WO2021040536A1

WO2021040536A1 - Biocontrol compositions and uses thereof

Info

Publication number: WO2021040536A1
Application number: PCT/NZ2020/050091
Authority: WO
Inventors: Stephen Reynold Ford; Michael TREADWELL; Ankita SIGDEL; Mildred Marsha ORMSKIRK; Ted KEY
Original assignee: Ecolibrium Biologicals Holdings Limited
Priority date: 2019-08-23
Filing date: 2020-08-24
Publication date: 2021-03-04

Abstract

The present invention relates to novel compositions derived from strains of Brevibacillius laterosporus, and methods of manufacturing and using such compositions, including for the biological control of pests such as insect pests and nematode pests.

Description

BIOCONTROL COMPOSITIONS AND USES THEREOF TECHNICAL FIELD

This invention relates to novel compositions derived from Brevibacillius laterosporus strains, and methods of manufacturing and using such compositions, including for the biological control of pests including agriculturally important pests such as insect and nematode pests.

BACKGROUND OF THE INVENTION

The following includes information that may be useful in understanding the present inventions.

It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

Insect pests represent a significant economic cost to modern agriculture. Current systems of agriculture often require one or a few crops or plant types to be grown over a large area. Such an ecologically unbalanced system is susceptible to insect pressure. However, even more integrated production systems, such as those more closely emulating naturally-occurring environments, are susceptible to insect pests.

Some insect pests are also harmful to animal health, including human health. For example, mosquitos are known to carry a variety of diseases, and act as vectors in the spread of disease. Control of insect vectors of disease has thus been explored as a mechanism to control the incidence and distribution of disease.

Traditionally, control of insect pests has been pursued through the use of chemical insecticides and pesticides. However, consumers are becoming increasingly concerned about chemical residues and their effects on animal and plant health, and the environment. Moreover, many insect pests are becoming resistant to pesticides and insecticides.

Biological control represents an alternative means of controlling insect pests which reduces dependence on chemicals. Such methods enjoy greater public acceptance, and may be more effective and sustainable than chemical control methods.

A wide range of biological control agents including bacteria, yeast and fungi have been investigated for use in controlling insect pests. One widely investigated genus of bacteria for insecticidal use is Bacillus.

Bacillus is a genus containing many diverse bacterial species with diverse properties, varying from detrimental to animal and plant health, to useful for insect control. For example, Bacillus thuringiensis (Bt) in particular, is a well known biocontrol agent commercially available in products such as Thuricide® and Dipel®.

However, there has been reports of insect resistance to Bt developing. See for example Tabashnik et al (1990); Baxter et al (2011); and Tabashnik et al (1998).

Accordingly, there remains a need for alternatives to existing insecticidal treatments, including existing biocontrol treatments. The B. laterosporus extracts and compositions provided herein go at least some way to meeting this need.

One object of the present invention is therefore to go some way towards overcoming one or more of the deficiencies identified above, and/or provide novel compositions from one or more B. laterosporus strains, including compositions useful as a biocontrol agent, and/or a method for producing and/or using such compositions, and/or to at least provide the public with a useful choice.

SUMMARY OF THE INVENTION

In a first aspect the invention relates to a cellular extract obtained from B. laterosporus NMI No. V12/001945 and/or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown.

In a second aspect the invention relates to a cellular extract obtained from B. laterosporus NMI No. V12/001944 and/or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown.

In a third aspect the invention relates to a cellular extract obtained from B. laterosporus NMI No. V12/001946 and/or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown.

In another aspect, the invention relates to a composition comprising a cellular extract obtained from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, and/or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is or has been grown and one or more agriculturally-acceptable carriers.

Any of the embodiments described herein can relate to any of the aspects presented herein.

In one embodiment, the cellular extract or the composition is substantially free of viable B. laterosporus NMI No. V12/001944 cells.

In one embodiment, the cellular extract or the composition is substantially free of viable B. laterosporus NMI No. V12/001945 cells.

In one embodiment, the cellular extract or the composition is substantially free of viable B. laterosporus NMI No. V12/001946 cells.

In one embodiment, the cellular extract or the composition is substantially free of protein, is substantially free of native protein, is substantially free of functional protein, and/or is substantially free of undenatured protein.

In one aspect the present invention relates to the use of a culture of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, in the manufacture of a composition suitable as a biopesticide.

In another aspect the invention relates to a method of producing a composition, such as a composition for application to a plant or its surroundings, the method comprising the steps of providing a culture of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, lysing substantially all of the cells of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, present in a sample from the culture to provide a culture extract composition, optionally formulating the culture extract with one or more agriculturally-acceptable carriers, and optionally diluting the composition prior to application.

In another aspect the invention relates to a method of producing a composition, such as a composition for application to a plant or its surroundings, the method comprising the steps of providing a composition as described herein comprising an extract of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, and optionally diluting the composition prior to application.

In a further aspect the present invention relates to a method of treating or protecting a plant or its surroundings, and/or plant derived materials, from pest infestation wherein the method comprises applying to the plant or its environment a composition comprising an effective amount of a cellular extract of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946.

According to a further aspect the present invention relates to a method of controlling and/or preventing a pest infestation characterised by the step of applying a composition comprising an effective amount of a cellular extract of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, to a surface.

In another aspect, the invention relates to a method for controlling one or more insect pests, comprising the step of applying to a plant or its surroundings or a locus at which insect pests are present a composition as described herein.

The invention further relates to methods of using the composition for the control of pests, particularly plant pests, such as insects or nematodes.

For example, the invention also relates to methods of controlling a pest population. The methods generally involve contacting the pests or the pest population with a pesticidally-effective amount of a composition as described herein. Such methods may be used to kill or reduce the numbers of target pests in a given area, or may be prophylactically applied to a locus, such as an environmental area, to prevent infestation by a susceptible pest.

In various embodiments, any of the methods described herein comprise the use of a water dispersible granule (WDG) formulation as herein described.

The invention further relates to the use of a composition of the invention for the control of one or more pests, including one or more insect or nematode pests.

The use of a composition produced by a method of the invention in the manufacture of a formulation for the control of one or more pests is similarly contemplated.

In agricultural and horticultural applications, the invention is applicable to any plant or its surroundings. Illustrative plants are monocotyledonous or dicotyledonous plants such as alfalfa, barley, canola, corn (maize), cotton, flax, kapok, peanut, potato, oat, rice, rye, sorghum, soybean, sugarbeet, sugarcane, sunflower, tobacco, tomato, wheat, turf grass, pasture grass, berry, fruit, legume, vegetable, for example, capsicum, a cucurbit such as cucumber, onion, ornamental plants, shrubs, cactuses, succulents, and trees.

In further illustrative embodiments, the plant may be any plant, including plants selected from the order Solanales, including plants from the following families: Convolvulaceae, Hydroleaceae, Montiniaceae, Solanaceae, and Sphenocleaceae, and plants from the order Asparagales, including plants from the following families:

Amaryllidaceae, Asparagaceae, Asteliaceae, Blandfordiaceae, Boryaceae, Doryanthaceae, Hypoxidaceae, Iridaceae, Ixioliriaceae, Lanariaceae, Orchidaceae, Tecophilaeaceae, Xanthorrhoeaceae, and Xeronemataceae.

In another aspect the invention relates to a plant or part thereof treated with, or to which has been applied, a composition of the invention. In one embodiment the plant or part thereof is reproductively viable, for example, a seed, bulb or cutting or other plant part capable of propagation.

In various embodiments, the extract is a cell filtrate, a supernatant, a cellular extract, or a fraction of any one or more thereof.

In one embodiment, the extract or composition comprises two or more fractions derived from a culture of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, a cellular extract thereof, or a growth media extract thereof.

In one embodiment, the extract or composition comprises a composition enriched in or to which has been added a sub-3 kDa fraction from B. laterosporus NMI No. V12/001944 or a culture thereof.

