MICROBIAL COMPOSITION, METHOD AND KIT FOR ENHANCING PLANT

GROWTH FIELD THIS INVENTION relates to plants. More particularly, this invention relates to a bacterial composition suitable for administration to plants and/or soil that enhances plant growth.

BACKGROUND

Microbial colonization of plants can provide plants with sources of nutrients and other growth-promoting substances. A well known example of such microbes is provided by Rhizobia, which are Gram-negative soil bacteria with the unique ability to establish a N2-fixing symbiosis on legume roots and on the stems of some aquatic legumes. During this interaction, the rhizobia are contained in intracellular compartments within a specialized organ, the nodule, where they fix N ₂ in a form available to the legume. In contrast, nitrogen-fixing Azorhizobium are non- nodulating bacteria that can exist as free-living bacteria or in a symbiotic interaction with plants (e.g Azorhizobium caulinodans and plants of the genus Sesbania).

Other important nitrogen-fixing bacteria are of the family Azotobacteraceae which contains aerobic diazotrophs within two genera, Azomonas and Azotobacter, which are free-living soil bacteria with an ability to form cysts. It has also been shown that some bacteria may exist as plant endophytes in a direct, symbiotic relationship with plants such as corn, sugarcane, coffee, sweet potato, mango, banana and other crop plants (Muthukumarasamy et al., 2002, Current Sci. 83 127).

Accordingly, bacterial compositions have been developed which, when applied to crop plant foliage or to soil, improve nitrogen fixation and thereby improve the health and vigour of crops. It has been proposed that use of these compositions may obviate or lessen the need for application of nitrogenous fertilizers, thereby reducing crop production costs and the impact of fertilizers on the environment. An example of this type of treatment is provided in International Publication WO2006/005100 where combinations of multiple nitrogen-fixing bacteria including some of those referred to above were formulated for delivery to plant foliage and soil. It was proposed that such bacterial treatments were suitable across a broad range of non-leguminous plants. SUMMARY

The present invention has arisen, at least in part, from the inventor's realization that microbial compositions for enhancing plant growth need to be selectively tailored for particular plant types by selecting particular bacteria for use in such compositions. Furthermore, the inventor has addressed problems that occur in obtaining microbial colonization of certain plants, particularly plants that have a waxy cuticle such as grasses. The inventor has also determined a need for improved monitoring of bacterial inoculation and colonization of plants and also more convenient and cost-efficient packaging of microbial compositions for enhancing plant growth.

It is therefore an object of the invention to at least partly ameliorate or address one or more deficiencies of the prior art, or at least provide a commercially useful alternative.

It is a preferred object of the invention to provide a microbial composition comprising one or more particular bacterial species that that enhances the growth of plants.

It is another preferred object of the invention to improve the ability of microbial components of microbial compositions to inoculate or colonize plants.

It is yet another preferred object of the invention to provide an improved packaging system for microbial compositions.

It is a further preferred object of the invention to provide an improved method of determining the level of bacterial inoculation artd/or colonization of a plant.

In a first aspect, the invention provides a plant growth-enhancing concentrate comprising Herbaspirillum seropedicae bacteria and at least one other bacterial species which upon dilution and administration to a plant or to soil, enhances plant growth.

Suitably, the bacteria are in a freeze-dried state.

Suitably, the concentrate may be reconstituted with a liquid diluent or medium.

In a second aspect, the invention provides a plant growth-enhancing composition administrable to a plant and/or to soil, said composition comprising: (a) Herbaspirillum seropedicae bacteria and at least one other bacterial species which upon administration to a plant or to soil, enhances plant growth; and (b) a liquid medium that facilitates administration of the plant growth-enhancing composition to a plant and/or to soil.

Preferably, the liquid medium is water.

Suitably, one or each of said at least one other bacterial species is nitrogen fixing and/or is capable of facilitating or enhancing nitrogen fixation by a plant.

