AU2007201792A1 - A release composition and method of preparation - Google Patents

Description

23/04 2007 16:41 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 004 e c

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicants: AgResearch Limited Invention Title: A RELEASE COMPOSITION AND METHOD OF PREPARATION The following statement is a full description of this invention, including the best method for performing it known to us; COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:41 FAX 61 3 92438333 GRIFFITH HACK -IPAUSTRALIA 0005s

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2 TITLE: A RELEASE COMPOSITION AND METHOD OF PREPARATION TECHNICAL FIELD SThe present invention relates to a release composition and method of preparation. More specifically the present invention relates to a composition preferably of a type that allows the release of a compound of biological materials contained within the composition once the composition is placed in water or other solvent 0 C BACKGROUND ART For the purpose of this specification the term "biological material" is used to encompass, but o is not limited to, any or all of the following: a bio-inoculant, a micro-organism, biological cells, a part or parts of biological cells, pharmaceuticals, enzymes, hormones, proteins and other bio-chemicals, unstable compounds and compositions (both biological and nonbiological); and a combination of these.

Two known problems are associated with the industrial or agricultural application of biological materials. Firstly, there is the difficulty of maintaining the biological materials in a viable state until they are used or required, and especially during the period in which they may be incorporated in a release mechanism for delivery of the biological material Secondly, there may be a need for further storage before the release of the biological material, once it has been applied or distributed to the intended substrate. The delay of release can be for a number of reasons. For example: the composition may need to be stored after manufacture so as to be ready when needed (convenience factor); there may need to be a time delay to the point at which release of the biological material starts, so that the material is released at the intended time for the intended purpose.

In this respect, an example is the release of a hormone for spring growth, intended for consumption by young steers or calves when they start grazing. The delay can also be intended to engender a weedicide, herbicide or insecticide with long-term action after the immediate release into the substrate.

1 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:41 FAX 61 3 92438333 GRIFFITH HACK IAUSTRALIA RooO

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For the purposes of this specification the term "substrate" is used to encompass, but is not Slimited to, agricultural, horticultual, forestty or other commercial substrates, such as grasses and crops, soils (etc); water, waste water, skins of animals and tissues of animals; and solids such as sands and gravels.

There have been numerous solutions disclosed for encapsulating products for fast release of o compounds to substrates.

o 10 An example of such a granule can be found in US patent No. 6087306. This patent discloses a granule, suitable for aqueous spray application, after mixing of the granules with waterin a tank, and application through a standard sprayer. The granule incorporates a wetable, dispersible, or water-soluble granule, with an agrochemical or other ingredient suitable for application by aqueous spray. The non-ionic, or predominately non-ionic, water-swellable gums and polymers are disclosed as including microbial polysaccharides such as dextrn, gellan gum, and xanthan gum, along with other polysaccharides. These gums and the active ingredient are combined along with an inert filler and extruded as a paste. This paste is dried and the result formed into granules in an extruder. The inert material is preferably waterswellable. The active ingredient is disclosed as a herbicide, insecticide, nematocide, fungicide or plant growth regulator. A wetter or other adjuvant is optionally added.

US Patent No. 4859377 discloses the use of starch to encapsulate a release granulation. The starch is used substantially as an agent without cross-linling. This also discloses, however, that the starch needs to be treated in order to ensure that it is insect digestible, such that the biological agent being carried within the starch capsule reaches the desired destination.

US Patent 4563344 discloses elements for both quick and slow release pesticides. The biological chemical is enclosed in a capsule incorporating peat, fine and coarse chaff and the woody portions of concobs. The combinations and proportions are combined in a predetermined manner which affect the rate of release.

2 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:42 FAX 61 3 92438333 GRIFFITH HACK -*IPAUSTRALIA 0007 0 O US Patent 4434231 discloses preparation of pellets which incorporate a bio-matrix gel, by the addition of crushed silica which is then homogenised, The resulting pellets are 3 mm in diameter, 33 mm in length and cylindrical in shape. However, there is no disclosure of n whether the resultant pellets are fast acting or slow acting or how they breakdown or are activated to break down.

