fixation from atmospheric air; (ii) forming biofilm on plant roots; (iii) solubilising phospholipids and/or phosphoproteins and/or phosphoesters and/or sugar phosphates and/or phytate; (iv) secretion of siderophores; and/or (v) inducing ethylene production. In some experiments, plant-beneficial Burkholderia-like species (which may include bacteria exhibiting similar physiological characteristics as B. australis Q208, Bacterium SOS1, Bacterium SOS2 and Bacterium SOS3 (denoted by B. australis Q208 and related species-BARS), and which may include other bacteria belonging to PBE clade of Burkholderia (referred to collectively as Burkholderia Related SpeciesBRS)) have been considered in further detail. It is expected that the physiological characteristics of BRS are similar to those of BARS. BARS and BRS can be identified via application of the following primers:OTU11burkhold_R: (GTTGGCAACCCTCTGTTC) and926F: (AAACTYAAAKGAATTGACGG (Y corresponds to C or T; K corresponds to G or T)) as described in Paungfoo-Lonhienne et al (ref. 10).

(7) Preliminary experiments conducted by the present inventors indicate that BARS and BRS enhance seed germination and growth of a wide variety of plants, including Solanaceae (e.g. tomato, potato) Poaceae (e.g. rice, wheat, and maize). Without being bound by theory, it is theorised that BRS and BARS have the ability to i) assimilate phosphorus from a number of hardly-degradable, inorganic and organic compounds, ii) trigger production of ethylene in plants, and iii) secrete siderophores.

(8) The applicants theorise that BARS and BRS have the ability to fix nitrogen from the air. BARS and BRS localize in the rhizosphere and fix atmospheric nitrogen via a new, not revealed yet molecular pathway.

(9) The applicants also theorise that BARS and BRS have the ability to extract phosphorus from organic fertilizers or phospholipids, phosphoproteins, phosphoesters, sugar phosphates, and phytate (commonly referred here as PRC). Plant tissues and organic fertilizers (i. e. manure and compost) contain high amount of hardly-degradable and highly phosphorus-enriched compounds. As PRC are highly abundant in manure and compost, the ability of BARS and BRS to utilize these compounds represents a great possibility to significantly reduce agricultural expenses worldwide, reducing the need for high amount of mineral phosphorus. BARS may also be able to solubilize some inorganic forms of phosphorus (i. e. calcium phosphate) that are hardly accessible for plants, but present in high concentration in soil (ref. 9).

(10) The applicants also theorise that BARS and BRS exhibit protective effect against pathogenic microorganisms, forming a biofilm around plant roots. This diminishes the possibility of root colonization by pathogenic microorganisms.

(11) The applicants also theorise that association of a plant with BARS or BRS triggers production of ethylene in plants. Ethylene is an efficient agent against nematodes. Nematodes are responsible for tremendous loss of crops. For example, Hirschmanniella oryzae, i.e. rice root nematode (RRN), is among the major pests of rice and is the most common plant-parasitic nematode found on irrigated rice. Accordingly, by inducing ethylene production in plants, treatment of plants with BARS or BRS renders plants more resilient to nematodes.

(12) Upon association with plants, it is believed that BARS and BRS secrete siderophores. Siderophores scavenge iron from minerals creating a soluble form of this element. Many plants are able to assimilate iron via these microbial siderophores (ref. 11). Thus, this is another important feature of BARS and BRS, which can be beneficial in soils poor for easily-accessible iron.

(13) Bacterium SOS1 has about 98% 16S rRNA sequence identity to the strain Burkholderia sp. Q208. Bacterium SOS2 has about 99% 16S rRNA sequence identity to the strain Burkholderia sp. Q208. Bacterium SOS3 has about 100% 16S rRNA sequence identity to the strain Burkholderia sp. Q208. Sequencing analyses conducted in the prior art have shown that these bacteria do not possess the determinants of pathogenicity, such as specific clusters of genes coding for T3SS3 and T4SS.