Suitably, according to the aforementioned aspects, said at least one other bacterial species is of a genus selected from (i) Azorhizobium; (ii) Azotobacter; (iii) Azoarcus; (iv) Clostridium; (v) Azospirillum; (vi) Bacillus; and (vii) Gluconacetobacter.

In particular embodiments, the at least one other bacterial species in (i)-(vii) is selected from Azorhizobium caulinodans, Azotobacter chroococcum, Azotobacter caulinodans, Azotobacter vinelandii, Azoarcus indigens, Azospirillum lipoferum, Bacillus subtilis and Gluconacetobacter diazotrophicus.

Preferably, said at least one other bacteral species is selected from Azospirillum lipoferum and Bacillus subtilus.

In a preferred embodiment, the plant growth-enhancing concentrate and composition comprises Herbaspirillum seropedicae, Azospirillum lipoferum and Bacillus subtilis bacteria.

The plant growth-enhancing composition may be suitable for foliar or soil application. In embodiments relating to foliar application, the plant growth- enhancing composition preferably comprises a surfactant.

In a third aspect, the invention provides a method of enhancing plant growth including the step of applying the plant growth-enhancing composition of the second aspect to a plant or to soil.

Preferably, the method includes rehydrating the concentrate of the first aspect in said liquid medium to thereby produce the composition of the second aspect.

In a fourth aspect, the invention provides a plant growth enhancing kit comprising a bacterial plant growth-enhancing concentrate and a container for rehydration of the concentrate, wherein the concentrate is provided in another container of reduced dimensions located inside said a container for rehydration of the concentrate.

Preferably, the bacterial plant growth-enhancing concentrate or composition is according to the first and second aspects, respectively. In one embodiment, the kit further comprises a surfactant, as hereinbefore described.

In a fifth aspect, the invention provides a method of determining the level of bacterial inoculation and/or colonization of a plant, said method including the step of detecting fluorescence emitted by a fluorescent molecule applied to a plant or a part thereof, wherein the plant or part thereof has been inoculated and/or colonized by bacteria.

Suitably, the method of this aspect correlates a level of detected fluorescence with a level of bacterial inoculation and/or colonization of the plant or plant part.

In a preferred embodiment, for bacterial inoculation and/or colonization of the plant or plant part, the bacteria are administered to the plant in a composition according to the second aspect.

Suitably, the plant of the aforementioned aspects is a crop plant.

Preferably, according to the aforementioned aspects the plant is of the Poaceae (formerly known as Graminae) family. Preferably, the plant is a cereal or sugarcane.

Other crop plants contemplated by the invention include potatoes, carrots, lettuce, canola, tomatoes, cotton and pineapples, although without limitation thereto.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

DETAILED DESCRIPTION

In certain aspects, the invention provides a plant growth-enhancing concentrate and composition comprising Herbaspirillum seropedicae bacteria and at least one other bacterium that is capable of enhancing plant growth and/or nitrogen fixation. Preferably, the bacteria are endophytes of any crop plant inclusive of grass crops such as cereals (e.g rice, wheat, barley, rye, oats, sorghum, corn) and/or sugarcane. Other crop plants contemplated by the invention include potatoes, carrots, lettuce, canola, tomatoes, cotton and pineapples, although without limitation thereto. More particularly, the invention provides a particular combination of Herbaspirillum seropedicae, Azospirillum lipoferum and Bacillus subtilis bacteria that upon administration to plants and/or soil enhances plant growth and may reduce the need for nitrogen fertiliser by up to 50% without significant reduction in yield.

Suitably, in a broad form the concentrate or composition comprises Herbaspirillum seropedicae bacteria and at least one other bacterial species which upon administration to a plant or to soil, enhances plant growth.

Preferably, the at least one other bacterial species is of a genus selected from: (i) Azorhizobium; (ii) Azotobacter; (iii) Azoarcus; (iv) Clostridium; (v) Azospirillum; (vi) Bacillus; and (vii) Gluconacetobacter.