However, all the above disclosed methods of conveying a biological agent to the soil or to a Ssubstrate (as herein before defined) disclose disadvantages: the matrix incorporating a biological material needs to be especially treated (as in US 4859377) in order to be adapted to break down at the desired end point and time (for example, inside an insect). Certain types of encapsulating material will only incorporate chemicals, not all types of biological materials (US 4563344). Some of the compositions are disclosed as being most appropriately applied in an aqueous spray for chemical insecticides like Lambda-cyhalodhrin and Pirimicarb and thus may not be useful for biological materials. (US 6087306). Thus many more steps are required to get the biological material to the intended substrate. This in turn increases the inconvenience and cost of application of the biological material.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION 3 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:42 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 1008 4 0 0 According to one aspect of the present invention there is provided a method of Spreparation of a bio-degradable composition containing microbiological material, wherein the microbiological material in the composition has less than one log loss in M viability when stored at room temperature for a time period of at least I month and I s wherein the composition is stored in an environment with no more than trace amounts of water comprising the steps of.

,I preparing a bio-matrix gel by mixing together i. at least one microbiological material containing bacteria or fungi or o both with, 10 ii- at least one polysaccharide material selected from xanthan gum, 0acacia gum, guar gum, gellan and, starch; and, (1 iii. adding oil, water or aqueous solutions of sodium hydroxide or combinations thereof to the mixture of and (ii); preparing a dry powder mixture comprising: talc; diatomaceous earth; bentonite; or combinations thereof, mixing the bio-matrix gel of step and the dry powder mixture of step (b) to form a bio-degradable homogenous dough composition; and then moulding or extruding the composition to form pellets before the cell concentration of the microbiological material reduces below one log loss in viability; and wherein, on mixing with aqueous solution, the composition releases the microbiological material into the aqueous solution.

According to a preferable aspect of the present invention there is provided a process for the preparation of a release composition as described above, wherein said biological material is combined with a chemical composition.

Advantageously, said organic material may be selected from agricultural materials or waste agricultural materials, for example: corn cobs; chaff; straw. Advantageously, said inert compounds may include peat and sands.

The present invention provides a process for the preparation of a release composition as described above and preferably includes a further step after step as follows: (ci) mixing the composition formed in step with at least one clay in powdered form.

N.%ltbn eClms iffMSD>((l^!!W 97B.AIre ~istMT97&AUSp wJJoaSa Z07.4-11.doc ISKJWQ7 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:42 FAX 61 3 92438333 GRIFFITH BACK -+IPAUSTRALIA 2 009 0 The present invention provides a process for the preparation of a release composition as described above and preferably includes a firther step, after step as follows: (cii) repeating steps to one or more times with a different bio-matrix and/or a different biological material; and mixing the homogenous mix resultant C' from each step to form a homogenous dough mixture.

The pellets or granules may have a size of 0.1 20 an diameter. The pellets may be up to 30 mm long.

N 10 Advantageously, the pellets resulting from step may be in the range of 0. 1 mam to mm in diameter, and up to 10 mm in length.

According to a preferable aspect of the present invention there is provided the method of preparation of a slow release or delayed release composition substantially as 1s described above wherein the method includes a further step of drying the composition at room temperature to form a dried composition with a water content of between 15 to by weight.

In the Applicant's experience, a slow release composition is bio-stable in that until the composition comes into contact with substantial amounts of water, it is both bio-stable and thermo-stable. However, once water is introduced the matrix breaks down slowly.

In pure water it takes at least 6 hours for the matrix to swell and breakdown. More preferably this process of breakdown takes 24 to 48 hours after which period, a gentle disturbance will complete collapse of the structure.

It will be appreciated by those skilled in the art that the release rate in soil may vary to that in water. It will also be appreciated that a disturbance in soil would occur from earth forms, larvae of soil dwelling organisms, livestock or similar such disturbances.

In practice, a combination of these factors causes gentle disturbances that collapse the matrix structure once combined with moisture and/or other environments.

It is to be noted that no additional drying of the composition of step is required where a liquid bio-matrix is used.

3s In the case of a fast release mixture, the granules and/or pellets formed are bio-stable, in that until they come into contact with substantial amounts of water, they are both biostable and thermo-stable. However, once water is introduced the structure of the N. 6'4 ao=mur m Ib$11000"4 a 9 =29T4 B97A isA Pe .AIJ.95 ificaWio 2007-4-13.dw 9t04/07 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:43 FAX 61 3 92438333 GRIFFITH HACK -*IPAUSTRALIA la010 O6 0 cgranules/pellet breaks down rapidly, within minutes, to release the biological material.