In one embodiment, the extract or composition comprises a composition enriched in or to which has been added a sub-3 kDa fraction from B. laterosporus NMI No. V12/001945 or a culture thereof.

In one embodiment, the extract or composition comprises a composition enriched in or to which has been added a sub-3 kDa fraction from B. laterosporus NMI No. V12/001946 or a culture thereof.

In one embodiment, the extract or composition comprises a composition enriched in or to which has been added a sub-3 kDa fraction from B. laterosporus NMI No. V12/001944 or a culture thereof, and/or from B. laterosporus NMI No. V12/001945 or a culture thereof, and/or from B. laterosporus NMI No. V12/001946 or a culture thereof.

In various embodiments, the extract is or has been sonicated, autoclaved, filtered, separated or fractionated by centrifugation, chromatography,

Another aspect of the present invention relates to a method of preparing a pesticidal composition, such as an insecticidal composition, the method comprising a) optionally growing a culture of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946; b) providing a cellular extract obtained from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, and/or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is or has been grown; c) admixing the cellular extract and/or composition with one or more agriculturally- acceptable carriers; to provide the insecticidal composition.

In one embodiment, the method additionally comprises lysing substantially all of the B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, present, for example, all of the B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, present in the cellular extract, all of the B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, present in the composition comprising or derived from media in which B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is or has been grown, or in the insecticidal composition.

In one embodiment, the method additionally comprises killing substantially all of the B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, present, for example, all of the B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, present in the cellular extract, all of the B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, present in the composition comprising or derived from media in which B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is or has been grown, or in the insecticidal composition.

In one example, the B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is lysed or killed by autoclaving, by UV sterilisation, by pasteurisation, by sonication, by chemical lysing or sterilisation, or any combination thereof.

In various embodiments, the composition comprises a cellular extract obtained from B. laterosporus NMI No. V12/001944, or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown, or both a cellular extract obtained from B. laterosporus NMI No. V12/001944 and a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown.

In various embodiments, the composition comprises: a) from about 1% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001944; or b) from about 1% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; or c) both a) and b) above.

In various embodiments, the composition comprises a cellular extract obtained from B. laterosporus NMI No. V12/001945, or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown, or both a cellular extract obtained from B. laterosporus NMI No. V12/001945 and a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown.

In various embodiments, the composition comprises: a) from about 1% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001945; or b) from about 1% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; or c) both a) and b) above.

In various embodiments, the composition comprises a cellular extract obtained from B. laterosporus NMI No. V12/001946, or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown, or both a cellular extract obtained from B. laterosporus NMI No. V12/001946 and a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown.

In various embodiments, the composition comprises: a) from about 1% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001946; or b) from about 1% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; or c) both a) and b) above. In various embodiments, the composition comprises a cellular extract obtained from B. laterosporus NMI No. V12/001944, or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown, or both a cellular extract obtained from B. laterosporus NMI No. V12/001944 and a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown, wherein the cellular extract, or the composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown, or both the cellular extract obtained from B. laterosporus NMI No. V12/001944 and the composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown is substantially free of viable B. laterosporus NMI No. V12/001944.

In various embodiments, the composition comprises: a) from about 1% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001944; or b) from about 1% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; or c) both a) and b) above; wherein the composition is substantially free of viable B. laterosporus NMI No. V12/001944.

In one embodiment, the composition is enriched in a sub-3 kDa fraction from a culture of B. laterosporus NMI No. V12/001944, such as a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001944, or a sub 3 kDa fraction of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown, or both. For example, the composition is enriched in components derived from a B. laterosporus NMI No. V12/001944 culture having a size below about 3 kDa. For example, a sub-3 kDa fraction from B. laterosporus NMI No. V12/001944 is added to the composition.

In one embodiment, the composition comprises: a) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001944; or b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; or c) both a) and b) above.

In one embodiment, the composition is substantially free of viable B. laterosporus NMI No. V12/001944.

In another embodiment, the composition comprises one or more of the following: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001944; b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001944; c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; or d) any combination of two or more of any of a) to c) above.

In another embodiment, the composition comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001944; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001944; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; d) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001944; or e) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; or f) any combination of two or more of any of a) to e) above.

In another embodiment, the composition comprises: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001944; and b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001944; and/or c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown.

In various embodiments, the composition comprises a cellular extract obtained from B. laterosporus NMI No. V12/001945, or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown, or both a cellular extract obtained from B. laterosporus NMI No. V12/001945 and a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown, wherein the cellular extract, or the composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown, or both the cellular extract obtained from B. laterosporus NMI No. V12/001945 and the composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown is substantially free of viable B. laterosporus NMI No. V12/001945.

In various embodiments, the composition comprises: a) from about 1% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001945; or b) from about 1% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; or c) both a) and b) above; wherein the composition is substantially free of viable B. laterosporus NMI No. V12/001945.

In one embodiment, the composition is enriched in a sub-3 kDa fraction from a culture of B. laterosporus NMI No. V12/001945, such as a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001945, or a sub 3 kDa fraction of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown, or both. For example, the composition is enriched in components derived from a B. laterosporus NMI No. V12/001945 culture having a size below about 3 kDa. For example, a sub-3 kDa fraction from B. laterosporus NMI No. V12/001945 is added to the composition.

In one embodiment, the composition comprises: a) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001945; or b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; or c) both a) and b) above.

In one embodiment, the composition is substantially free of viable B. laterosporus NMI No. V12/001945.

In another embodiment, the composition comprises one or more of the following: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001945; b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001945; c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; or d) any combination of two or more of any of a) to c) above.

In another embodiment, the composition comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001945; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

V12/001945; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; d) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001945; or e) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; or f) any combination of two or more of any of a) to e) above.

In another embodiment, the composition comprises: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001945; and b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001945; and/or c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown.

In various embodiments, the composition comprises a cellular extract obtained from B. laterosporus NMI No. V12/001946, or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown, or both a cellular extract obtained from B. laterosporus NMI No. V12/001946 and a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown, wherein the cellular extract, or the composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown, or both the cellular extract obtained from B. laterosporus NMI No. V12/001946 and the composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown is substantially free of viable B. laterosporus NMI No. V12/001946.

In various embodiments, the composition comprises: a) from about 1% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001946; or b) from about 1% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; or c) both a) and b) above; wherein the composition is substantially free of viable B. laterosporus NMI No. V12/001946.

In one embodiment, the composition is enriched in a sub-3 kDa fraction from a culture of B. laterosporus NMI No. V12/001946, such as a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001946, or a sub 3 kDa fraction of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown, or both. For example, the composition is enriched in components derived from a B. laterosporus NMI No. V12/001946 culture having a size below about 3 kDa. For example, a sub-3 kDa fraction from B. laterosporus NMI No. V12/001946 is added to the composition.

In one embodiment, the composition comprises: a) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001946; or b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; or c) both a) and b) above.

In one embodiment, the composition is substantially free of viable B. laterosporus NMI No. V12/001946.

In another embodiment, the composition comprises one or more of the following: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001946; b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001946; c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; or d) any combination of two or more of any of a) to c) above.

In another embodiment, the composition comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001946; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

V12/001946; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; d) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001946; or e) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; or f) any combination of two or more of any of a) to e) above.

In another embodiment, the composition comprises: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001946; and b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001946; and/or c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown.

In one embodiment, the extract or composition is substantially non-proteinaceous.

In one embodiment, the extract or composition is one to which has been added an undenatured proteinaceous extract or fraction from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946.

In one embodiment, the extract or composition is one which is enriched in undenatured protein from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946.

In one embodiment, the extract or composition is one which is enriched in undenatured protein from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, and enriched in a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, and/or from media in which B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is or has been grown.