In particular embodiments, the bacterial species in (i)-(vii) are selected from Azorhizobium caulinodans, Azotobacter chroococcum, Azotobacter caulinodans, Azotobacter vinelandii, Azoarcus indigens, Azospirillum lipoferum, Bacillus subtilis and Gluconacetobacter diazotrophicus.

Preferably, the at least one other bacterial species is of a genus selected from Azospirillum and Bacillus.

In a preferred embodiment, the plant growth-enhancing concentrate and composition comprises Herbaspirillum seropedicae, Azospirillum lipoferum and Bacillus subtilis bacteria.

It will be appreciated that in the context of the bacterial species disclosed herein, the composition or concentrate may comprise one or more strains of the said bacterial species.

The concentrate preferably comprises a total 10 ⁸ -10 ^u , or more preferably 2 x 10 ⁹ - 2 x 10 ¹⁰ , CFU bacteria per gram (g). Preferably, the composition is suitable for application at a rate of 2.5 g (i.e. of original concentrate) per hectare (ha) or 1 g per acre of plants or soil for intended use. For example, the concentrate may comprise 10 ¹⁰ CFU bacteria per gram, wherein 2.5 g or 2.5 x 10 ¹⁰ CFU bacteria are suitable for administration as a diluted composition to 1 ha, 25 g or 2.5 x 10 ¹¹ CFU bacteria are suitable for administration to 10 ha or 1 g or 10 ¹⁰ CFU bacteria are suitable for administration to 1 acre.

Suitably, the bacteria in the concentrate have been cultured, fermented and then freeze-dried to facilitate storage and subsequent rehydration. Preferably, the concentrate further comprises one or more agents that assist storage and/or rehydration. The one or more agents that assist storage and/or rehydration may comprise protein(s) and/or carbohydrate(s) inclusive of sugars and sugar alcohols. In a particular embodiment, the one or more agents comprise milk powder and glucose. Typically, the mass ratio of milk powder and glucose is about 1 :1. Preferably, the mass ratio of milk powder and glucose to freeze-dried bacteria is about 1 :1.

The plant growth-enhancing composition may be suitable for foliar or soil application. In embodiments relating to foliar application, the plant growth- enhancing composition preferably comprises a surfactant. The surfactant may facilitate application of the composition to a plant leaf surface and/or assist the applied bacteria to enter stomata and/or internal plant cavities. This may be particularly useful in embodiments where the plant leaf comprises a thick cuticle.

Preferably, the surfactant is an organosilicone such as a siloxane, inclusive of polyether siloxanes including a polyether modified polysiloxane (e.g a polyether modified trisiloxane). Suitably, the surfactant is provided "neat" but it is also possible that a diluted surfactant could be provided.

In some embodiments, the surfactant may further comprise trace elements UV protectants or stimulants to enhance colonisation, multiplication and plant growth promoting effects. Non limiting examples of trace elements include molybdenum, cobalt and iron. Non-limiting examples of stimulants include simple such as simple sugars such as maltose.

In use, the composition is applied to soil or leaves in a further diluted form in from a delivery apparatus such as a crop spraying apparatus, drip irrigator, water injector, backpack sprayer, centre pivot application, boomspray, spray bottle, watering can or the like.

Preferably, in embodiments where a surfactant is provided for plant (i.e foliar) administration, the surfactant is added to the diluted composition in the spraying apparatus (e.g in a tank or other liquid storage vessel of the spraying apparatus).

Suitably, the concentrate is diluted according to whether surfactant is to be included and/or whether the composition is to be used for foliar or soil administration. Rates of application are generally lower for compositions comprising a surfactant. This would typically be about 50-100 L per hectare. Typically, for foliar administration 20-100 mL of the composition is diluted to achieve 100 L administered per hectare, or preferably about 50 mL composition per 100 L administered per hectare. Preferably, 20-100 mL of the surfactant is included per 100 L or more preferably about 50 mL per 100 L. Rates of application are generally higher for compositions not comprising a surfactant. This would typically be about 300-1000 L per hectare.