This rapid breakdown optimally occurs in less than one minute.

M In the case of fast release compositions, it has been found that the pellets break down faster than composition formed in step of the above process.

According to a further preferable aspect of the present invention there is provided a method of preparing a slow release or delayed release composition substantially as o described above wherein said composition is a thermo-stable gel and the composition is o capable of storage of the biological materials for a period of time. On contact with osubstantial amounts of water, the composition slowly releases the biological material.

The liquid bio-degradable bio-matrix can be obtained in a number of ways or arrived at through a number of methods of.preparation. An example of this is the use of acacia gum mixed with distilled water and agitated; and the further addition of concentrated biological material- This mix can be left to stand for up to three hours before being mixed with the preparation of step Other alternative liquid bio-matrixes which are also bio-degradable and/or thermostable can be used. For example the liquid bio-matrix disclosed in specification of US Patent No. 5292507.

The concentration of such biological material at the end of step is hereafter referred to as the "cell concentration". Preferably, the cell concentration is in the range 10 5 cells to 1012 cells more preferably in the range of 108 to 1012 cells more preferably in the range of 109 to 10 cells Preferably, the biological material may be present in step in a broth, or on a growing medium.

According to an optional aspect of the present invention there is provided a method of preparing a slow-release or delayed release composition substantially as described above wherein the biological material includes: a pesticide; a viricide; a bacteriacide; a fungicide; and a combination thereof.

According to a further optional aspect of the present invention there is provided a method for producing a thermo-stable biodegradable medium for storage of biological materials wherein the biological material is a vaccine selected from: a live vaccine; an oral attenuated vaccine; an encapsulated mycobacterium vaccine; and a combination ?J*{etoumass €tern ifaooo' l 9pqBsg A W0 97 L \i9P AU rgitiiein 107--I.d 0 e 19M4167 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:43 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA Ill0 7 0.1 thereof. Examples of the vaccine include Bacille Calmette and Guerin For the purposes of this specification, the term "storage" means a stability of better than cn

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5 0 with respect to the cell concentration of the biological material. That is, more than C. s 50% of the cells (if cells are the biological material) are viable at the end of the storage period; or more than 50% of the non-living material is viable at the end of the storage l period. Advantageously, LT5 0 may be achievable after two months, four to six months, or 12 to 18 months.

0 For the purposes of this specification, the term "thenno-stable" means a range of o temperatures in which the combination of bio-polymer and biological material is stable.

O This temperature range is 4°C to 40°C, and preferably between 5°C to 30 °C.

According to a preferable aspect of the present invention there is provided a method of is preparing a slow-release or delayed release composition substantially as described above wherein the biological material is a micro-organism. Said micro-organism may be selected form the group Serrafia, Psudomonas, Xanthomonas, Rhizobtum. Most preferably the micro-organism is Serratia entomophila.

According to another preferable aspect of the present invention there is provided a slow-release or delayed release composition incorporating a biodegradable thermostable bio-matrix with a biological material therein, said composition being formed by the above described method.

According to a fitrther preferable aspect of the present invention there is provided a fast release composition, suitable for application to a substrate (as defined above), the composition being produced by steps to or steps to (ci) and (cii) of the method as described above.

According to a further aspect of the present invention there is provided a method of producing a fast release composition as described above wherein the steps (cii) and the step of drying the composition occur a substantial time after the immediately preceding step. Optimally, the steps (cii) and drying the composition can occur immediately before the composition is applied to a substrate.

According to a preferable aspect of the present invention there is provided a composition, in granular form, produced in accordance with steps to or steps (a) NB 9 p 9 ifcmio 2001-13 Anc IP)0W9D COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:43 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA [i 012 0 0 ~to (ci) and (cii) of the method of preparation as described above, characterised in that to the composition can be added one or more clays or/and other dry powders or/and sufficient water, and that the composition is capable of being formed into pellets of the type described above.

ci According to further preferable aspect of the present invention there is provided a "I release composition, produced in accordance with steps to (ci) of the method of r^ preparation as described above.

0 C 10 According to another preferable aspect of the present invention there is provided a Sslow-release composition in the form of a dough incorporating a biodegradable thermno- O stable bio-matrix and biological material, produced by the steps to (ci) and (cii) of the above described method, wherein said dough can be passed through a pelletiser and dried.