In one embodiment, the undenatured protein comprises, consists essentially of, or consists of an S-layer protein or a functional fragment thereof, an Adhesin-like protein or functional fragment thereof, or an Efflux pump protein or functional fragment thereof, as described in the Applicant's co pending NZ patent application NZ 756630. In one embodiment, the undenatured protein comprises, consists essentially of, or consists of a polypeptide having the amino acid sequence of any one of SEQ ID No.s 1 to 87 as disclosed in Applicant's co-pending NZ patent application NZ 756630. In another embodiment, the undenatured protein comprises, consists essentially of, or consists of a polypeptide encoded by the nucleotide sequence capable of encoding any one of the amino acid sequences of SEQ ID No.s 1 to 87 as disclosed in Applicant's co-pending NZ patent application NZ 756630.

In various embodiments, the composition provided herein, for example, the insecticidal composition and/or the composition to be applied to control of pests, is formulated as a water dispersible granule (WDG).

In various embodiments, the water dispersible granule formulation comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001945; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

V12/001945; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; d) from about 1% to about 50% w/w one or more wetting agent; e) from about 1% to about 50% w/w one or more dispersant; f) from about 2% to about 50% w/w one or more humectant or agent to control water activity; g) from about 0% to about 50% w/w one or more protectants; h) from about 0% to about 50% w/w one or more nutrients or mixture thereof; i) from about 5% to about 80% w/w one or more filler; j) from about 0% to about 20% w/w one or more antioxidant or UV radiation protectant; k) from about 1% to about 50% w/w one or more binding agent;

L) from about 0% to about 50% one or more disintegrating agent; m) from about 0% to about 10% w/w water; n) any combination of two or more of any of a) to m) above.

In one embodiment, when the WDG formulation comprises viable B. laterosporus NMI No. V12/001945, the B. laterosporus NMI No. V12/001945 is present at a concentration of at least about 1 x 10^s cells per g, for example, about 5 x 10^s cells per g.

In one embodiment, when the WDG formulation comprises viable B. laterosporus NMI No. V12/001945, the formulation comprises one or more of the following; a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001945; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

V12/001945; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; d) from about 1% to about 50% w/w one or more wetting agent; e) from about 1% to about 50% w/w one or more dispersant; f) from about 2% to about 50% w/w one or more humectant or agent to control water activity; g) from about 0% to about 50% w/w one or more protectants; h) from about 2% to about 50% w/w one or more nutrients or mixture thereof; i) from about 5% to about 80% w/w one or more filler; j) from about 0.1% to about 20% w/w one or more antioxidant or UV radiation protectant; k) from about 1% to about 50% w/w one or more binding agent;

In one embodiment, the cellular extract obtained from B. laterosporus NMI No. V12/001945, or the composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown, comprises, consists essentially of, or consists of a sub-3 kDa fraction. It will be understood that such a fraction can be prepared by fractionating a sample to remove all components above 3 kDa, for example, by filtration through a suitable filter having a size cutoff of 3 kDa, or by size-based chromatography, or related methods.

In one example, the sub-3 kDa fraction comprises a fraction prepared by fractionating a lysed cell culture of B. laterosporus NMI No. V12/001945 together with the growth media in which it was grown, to provide the sub-3 kDa fraction.

In one embodiment, the cellular extract obtained from B. laterosporus NMI No. V12/001945, or the composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown, is enriched in components having a size below about 3 kDa. For example, a sub-3 kDa fraction is added to the cellular extract or composition.

In various embodiments, the water dispersible granule formulation comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001945; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001945; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; d) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001945; or e) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; or f) from about 1% to about 50% w/w one or more wetting agent; g) from about 1% to about 50% w/w one or more dispersant; h) from about 2% to about 50% w/w one or more humectant or agent to control water activity; i) from about 0% to about 50% w/w one or more protectants; j) from about 0% to about 50% w/w one or more nutrients or mixture thereof; k) from about 5% to about 80% w/w one or more filler;

L) from about 0% to about 20% w/w one or more antioxidant or UV radiation protectant; m) from about 1% to about 50% w/w one or more binding agent; n) from about 0% to about 50% one or more disintegrating agent; o) from about 0% to about 10% w/w water; р) any combination of two or more of any of a) to o) above.

In one embodiment, the water dispersible granule formulation comprises: a) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001945; or b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; or с) both a) and b) above.

In one embodiment, the water dispersible granule formulation is substantially free of viable B. laterosporus NMI No. V12/001945.

In another embodiment, the water dispersible granule formulation comprises one or more of the following: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001945; b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001945; c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; or d) any combination of two or more of any of a) to c) above.

In another embodiment, the water dispersible granule formulation comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001945; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

In another embodiment, the water dispersible granule formulation comprises: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001945; and b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001945; and/or c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown.

In various embodiments, the water dispersible granule formulation comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001944; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001944; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; d) from about 1% to about 50% w/w one or more wetting agent; e) from about 1% to about 50% w/w one or more dispersant; f) from about 2% to about 50% w/w one or more humectant or agent to control water activity; g) from about 0% to about 50% w/w one or more protectants; h) from about 0% to about 50% w/w one or more nutrients or mixture thereof; i) from about 5% to about 80% w/w one or more filler; j) from about 0% to about 20% w/w one or more antioxidant or UV radiation protectant; k) from about 1% to about 50% w/w one or more binding agent;

In one embodiment, when the WDG formulation comprises viable B. laterosporus NMI No. V12/001944, the B. laterosporus NMI No. V12/001944 is present at a concentration of at least about 1 x 10^s cells per g, for example, about 5 x 10^s cells per g.

In one embodiment, when the WDG formulation comprises viable B. laterosporus NMI No. V12/001944, the formulation comprises one or more of the following; a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001944; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

V12/001944; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; d) from about 1% to about 50% w/w one or more wetting agent; e) from about 1% to about 50% w/w one or more dispersant; f) from about 2% to about 50% w/w one or more humectant or agent to control water activity; g) from about 0% to about 50% w/w one or more protectants; h) from about 2% to about 50% w/w one or more nutrients or mixture thereof; i) from about 5% to about 80% w/w one or more filler; j) from about 0.1% to about 20% w/w one or more antioxidant or UV radiation protectant; k) from about 1% to about 50% w/w one or more binding agent;

In one embodiment, when the WDG formulation comprises viable B. laterosporus NMI No. V12/001944, the formulation comprises one or more of the following; a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001944; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001944; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; d) from about 1% to about 50% w/w one or more wetting agent; e) from about 1% to about 50% w/w one or more dispersant; f) from about 2% to about 50% w/w one or more humectant or agent to control water activity; g) from about 0% to about 50% w/w one or more protectants; h) from about 2% to about 50% w/w one or more nutrients or mixture thereof; i) from about 5% to about 80% w/w one or more filler; j) from about 0.1% to about 20% w/w one or more antioxidant or UV radiation protectant; k) from about 1% to about 50% w/w one or more binding agent;

In one embodiment, the cellular extract obtained from B. laterosporus NMI No. V12/001944, or the composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown, comprises, consists essentially of, or consists of a sub-3 kDa fraction. It will be understood that such a fraction can be prepared by fractionating a sample to remove all components above 3 kDa, for example, by filtration through a suitable filter having a size cutoff of 3 kDa, or by size-based chromatography, or related methods.

In one example, the sub-3 kDa fraction comprises a fraction prepared by fractionating a lysed cell culture of B. laterosporus NMI No. V12/001944 together with the growth media in which it was grown, to provide the sub-3 kDa fraction.

In one embodiment, the cellular extract obtained from B. laterosporus NMI No. V12/001944, or the composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown, is enriched in components having a size below about 3 kDa. For example, a sub-3 kDa fraction is added to the cellular extract or composition.