Typically, for foliar administration 5-50 mL of the composition is diluted to achieve 500 L administered per hectare, or preferably about 20mL per 500 L (without a surfactant).

Typically, for soil administration 10-150 mL of the composition (without a surfactant) is diluted to achieve 300 L administered per hectare, or preferably about 30 mL per 300 L (without a surfactant).

In another aspect the invention provides a kit comprising the concentrate and a container suitable for storage, transport and rehydration of the concentrate to produce a composition administrable to plants and/or soil. Suitably, the concentrate is in a rehydratable, freeze-dried form packaged in the container. Preferably, the container is a water-proof, flexible pouch having a base that allows the pouch to "stand-up" when adding a rehydrating medium such as non-chlorinated water. For storage and transport, the container also suitably contains another container of relatively reduced dimensions (e.g a sachet) comprising the freeze-dried bacteria and preferably the one or more agents such as glucose and milk powder, as hereinbefore described, to assist with the rehydration of the freeze-dried bacteria. Suitably, in embodiments where a surfactant is provided, it is packaged in a separate sachet. Suitably, the kit further comprises instructions for use, typically in the form of a leaflet, or alternatively printed on the container or sachet(s).

To facilitate storage and transport, the container (e.g the flexible pouch) and the other container(s) comprising the freeze-dried bacteria and, optionally the surfactant, are made of a flexible, synthetic material (e.g metalized foil plastic) and are of minimal thickness so that the kit can be packed "flat" and thereby readily transported and stored while occupying minimal volume, particularly compared to a non-flexible, cylindrical container. This reduces storage and transportation costs.

In particular embodiments, the kit may be in a form suitable for rehydration of the concentrate whereby the composition is administrable to 1 acre, 1 ha, 5 ha or 10 ha or plants and/or soil, although without limitation thereto.

Also provided is a method for monitoring administration of the composition to plants. More particularly, the method detects the ability of the bacteria in the composition to inoculate or colonize the plant. Preferably, the method is performed on a particular plant or group of plants to initially determine the ability of bacteria in the composition to inoculate or colonize the plant before the composition is applied en masse. The method may also facilitate determining which plant parts {e.g. leaves, stems, petioles) or surfaces or structures thereof (e.g. leaf upper epidermis, leaf lower epidermis, stomata etc) that are best inoculated or colonized by the bacteria.

According to the method, there is a direct correlation between the amount of fluorescence detected and the concentration of bacteria in the plant after administration of the composition. Therefore, a relative level of fluorescence may correlate with a level of endophytic colonisation. In principle, a skilled person may be able to accurately correlate florescence detected from the plant and endophytic bacterial colonisation (e.g as CFU/g plant material). This may enable the skilled person to calculate an appropriate rate of application of the composition to a particular plant type depending on the level of bacterial colonization or inoculation determined by the method for that plant type.

In one particular embodiment, the method may be suited for determining whether use of a surfactant is optimal for administration of the composition to plant foliage.

In one embodiment the detected fluorescence may be emitted by a molecule that binds bacteria non-specifically, such as a fluorescein or other fluorescent compounds that binds biological material such as bacteria. An example is fluorescein sodium (biological stain CI 45350), which is preferably used at 1 g/100 mL. Other fluorescent compounds that may be useful according to the invention may be found in publications such as Conn's Biological Stains 10 ^th Ed. Bios Scientific Publishers, London UK, for example.

In an alternative embodiment, the detected fluorescence may be emitted by a molecule that binds bacteria specifically. Suitably, the molecule is an antibody that binds a bacterium as hereinbefore described. The antibody may be conjugated with a fluorescent molecule or may be detectable by a secondary antibody conjugated with a fluorescent molecule. By way of example, the antibody may be a monoclonal or polyclonal antibody which binds Herbaspirillum seropedicae, Azorhizobium caulinodans, Azotobacter chroococcum, Azotobacter caulinodans, Azotobacter vinelandii, Azoarcus indigens, Azospirillum lipoferum, Bacillus subtilis or Gluconacetobacter diazotrophicus. It will also be appreciated that antibodies to specific bacteria may be used in combination according to the method. Detection of fluorescence emitted by the molecule may be performed by any fluorescence detector known in the art. Preferably, the fluorescence detector is a portable or hand-held detector that can be used in the field to provide rapid assessment of the level of bacterial inoculation or colonization of a plant. One particular example of a hand-held fluorescence detector is a SARDI™ UV light- based detector from Croplands.

