NZ614079B2 - Microbial inoculants and fertilizer compositions comprising the same - Google Patents

Abstract

Disclosed herein are microbial inoculants for use in increasing plant growth, plant productivity and/or soil quality, comprising strains of one or more bacterial species selected from Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapi and Lactobacillus zeae. Optionally the microbial inoculants also comprise a strain of Acetobacter fabarum and/or a strain of Candida ethanolica. Also provided are fertilizer compositions comprising said microbial inoculants. obial inoculants also comprise a strain of Acetobacter fabarum and/or a strain of Candida ethanolica. Also provided are fertilizer compositions comprising said microbial inoculants.

Description

Microbial inoculants and fertilizer compositions comprising the same Field of the Art The present sure relates lly to microbial inoculants, particularly for use as izers, comprising one or more microbial species or strains as described herein, a d to fertilizer compositions comprising such sms. The disclosure also relates to methods of promoting plant growth, sing availability of nutrients in the soil and remediating degraded soils and pastures using ial inoculants and fertilizer compositions of the present disclosure.

Background The use of fertilizers to enhance plant and crop production and overcome poor soil quality is widespread. Most commonly employed commercially available fertilizers are inorganic chemical fertilizers. Such chemical fertilizers can be expensive to produce, can be hazardous to use and are often associated with environmentally damaging consequences, such as nitrate contamination in run off and ground water. Environmental sustainability can be promoted by limiting the use of al fertilizers.

Fertilizer compositions comprising microorganisms (so-called rtilizers") are increasingly considered as atives to tional al fertilizers. The y of specific bacterial species to promote plant growth has long been recognised. For example, nitrogen-fixing bacteria such as Rhizobium species provide plants with essential nitrogenous compounds. Species of Azotobacter and Azospirillum have also been shown to promote plant growth and increase crop yield, promoting the accumulation of nutrients in . However bacteria of these genera are often unable to compete effectively with native soil and plant flora, thereby requiring the application of impractically large volumes of inoculum. Various Bacillus and Pseudomonas species have also found application in microbial-based fertilizers.

To date, biofertilizers have typically met with limited success, often not proving to be efficacious under real farming conditions. There remains a need for improved microbial-based fertilizers that are effective in providing nts for plant growth and are environmentally safe and nonhazardous.

Summary of the Disclosure A first aspect of the t disclosure provides a microbial inoculant for use in increasing plant growth, plant productivity and/or soil quality, comprising s of one or more bacterial species selected from Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapi and acillus zeae.

In a particular ments the inoculant comprises two of said acillus species, three of said Lactobacillus species or all of said Lactobacillus species. The ant may represent a symbiotic combination of two or more or three or more of said Lactobacillus species.

The Lactobacillus parafarraginis strain may be Lactobacillus parafarraginis Lpl8.

In a particular embodiment the Lactobacillus parafarraginis strain is Lactobacillus parafarraginis Lpl8 deposited with National Measurement Institute, Australia on 27 October 20 1 under Accession Number V1 /022945 .

The Lactobacillus buchneri strain may be Lactobacillus buchneri Lb23. In a particular embodiment the Lactobacillus buchneri strain is Lactobacillus buchneri Lb23 deposited with National Measurement Institute, lia on 27 October 201 1 under Accession Number VI 1/022946.

The Lactobacillus rapi strain may be Lactobacillus rapi Lr24. In a particular embodiment the Lactobacillus rapi strain is Lactobacillus rapi Lr24 ted with National Measurement Institute, Australia on 27 October 201 1 under Accession Number VI 1/022947.

The Lactobacillus zeae strain may be Lactobacillus zeae Lz26. In a particular ment the Lactobacillus zeae strain is Lactobacillus zeae Lz26 deposited with National ement ute, Australia on 27 October 201 under Accession Number V 1/022948. [001 ] An inoculant of the first aspect may further comprise a strain of Acetobacter fabarum. The acter fabarum strain may be Acetobacter fabarum Afl5. In a particular embodiment the Acetobacter fabarum strain is Acetobacter fabarum Afl5 deposited with the National Measurement Institute, lia on 27 October 201 1 under ion Number VI 1/022943.

An inoculant of the first aspect may further comprise a yeast. The yeast may be a strain of Candida ethanolica. The Candida ethanolica strain may be Candida ethanolica Ce31. In a particular embodiment the Candida ethanolica strain is Candida ethanolica Ce31 deposited with the National ement Institute, Australia on 27 r 2011 under Accession Number V I 44.

One or more of the strains in the inoculant may be encapsulated. Where multiple strains are encapsulated, the strains may be individually ' encapsulated or combined in a single ulation.

A second aspect of the present disclosure provides a microbial inoculant comprising at least one Lactobacillus species, at least one Acetobacter species and at least one Candida species.

In a particular embodiment the at least one Lactobacillus species is selected from Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapt and Lactobacillus zeae. In a further particular embodiment, the microbial ant comprises at least one strain of each of said Lactobacillus species. In a further particular embodiment, the Lactobacillus parafarraginis is strain Lpl8 (deposited under Accession Number V I 1/022945), Lactobacillus buchneri is strain Lb23 (deposited under Accession Number V 1/022946), Lactobacillus rapi is strain Lr24 (deposited under Accession Number VI 1/022947) and Lactobacillus zeae is strain Lz26 (deposited under Accession Number V I 1/022948).

In a particular embodiment the at least one Acetobacter s is Acetobacter fabarum. In a further ular embodiment the Acetobacter fabarum is Afl5 (deposited under Accession Number V I 1/022943).

In a particular embodiment the at least one Candida species is Candida ethanolica.