In various embodiments, the water dispersible granule formulation comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001944; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

V12/001944; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; d) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001944; or e) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; or f) from about 1% to about 50% w/w one or more wetting agent; g) from about 1% to about 50% w/w one or more dispersant; h) from about 2% to about 50% w/w one or more humectant or agent to control water activity; i) from about 0% to about 50% w/w one or more protectants; j) from about 0% to about 50% w/w one or more nutrients or mixture thereof; k) from about 5% to about 80% w/w one or more filler;

L) from about 0% to about 20% w/w one or more antioxidant or UV radiation protectant; m) from about 1% to about 50% w/w one or more binding agent; n) from about 0% to about 50% one or more disintegrating agent; o) from about 0% to about 10% w/w water; p) any combination of two or more of any of a) to o) above.

In one embodiment, the water dispersible granule formulation comprises: a) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001944; or b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; or c) both a) and b) above.

In one embodiment, the water dispersible granule formulation is substantially free of viable B. laterosporus NMI No. V12/001944.

In another embodiment, the water dispersible granule formulation comprises one or more of the following: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001944; b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001944; c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; or d) any combination of two or more of any of a) to c) above.

In another embodiment, the water dispersible granule formulation comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001944; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

V12/001944; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; d) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001944; or e) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; or f) any combination of two or more of any of a) to e) above.

In another embodiment, the water dispersible granule formulation comprises: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001944; and b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001944; and/or c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown.

In various embodiments, the water dispersible granule formulation comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001946; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

V12/001946; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; d) from about 1% to about 50% w/w one or more wetting agent; e) from about 1% to about 50% w/w one or more dispersant; f) from about 2% to about 50% w/w one or more humectant or agent to control water activity; g) from about 0% to about 50% w/w one or more protectants; h) from about 0% to about 50% w/w one or more nutrients or mixture thereof; i) from about 5% to about 80% w/w one or more filler; j) from about 0% to about 20% w/w one or more antioxidant or UV radiation protectant; k) from about 1% to about 50% w/w one or more binding agent;

In one embodiment, when the WDG formulation comprises viable B. laterosporus NMI No. V12/001946, the B. laterosporus NMI No. V12/001946 is present at a concentration of at least about 1 x 10^s cells per g, for example, about 5 x 10^s cells per g.

In one embodiment, when the WDG formulation comprises viable B. laterosporus NMI No. V12/001946, the formulation comprises one or more of the following; a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001946; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

V12/001946; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; d) from about 1% to about 50% w/w one or more wetting agent; e) from about 1% to about 50% w/w one or more dispersant; f) from about 2% to about 50% w/w one or more humectant or agent to control water activity; g) from about 0% to about 50% w/w one or more protectants; h) from about 2% to about 50% w/w one or more nutrients or mixture thereof; i) from about 5% to about 80% w/w one or more filler; j) from about 0.1% to about 20% w/w one or more antioxidant or UV radiation protectant; k) from about 1% to about 50% w/w one or more binding agent; L) from about 0% to about 50% one or more disintegrating agent; m) from about 0% to about 10% w/w water; n) any combination of two or more of any of a) to m) above.

V12/001946; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; d) from about 1% to about 50% w/w one or more wetting agent; e) from about 1% to about 50% w/w one or more dispersant; f) from about 2% to about 50% w/w one or more humectant or agent to control water activity; g) from about 0% to about 50% w/w one or more protectants; h) from about 2% to about 50% w/w one or more nutrients or mixture thereof; i) from about 5% to about 80% w/w one or more filler; j) from about 0.1% to about 20% w/w one or more antioxidant or UV radiation protectant; k) from about 1% to about 50% w/w one or more binding agent;

In one embodiment, the cellular extract obtained from B. laterosporus NMI No. V12/001946, or the composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown, comprises, consists essentially of, or consists of a sub-3 kDa fraction. It will be understood that such a fraction can be prepared by fractionating a sample to remove all components above 3 kDa, for example, by filtration through a suitable filter having a size cutoff of 3 kDa, or by size-based chromatography, or related methods.

In one example, the sub-3 kDa fraction comprises a fraction prepared by fractionating a lysed cell culture of B. laterosporus NMI No. V12/001946 together with the growth media in which it was grown, to provide the sub-3 kDa fraction.

In one embodiment, the cellular extract obtained from B. laterosporus NMI No. V12/001946, or the composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown, is enriched in components having a size below about 3 kDa. For example, a sub-3 kDa fraction is added to the cellular extract or composition.

In various embodiments, the water dispersible granule formulation comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001946; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001946; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; d) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001946; or e) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; or f) from about 1% to about 50% w/w one or more wetting agent; g) from about 1% to about 50% w/w one or more dispersant; h) from about 2% to about 50% w/w one or more humectant or agent to control water activity; i) from about 0% to about 50% w/w one or more protectants; j) from about 0% to about 50% w/w one or more nutrients or mixture thereof; k) from about 5% to about 80% w/w one or more filler;

In one embodiment, the water dispersible granule formulation comprises: a) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001946; or b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; or c) both a) and b) above.

In one embodiment, the water dispersible granule formulation is substantially free of viable B. laterosporus NMI No. V12/001946.

In another embodiment, the water dispersible granule formulation comprises one or more of the following: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001946; b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001946; c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; or d) any combination of two or more of any of a) to c) above.

In another embodiment, the water dispersible granule formulation comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001946; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

In another embodiment, the water dispersible granule formulation comprises: a) from about 1% to about 80% w/w viable B. laterosporus NMI No. V12/001946; and b) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a cellular extract obtained from B. laterosporus NMI No. V12/001946; and/or c) from about 0.1% to about 80% w/w of a sub-3 kDa fraction from a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown.

In various embodiments, the composition comprises piperonyl butoxide, safrole, apiole, parsley oil, or dillapiole. In one example, the composition is a synergistic composition comprising dillapiole.

It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5,

6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7). These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

Those skilled in the art will appreciate the meaning of various terms of degree used herein. For example, as used herein in the context of referring to an amount (e.g., "about 9%"), the term "about" represents an amount close to and including the stated amount that still performs a desired function or achieves a desired result, e.g. "about 9%" can include 9% and amounts close to 9% that still perform a desired function or achieve a desired result. For example, the term "about" can refer to an amount that is within less than 10% of, within less than 5% of, within less than 1% of, within less than 0.1% of, or within less than 0.01% of the stated amount. It is also intended that where the term "about" is used, for example with reference to a figure, concentration, amount, integer or value, the exact figure, concentration, amount, integer or value is also specifically contemplated.

Other objects, aspects, features and advantages of the present invention will become apparent from the following description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing % mortality in target species observed with different formulations comprising extracts from B. laterosporus NMI No. V12/001945, as described in Example 1 herein. Figure 2 is a graph showing % mortality in target species observed with different formulations comprising extracts from B. laterosporus NMI No. V12/001945, as described in Example 2 herein.

Figure 3 is a graph showing % mortality in target species observed with different formulations comprising B. laterosporus NMI No. V12/001945, including formulations comprising the additional, synergistic agent d ilia piole, as described in Example 3 herein.

Figure 4 is a graph showing % mortality in target species observed with different formulations comprising B. laterosporus NMI No. V12/001945, including formulations comprising the additional, synergistic agent d ilia piole, as described in Example 3 herein.

Figure 5 is a graph showing % mortality in target species observed with different formulations comprising extracts from B. laterosporus NMI No. V12/001945, including formulations comprising additional agents, as described in Example 4 herein.

Figure 6 is a graph showing % mortality in target species observed with different formulations comprising extracts from B. laterosporus NMI No. V12/001945, including representative WDG formulations, as described in Example 5 herein.

Figure 7 is a chromatograph of High performance liquid size exclusion chromatography of B. laterosporus NMI No. V12/001945 (also referred to herein as BI45) culture supernatant ammonium sulphate precipitate, as described herein in Example 6. Four distinct areas were selected in the first peak for protein analysis by SDS-PAGE and Native PAGE (see Figures 8 8i 9). These were fractions 23-28, fractions 29-32, fractions 33-38 and fractions 39-46. Fractions 47-69 were selected for protein analysis in the second peak.