According to a further preferable aspect of the present invention there is provided a bulk dough release composition produced in accordance with steps to (ci) and (cii) of the method of preparation as described above, and wherein said dough is processed through a pelletiser to form pellets.

According to a further preferable aspect of the present invention there is provided pellets produced in accordance with steps to (ci) and (cii) and the step of drying the composition of the method described above.

Preferably, the pellets are of a size that can easily be drilled into soil or other substrate, either alone or in combination with seeds.

Optionally, the bio-matrix of step will be between 0-50% water by weight. It will be appreciated that the powdered mix after step of the method, or the dough formed after step (cii) of the method, is also suitable for storage of the biological material until it is required to be applied to the substrate.

Optionally, the release composition can be used to stabilize biological material for transportation and/or storage and/or delivery.

N:4rlbcrmmSCucc tMSOOO-4I99497sn AU' 4t.IpE 4971AU SzctifWicin 007-4-13.doe 19/t7 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:44 FAX 61 3 92438333 GRIFFITH HACK 4IPAUSTRALIA 013 9 C0 According to a further aspect of the present invention there is provided the use of the release component for delivery of biological material via a spray for application to plants and/or animals, characterised in that the biological material includes an active M* ingredient to be sprayed over plants and/or animals.

ci According to another aspect of the present invention there is provided a spray solution c,1 which includes at least one release composition.

1 According to a still further aspect of the present invention there is provided a method of inoculating a plant seed, cutting, or part thereof with microbiological material including othe steps of: 0 selecting at least one microbiological material to be used as an inoculant; preparing the composition by the method as claimed in any one of claims I to 15 using the microbiological material of step adding the composition of step to water and mixing to release the microbiological material into the solution forming an aqueous solution; and soaking the plant seed, cutting, or part thereof in the aqueous solution to allow the microbiological material to coat the plant seed, cutting, or part thereof.

According to a further aspect of the present invention there is provided a method of inoculating a plant seed with a biological material substantially as described above, said method preferably includes, a further step after step of adding a powdered compound to the composition, said powdered compound being selected from the group: a second micro biological material, a dried and powdered composition, a dried and powdered bio-polymer matrix containing at least a second micro biological material, a chemical, and a combination of these.

Preferably, the plant seed can be dried at room temperature before drilling or seed broadcast. Preferably, more than one inoculant may be used in step above, each inoculant being for a different purpose. As the bio-matrix is thermo-stable and biostable, the seeds need not be drilled or sown immediately after the inoculation process.

According to a further aspect of the present invention there is provided seed inoculated by the method as described above.

N 5M'db re C- vtWllD.4 9M)P99 $Ar pe4 P4 .AU Spedllcaiiot20T74-1.da 19/04A7? COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:44 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA I0014 0 CN According to a further aspect of the present invention there is provided a composition p for application to a substrate (a hereinbefore defined) wherein said composition includes: n one or more fast release compositions as produced by a preparation method s described above; and one or more slow release compositions as produced by a preparation method described above.

o BEST MODES FOR CARRYING OUT THE INVENTION 0 Example I A slow release gel is made with xanthan gum as the bio-polymer and Serratia entomophila as the biological material. The cell concentrations are set out in Table 1.

is To 5 grams of dry xanthan gum is added 5 grams monounsaturated oil, The mixture is agitated at room temperature for between 5-10 minutes to form a suspension.

grams of the micro-organism concentrate is added to the suspension. The mix is agitated for a further 10 minutes at room temperature. The result is a gel matrix.

Equal portions of diatomaceous earth and talc are mixed to form 200 grams of powder.

To this powder is added the 100 grams of gel. This can be done, for example, by drying and crumbling the blend of the gel and diatomaceous earth and talc, or by other known means.

THE NEXT PAGE IS PAGE 12.

COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:44 FAX 61 3 92438333GRFIhHCIAUTLA l01 GRIFFITH HACK 4 IPAUSTRALIA Q015 en ci To 300 grams of this mixed powder is added 27 grams of bentonite and up to 180 grams of distille water. This mixture is homogenised and forms a dough. The, mixtur is passed through a pelletiser (Of known type) and/or a die to formn pellets of predetermined, size and thickness, These pellets are then air dried to between 10-40% moisture content.