In a further particular embodiment the Candida ethanolica is Ce31 (deposited under Accession Number VI 1/022944).

A third aspect of the present disclosure provides a microbial inoculant comprising at least one bacterial strain ed from Lactobacillus rraginis Lpl 8, Lactobacillus buchneri Lb23, Lactobacillus rapi Lr24 and Lactobacillus zeae Lz26.

An inoculant of the third aspect optionally further comprises Acetobacter fabarum Afl5 and/or Candida ethanolica Ce31.

An inoculant of the first, second or third aspect may be used as a fertilizer.

A fourth aspect of the present disclosure provides a fertilizer composition comprising a microbial inoculant of the first, second or third aspect. The fertilizer ition may optionally se one or more additional components such as organic material, humic nces, penetrants, utrients, micronutrients and other soil and/or plant additives.

A fifth aspect of the present disclosure provides a method for increasing plant growth and/or productivity, the method comprising applying to the plant, plant seeds or to the soil in which the plant or plant seeds are grown an effective amount of a microbial inoculant of the first, second or third aspect or a fertilizer composition of the fourth aspect.

A sixth aspect of the present disclosure provides a method for improving soil quality, the method comprising applying to soil or to the plants or plant seeds in said soil an effective amount of a microbial inoculant of the first, second or third aspect or a izer ition of the fourth aspect.

In accordance with the above aspects the plant may be, for example, a pasture plant, crop plant (including fruit and vegetable ) or ornamental plant. The crop may be, for e, any human or animal food crop or crop for use as fuel or for pharmaceutical production. The food crop may be, for example, a fruit, vegetable, nut, seed or grain.

A seventh aspect of the present disclosure provides a method for remediating degraded soil or pasture, the method comprising applying to the soil or pasture an effective amount of a microbial inoculant of the first, second or third aspect or a fertilizer composition of the fourth aspect.

Brief Description of the Drawings Aspects and embodiments of the present disclosure are described , by way of non-limiting example only, with reference to the following gs.

Figure 1. Root pment in tick bean plants, treated as described in Example 5.

A, control group; B, T40 treatment group; C, SGL40 treatment group; D, T25%GL40 ent group; E, GL40 treatment group.

Figure 2. Average rate of change of growth (height) of tomato plants over a 20 day treatment period in three different soils (A-C), treated as described in Example 6.

Squares ent IMP Bio treated seedlings, diamonds represent FlowPhos treated ngs, triangles ent IMP Bio plus FlowPhos treated seedlings, crosses ('c') represent ted (water only) seedlings.

Figure 3. Comparison of plant height, foliage size and root development in tomato seedlings, treated as described in Example 6. GreatLand = IMP Bio treated seedling.

Figure 4. Comparison of vegetative growth (and density of growth) of strawberry plants, treated as described in Example 8. A, conventional fertilizer treated plants after 3 months. B, IMP Bio treated plants after 3 months.

Detailed Description Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ry skill in the art to which the disclosure belongs. Although any methods and materials similar or lent to those described herein can be used in the practice or testing of the present disclosure, typical methods and materials are described.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. ~ o at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

In the context of this specification, the term "about," is understood to refer to range of numbers that a person of skill in the art would consider lent to the recited value in the context of achieving the same function or result.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The term "plant productivity" as used herein refers to any aspect of growth or development of a plant, that is a reason for which the plant is grown. Thus, for purposes of the present sure, improved or increased plant tivity refers broadly to improvements in biomass or yield of leaves, stems, grain, fruit, vegetables, s, or other plant parts harvested or used for various purposes, and improvements in growth of plant parts, including stems, leaves and roots. For example, when referring to food crops, such as grains, fruits or vegetables, plant tivity may refer to the yield of grain, fruit, bles or seeds harvested from a particular crop. For crops such as e, plant productivity may refer to growth rate, plant density or the extent of groundcover. "Plant growth" refers to the growth of any plant part, including stems, leaves and roots. Growth may refer to the rate of growth of any one of these plant parts.

The term "yield" refers to the amount of produced biological material and may be used interchangeably with "biomass". For crop plants, "yield" may also mean the amount of harvested al per unit of production or per area (e.g. hectare). Yield may be defined in terms of quantity or quality. The harvested material may vary from crop to crop, for e, it may be seeds, above-ground s, below-ground s (e.g. potatoes), roots, fruits, or any other part of the plant which is of economic value. "Yield" also encompasses yield ity of the . "Yield" also asses yield potential, which is the m obtainable yield under optimal growth conditions. Yield may be dependent on a number of yield components, which may be monitored by certain parameters. These parameters are well known to persons d in the art and vary from crop to crop. For example, breeders are well aware of the specific yield components and the corresponding parameters for the crop they are aiming to improve. For e, key yield parameters for potato include tuber weight, number of tubers, and number of stems per plant.

By "improving soil quality" is meant increasing the amount and/or bility of nutrients required by, or beneficial to plants, for growth. By way of example only, such nutrients include en, phosphorous, potassium, copper, zinc, boron and molybdenum.

Also encompassed by the term "improving soil quality" is reducing or minimising the amount of an element that may be detrimental to plant growth or development such as, for example iron and manganese. Thus, improving soil quality by use of microbial inoculants and fertilizer compositions of the present disclosure thereby assists and promotes the growth of plants in the soil.

The term "remediating" as used herein in relation to degraded pasture or soil refers to the improvement in plant nutrient content in the soil to facilitate improved plant growth and/or yield. Degraded pasture includes overgrazed pasture.