(14) BRS may be isolated by screening bacteria in plant rizosphere or soil, or other media, by using 16S rRNA primers specific to Burkholderia sp. Q208.

(15) BARS and BRS may be exposed to seeds or seedlings, for a brief period of time before transferring the plants into the soil or other growth media. In one embodiment, the seeds or seedlings are soaked in a suspension containing BRS or BARS. The brief soaking of seeds or seedlings into the bacterial suspension for an initial time period is sufficient to initiate improved growth and disease-suppressive characteristics. The seeds treated with BRS or BARS may be germinated in soil or in a soil-free growth medium as discussed in previous sections.

(16) The method of promoting plant growth as discussed in previous sections in some embodiments relies on protecting plant roots from pathogens. This embodiment is based on an expected ability of BARS and BRS to form biofilm on plant roots and stimulate production of ethylene in plants. The formation of the biofilm impedes other microorganisms such as bacteria and fungi to colonize the roots. The method may also promote the production of ethylene which assists in protecting plants from nematodes.

(17) The present disclosure provides a number of methods for treatment of seeds, seedlings, plantlets, and plants with BRS and BARS, and compositions for delivery of BRS and BARS into soil and hydroculture media. In an embodiment, seedlings may be grown in a soil-free media containing BRS or BARS for a period of 2-6 weeks and then transplanted to field or greenhouse conditions. This method may allow seedlings to grow in an optimized growth medium, which is chosen in function of the plant's specific requirement. PRC may be an additive that strongly increases the efficiency of the plant-Burkholderia (or, more specifically, plant-BRS) association. This method may provide young plants with improved physical characteristics, such as greater height and weight, increased root system, and increased health, compared to control plants. The outcome may be that plants reach a mature stage earlier and provide better yield.

(18) Seeds may be treated with BRS or BARS via spray or dipping, such as spraying or dipping a bacterial suspension onto the seeds, before transferring the seeds into the growth media. This method may stimulate growth at the early seedling stage which increases the seeding's vigour, ultimately leading to higher yields.

(19) Alternatively, seeds may be coated with a solution of alginate (2% w/v) or CMC (4% w/v). The solutions can be prepared by dissolving powder in distilled water at room temperature, agitating the solution and autoclaving at 121 C. for 20 minutes. Seeds may then be coated in the solution containing BRS and are subsequently air dried.

(20) Seeds and seedling which may be treated, include the whole spectrum of both monocotyledonous and dicotyledonous plant species selected from rice, cotton, wheat, corn, tomato, cucumber, beans, pea, broccoli, cabbage, soybean, cauliflower, lettuce, radish; strawberry etc. In general, any seed or seedling which responds to the acting component (BRS or BARS) of the invention may be treated in accordance with the invention.

(21) The present disclosure, in another embodiment, offers the methods of combined treatment of soil and hydroculture with BRS or BARS, and manure or compost. Applying animal manure or compost to farmland constitutes environmentally sound management of soil. Bacteria require nitrogen, phosphorus and other chemical elements for their metabolism. BRS and BARS, which have the ability to metabolise organic molecules that contain high concentration of these elements, not only use the product for their own development, but also render organic chemical elements available for the plant. The applicants have found that addition of manure to the growth medium containing BARS or BRS greatly enhances plant growth and fitness, compared to the plants grown only in the presence of manure.

(22) BRS or BARS may be mixed with manure and compost according to known agricultural practises in the form of dry pellets. This method may enrich the soil with BRS or BARS durably, stimulating growth and fitness of plants for their entire life cycle.

(23) Another compound that may be used for co-treatment with BARS or BRS is PRC (phospholipids, phosphoproteins, phosphoesters, sugar phosphates, and phytate). Any of the PRC compounds may be used separately or in different combinations. Addition of PRC to the growth medium containing BARS or BRS may enhance plant growth and fitness at the early stage of plant growth. The combination of BRS or BARS with one or few compounds from the PRC list may be included in the feeding media for growth of hydroponic culture. It is envisaged that the present disclosure will result in great economical effect. PRC compounds are relatively cheap components and represent a renewable source of phosphorus. PRC which are present in high amounts in plant tissues and acting as phosphorus storage are highly available in compost of animal manure. Its use is an environmentally friendly process, occurring without damaging of phosphate rocks.