Figure 8 is a photo of SDS-PAGE of BI45 supernatant size exclusion chromatography fractions, as described herein in Example 6.

Figure 9 is a photo of native PAGE of BI45 size exclusion chromatography fractions, as described herein in Example 6. A lower number of bands were visible in most samples compared to the SDS-PAGE gel (see Figure 8). Fractions 26-36, 37-39, 40-42, 43-47 and 48-67 were pooled to test for DBM larvae activity as described in Example 6.

Figure 10 is a photo of native PAGE of the size exclusion chromatography bioassay treatments, as described herein in Example 6. All size exclusion fractions had different protein profiles. The last size exclusion fractions 47 to 67 were derived from the second peak in the chromatograph shown in Figure 7.

Figure 11 is a graph showing the mean mortality rate of L3 larvae after infection of young maize leaves with different concentration of EBJ2.

Figure 12 is a graph showing LC50 values after inoculation of S. frugiperda larvae: L2

Figure 13 is a graph showing LC50 values after inoculation of S. frugiperda larvae: L3

Figure 14 is a graph showing LC50 values after inoculation of S. frugiperda larvae: L4

Figure 15 is a graph showing LC50 values after inoculation of S. frugiperda larvae: L5

Figure 16 is a graph showing % mortality in Diamondback moth observed with different formulations comprising extracts from B. laterosporus NMI No. V12/001946, as described in Example 9.

Figure 17 is a graph showing % mortality in Diamondback moth observed with different formulations comprising extracts from B. laterosporus NMI No. V12/001944, as described in Example 10.

Figure 18 is a graph showing % mortality in Diamondback moth observed with different formulations comprising extracts from B. laterosporus NMI No. V12/001945, as described in Example 11. Figure 19 is a graph showing % mortality in Diamondback moth observed with different formulations comprising extracts from B. laterosporus NMI No. V12/001945, as described in Example 12.

DETAILED DESCRIPTION

In one aspect, the present invention is directed to compositions derived from a Brevibadllius laterosporus strain, wherein the composition has activity against one or more insect pests, and particularly to insecticide compositions, including a composition that has insecticidal activity against one or more insect pests of agricultural and horticultural significance.

In various embodiments, the compositions comprise cellular extracts from the B. laterosporus strain, or growth media in which the B. laterosporus strain is or has been grown.

The term "and/or" can mean "and" or "or".

The term "agriculturally acceptable carrier" covers all liquid and solid carriers known in the art such as water and oils, as well as adjuvants, dispersants, binders, wettants, surfactants, humectants, protectants, UV protectants and/or stabilisers, tackifiers, and the like that are ordinarily known for use in the preparation of agricultural compositions, including insecticide compositions.

The term "biologically pure culture" or "biologically pure isolate" as used herein refers to a culture, for example of a B. laterosporus strain as described herein, comprising at least 99% and more preferably at least 99.5% cells of the specified strain. Typically, a biologically pure culture or a biologically pure isolate is an axenic culture or an axenic isolate.

As used herein the term "cellular extract" refers to a substance or mixture of substances obtained from a cell, typically in this description a bacterial cell.

It should be appreciated that the 'cellular extract' may be obtained in a variety of different ways, and may come in a variety of different forms without departing from the scope of the present invention.

In some embodiments the cellular extract may be a crude extract of the contents of the cell. For example, in certain embodiments the crude extract is obtained via concentration of the cells, for example by centrifugation of a whole broth culture, followed by resuspension in a suitable buffer, typically followed by cellular lysis.

Such an extract may have been derived by various well known methods of cell lysis, including, for example, sonication, osmotic lysis, enzymatic lysis, lysis using a French press or a Mantin gaulin press, or particle or bead-mediated lysis.

As used herein the term "sonicate" or grammatical variants thereof refers to subjecting a cell to ultrasonic vibrations in order to fragment the cell wall to release the contents of the cell.

In other embodiments the cellular extract is a freeze dried or a spray dried extract. In certain embodiments, the freeze or spray dried extract is obtained via any cellular extract which has also been subjected to a freeze-or spray drying process as are well known in the art.

In preferred embodiments the cellular extract may be derived from the aforementioned methods via sonication; French press; Mantin gaulin press, bead basher, bead mill mincer osmotic lysis or enzyme related lysis.

The term "comprising" as used in this specification means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises", and the terms "including", "include" and "includes" are to be interpreted in the same manner.

The term "consisting essentially of" when used in this specification refers to the features stated and allows for the presence of other features that do not materially alter the basic characteristics of the features specified.

The term "contacting" as used herein refers to the provision of a composition or strain(s) of the invention to a pest in a manner useful to effect pest control. Most commonly contacting will involve the pest feeding on material comprising a composition or strain(s) of the invention but is not limited thereto. Accordingly, "contacting" includes feeding.

The term "control" or "controlling" as used herein generally comprehends preventing an increase in, reducing, or eradicating a population or one or more members of a population, or preventing, reducing or eradicating infection or infestation by one or more pests or pathogens, such as infection by one or more phytopathogens or pests, or inhibiting the rate and extent of such infection, such as reducing a pest population at a locus, for example in or on a plant or its surround ings, wherein such prevention or reduction in the infection(s) or population(s) is statistically significant with respect to untreated infection(s) or population(s). Curative treatment is also contemplated. Preferably, such control is achieved by increased mortality amongst the pest or pathogen population.

It will be appreciated that control may be via antagonism, which may take a number of forms.

In one form, the compositions contemplated herein may simply act as a repellent. In another form, the compositions contemplated herein may render the environment unsuitable or unfavourable for the pest or pathogen. In a further, preferred form, the compositions contemplated herein may incapacitate, render infertile, impede the growth of, impede the spread or distribution of, and/or kill the pest or pathogen. Accordingly, the antagonistic mechanisms include but are not limited to antibiosis, immobilisation, infertility, and toxicity. Therefore, compositions which act as antagonists of one or more pests, such that such compositions are useful in the control of a pest, can be said to have pesticidal activity. For example, compositions that act as antagonists of one or more insects can be said to have insecticidal efficacy. Furthermore, an agent or composition that is or comprises an antagonist of a pest can be said to be a pesticidal agent or a pesticidal composition, for example, an agent that is an antagonist of an insect can be said to be an insecticidal agent. Likewise, a composition that is or comprises an antagonist of an insect can be said to be an insecticida I composition.

Accordingly, as used herein a "pesticidal composition" is a composition which comprises or includes at least one agent that has pesticidal efficacy.

In various embodiments, said pesticidal efficacy is the ability to repel, incapacitate, render infertile, impede the growth of, or kill one or more pests, including insects or nematodes, for example within 14 days of contact with the pest, such as within 7 days. Particularly contemplated pesticidal efficacy is the ability to kill one or more insect pests of plants within 7 days.

Accordingly, as used herein an "insecticidal composition" is a composition which comprises or includes at least one agent that has insecticidal efficacy.

As used herein the term "culture" refers to a population of microbes, in particular in the context of this disclosure bacteria, together with the media in or on which the population was propagated (i.e. grown) or maintained. For example, the term "whole broth culture" refers to a liquid media and the bacteria therein, for example the population of viable bacteria therein. It will be appreciated that, in certain embodiments contemplated herein, the whole broth culture is one in which substantially all of the bacteria are killed or attenuated, for example, are no longer reproductively viable.

The term "effective amount" as used herein means an amount effective to control or eradicate pests, particularly insect pests.

The term "insecticide" as used herein refers to agents which act to kill or control the growth of insects, including insects at any developmental stage. The related term "insecticidal" will be understood accordingly.