As used herein, the term "effective amount" refers to an amount of microbial inoculant or fertilizer composition applied to a given area of soil or vegetation that is ient to effect one or more beneficial or desired outcomes, for example, in terms of plant growth rates, crop yields, or nutrient availability in the soil. An "effective amount" can be provided in one or more administrations. The exact amount required will vary depending on factors such as the identity and number of individual strains employed, the plant species being treated, the nature and condition of the soil to be treated, the exact nature of the ial inoculant or fertilizer composition to be applied, the form in which the inoculant or fertilizer is applied and the means by which it is applied, and the stage of the plant growing season during which application takes place. Thus, it is not le to specify an exact "effective amount". r, for any given case, an appropriate "effective " may be determined by one of ordinary skill in the art using only routine experimentation.

The term "crop" as used herein refers to any plant grown to be harvested or used for any economic purpose, including for example human foods, livestock fodder, fuel or pharmaceutical production (e.g. poppies).

The term "optionally" is used herein to mean that the uently described feature may or may not be present or that the subsequently bed event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiments in which the event or circumstance occurs as well as embodiments in which it does not.

In accordance with the present disclosure, novel microbial ants and microbial fertilizer compositions are presented which find application in increasing plant productivity and improving soil quality. In particular embodiments the microbial species present in the microbial inoculant or fertilizer composition provide a symbiotic combination of sms.

In the broadest embodiments, a microbial ant of the present disclosure comprises strains of one or more bacterial Lactobacillus species. The Lactobacillus species may be selected from Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapt and Lactobacillus zeae. The inoculant may further comprise at least one Acetobacter s and at least one a species.

The Lactobacillus parafarraginis strain may be Lactobacillus parafarraginis Lpl8.

In a particular embodiment the Lactobacillus parafarraginis strain is acillus parafarraginis Lpl8 deposited with National Measurement Institute, Australia on 27 r 201 1 under Accession Number V 1/022945. The Lactobacillus buchneri strain may be Lactobacillus buchneri Lb23. In a particular embodiment Lactobacillus buchneri strain is Lactobacillus buchneri Lb23 deposited with National Measurement Institute, Australia on 27 October 2011 under Accession Number V I 1/022946. The Lactobacillus rapi strain may be Lactobacillus rapi Lr24. In a particular embodiment the acillus rapi strain is Lactobacillus rapi Lr24 deposited with National Measurement ute, Australia on 27 October 201 under ion Number V 47. The Lactobacillus zeae strain may be acillus zeae Lz26. In a particular embodiment the Lactobacillus zeae strain is Lactobacillus zeae Lz26 deposited with National Measurement Institute, Australia on 27 October 201 1 under Accession Number V 1/022948.

The inoculant may further comprise a strain of Acetobacter fabarum. The Acetobacterfabarum strain may be Acetobacterfabarum Afl5. In a particular ment the Acetobacter fabarum strain is Acetobacter fabarum Afl5 deposited with the National Measurement Institute, Australia on 27 October 201 1 under Accession Number VI 1/022943.

The inoculant may further comprise a yeast. The yeast may be a strain of Candida ethanolica. The Candida lica strain may be Candida ethanolica Ce31. In a particular embodiment the Candida ethanolica strain is Candida ethanolica Ce31 deposited with the National Measurement Institute, Australia on 27 October 2011 under Accession Number VI 1/022944.

The concentrations of each microbial strain to be added to ial inoculants and fertilizer compositions as disclosed herein will depend on a variety of factors including the identity and number of individual strains ed, the plant species being treated, the nature and condition of the soil to be treated, the exact nature of the microbial inoculant or fertilizer composition to be applied, the form in which the inoculant or fertilizer is applied and the means by which it is applied, and the stage of the plant growing season during which application takes place. For any given case, appropriate concentrations may be determined by one of ordinary skill in. the art using only routine experimentation. By way of example only, the concentration of each strain present in the ant or fertilizer composition may be from about 1 x 102 cfu/ml to about 1 x 10 10 cfu/ml, and may be about 1 x 103 cfu ml, about 2.5 x 103 , about 5 x 103 cfu/ml, 1 x 104 cfu/ml, about 2.5 x 104 cfu/ml, about 5 x 104 cfu/ml, 1 x 10 cfu/ml, about 2.5 x 10 , about 5 x 10 cfu/ml, 1 x 106 cfu/ml, about 2.5 x 106 cfu/ml, about 5 x 106 , 1 x 107 cfu/ml, about 2.5 x 107 cfu/ml, about 5 x 107 cfu/ml, 1 x 10 cfu/ml, about 2.5 x 08 cfu/ml, about 5 x 108 cfu/ml, 1 x 109 cfu/ml, about 2.5 x 109 cfu/ml, or about 5 x 109 cfu/ml. In particular exemplary ments the final concentration of the Lactobacillus strains is about 2.5 x 10s cfu/ml, the final concentration of Acetobacter fabarum may be about 1 x 106 cfu/ml and the final concentration of Candida ethanolica may be about 1 x 10 cfu/ml.

Also plated by the present disclosure are variants of the microbial strains described herein. As used herein, the term "variant" refers to both naturally occurring and specifically developed variants or s of the microbial s disclosed and exemplified herein. Variants may or may not have the same identifying biological characteristics of the specific strains exemplified herein, provided they share similar advantageous properties in terms of promoting plant growth and providing nutrients for plant growth in the soil. Illustrative examples of suitable methods for preparing variants of the microbial strains exemplified herein include, but are not d to, gene ation techniques such as those ed by inseftional elements or transposons or by homologous recombination, other recombinant DNA techniques for modifying, inserting, deleting, activating or silencing genes, intraspecific protoplast , mutagenesis by irradiation with ultraviolet light or X-rays, or by treatment with a chemical mutagen such as nitrosoguanidine, methylmethane sulfonate, nitrogen mustard and the like, and iophage-mediated transduction. Suitable and applicable methods are well known in the art and are described, for example, in J . H . Miller, Experiments in lar Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1972); J. H. Miller, A Short Course in Bacterial Genetics, Cold Spring Harbor tory Press, Cold Spring Harbor, N.Y. (1992); and J. Sambrook, D. Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), inter alia.