(24) The diverse group of Burkholderia may be re-classified and a separate genus may appear in the bacterial taxonomy which will include the microorganisms currently belonging to the PBE Burkholderia clade. The PBE group of Burkholderia is separate from the pathogenic group (see FIG. 1), and the bacteria having at least 96% 16S rRNA sequence identity to the strain Burkholderia sp. Q208 and Bacterium SOS3 belong to the PBE, clade. Plant-beneficial Burkholderia-like species include such bacteria. Currently this environmental Blade includes such representatives as: Bacterium SOS1, Bacterium SOS2, Bacterium SOS3, Burkholderia sp. Q208, Burkholderia oxyphila, Burkholderia sacchari, Burkholderia ferrariae, Burkholderia silvatlantica, Burkholderia heleia, Burkholderia nodosa, Burkholderia bannensis, Burkholderia tropica, Burkholderia unamae, Burkholderia kururiensis, Burkholderia diazotrophica, Burkholderia tuberum, Burkholderia acidipaludis, Burkholderia caribensis, Burkholderia hospita, Burkholderia terrae, Burkholderia phymatum, Burkholderia sabiae, Burkholderia sartisoli, Burkholderia phenazinium, Burkholderia sediminicola, Burkholderia phytofirmans, Burkholderia ginsengisoli, Burkholderia fungorum, Burkholderia megapolitana, Burkholderia bryophila, Burkholderia terricola, Burkholderia graminis, Burkholderia phenoliruptrix, Burkholderia xenovorans, and Burkholderia caledonica. FIG. 1 provides a phylogenetic tree of 16S rRNA gene sequences showing the position of strains Bacterium SOS1, SOS2, and SO3 within the PBE clade of Burkholderia. The consensus tree topology was inferred using neighbour-joining with nonparametric bootstrap based on maximum composite likelihood (1000 replicates; MEGA v5.05). Bootstrap support value 50% is shown a teach internal node. Thick branches indicate Bayesian posterior probabilities 0.90. Unit of branch length is in number of substitutions per site. Boxed area indicates Burkholderia australis Q208-related strains, which have 100% sequence identity by 16S rRNA sequence comparison, this group includes Bacterium SOS3. All other PBE bacteria which have at least 96% 16S rRNA sequence identity to Burkholderia australis Q208 are indicated within the text as BRS (Burkholderia-related strains), and Bacterium SOS1 and Bacterium SOS2 belong to this group.

(25) In the present specification and claims (if any), the word comprising and its derivatives including comprises and comprise include each of the stated integers but does not exclude the inclusion of one or more further integers.

(26) Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases in one embodiment or in an embodiment in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.

(27) In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims (if any) appropriately interpreted by those skilled in the art.

EXAMPLES

(28) The following examples are illustrative of one or more exemplary embodiments of the invention and should not be construed as limiting in any way the general nature of the disclosure of the description throughout this specification.

Example 1Microbial Strains and Maintenance of Culture

(29) Microbial Strains.