The results of six tests using the method of example 1 ane shown in table 1 Meow. A comparison is shown of the survival of Sen-adia entomnophila in broth at 200C.

TABLE 1 Exaimple 1 Vt concentration eca ge Surivival 1 mouath cf-a e surviva S urvival 2 monfth 4 moanths au' e ug' survival 6f moth LT4 days 6OXlO' 2.72x10 9 U6x1 5.101 L52X10 8 60-120

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6 1 63 64 213 I I.32xI3 E.98x10 9 3-05x10 9 1.23x10' 6-SlxlO' 120-180 2.77x1'694x0' 753xI0' 4.42,406 6.6891(0' 60-12 3-53x10 9 4.59xl0' 12410 4.98x10 9 3.87x]0 9 ;>180 8.2540' 3.2x109 1-75X1C9 3.78X0' L.7SxiO >180 4.65 x W0 3.72xc1O9' 2.04x10' -165 Compartion Example 2 A slow release gel is made with xanthan gum as the bio-polymet and Serrtzta entomophila as, the biological material. The el-l concentrations are set out in Table 2.

COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:44 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA zot61

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2 To 7.5 grams of dry xanthan gum is added 42.5 grams distilled water. The mixture is agitated at room temperature for between 5-10 minutes to form a suspension. Alternatively, a C) 50% solution of xanthan gum medium may be used for the gel.

l 5 50 grams of the micro-organism concentrate is added to the suspension. The mix is agitated for a further 10 minutes at room temperature. The result is a gel matrix.

0 Equal portions of diatomaceous earth and talc mixed to form 100 grams of powder. To this Spowder is added 100 grams of gel. This can be done, for example, by drying and crumbling the blend of the gel and diatomaceous earth and talc, or by other known means.

To 200 grams of this mixed powder is added 20 grams of bentonite and between 75-95 grams of distilled water. This mixture is homogenised and forns a dough. The mixture is passed through a pelletiser (of kown type) and/or a die to form pellets of predetermined size and thickness. These pellets are then air dried to between 10-40% moisture content.

The pellets, depending on the die, may vary in size from 0.1-20 mm in diameter and from 0.1-20 mm in length. Preferably, if the pellets are to be drilled, a diameter of less than 3 mm is used. The pellets are stored in the absence of moisture at room temperature, until required.

Data for the survival of micro-organisms in a slow release pellet form over a period of months from 1-6 is shown in Table 2.

Alternatively, the powdered mix before the addition of the bentonite, or the dough after the addition of water, may be stored in the absence of moisture. The additional steps described above can be performed in a one or two stage process, depending on when the pellets or the composition is needed. Thus the next step can be separated in time from the immediately preceding mixing step and also from the step of addition of water. Alternatively, these steps can be performed immediately one after the other, and immediately before the pellets are to be used.

13 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:45 FAX 61 3 92438333 GRIFFITH HACK 4IPAUTSTRALIA lih(17 0 0 (i SThe pellets, if small, can be mixed with seed and passed through a drill in known manner and directly drilled into the soil. Altenatively, granules or pllets from the composition may mc be broadcast in known manner to a substrate of plants or soils or a combination thereof.

TABLE 2 Example 2 Sanmple Intial Snrvdal- Survival Survival -Survival LT- Concentraton 1 month 2 aonths 4 nmaths 6mouths days eN ge efu r dug 4 ia O 14 1.46x10' 0 2.63x10 4.76x10 9 2.76x10' 7.90x1l 60-120 Example 3 A slow release gel is made with guar gum as the bio-polymer and Serratia entomophila as the biological material. The cell concentrations are set out in Table 3.

To 5.6 grams of dry guar gum is added 0.5 grams of 0.5M sodium hydroxide, The mixture is agitated at room temperature for between 5-10 minutes to form a suspension.

100 grams of the micro-organism conceatrate is added to the suspension. The mix is agitated for a further 10 minutes at room temperature. The result is a gel matrix.

Equal portions of diatomaceous earth and talc are mixed to form 150 grams of powder. To this powder is added 106.1 grams of gel. This can be done, for example, by drying and crumbling the blend of the gel and diatomaceous earth and talc, or by other known means.