Also assed by the term "variant" as used herein are microbial strains phylogenetically closely related to strains disclosed herein and strains possessing substantial sequence identity with the strains disclosed herein at one or more phylogenetically informative markers such as rRNA genes, elongation and initiation factor genes, RNA polymerase subunit genes, DNA gyrase genes, heat shock protein genes and recA genes. For example, the 16S rRNA genes of a "variant" strain as contemplated herein may share about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a strain disclosed herein.

Microbial inoculants and fertilizer compositions of the present disclosure may optionally further comprise one or more additional microbial organisms, for example, additional agronomically beneficial microorganisms. Such agronomically beneficial microorganisms may act in synergy or t with, or otherwise cooperate with the organisms of the t disclosure in the inoculant or fertilizer. Examples of agronomically beneficial microorganisms include Bacillus sp., Pseudomonas sp., Rhizobium sp., Azospirillum sp., Azotobacter sp., phototrophic and cellulose degrading ia, Clostridium sp., Trichoderma sp. and the like. Those skilled in the art will appreciate that this list is merely exemplary only, and is not limited by reference to the specific examples here provided.

In the soil nment, inoculated bacteria can find survival ult among naturally occurring itor and predator organisms. To aid in survival of microorganisms present in microbial inoculants and fertilizer itions of the present disclosure upon application in the environment, one or more of the s may be encapsulated in, for example, a suitable polymeric matrix. In one example, encapsulation may se alginate beads such as has been described by Young et al, 2006, Encapsulation of plant growth-promoting bacteria in alginate beads enriched with humic acid, Biotechnology and Bioengineering 95:76-83, the disclosure of which is incorporated herein by reference in its ty. Those skilled in the art will appreciate that any suitable encapsulation material or matrix may be used. Encapsulation may be achieved using methods and techniques known to those skilled in the art. Encapsulated rganisms can include nutrients or other components of the inoculant or fertilizer composition in addition to the microorganisms.

Those d in the art will appreciate that any plant may benefit from the application of microbial inoculants and fertilizer compositions of the present disclosure to soil, seeds and/or vegetation. Particular embodiments are employed to aid the growth, development, yield or productivity of crops and pastures or other plants of economic value, including ntals and plants grown for oils or biofuel. The crop plant may be, for example, a food crop (for humans or other animals) such as any fruit, vegetable, nut, seed or grain producing plant. Exemplary crop plants include, but are not limited to, tubers and other below-ground vegetables (such as potatoes, beetroots, radishes, carrots, onions, etc.), ground-growing or vine vegetables (such as pumpkin and other members of the squash family, beans, peas, asparagus, etc.), leaf vegetables (such as lettuces, chard, spinach, alfalfa, etc.), other vegetables (such as tomatoes, brassica including broccoli, avocadoes, etc.), fruits (such as berries, , stone fruits including nectarines and peaches, tropical fruits including mangoes and s, apples, pears, mandarins, oranges, mandarins, kiwi fruit, coconut, etc.), cereals (such as rice, maize, wheat, barley, millet, oats, rye etc.), nuts (such as macadamia nuts, peanuts, brazil nuts, hazel nuts, walnuts, almonds, etc.), and other economically valuable crops and plants (such as sugar cane, soybeans, sunflower, , sorghum, pastures, turf grass, etc).

Microbial inoculants and fertilizer compositions of the present disclosure may be applied directly to plants, plant parts (such as foliage) or seeds, or alternatively may be applied to soil in which the plants are growing or to be grown or in which seeds have been or are to be sown. Application may be by any suitable means and may be on any suitable scale. For example, application may comprise g, spreading or spraying, including broad scale or bulk spreading or ng, g of seeds before planting, and/or drenching of seeds after planting or seedlings. Those skilled in the art will appreciate that le means of application may be used in combination (for example soaking of seeds prior to ng followed by drenching of planted seeds and/or application to seedlings or mature plants). Seeds, ngs or mature plants may be treated as many times as appropriate. The number of applications ed can readily be determined by those skilled in the art depending on, for example, the plant in question, the stage of development of the plant at which treatment is initiated, the state of health of the plant, the growth, environmental and/or climatic conditions in which the plant is grown and the purpose for which the plant is grown. For example, in the case of flowering crops such as tomatoes, it may be desirable to apply the microbial inoculant or fertilizer composition once or more than once during the flowering .

Thus, in accordance with the present disclosure, microbial inoculants and fertilizer / ts as disclosed herein may be prepared in any suitable form depending on the means by which the inoculant or izer composition is to be applied to the soil or to plant seeds or vegetation. Suitable forms can include, for example, slurries, liquids, and solid forms.

Solid forms include powders, granules, larger particulate forms and pellets. Solid form fertilizer particles can be encapsulated in water soluble coatings (for example dyed or undyed gelatin spheres or capsules), extended release coatings, or by micro-encapsulation to a free flowing powder using one or more of, for example, gelatin, polyvinyl alcohol, ellulose, cellulose acetate phthalate, or styrene maleic anhydride. Liquids may e aqueous ons and aqueous suspensions, and emulsifiable concentrates.