(30) The following microbial strains were used for treatment of seeds, seedlings and plants, and in the production of biofertilisers: Burkholderia australis Q208, Bacterium SOS1, Bacterium SOS2 and Bacterium SOS3 (BARS). Bacterium SOS1, Bacterium SOS2 and Bacterium SOS3 were isolated from the sugar cane and rice rhizospheres (in the case of Burkholderia australis Q208) and tomato rhizospheres (in the case of Bacterium SOS1, Bacterium SOS2 and Bacterium SOS3). Partial 16S rRNA sequencing indicated that the strains belong to the Plant-Beneficial-Environment (PBE) Burkholderia clade. Full sequencing of DNA confirmed that these bacteria are non-pathogenic microorganisms as they lack necessary genes for pathogenicity such as coding for components of T3SS and T4SS systems. When cultured aerobically on R2A media with the addition of phytate for 2 days at 28 C., BARS produce non-mucoid small and mucoid big colonies. Cells are Gram-negative, non-spore-forming and ovoids to short rods (0.4-0.5 m in width and 1.01.3 m in length), and occur singly or in pairs. Physiologically the bacteria are -Galactosidase, catalase and oxidase positive, not producing indole, not hydrolysing gelatin and not fermenting glucose. The DNA G+C content is around 63.3 mol %. Bacterium SOS1, Bacterium SOS2 and Bacterium SOS3 represent rod-shaped cells, form white colonies on R2A medium, entire margin, and are Gram negative. The tested morphological and physiological characteristics of Bacterium SOS1, Bacterium SOS2 and Bacterium SOS3 are similar to the other representatives of the genus Burkholderia: the cells are motile, non-fermentative and non-fluorescent, and are resistant to ampicillin, but not to kanamycin. Bacterium SOS1, Bacterium SOS2 and Bacterium SOS3 are negative in the arginine dihydrolase test. The cDNA sequence corresponding to the 16S rRNA sequence of Bacterium SOS1 is provided in SEQ ID No: 1, the cDNA sequence corresponding to the 16S rRNA sequence of Bacterium SOS2 is provided in SEQ ID No: 2 and the cDNA sequence corresponding to the 16S rRNA sequence of Bacterium SOS3 is provided in SEQ ID No: 3.