As used herein the term "isolated" means removed from the natural environment in which the subject, typically in this case the B. laterosporus NMI No. V12/001945 bacteria, naturally occurs, such that the subject is separated from some or all of the coexisting materials in the natural system from which the subject has been obtained.

The term "pest" as used herein refers to organisms that are of inconvenience to, or deleterious to, another organism, such as a plant or animal, including a human, whether directly or indirectly. In one embodiment the term refers to organisms that cause damage to animals, including humans, or plants. The damage may relate to plant or animal health, growth, yield, reproduction or viability, and may be cosmetic damage. In certain particularly contemplated embodiments, the damage is of commercial significance. As will be apparent from the context, the term "pest" as used herein will typically refer to one or more organisms that cause damage to plants, for example, cultivated plants, including horticulturally or agriculturally important plants.

The term "plant" as used herein encompasses not only whole plants, but extends to plant parts, cuttings as well as plant products including roots, shoots, leaves, bark, pods, flowers, seeds, stems, callus tissue, nuts and fruit, bulbs, tubers, corms, grains, cuttings, root stock, or scions, and includes any plant material whether pre-planting, during growth, and at or post harvest. Plants that may benefit from the application of the present invention cover a broad range of agricultural and horticultural crops. The compositions described herein are also especially suitable for application in organic production systems.

The term 'plant derived materials' refers to products that may be produced from a plant or part thereof. It will be appreciated that a person skilled in the art will know of various examples of plant derived products, such as hay, silage or other types of feed or products.

The term "surroundings" when used in reference to a plant subject to the methods and compositions of the present invention includes water, leaf litter, and/or growth med ia adjacent to or a round the plant or the roots, tubers or the like thereof, adjacent plants, cuttings of said pla nt, supports, water to be administered to the plant, and coatings includ ing seed coatings. It further includes storage, packaging or processing materials such as protective coatings, boxes and wrappers, and planting, maintena nee or harvesting equipment.

Brevibadllus laterosporus is an aerobic spore-forming bacterium, strains of which have been reported to be pathogenic to some insect species. B. laterosporus is characterized by the formulation of a typical canoe-shaped parasporal body (CSPB) created on one side of the spore after the sporangium lysis.

Surprisingly, the applicants have now identified compositions derived from a strain of Brevibadllius laterosporus, B. laterosporus NMI No. V12/001945, have insecticidal activity without the need for viable bacteria to be present. B. laterosporus NMI No. V12/001945 was deposited in the National Measurement Institute Laboratories (NMI), Suakin Street, Pymble, New South Wales, Australia on 12 September 2012 according to the Budapest Treaty for the purposes of patent procedure and was accorded deposit number NMI No. V12/001945.

Accordingly, in one aspect the invention relates to compositions comprising or derived from B. laterosporus NMI No. V12/001945.

In certain embodiments, the composition derived from B. laterosporus NMI No. V12/001945 is a cell extract prepared from B. laterosporus NMI No. V12/001945 cells or spores.

In certain embodiments, the composition derived from B. laterosporus NMI No. V12/001945 is a culture extract prepared from a B. laterosporus NMI No. V12/001945 culture.

In one embodiment, the compositions is substantially free of viable B. laterosporus NMI No. V12/001945.

In one embodiment, the culture extract is substantially free of viable B. laterosporus NMI No. V12/001945.

In one embodiment, the composition, cell extract, culture extract, or the like substantially free of viable B. laterosporus NMI No. V12/001945 is substantially free of viable B. laterosporus NMI No. V12/001945 spores.

Compositions derived from B. laterosporus NMI No. V12/001945, as is shown herein in the Examples, have insecticidal activity against diamondback moth (DBM). Diamondback moth is an example of insect pests that are particularly problematic, as moth species in particular are responsible for significant economic loss in agricultural and horticultural crops. For example, currently over US$1 billion is spent annually on diamondback moth (DBM) control worldwide.

In one embodiment the composition derived from B. laterosporus NMI No. V12/001945 has insecticidal activity against at least one Lepidoptera species. In one embodiment the Lepidoptera species is from a family selected from Tortricidae, Plutellidae, Nocudiae, and Geometridae.

In certain embodiments, the Tortricidae species include: codling moth {Cydia pomonella), light brown apple moth {Epiphyas postvittana), blacklegged leaf roller {Planotortrix notophaea) and black lyre leaf roller {Cnepasia jactatana).

In one embodiment, the Plutellidae species is diamond back moth ( Plutella xyostella ).

In one embodiment, the Nocudiae species is cabbage looper moth ( Trichoplusia ni) or cotton boll worm moth ( Helicoverpa armigera ).

In one embodiment, the Geometridae species is common forest looper ( Pseudocoremia suavis).

Compositions derived from two further strains of Brevibacillius laterosporus, B. laterosporus NMI No. V12/001944, and B. laterosporus NMI No. V12/001946, have insecticidal activity without the need for viable bacteria to be present.

B. laterosporus NMI No. V12/001944 was deposited in the National Measurement Institute Laboratories (NMI), Suakin Street, Pymble, New South Wales, Australia on 12 September 2012 according to the Budapest Treaty for the purposes of patent procedure and was accorded deposit number NMI No. V12/001944.

B. laterosporus NMI No. V12/001944 was deposited in the National Measurement Institute Laboratories (NMI), Suakin Street, Pymble, New South Wales, Australia on 12 September 2012 according to the Budapest Treaty for the purposes of patent procedure and was accorded deposit number NMI No. V12/001946. Accordingly, in one aspect the invention relates to compositions comprising or derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946.

In certain embodiments, the composition derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is a cell extract prepared from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, cells or spores.

In certain embodiments, the composition derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is a culture extract prepared from a B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, culture.

In one embodiment, the compositions is substantially free of viable B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946.

In one embodiment, the culture extract is substantially free of viable B. laterosporus NMI No. V12/001944.

In one embodiment, the composition, cell extract, culture extract, or the like substantially free of viable B. laterosporus NMI No. V12/001944 is substantially free of viable B. laterosporus NMI No. V12/001944 spores.

In one embodiment, the culture extract is substantially free of viable B. laterosporus NMI No. V12/001946.

In one embodiment, the composition, cell extract, culture extract, or the like substantially free of viable B. laterosporus NMI No. V12/001946 is substantially free of viable B. laterosporus NMI No. V12/001946 spores.

The compositions contemplated herein in certain embodiments comprise an effective amount of a cell extract or culture extract from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, or a fraction from a cell extract or culture extract from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, in an amount effective to control the pest of interest. The effective concentration may vary depending on the nature of the composition, the nature of the composition derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, the environment to which the composition is to be applied, the type, concentration and degree of pest infestation; temperature; season; humidity; stage in plant growing season; age of plant; method, rate and frequency of application; number and type of conventional fungicides, pesticides and the like being applied, and plant treatments (for example pruning, grazing, and irrigation). All factors may be taken into account in formulating the composition. The compositions contemplated herein for the control on one or more insect pests are in certain embodiments made by mixing one or more extracts derived from B. laterosporus NMI No.

V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, including one or more cellular extracts, and/or one or more culture extracts, and/or one or more fractions therefrom, with one or more desired agriculturally-acceptable carrier.

Production of compositions derived from B. laterosporus

It will be appreciated that the preparation of extracts derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, suitable for formulation into compositions as contemplated herein will generally involve techniques well known in the art. For example, cultures of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, will in certain embodiments be prepared for use in the compositions contemplated herein using standard drying and fermentation techniques known in the art.

Growth is commonly effected under aerobic conditions in a bioreactor at suitable temperatures and pH for growth. Typical growth temperatures are from 15 to 37°C, commonly 27°C to 32°C.

Growth medium may be any known art medium suitable for B. laterosporus culture. For example Nutrient Yeast Extract Salt Medium (NYSM) (Favret, and Youstein 1985).