In order to achieve effective dispersion, on and/or conservation or stability within the environment of inoculants and izer compositions disclosed herein, it may be advantageous to formulate the inoculants and compositions with suitable carrier components that aid sion, adhesion and conservation/stability. Suitable carriers will be known to those skilled in the art and include, for example, chitosan, vermiculite, compost, talc, milk powder, gels and the like.

Additional components may be incorporated into ants and fertilizer compositions of the present disclosure, such as humic substances, trace elements, organic material, penetrants, utrients, micronutrients and other soil and or plant additives.

Humus or humic substances that may be incorporated may include, but are not limited to, humic acid derived from, for example oxidised lignite or leonardite, fulvic acid and humates such as potassium humate.

Organic material added may include, but is not limited to, ids, animal manure, compost or composted organic byproducts, activated sludge or processed animal- or ble byproducts (including blood meal, feather meal, cottonseed meal, ocean kelp meal, seaweed extract, fish emulsions and fish meal).

Penetrants include, but are not limited to, non-ionic wetting agents, detergent based tants, silicones, and/or organo-silicones. le penetrants will be known to those skilled in the art, non-limiting examples including polymeric polyoxyalkylenes, allinol, nonoxynol, nol, oxycastrol, TRITON, TWEEN, Sylgard 309, Silwet L-77, and Herbex (silicone/surfactant blend). ary trace elements for inclusion in microbial inoculants and fertilizer compositions are provided in Example 1. However those skilled in the art will recognise that suitable trace elements are not limited thereto, and that any trace elements (natural or synthetic) amy be employed.

Optional further soil and/or plant additives that can be added to inoculants and fertilizer compositions of the t disclosure include, for example, water ng agents such as zeolites, enzymes, plant growth hormones such as ellins, and pest control agents such as acaracides, insecticides, fungicides and cides.

The reference in this ication to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common l knowledge in the field of endeavour to which this specification relates.

The present disclosure will now be described with reference to the following ic examples, which should not be construed as in any way limiting the scope of the invention.

Examples The following examples are illustrative 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 1- Microbial strains The following microbial strains were used in the production of a tilizer.

Lactobacillus parafarraginis Lpl8 was isolated from an environmental source.

Partial 16S rRNA sequencing indicated 100% similarity to acillus parafarraginis AB 262735 which has a risk group of 1 (TRBA). When cultured on MRS media for 3 days at 34°C, anaerobically, Lpl8 produces cream, round, slight sheen, convex, colony diameter l-2mm (facultative be). Its microscopic appearance is Gram positive, non-motile, short rods rectangular, mainly diploid. Lactobacillus parafarraginis Lpl8 was deposited with the National Measurement Institute, Australia on 27 October 201 1 under Accession Number V I 1/022945.

Lactobacillus buchneri Lb23 was isolated from an environmental . l 16S rRNA sequencing indicated 99% similarity to Lactobacillus buchneri AB 429368 which has a risk group of 1 . When cultured on MRS media for 4 days at 34°C, anaerobically, Lb23 produces cream, shiny, , colony diameter l-2mm (facultative anaerobe). Its microscopic appearance is Gram positive, non-motile, rods in chains.

Lactobacillus buchneri Lb23 was deposited with the National Measurement Institute, Australia on 27 October 201 1 under Accession Number V I 1/022946.

Lactobacillus rapi Lr24 was isolated from an environmental source. Partial 16S rRNA sequencing indicated 99% similarity to Lactobacillus rapi AB 366389 which has a risk group of 1 (DSMZ). When cultured on MRS media for 4 days at 34°C, anaerobically, Lr24 es cream, round, shiny colonies with a diameter of 0.5mm (facultative anaerobe). Its microscopic appearance is Gram positive, non-motile, short rods single or d. Lactobacillus rapi Lr24 was deposited with the National Measurement Institute, Australia on 27 October 201 1under Accession Number VI 1/022947.

Lactobacillus zeae Lz26 was isolated from an nmental source. Partial 16S rRNA cing indicated 99% similarity to Lactobacillus zeae AB .1 which has a risk group of 1 (TRBA). When cultured on MRS media for 48 hours at 34°C, anaerobically, Lz26 produces white, round, shiny, convex, colonies with a er of lmm (facultative anaerobe). Its microscopic appearance is Gram positive, non-motile, short rods almost coccoid, diploid and some chains. Lactobacillus zeae 2 6 was deposited with the National Measurement ute, Australia on 27 October 201 1 under Accession Number V I 1/022948.

Acetobacter fabarum Afl5 was isolated from an environmental source. Partial 16S rRNA sequencing indicated 100% similarity to Acetobacter fabarum AM 905849 which has a risk group of 1 (DSMZ). When ed on Malt extract media for 3 days at 34°C, AF15 produces opaque, round, shiny, convex, colony diameter lmm (aerobic). Its copic appearance is Gram negative, rods single or diploid. Acetobacter fabarum Afl5 was ted with the National Measurement Institute, Australia on 27 October 201 1 under Accession Number V I 1/022943.

Candida ethanolica Ce31 was isolated from an nmental source. l 16S rRNA cing indicated 89% similarity to Candida ethanolica AB534618. When cultured on Malt extract media for 2 days at 34°C, Ce31 produces cream, flat, dull, roundish, colony diameter 2-3mm (aerobic). Its microscopic appearance is budding, ovoid yeast. Candida ethanolica Ce3 1 was deposited with the National Measurement Institute, Australia on 27 r 201 1 under Accession Number V 1/022944.

Maintenance of cultures 30% glycerol stocks were made of each isolate and maintained at -80°C for longterm culture storage. Short-term storage of the cultures are maintained at 4°C on agar slopes (3 month storage) and on agar plates which are subcultured monthly. To maintain the isolates original traits, a fresh plate is made from the -80°C stock following three plate subcultures.