(31) TABLE-US-00001 SEQIDNo:1 BacteriumSOS1 AAGTCGGACGGCAGCGCGGGGGCAACCCTGGCGGCGAGTGGCGAACGGGT GAGTAATACATCGGAACGTGTCCTGGAGTGGGGGATAGCCGGCGAAAGCC GGATTAATACCGATACGCTCTGTGGAGGAAAGCGGGGGATCTTCGGACCT CGCGCTCAAGGGGCGGCCGATGGCAGATTAGCTAGTTGGTGGGGTAAAGG CCTACCAAGGCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACAC TGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATT TTGGACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGTGTGAAGAAG GCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAAACCGCTTCTCTAA TACAGGGGCGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGT GCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTG GGCGTAAAGCGTGCGCAGGCGGTTCGCTAAGACCGATGTGAAATCCCCGG GCTTAACCTGGGAACTGCATTGGTGACTGGCGGGCTAGAGTATGGCAGAG GGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAA TACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACGCTCATGCACGA AAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAA CGATGTCAACTAGTTGTCGGGTCTTCATTGACTTGGTAACGAAGCTAACG CGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGG AATTGACGGGGACCCGCACAAGCGTGGATGATGTGGATTAATTCGATGCA ACGCGAAAAACCTTACCTACCCTTGACATGTACGGAACCTTGCTGAGAGG TGAGGGTGCCCGAAAGGGAGCCGTAACACAGGTGCTGCATGGCTGTCGTC AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT GTCCCTAGTTCGTACGCAAGAGCACTCTAGGGAGACTGCCGGTGACAAAC CGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGG CTTCACACGTCATACAATGGTCGGAACAGAGGGTTGCNAAGCCGCGAGGT GGAGCCAATCCCAGAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTC GACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGT GAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGG GTTTTACCAGAAGTGGCTAGTCTAACCGCAAG SEQIDNo:2 BacteriumSOS2 GCTGGCGGCATGCTTTACACATGCAAGTCGAACGGCAGCACGGGGGCAAC CCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACGTGTCCTGG AGTGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGCTCGGGA GAGGAAAGCGGGGGACCTTCGGGCCTCGCGCTCAAGGGGCGGCCGATGGC GGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCGTAGC TGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACT CCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGAT CCAGCAATGCCGCGTGTGCGAAGAAGGCCTTCGGGTTGTAAAGCACTTTT GTCCGGAAAGAAATCCTGCCTGATAATACCGGGCGGGGATGACGGTACCG GAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG GGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTT CGCTAAGACCGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTGGT GACTGGCGGGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCA GTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCT GGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTA GATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGAT TCATTTCCTTAGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGGAGT ACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCG GTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCT TGACATGGACGGAATCCCGCTGAGAGGTGGGAGTGCTCGAAAGAGAACCG TCGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGG TTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGC ACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTC AAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCG GAACAGAGGGTCGCCAACCCGCGAGGGGGAGCCAATCCCAGAAAACCGAT CGTAGTCCGGATTGCACTCTGCAACTCGAGTGCATGAAGCTGGAATCGCT AGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTAC ACACCGCCCGTCACACCATGGGAGTGGGTTTTACCAGAAGTGGCTAGTCT AACCGCAAGGAGGACGGTCACCACGGTAGGATTCATGACT SEQIDNo:3 BacteriumSOS3 AGTGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGATCTGAG GATGAAAGCGGGGGACCGCAAGGCCTCGCGCTCAAGGAGCGGCCGATGGC GGATTAGCTAGTTGGTGGGGTAAAGGCCCACCAAGGCGACGATCCGTAGC TGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACT CCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGAT CCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTT GTCCGGAAAGAAAACTTCGTCCCTAATATGGATGGAGGATGACGGTACCG GAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG GGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTG ATGTAAGACCGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTGGT GACTGCATTGCTGGAGTATGGCAGAGGGGGGTGGAATTCCACGTGTAGCA GTGAAATGCGTAGAGATGTGGAGGAACACCGATGGCGAAGGCAGCCCCCT GGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTA GATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTCGGGCCT TCATTGGCTTGGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGT ACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCG GTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCT TGACATGGACGGAACCTCGATGAGAGTTGAGGGTGCCCGAAAGGGAGCCG TCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGG TTAAGTCCCGCAACGAGCGCAACCCTTGTCCCTGGTTGCTACGCAAGAGC ACTCCAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTC AAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCG GAACAGAGGGTTGCCAAGCCGCGAGGTGGAGCCAATCCCAGAAAACCGAT CGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCT AGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTAC ACACCGCCCGTCACACCATGGGAGTGGGTTTTACCAGA
Maintenance of Culture.

(32) The strains are stored as a 30% glycerol stock at 80 C. For short-term storage the strain is maintained on R2A-agar plates. The cultures are routinely grown at 28 C. for 2 days and the freshly grown colonies on the plate are kept at 4 C. with subculturing every 4 weeks.

Example 2Isolation of Plant-Beneficial Burkholderia-Like Species

(33) Burkholderia australis Q208 related strains (BARS) were isolated as follows: 10 g of pooled roots of sugar cane or rice (for Burkholderia australis Q208) or 10 g of pooled roots of tomato (for Bacterium SOS1, Bacterium SOS2 and Bacterium SOS3) were homogenised in a vegetable juicer (Breville) with 10 ml sterile phosphate-buffered saline, pH 7.2 for 1 min at high speed. The homogenized sample was filtered through Whatman paper number 1, and serial dilutions (up to 10.sup.9) were made with the filtrate. 100 l of each dilution was spread on agar plates containing R2A minimal medium and, in the case of BRS, phytate at a concentration of 4 g/L. Plates were incubated at 28 C. for 5 days. The plates containing separated individual bacterial colonies were used for PCR screening. For identification of BRS 16S rRNA specific primers for Burkholderia OTU11burkhold_R: GTTGGCAACCCTCTGTTC and 926F: AAACTYAAAKGAATTGACGG were applied. Using this method 10 independent colonies were isolated which had 96%-100% 16S rRNA sequence identity to Burkholderia sp. Q208 (FIG. 2). After laboratory and plant growth experiments three species showed the best plant growth-promotion results: Bacterium SOS1 (96% sequence identity to Burkholderia sp. Q208), Bacterium SOS2 (98% sequence identity to Burkholderia sp. Q208) and Bacterium SOS3 (100% sequence identity to Burkholderia sp. Q208) (FIG. 2). All three bacteria (Bacteria SOS1-SOS3) are able to fix nitrogen, solubilize phytate, and form biofilm on the plant roots.