To 256.1 grams of this mixed powder is added 18g of bentonite and up to 70 grams of distilled water. This mixture is homogenised and forms a dough. The mixture is passed through a pelletiser (of known type) and/or a die to form pellets of predeteamined size and thickness. These pellets are then air dried to between 10-40% moisture content.

A second sample is made by comparison without sodium hydroxide. The same method as above is used, however the quantities used are: 2.1 grams of dry guar gum added to 60 grams 14 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:45 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA a 018 e c of micwo-organism concentrate. The mix is agitated for 10 minutes at room temperature. The result is a gel matrix. A furtber 110-grams of diatomaceous earth-tal powder (mixed in equal proportions) is added to the ge matdx. After this, a further 10 grams of bentonite and 135 grams of distilled water is added This mixture is formed into a dough and pelletised as above.

The results for both treatments are shown in Table 3 below.

TABLE 3 0 Example 3 Example 4 The same composition and method was used as for Example 1, however the microbe Pseudomonarftluor2 e ce was used in the microbial concenrate.

Results were taken of the cell concentration initially and after two months. The results are shown below in Table 4.

TABLE 4 Example 4 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:45 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 0

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ci Example en A fast release composition is made by combining 20 grams of acacia gum with 30 grams of distilled water. The mix is agitated at room temperature. To this mix is added 50 grams of concentrated biological material containing Bacillus mycoides. This mixture is agitated at croom temperature and left to stand for between 1% to 3 hours at room temperature.

o A powder is formed of diatomaceous earth and talc in the ratio 50:50 (by weight). The solution of acacia gum and biological material is added to 200 grams of the powdered mix S0 and mixed thoroughly.

A second mixture was prepared using the above method. However, the quantities used were 16 grams of acacia gum, 8 grams of 0.5% yeast, 12 grams of distilled water, 36 grams of Trichoderma spores and 130 grams of a 50:50 mix of diatomaceous earth and talc (by weight).

Either mixture can either be stored at room temperature until equired.

The survival rates for the biological materials of Example 5 were tested and the results are set out in Table 5 below.

TABLE

Example Sample Organsm itl 3 months 6 months s moths LT du g g 1 cfig- 1 cu g days 186 Bacillus Mycoldes 8 .47x10 8.2Sr40 8.00x10 43x10 >240 171 Tichoderma 2.96xi 2.02x1 1.81x0 7.81x0 120-180 Example 6 A fast release composition was made by combining 2.5 grams of gellan gum with 50 grams of concentrated biological material containing Bacillus mycoides. This mixture is agitated at room temperature and left to stand for between 1 to 3 hours at room temperature.

16 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:46 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA Z 020 0

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A powder is formed of diatomaceous earth and talc in the ratio 60:40 (by weight). The en solution of gellan gum and biological material is added to 100 grams of the powdered mix and mixed thoroughly. To this resulting mixture a further 15 grams of bentonite and 100 grams of distilled water is added.

cN A second mixture was prepared using the same composition and method as used above, o however the microbe Pseudomonas fluorescence was used in the microbial concentrate.

0 10 A third mixture was prepared using the above method, however the quantities used were 3 grams of gellan gum, 9 grams of 0.5% yeast, 18-grams of distilled water, 28 grams of richoderma spores and 120 grams of a 50:50 mix of diatomaceous earth and talc (by weight), 15 grams of bentonite and 110 grams of distilled water.

Results were taken of the cell concentration initially and after various monthly intervals thereafter. The results are shown below in Table 6.

TABLE 6 Example 6 Sample Organism Initial 2 months 3 months 6 months 8 months LT rcf 1 c fU g cftig cfi g-4 days 185 Bacius 1.61x1 02x10 i-.03x15 >240 amycodes 289 Pseudamonas 1.73x10 1.86x10 fluoresence 172 Trichodsrna 12xl0 1.3x0 4.6x10 6.7x 90-180 Example 7 A fast release composition is made by combining 3.5 grams of starch with 50 grams of hot water (70°C to 80 0 The mix is held in this state for 10 minutes and then cooled to room temperature. To this mix is added 50 grams of concentrated biological material (in this 17 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:46 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 1021 0

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k example Serratia enbomophila). This mixture is agitated at room temperature and left to stand for between 1 V to 3 hours at room temperature.