Inoculum and growth media The Lactobacillus s were grown with or without air (I. rapi prefers anaerobic) either in MRS broth (Difco) or on MRS agar plates depending on application.

The cultures were routinely grown for 2 days at a mesophilic temperature of 30-34°C. The Acetobacter and Ethanolica strains are grown aerobically either in Malt extract broth (Oxoid) or on Malt extract agar plates depending on application. The cultures are routinely grown for 2 days at a mesophilic temperature of 30-34°C.

Fermenter 'seed'preparation For individual strains, using a sterile nichrome wire a single colony is removed from a fresh culture plate and transferred to a universal bottle containing 15mL of sterile media. The bottle is securely placed in a g tor set at 30°C, 140rpm for 48hrs (L.rapi is not shaken). After incubation a cloudy bacterial growth should be visible. 'Seed' inoculation bottles are stored at 4°C until required (maximum 1 week).

Typically a 5% bacterial inoculation is required for a fermenter run. The stored 15ml culture seed is added to a Schott bottle containing a volume of e media which is % of the total fermenter working volume. The culture is incubated and shaken in the same way as the 15ml seed. Large scale automatic ters are used to grow pure es of each isolate. There is an automatic feed of , antifoam and glucose. Typically the temperature is ined at 30-34°C, pH 5.5 but the oxygen and agitation varies depending on the microorganism.

Sample analysis After each large scale culturing of an isolate a sample is aseptically withdrawn and a viability count undertaken using 0 fold serial dilutions, performed in a laminar flow hood. A wet slide is also prepared and purity observed using a phase contrast microscope to double check for contaminants that may be present but unable to grow on the culture media. After 48 hours the viability plates are d for a pure culture (same colony morphology) and the colonies counted to produce a colony forming unit per ml (cfu/ml) value. A Grams stain is also performed.

Example 2- Pasture trials Field trials on pasture were conducted using a tlizer as sed herein, in comparison to untreated pasture and pasture treated with conventional inorganic fertilizers.

The biofertilizer (hereinafter "IMP Bio") comprised the six microbial strains listed in Example 1, at final concentrations of 2.5 x 10 c u ml for each of the Lactobacillus strains, 1.0 x 10 cfu/ml for Candida ethanolica and 1.0 x 106 cfu/ml for Acetobacter m. The strains were grown as described in Example 1 and mixed with 2% trace elements, 0.3% humate (Soluble Humate, LawrieCo), 3% molasses and 0.1-0.2% phosphoric acid. Phosphoric acid was added to the point where pH was in the range 3.8 to 4.0. The trace elements ent typically comprised the following (per 1,000L): Table 1 Trace ts component of biofertilizer Conventional inorganic fertilizers used as comparators were Spray Gro Liquid Urea, DAP (diammonium phosphate), and :16:11 commercial NPK mix.

Sites for the pasture trials were ed based on ll levels, soil type, pasture composition and past izing practices. The following locations in Tasmania were used: Nabageena (high rainfall; rye grass, cocksfoot, Yorkshire fog and other grasses), Cuprona (high rainfall; rye grass), West lle/Upper Burnie (high rainfall; rye , Connorville (dryland pasture; degraded) and Connorville (irrigated pasture; rye grass).

At each location, multiple 4 x 10m strips of pasture were prepared by mowing to a height of 45mm (and removal of clipped plant material prior to fertilizing). At West Moorville/Upper Burnie and Nabageena, IMP Bio was applied to replicate plots at 20L/ha, 30L/ha or 50L/ha, and 14:16:11 NPK mi was applied to replicate plots at 250kg/ha. At West Moorville, DAP was also applied to replicate plots at 125kg/ha. At Cuprona, IMP Bio was applied to replicate plots at 20L/ha, 30L/ha or 50L/ha, and Spray Gro Liquid Urea was applied at 50L/ha. At Connorville, IMP Bio was d to replicate plots at 20L/ha, 30L/ha or 50L/ha, and DAP was applied to replicate plots at 125kg/ha. IMP Bio and SprayGro Urea were applied as large ts through 2m backpack boom sprays in a single pass. 14:16:11 NPK mix and DAP were applied by hand distribution. At each location, ate control (unfertilized) plots were set aside.

Plant yield and leaf nutrient content were analysed 6-8 weeks after treatment.

Results for plant yield are shown in Table 2 below. These s indicate that the IMP Bio fertilizer produced yields at least similar to, and in some cases superior to, conventional inorganic fertilizers.

Table 2. Yield (kg/ha/day) Plant material nutrient analysis was conducted, as shown in Table 3 below. Key elements ed by, or beneficial to, the pasture for growth (such as nitrogen, phosphorous, postassium, calcium, copper, zinc, boron, molybdenum) were present in plant material from the IMP Bio treated plots at levels equivalent to or higher than those plots d with the comparator conventional inorganic fertilizer, despite these nutrients not being added in the IMP Bio fertilizer.

Table 3able 3 Nutrient Control . Conventional IMP Bio ' fertilizer. ‘ 30L/ha Connorville irriated Nigerian (8/22 .1. VVVVV _____ Phoghgrus (%) ' .SELQQQK‘ZELWW--W.. ..

Calcium (%) Ma ; esium % sodium..(?_/.9L_.W.......-.

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Eflmem)_-w-._.,.w - 172 114 120 ......................... ______ _Boronggp_m2 7.7 8.3 _____m ____1.9_mmmmm -.-,_ _.

M01 b69291 m 1-3 1.0 ..........._ _ ....... ........;........111 Cobaltgmemwm ._.n.~.__9;1.................................. <0-‘1 <0-1 ~~~~~~ Silicon $2221.) .m,~._‘__._._... 316 244 ' W_-__.. 35,8 .