Example 3Treatment of Seeds and Plantlets with Plant-Beneficial Burkholderia-Like Species

(34) Seeds of tomato, rice, or lettuce were soaked into the solution containing BARS at the concentration of 110.sup.9 cell per ml for 15 minutes. Control seeds were soaked in pure water for 15 mins prior to planting. Three replicas of each treatment group, including control, were used. The seeds or seedlings were planted according to the established methods used for hydroponic, greenhouse or field trials.

Example 4Composition of the Hydroponic and Greenhouse Fertilisers

(35) Composition of the Organic Phytate Biofertiliser (Phytoliser).

(36) The biofertiliser for hydroponic and greenhouse applications (phytoliser) is a two-component system. It comprises of BARS at final concentration of 110.sup.9 cell per ml, and of the organic matter described below, as liquid solution. The bacterial strains were grown and maintained as described in Example 1, and delivered to the trial spot as lyophilised powder. At the trial spot the bacteria were transferred to 100 ml of pure water (making 110.sup.6 concentration) and left for 1 hr for their recovery. The organic matter (phytate) containing necessary chemical elements is manufactured from the plants, or from pig or chicken manure. The major component of the phytoliser is phytate, which represents the organic phosphorus stored by plants and abundantly found in different kinds of manures, plants and seeds. The phytoliser also contains a number of other chemical elements in the form of organic molecules, which are necessary for plant growth. Thus, all the inorganic nutrients in the recipe are substituted with organic nutrients, where the necessary chemical elements represent the parts of the organic molecules. Concentration of the chemical elements in the biofertiliser corresponds to the average concentration used for hydroponic applications in current practices, as shown in Table 1.

(37) TABLE-US-00002 TABLE 1 Concentration of chemical elements in the phytoliser. Delivered nutrient concentration (ppm).sup.z Nutrient Minimal Maximum N 0 70 P 50 150 K 120 200 Ca 100 200 Mg 30 60 S 30 60 Fe 2.0 3.0 Cu 0.1 0.3 Mn 0.5 1.0 Zn 0.2 0.5 B 0.5 1.0 Mo 0.05 0.1 1 ppm = 1 mg/liter
Composition of the Partially Organic Phytate Fertiliser.

(38) In separate trials, highly purified phytate (98% purity) was mixed with the other 10 chemical elements (see Table 1) necessary for plant growth (K, Ca, Mg, S, Fe, Cu, Mn, Zn, B, and Mo). The roots of rice plantlets were soaked in a solution containing BARS and then left growing in the medium containing crude phytate solution. The plantlets grew faster than the control plants incubated with a standard hydroponic medium and their roots appeared thicker (FIG. 3). In FIG. 3, bars indicate % of increase compared to non-inoculated plants. Error bars are SE from 10 replicas per same treatment. 1-root length; 2-root dry mass; 3-shoot length; 4-shoot dry mass; 5-seed germination. The trials were conducted whether in soil or hydroponic conditions. This fertiliser provides the opportunity to enhance plant growth, however, those conditions potentially may not be considered as fully organic. This recipe also allows avoiding the usage of the mineral phosphate and nitrogen-based fertilisers, thus minimizing concentration of nitrates in the final product.

Example 5Composition and Preparation of Biofertilisers for Field Applications

(39) Preparation of Biofertilizer.

(40) BARS are mixed thoroughly with Alginate-humic acid mixture. This suspension was capsulated in the presence of solution of CaCl.sub.2, converting water-soluble sodium alginate into the water-insoluble calcium alginate beads. The beads are then coated with a mixture of phytate and manure, and air dried. Such biofertilizer pellets can be stored for months at room temperature. When pellets are applied to the wet soil, the bacteria start reviving and proliferating. Then, by direct contact with the roots the bacteria form biofilm on the roots, providing nutrients to the plants.