A powder is formed of diatomaceous earth and talc in the ratio 60:40 (by weight). The solution of starch and biological material is added to 175 grams of the powdered mix, 26 grams of bentonite and 150 grams of distilled water, and the resulting solution is mixed thoroughly.

A similar fast relase composition is made by combining 6 grams of starch with 100 grams 0 10 of concentrated biological material (Serraia entomophila). A powder is formed of diatomaceous earth and talc in the ratio 60:40 (by weight). The solution of starch and biological material is added to 175 grams of the powdered mix, 12 grams of bentonite and 180 grams of distilled water and the resulting solution is mixed thoroughly.

The survival rates for both of the above options were tested at 2 H months, 3 months and months. The results are set out in Table 7 below.

TABLE 7 Example 7 Example 8 The success or otherwise of the biomatrix (biofungicide) in aiding toot rot disease control was compared against a control sample where no treatment occurred and a seed treated with a known chemical fungicide.

18 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:46 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 1022 0 0 The seeds chosen were pea seeds which are prone to Aphanomyces root rot. The microbe Bacillus mycoides is known to control this disease, as is the chemical fungicide used in the.

n trial as a positive control. It was expected that the positive control fungicide would have a heightened effect as the fungicide has a broad range of fungicidal properties.

NA matrix treated according to Example 6 was placed at the bottom of each drilled hole, upon rwhich the pea seed was placed.

O

The results of the test are shown below in Table 8 where the better yield of the plant relates o 10 to the lower presence of Aphanomyces root rot.

TABLE 8 Example 8 Treatent Initial Plot Pods per Pod Weight Yield of Pods Sport e- Stand plant per plant per Plot (g) Chemical 870 92.78 2.78 15.07 503.15 fungicide Control 4.0 x 10 77.22 2.59 14.37 399.62 Bofungicide 1-.5 x 10 79.44 3-26 16.86 482.20 It will be appreciated by those skilled in the art that a number of compounds, biological material or other chemicals can be added to the suspension prior to the inoculation of the plant seed. Such compounds can be for the promotion of plant growth, the promotion of growth of the animal feeding off the plant, etc.

It will also be appreciated that the suspension generated in the Example 8 above may be sprayed in known manner, to broadcast the biological material onto a substrate. Such spraying can be done either before or after planting.

It will also be further appreciated that the seeds in Example 8 above may be inoculated with the bio-matrix by soaking the seeds in the suspension prior to drilling.

19 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:46 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA Z 023

O

O

Example9 SA further expeiment to assess the success or otherwise of the bio-matrix in control of grass CC grubs at filed level was compared against a control sample where no treatment occurred and a field drilled with two separate release formulations containing Serratia entomyphila.

Serratia entomyphila is a microbe that is efficient at controlling grass grub, a common Sdisease in pasture grass.

o The above samples were compared to positive controls where a liquid, product was applied C .through a modified see drill onto the paddock 0

I

The first release solution is that produced by example 2 and the second, by example 7.

The paddock kept isolated via a fence. Presence of the disease in grass grubs was tested after 6 weeks in the case of the fast release formulation and after 10 weeks in the case of the slow release formulation. The results are shown below in Table 9. Ideally the higher the percentage of disease prevalence in the grass grub, the better the rate of success of the formulation i.e. the higher the occurrence of the disease in the grass grub relates to the presence of Serratia.

TABLE 9 Example 9 Treatment Sample Set 1 Sample Set 2 Trial Average Average Average Contro 9.2% 2-7% Drilled PasRelease 25.2% 5.0% 15.1% Drilled Slow Release 24.7% 18-4% 21.6% ~Lquid (into SR) 233% 323% 27.9% Liquid (into FR) 25.7% 9.1% 17 4% Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof.

COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23

Claims (14)

1. A method of preparation of a bio-degradable composition containing microbiological material, wherein the microbiological material in the composition has less than one log loss in viability when stored at room temperature for a time period of at least 1 month and wherein the composition is stored in an environment with no more than trace amounts of water comprising the steps of: preparing a bio-matrix gel by mixing together oi. at least one microbiological material containing bacteria or o fungi or both with, o ii. at least one polysaccharide material selected from xanthan C, gum, acacia gum, guar gum, gellan and, starch; and, iii. adding oil, water or aqueous solutions of sodium hydroxide or combinations thereof to the mixture of and (ii); preparing a dry powder mixture comprising: talc; diatomrnaceous earth; bentonite; or combinations thereof, mixing the bio-matrix gel of step and the dry powder mixture of step to form a bio-degradable homogenous dough composition; and then moulding or extruding the composition to form pellets before the cell concentration of the microbiological material reduces below one log loss in viability; and wherein, on mixing with aqueous solution, the composition releases the microbiological material into the aqueous solution.