Nitrossy Sumhugflio 7 0 , .,,__§.2.4.___M “WA?_____________ W. gnihepgheflls ratio 5 3 ...............ESMMMAJ......... W..- M§9.i9£a_8§i1m§.tj9.m 41.7. ._._w_,_‘2_-§_M Vania!..............m Carbonflsmiflitmgsyfiig..........................................Z82___;8_1.................-............26.43.........a Crude n %Nx6.25 9.8 9.8 10.2 .Nitrogeqm 3 68 ......... _ M. ‘ W W_MMWW..................., - mmwgw.4239 __ _ Potassigm (%) _} __, 3:13" Sul h95_§‘122_.~.._..,wwm “WEE? Carboewgfyo) 436 - ‘ a........................ .............

Calcium (%) W,WW*051 Mamgigrfl’élmn..flm._~,._#______-.-m_024__._ Sodium,_,(%) __, , -026.......... 00231922911.....................................1.3J..-” 2m ‘3me M25: - Mm_mm,... ' -106 -m......... .m)._m_____________ H.491............... .,,.§9322.,:92m.2.......... l 6.2 ................ ...........

M‘Mgbzbmmgpm) 0-3 ,..... , __ ..........................

ML...“........... <0-1 ..,_Sfli_cgn_£22m)_ _......___ _._2_6_7w_. @3313 Phggshorpmggtio 4 9.5 _ “““““ Nitrogen : Potassium ratio 1.1 1.1 1.3 Carbon : Nitrogen ratio 11.8 12.3 11.9 Crude protein (%N x 6.25) 23.0 22.5 23.0 Example 3- Soil quality To determine the effect of a biofertilizer as disclosed herein on soil quality, 2 x 150g of soil from a farm in Tasmania were each weighed into 2 x clean 150ml Schott bottle. lOmls of a 1:10 dilution of IMP Bio fertilizer (see Examples 1 and 2) was dripped over the top of the soil in one bottle and the lid replaced and incubated at 34°C for one week. The second bottle had no biofertilizer added was incubated 34°C. The soil from both bottles was analysed by nmental Analytical Laboratories (EAL, rn Cross University Lismore, NSW) using standard soil testing methods.

The results for the one week treatment of soil with IMP Bio are summarised in Table 4. Soil tests on the untreated ted sample are not shown as these were substantially the same as the initial untreated soil test. It is clear from the soil tests on the two treated samples that there is a marked difference in the soil after incubation with IMP Bio. The second sample analysed, shows a general trend of increasing the levels of available cations um, magnesium, potassium, sodium and all trace elements - zinc, manganese, iron and copper) and ammonium nitrogen, while the total levels under the acid extractions were slightly lower across all nutrients. Organic matter increased by 1% (14.6% to 15.5%) between the samples dates. The overall decrease in total nts does not appear to be significant.

There was a greater than three-fold se in ammonium nitrogen, although no increase in nitrates. This indicates an increase in mineralisation of en from the organic en pool, and may be linked to the transformation of organic material, the level of which in this soil is particularly high. This could also indicate nitrogen fixation.

Table 4 Example 4 - Potato trials A field trial was conducted in which Bondi y potatoes were treated with the IMP Bio biofertilizer (see Example 2) at planting. The trial was conducted at ouse, Tasmania. IMP Bio was applied in furrows to rows 30m long at a rate of 50L ha, either alone, or together with the conventional chemical fertilizer 516 at either 650kg/ha (delivering 32kg/ha en, 63kg/ha phosphorus and lOOkg/ha potassium) or 1250kg/ha (delivering 63kg/ha nitrogen, 125kg/ha phosphorus and 200kg/ha potassium). In a fourth replicate, 516 was applied at /ha together with the fungicide Amistar. Four plots of 4m length were dug from each treatment and tubers assessed for size and yield.

The results are shown in Table 5.

Table 5. Potato yield There was an increase in stem s per plant in the IMP Bio treatment, which is desirable r stem numbers typically correlating with higher tuber numbers). The reduction in large (>350g) tubers observed with IMP Bio treatment is also significant as larger tubers have lower commercial value than seed sized tubers (45-350g). onally, the 14% se (5 tonnes/ha) in seed weight in the IMP Bio compared to the 516 + Amistar treatment is also of significant economic value. The IMP Bio treated potato plants were also observed to be approximately three weeks more developed (in terms of maturity) than those treated with 516.

Example 5 - Tick bean trials A greenhouse experiment was ted to establish the effect of IMP Bio biofertilizer (see Example 2) on tick bean plant growth, compared to the commercial fertilizer Baileys TriStar (8.3%N, 0% P, 16% K, 14% S, 1% Fe, 2% Mg).

The treatment groups and regimes employed for seedlings post-germination were as follows: Control: 300 mΐ water "T40": 300 m TriStar at 40L/ha "SGL40": 300 m IMP Bio at 40L/ha "T25%GL40": 300 m TriStar 25% plus IMP Bio at 40L/ha "GL40": 300 mΐ IMP Bio at 40L/ha Seeds in the T40, SGL40 and 40 groups were soaked for 1 hour in 100ml of a 1:10 dilution of IMP Bio on prior to planting. Control and GL40 seeds remained dry prior to planting. Three replicates of each treatment group (and two replicates of the control group) were used. Seeds were planted 5mm deep in the middle of each pot and the pots placed in a temperature-controlled greenhouse at 16-18°C under hydroponic lights.

After germination, all seedlings were d every two weeks (for a total of four weeks) using the ents described above. Seedlings were watered once a day.