(41) A further exemplary method for producing the biofertiliser is as follows. Alginate beads were prepared according to the methods outlined in Young et al. (Young C C, Rekha P D, Lai W A, Arun A B. Encapsulation of plant growth-promoting bacteria in alginate beads enriched with humic acid. Biotechnol Bioeng. 2006 Sep. 5; 95(1):76-83). A mixture of 2.5 ml of 10% humic acid and 750 ml of 30% glycerol were added to 2% sodium alginate solution to obtain a final volume of 25 ml. The bacterial culture (250 ml) was centrifuged, the cell pellet was washed with saline (0.85% NaCl, w/v) and suspended in 25 ml of alginate-humic acid mixture and mixed thoroughly. This suspension was extruded through a 26-gauze needle drop wise into a pre-cooled sterile 1.5% (w/v) aqueous solution of CaCl.sub.2 under mild agitation. The water-soluble sodium alginate was converted into water-insoluble calcium alginate beads. Thus instantaneously formed beads were allowed to harden for 3-6 h at room temperature. Beads were collected by sieving and were washed several times with sterile water.

Example 6Tomato, Rice and Lettuce Trials

(42) Hydroponic and Greenhouse Trials.

(43) The phytoliser compositions are described in Example 4. The seedlings or plantlets of tomato, rice and lettuce were treated with BARS as described in Example 3 before planting. Non-treated plants were used as a control. Other controls included: for hydroponic trials, water only, and commercially available inorganic fertilisers; for greenhouse trials, water only and the commercially available fertiliser FlowPhos (Yara Nipro). In all experiments the experimental plants grew faster than the control plants (FIG. 3) and dry mass of plants grown with addition of BRS was significantly higher (up to 30%) than that of the control plants.

(44) Field Trials.

(45) Tomato seeds and plantlets were used for field trials. They were used either treated as in Example 3 or untreated. The fields were prepared as follow: cow, chicken, or pig manure, or different combinations of these manures is mixed with soil (upper 20-50 cm layer) in a range of concentration varying from 0.1 to 1% w/w, in order to provide adequate concentration of the organic nutrients, including phytate. Then biofertilizer pellets (Example 5) are applied to the soil (upper 20-50 cm layer) in a concentration range varying between 0.01 to 0.1% w/w. The pellets allow retaining bacteria in the soil for a long period of time, keeping their concentration relatively high, compared to other microorganisms. This soil is used for planting of the bacteria-treated and untreated seeds or plantlets (Example 3). For those trials controls were untreated seeds and plantlets with commercial inorganic fertiliser used in conventional practice, and untreated seeds and plantlets with pure organic fertilisers. The results of the field trials (FIG. 3) were similar to those obtained in the glasshouse conditions with average dry mass increase of 10-30%.

Example 6. Seed Germination Enhancement

(46) Tomato and corn seeds were coated with Bacterium SOS' or Bacterium SOS3 in a solution of CMC (4% w/v). The control seeds were treated with CMC (4% w/v) only. After placing the seeds in the soil conditions the number of appeared plants were counted and significant improvement of the germination rate was detected (FIG. 3).

(47) Hydroponics Trials:

(48) Pre-germination of lettuce or rice seeds was conducted for 4 to 5 days in trays lined with damp paper and covered with plastic. When the roots reached 1-2 cm in length the seedlings were dipped in a solution containing bacteria in the concentration of 107 cells/ml and relocated into small pots with vermiculite. Growth solution was supplied via capillary action from below. The measurements of the plant roots and shoots were conducted after 4 weeks post transplantation. The hydroponic medium contained 0.1% phytate solution (50% phytate purity, Lvyu Chemical Co., Ltd or Henan Kingway Chemicals Co., Ltd.).

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