2. The method as claimed in claim I wherein the method includes a further step, after step as follows: (ci) repeating steps to at least once more with a different bio-matrix and/or a different microbiological material; and mixing together the dough resultant from all steps

3. The method as claimed in any one of the above claims wherein the method includes a further step, after step as follows: (cii) mixing the dough formed in step with powdered bentonite and water.

4. The method as claimed in claim 3 wherein step (cii) occurs at any time after step and before cell concentration reduces below one log loss in viability 4M taieM:« re ivAlWM8 994F'4897AI ri 489774-.AU ;.li l 713 dov tJdhir COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:47 FAX 61 3 92438333 GRIFFITH HACK -IPAUSTRALIA O 22 O i,

5. The method as claimed in claim 3 wherein step (cii) occurs immediately before the composition is applied to a substrate. en

6. The method as claimed in any one of the above claims wherein the pellets have a s size of 0.1 20 mm diameter and up to 30 mm long and are of a shape selected from round, ovoid, cylindrical, spherical and rectangular.

7. The method as claimed in claim 6 wherein the pellets are 0.1 mm to 10 mm in O diameter and up to 10 mm long. 1o S8. The method as claimed in claim 6 or claim 7 wherein pellet formation occurs C immediately before the composition is applied to a substrate.

9. The method as claimed in any one of the above claims wherein the room is temperature refers to any temperature and the range 4°C to 40 0 C. The method as claimed in any one of the above claims wherein the microbiological material is selected from: Serratia, Pseudomonas, Xanthomonas, Rhizobium, and combinations thereof.

11. The method as claimed in any one of the above claims wherein, when the composition formed is introduced to an aqueous solution, the physical structure of the composition disintegrates in less than 10 minutes and releases the microbiological material into the aqueous solution.

12. The method of claim 11 wherein the disintegration time is less than one minute.

13. The method as claimed in any one of claims 1 to 10 wherein the method includes the further step of drying the composition at room temperature to form a dried composition with a water content of between 15 and 40% wt.

14. The method as claimed in claim 13 wherein the resulting dried composition on mixing with an aqueous solution disintegrates and releases microbiological material and the aqueous solution over a time period of longer than 10 minutes. The method as claimed in claim 13 or claim 14 wherein the microbiological material is Serratia entomophila. NMIMbumt\aslarent g4(9pg ,49T97A ,Spa 9 T ASP97 U aSPiOnic 2 (n2007-4-1ldc I/Af7 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23 23/04 2007 16:47 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA @026 o 23 A bidegradable compositio foed by the ethod of any one of claims I to 12. s 16. A biodegradable composition formed by the method of any one of claims to 12. S17_ A biodegradable composition formed by the method of any one of claims 13to

18. A method of inoculating a plant seed, cutting, or part thereof with microbiologicaj material including the steps of: S(a) selecting at least one bacterial or fungal microbiological material to be used as an inoculant; 10 preparing the composition by the method as claimed in any one of claims 1 Sto 15 using the microbiological material of step C adding the composition of step to water and mixing to release the microbiological material into the solution forming an aqueous solution; and soaking the plant seed, cutting, or part thereof in the aqueous solution to allow the microbiological material to coat the plant seed, cutting, or part thereof.

19. The method of claim 18 wherein the method includes a further step after step (b) and before step of: o2 (bi) adding a further powdered compound to said composition, said powdered compound being selected from the group comprising: a second microbiological material, a dried and powdered composition, a dried and powdered bio-matrix containing at least a second microbiological material, a chemical, and combinations thereof. The method as claimed in either claim 18 or claim 19 wherein the plant seed is dried at room temperature after inoculation before drilling or seed broadcast. N:.Wb Mebomer'LPatmataMgiOM9994M.Ai97VpViAP4I97AL Speificmaln2007-4-13.doee 19 COMS ID No: SBMI-07100716 Received by IP Australia: Time 16:48 Date 2007-04-23