In certain embodiments, cultures greater than two days old, typically four days old or older are used to prepare the composition derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, such as the cell extract or culture extract. In embodiments where viable, or attenuated or killed whole cells of B. laterosporus in present in the compositions, such as in a WDG formulation as herein described, the composition will typically be prepared as a cell suspension, to be combined with other formulation ingredients, such as one or more desired agricultural carriers.

Typical concentration ranges for the B. laterosporus, when present in the composition in the form of intact cells, is from 1 x 10³ to 1 x 10¹⁴, preferably 1 x 10⁴ to 1 x 10¹⁰, more preferably 1 x 10⁶ to 1 x 10^s cells/mg. It will be appreciated that compositions with cell concentrates in the order of 1 x 10¹¹ to 1 x 10¹⁴ may be prepared and diluted before application if required.

Generally, in embodiments in which a culture extract or growth media component is not to be present, for example, the composition derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, consists of a cellular extract, the cells used to prepare the composition derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, will typically be harvested using conventional washing, filtering or sedimentary techniques such as centrifugation, or may be harvested using a cyclone system. Harvested cells can be used immediately for further processing, or stored under chilled conditions (for example at 4°C) or may be freeze dried. In such cases, the B. laterosporus cells can be processed prior to use, whether or not that use is in conjunction with a media component or secreted extract, to produce one or more compositions derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, such as cell extracts, cell suspensions, cell homogenates, cell lysates, cell supernatants, cell filtrates, cell pellets or may be used as whole cell preparations.

Typically, however, compositions derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, will include a fraction, such as a growth media component or fraction, that comprises one or more components secreted by the B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, cells.

Similarly, the media in which from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is or has been grown can be used, optionally in conjunction with one or more other compositions derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, in the preparation of a composition as contemplated herein.

Representative examples of media extracts or fractions are exemplified herein in the Examples. In one embodiment, the composition derived from B. laterosporus NMI No. V12/001945 is a cellular and/or culture extract comprising, consisting essentially of, or consisting of a sub-3 kDa fraction from B. laterosporus NMI No. V12/001945 of a culture thereof. In one example, the sub-3 kDa fraction comprises a fraction prepared by fractionating a lysed cell culture of B. laterosporus NMI No. V12/001945, together with the growth media in which it was grown, to provide a sub-3 kDa fraction. Methods to fractionate cell cultures, such as a lysed cell culture referred to above, to provide such size-selected fractions are well known. For example, a sub-3 kDa fraction can readily be prepared using a finite 3 kDa centrifuge filter, such as an Amicon Ultra filter (Merck), or the like.

Agricultural Compositions

The compositions for agricultural application, such as in the control of one or more plant pests will typically include one or more agriculturally-acceptable carrier, such as one or more humectants, spreaders, stickers, stabilisers, penetrants, emulsifiers, dispersants, surfactants, buffers, binders, protectants, and other components typically employed in agricultural compositions, or in insecticidal or pesitcidal compositions.

Compositions contemplated herein may be formulated in a variety of different ways without departing from the scope of the present invention. The composition of the invention may be in liquid or solid form. In general the formulation chosen will be dependent on the end application. For example, possible formulations include, but should not be limited to matrixes, soluble powders, granules including water dispersible granules, encapsulations including micro-encapsulations, aqueous solutions, aqueous suspensions, non-aqueous solutions, non-aqueous suspensions, emulsions including microemulsions, pastes, emulsifiable concentrations, and baits.

In various embodiments, the agricultural composition is a liquid composition. Liquid compositions typically include water, saline or oils such as vegetable or mineral oils. Examples of vegetable oils useful in the invention are soy bean oil and coconut oil. The compositions may be in the form of sprays, suspensions, concentrates, foams, drenches, slurries, injectables, gels, dips, pastes and the like. Liquid compositions may be prepared by mixing the liquid agriculturally acceptable carrier with the composition(s) derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, such as the cellular extract or fraction, or the compositions or fractions derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, growth media. Conventional formulation techniques suitable for the production of liquid compositions are well known in the art.

In various embodiments the composition is in solid form. In one example, a solid composition is produced by drying a liquid composition derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946. Alternatively, a solid composition useful as described herein is prepared by mixing one or more compositions derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, with a variety of inorganic, organic, and/or biological materials. For example, solid inorganic agricultural carriers suitable for use include carbonates, sulphates, phosphates or silicates, pumice, lime, bentonite, or mixtures thereof. Solid biological materials suitable for use include powdered palm husks, corncob hulls, and nut shells.

Exemplary solid agricultural compositions include those formulated as dusts, granules induing water dispersible granules, seed coatings, wettable powders or the like. As is understood in the art, certain solid compositions are applied in solid form, while others are formulated to be admixed with a liquid prior to application, so as to provide a liquid agricultural composition for application.

The compositions contemplated herein are in certain embodiments in the form of controlled release, or sustained release formulations. The compositions contemplated herein in certain embodiments also include other control agents such as pesticides, insecticides, fungicides, nematocides, virucides, growth promoters, nutrients, germination promoters and the like, provided they are compatible with the activity of the composition derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946.

In embodiments of particular compositions, for example, of WDG compositions described herein, where viable B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, are present, the same considerations with regard to combinations of components, preparation and application as are discussed above will generally apply.

In certain embodiments, the composition comprises an anti-caking agent, for example, an anti caking agent selected from talc, silicon dioxide, calcium silicate, or kaelin clay.

In certain embodiments, the composition comprises a wetting agent, such as skimmed milk powder.

In certain embodiments, the composition comprises an emulsifier, such as a soy-based emulsifier such as lecithin, or a vegetable-based emulsifier such as monodiglyceride.

However, other examples of agriculturally acceptable carriers are well known in the art and may be substituted, provided the efficacy of the composition is maintained.

In various embodiments, a desiccation protection agent, such as Deep Fried™, Fortune™, or Fortune Plus™, is admixed to a final concentration of about 1 ml/L prior to application.

In one exemplary embodiment, the composition comprises an oil flowable suspension, such as an oil flowable suspension of one or more extracts from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, as described herein.

In a second exemplary embodiment, the composition comprises a wettable powder, dust, pellet, or colloidal concentrate. Such dry forms of the compositions may be formulated to dissolve immediately upon wetting, or alternatively, dissolve in a controlled-release, sustained-release, or other time-dependent manner.

In a third exemplary embodiment, the composition comprises an aqueous solution or suspension of one or more active agents, for example of the extract from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, as described herein. Such aqueous solutions or suspensions are in certain embodiments provided as a concentrated stock solution which is diluted prior to application, or alternatively, as a diluted solution ready-to-apply.

In a further exemplary embodiment, the composition comprises a microemulsion.

In various specifically contemplated embodiments, the compositions contemplated herein are formulated as a water dispersible granule (WDG). Water dispersible granule formulations offer advantages over other types of formulations that are agriculturally applied in liquid form. These include simplicity in packaging, ease of handling, and safety. Typically, water dispersible granule formulations are free flowing, low dusting, and readily disperse in water to form either a solution or a homogenous suspension of very small particles suitable for application via conventional techniques and machinery, such as conventional spray equipment and spray nozzles.

The present disclosure provides water dispersible granule formulations comprising from about 2% to about 80% (w/w) of a composition derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, such as a cellular extract or a fraction thereof, a culture extract or fraction thereof, or a combination of both. In certain embodiments, viable B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is present. However, embodiments in which no viable B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is present are specifically contemplated.

In certain embodiments, the WDG formulation additionally comprises one or more of the following: a) from about 0% to about 20% (w/w) of one or more surfactants; b) from about 0% to about 30% (w/w) of one or more binders; c) from about 0% to about 90% (w/w) of one or more fillers; d) any combination of a) to c) above, including any combination of two or more of a) to c) above.