At the conclusion of the experiment it was observed that the tallest plants, and the plants with the strongest main stem were those of the T25%GL40 treatment group.

Overall, the best growth was observed in the T25%GL40 and SGL40 groups (data not shown). However the most noticeable ences observed were in root development (see Figure 1). Roots of the control plants were the least dense and the shortest (Figure 1A).

Roots of the T40 plants had good root density and length (Figure IB), however pment was not as extensive as in the plants treated with IMP Bio. In the SGL40 plants the root system shown good desnity and length (Figure 1C). Root s were present as were black nodule-like growths. In the T25%GL40 plants the root system was more dense and longer than in other treatment groups (Figure ID). Root nodules were present but black nodule-like growth was not seen. In the GL40 plants the root system was similarly dense, long and well developed (Figure IE). Root nodules were present as were black nodule-like growths.

Example 6 - Tomato trials A ouse experiment was conducted to investigate the effect of IMP Bio biofertilizer (see Example 2) on the growth rate of tomato plants over a 20 day period.

Tomato seedlings were provided by Cedenco. Water only was used as a control, and the commercial izer FlowPhos (Yara Nipro) used as a comparator. Seedlings were potted into 50 mm pots in one of three different soils obtained from different locations (Cedenco) and drenched once with either: (i) 10 ml water; (ii) 0 ml of IMP Bio (100 ml in 900 ml water); (iii) 0 ml of FlowPhos (7.5 ml in 900 ml water); or (iv) 10 ml of FlowPhos plus IMP Bio (7.5 ml FlowPhos and 100 ml IMP Bio made to a total volume of 1000ml with water). Three replicates of the control (water) group and eight replicates of each of the ent groups. Plants were d twice a day with 30 ml water. Plant height was measured every third day over the 20 day period of the ment.

The e rate of change of growth (height) of tomato seedlings over the 20 day period for all treatment groups, in each of the three soils, is shown in Figure 2. As can be seen, the IMP Bio treated plants were the only plants that tently showed increases in growth over the course of the experiment, resulting in taller plants. Figure 3 shows an exemplary comparison of difference in plant height, foliage and root system development in control plants, FlowPhos treated plants and IMP Bio (GreatLand) treated plants, in which the advantages of IMP Bio treatment are clearly evident.

A field trial was then conducted at Timmering, Victoria in which tomato plants were treated with IMP Bio by foliar application during flowering, either at a rate of 80L/ha or 40L ha during early flowering followed by 40L ha during mid flowering. Yield of tomato fruit was determined and compared to the yield from the same number of untreated plants. For the plants that received 80L ha IMP Bio, total fruit yield was 149.87 tonnes/ha, compared to 128.87 tonnes/ha for the untreated plants. For the plants that received two applications of 40L/ha IMP Bio, total fruit yield was 130.15 /ha, compared to 103.05 tonnes/ha for the untreated plants.

Example 7 - Macadamia trials A field trial was conducted in which macadamia trees in a 100 ha farm in Lismore, NSW were treated with the IMP Bio biofertilizer (see Example 2) by spraying at the rate of 40L/ha, every 2-3 months for a period of 12 . IMP Bio was applied in conjunction with chemical izer (Easy N Fertilizer), the same fertilizer used for at least the previous four years. The yield of macadamia nuts following the 12 month treatment was approximately 70 tonnes, compared to an average yield of 35 tonnes per year over the previous four years. The benefits offered by the IMP Bio biofertilizer d for a significant reduction in the application of al fertilizer.

Leaf and soil analysis was also conducted at four sites across the farm after 45 days of IMP Bio use. Significant increases were observed in levels of zinc, manganese, iron and boron in macadamia leaves, and in ammonium nitrogen, nitrate nitrogen,' phosphorus, ium, calcium, copper and boron in the soil.

Example 8 · Strawberry trials A field trial was conducted in Beerwah, Qld to establish the effect of IMP Bio biofertilizer (see Example 2) on strawberry plant growth and fruit yield over an 8 ha plot.

The IMP Bio was applied at a rate of 40L ha to the soil pre-planting, again at the same rate at planting, and weekly during the vegetative growth and flowering stage (weeks 2-4), during the fruiting stage (weeks 5-8) and during the g stage (weeks 9-16). In comparison to conventional fertilizer (NitroPhoska(blue) applied preplanting at 1000 kg ha), plant growth rate was icantly increased and plants showed increased vegetative growth and leaf area (Figure 4). Fruit yield was also significantly increased (38,000 kg as compared to 20,000 kg). e 9 - Other trials Preliminary trials have also been conducted on sugar cane, lettuce, raspberries, roses, wheat, basil and turf grass (golf course green). In each case IMP Bio biofertilizer (see e 2) was observed to result in increased rate of growth of plants compared to untreated plants (data not shown).

Claims (6)

Claims

1. A microbial fertilizer, comprising strains of three or more bacterial species selected from Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapi and Lactobacillus zeae.

2. A microbial fertilizer according to claim 1 n the inoculant comprises three of said acillus species.

3. A microbial fertilizer according to claim 1 wherein the inoculant comprises all of said Lactobacillus species.

4. A microbial fertilizer according to any one of claims 1 to 3 sing a symbiotic combination of said three or more acillus species.

5. A microbial fertilizer according to any one of claims 1 to 4 wherein the Lactobacillus rraginis strain is Lactobacillus parafarraginis Lp18.

6. A microbial fertilizer according to claim 5 wherein the Lactobacillus parafarraginis strain is Lactobacillus parafarraginis Lp18 deposited with National Measurement Institute, Australia on 27 October 2011 under Accession Number V