In one particular embodiment, Bacillus spores may be detected by terbium fluorescence to determine successful colonisation of the leaf surface and/or internal colonisation post-application.

So that preferred embodiments of the invention may be fully understood and put into practical effect, reference is made to the following non-limiting Examples.

EXAMPLES

Example 1

Foliar administration of bacterial composition with surfactant

In this embodiment, the concentrate is for foliar application to 1 acre (~0.40 ha). The concentrate comprises freeze-dried Herbaspirillum seropedicae, Azospirillum lipoferum and Bacillus subtilis bacteria at about 2.5 x 10 ¹⁰ CFU (total) bacteria per hectare (ha). The freeze-dried bacterial concentrate is provided in a sachet within a flexible, stand up pouch that can be used for reconstituting the concentrate. The organosilicate surfactant is provided in a separate sachet.

1. Remove the sachet comprising the freeze-dried microbial concentrate from the stand up pouch and fill the pouch with 100 mL of non-chlorinated water.

2. Add the microbial contents of the sachet to the 100 mL water.

3. Mix thoroughly.

4. Allow the composition to stand for 5 minutes and use within 24 hrs.

5. Add the composition to the storage tank of the spraying apparatus and add further water to achieve a desired application rate (see Table 1).

6. The organosilicate surfactant is finally added to the diluted composition in the tank. A preferred application rate (per hectare) is in Table 1.

Example 2

Foliar and soil administration of bacterial composition without surfactant

In this embodiment, the composition is for application to 1 ha. The concentrate comprises freeze-dried Herbaspirillum seropedicae, Azospirillum lipoferum and Bacillus subtilis bacteria at about 2.5 x 10 ¹⁰ CFU (total) bacteria per hectare (ha). The freeze-dried bacterial concentrate is provided in a sachet within a flexible pouch that can be used for reconstituting the concentrate.

1. Remove the sachet comprising the freeze-dried microbial concentrate and fill the stand-up pouch with 50 mL of non-chlorinated water.

2. Add the microbial contents of the sachet to the 50 mL water.

3. Mix thoroughly.

4. Allow the composition to stand for 5 minutes and use within 24 hrs.

5. Add the composition to the storage tank of the spraying apparatus and add further water to achieve a desired application rate (see Table 2). A preferred application rate is in Table 2.

For liquid inject application, the composition is administered to moist soil directly on top of planted seeds.

Field trials using the composition described in Examples 1 and 2 have shown that wheat and potatoes displayed enhanced growth over a 4 month period as measured by comparing plant biomass during the vegetative stage and monitoring applied nitrogen fertiliser efficiency be comparing tuber weight and grain yield (kg ha) respectively. Under ideal application and plant growth conditions nitrogen fertiliser can be reduced by up to 50% without significant reductions in yield. This equates to reductions between 30-60kg of nitrogen per hectare. Yield increases between 5-15% without the reduction of nitrogen have also been witnessed.

Similar field trials are currently underway with canola, tomatoes and sugarcane.

It will be appreciated from the foregoing that the invention at least partly ameliorates or addresses one or more deficiencies of the prior art, or at least provides a commercially useful alternative. In this regard, the invention provides a microbial composition that enhances the growth of plants, including those of the Poaceae family. The invention also improves the ability of microbial components of microbial compositions to inoculate or colonize plants, including those of the Poaceae family. The invention also provides an improved packaging system for microbial compositions. Furthermore, the invention provides an improved method of determining the level of bacterial inoculation and/or colonization of a plant.

Throughout the specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated without departing from the present invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.