In certain embodiments, the WDG formulation additionally comprises water, for example, from about 1% to about 5% (w/w) water, for example, up to about 2% (w/w) water.

In one example, the WDG formulation comprises from about 5% to about 80% (w/w) of bacterial extract or a fraction thereof, and comprises one or more of the following: a) from about 1% to about 20% (w/w) of one or more surfactants; b) from about 1% to about 30% (w/w) of one or more binders; c) from about 1% to about 90% (w/w) of one or more fillers; d) any combination of a) to c) above, including any combination of two or more of a) to c) above.

In one example, the WDG formulation comprises from about 5% to about 80% (w/w) of bacterial extract or a fraction thereof, and from about 1% to about 20% (w/w) of one or more surfactants; from about 1% to about 30% (w/w) of one or more binders; and from about 1% to about 90% (w/w) of one or more fillers.

In various embodiments, the water dispersible granule formulation comprises one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001945; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001945; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; d) from about 1% to about 50% w/w one or more wetting agent; e) from about 1% to about 50% w/w one or more dispersant; f) from about 2% to about 50% w/w one or more humectant or agent to control water activity; g) from about 0% to about 50% w/w one or more protectants; h) from about 0% to about 50% w/w one or more nutrients or mixture thereof; i) from about 5% to about 80% w/w one or more filler; j) from about 0% to about 20% w/w one or more antioxidant or UV radiation protectant; k) from about 1% to about 50% w/w one or more binding agent;

V12/001944; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; d) from about 1% to about 50% w/w one or more wetting agent; e) from about 1% to about 50% w/w one or more dispersant; f) from about 2% to about 50% w/w one or more humectant or agent to control water activity; g) from about 0% to about 50% w/w one or more protectants; h) from about 0% to about 50% w/w one or more nutrients or mixture thereof; i) from about 5% to about 80% w/w one or more filler; j) from about 0% to about 20% w/w one or more antioxidant or UV radiation protectant; k) from about 1% to about 50% w/w one or more binding agent;

V12/001944; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; d) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001945; e) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

V12/001945; f) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; g) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001946; h) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No.

V12/001946; i) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; j) from about 1% to about 50% w/w one or more wetting agent; k) from about 1% to about 50% w/w one or more dispersant;

L) from about 2% to about 50% w/w one or more humectant or agent to control water activity; m) from about 0% to about 50% w/w one or more protectants; n) from about 0% to about 50% w/w one or more nutrients or mixture thereof; o) from about 5% to about 80% w/w one or more filler; p) from about 0% to about 20% w/w one or more antioxidant or UV radiation protectant; q) from about 1% to about 50% w/w one or more binding agent; r) from about 0% to about 50% one or more disintegrating agent; s) from about 0% to about 10% w/w water; t) any combination of two or more of any of a) to s) above.

In various embodiments, the wetting agent or dispersant is selected from the group comprising Sodium lignosulphonate, Sodium methoxy-lignosulphonate, Sodium polycarboxylate, Potassium polycarboxylate, Phosphate ester surfactants, including ethoxylated alcohol ether phosphate esters, Sodium aryl sulphonates, Ethoxylated linear alcohols, alkyl phenol alcohols, Alkyl polyglucoside, Alkali salts of dioctyl sulphosuccinate, including sodium dioctyl sulphosuccinate, and any combination of any two or more thereof.

In various embodiments, the filler is selected from the group comprising Kaolin, Talc, Bentonite, Atapulgite, Sepiolite, Vermiculite, Silica, including ground silica, fumed silica, and precipitated silica, Perlite, Cellulosic fibre, such as ground nut shells, husks, and the like, and any combination of any two or more thereof.

In various embodiments, the binding agent is selected from the group comprising Sugars, such as sucrose, fructose, maltodextrin, and the like, Acrylic or maleic acid polymers or copolymers, Polyvinylpyrrolidone, Starch and modified starch, Cellulosic gums, such as CMC, HEC, HMC, Polysaccharide gums, such as guar, Xanthan, pullulan, carrageenan, gellan, agar, alginate, chitin and chitosan, and the like, and any combination of any two or more thereof.

In various embodiments, the protectant is selected from the group comprising antioxidants, UV protectants, preservatives, antidessicants, and emollients, and any combination of any two or more thereof.

In various embodiments, the antioxidant is selected from the group comprising water soluble antioxidants, oil soluble antioxidants, including antioxidants such as ascorbic acid and salts thereof, such as sodium ascorbate, calcium ascorbate, etc., vitamin E and other phenolic antioxidants, TBHQ, Propyl ga Mate and other gallic acid esters, tert-butylhydroquinone (TBHQ), and any combination of any two or more thereof.

In various embodiments, the emollient is selected from the group comprising vegetable oils, waxes, or greases, mineral oils, waxes or greases, mono and diglycerides of longer chain fatty acids, and any combination of any two or more thereof.

In various embodiments, the humectant of agent to control water activity is selected from the group comprising one or more sugars, such as glucose, glycerol, propylene glycol, betaine, one or more salts that can serve to limit water activity, and any combination of any two or more thereof.

In particular embodiments, WDG formulations contemplated herein, for example those prepared via wet granulation processes, do not require a disintegrant. The present disclosure also relates to liquid formulations comprising water dispersible granule formulations dispersed in water, processes for the preparation of water dispersible granule formulations using wet granulation processes, and methods of administering an effective amount of water dispersible granule formulations to a plant or its surroundings, for example to control one or more insect pests.

In one embodiment, a representative WDG formulation is prepared as below:

* biomass and residual media One suitable method for preparing WDG formulations of which the above formulation is an example is a direct granulation method, in which the liquid fermentation is directly applied to the dry ingredients to form an extrudable paste. The paste is then formed into an elongate extrudate. In one embodiment, the extrudate is dried, and may then be cut or granulated when dry, while in another embodiment the extrudate is agitated or cut to form granules in a granulating mixer before being dried. Typically, the damp granules are dried in a fluid bed drier to achieve the desired moisture content.

It will be appreciated that the moisture content can vary depending on the uses to which the WDG is to be put, the storage expectations for the WDG product, or whether viable cells or spores are present in the final product or not.

It will be appreciated that this method advantageously employs a single drying step to produce the final product.

Another representative WDG formulation is prepared as below:

* biomass and residual media

One suitable method for preparing WDG formulations of which the above formulation is an example is an indirect granulation method in which the liquid fermentation product is first dried to the desired moisture content/non-volatile material content (in this case, 20% non-volatiles) before addition to the other WDG ingredients. Additional water is normally required to provide enough moisture to form an extrudable paste and this in turn has to be dried off in the final drying process.

The initial drying of the liquid fermentate can be achieved by any suitable drying method, such as batch drying, vacuum falling film evaporating, spray drying or freeze drying. In formulations in which viable cells or spores are to be present, freeze drying and vacuum spray drying will typically be used, as the gentle conditions achievable with these methods help maximise viability.

This method has the advantage of reducing the water activity of the product to a low level that improves the stability until it is ready for incorporation into WD granules and can also be used to increase the level of active material in the final granule.

Another representative WDG formulation is prepared as below:

* biomass and residual media

One suitable method for preparing WDG formulations of which the above formulation is an example is the so-called 'Sorbie' process in which absorbent dispersible granules are produced using inert materials and a binder in the absence of fermentation product. Subsequently, the liquid fermentation product is sprayed onto the absorbent granules, typically while the absorbent granules are fluidized, for example, in a fluid bed drier, followed by gentle heating to dry the granules.

This method has the advantage of allowing very gentle final drying conditions, for example, for formulations comprising heat-sensitive ingredients, such as viable spores, while more aggressive conditions can be used to produce the inert 'sorbie' particles. This allows a degree of flexibility in process control, in which bulk 'sorbie' particles can be produced independently of the fermentation product. It will be appreciated that fermentation will often be the rate-limiting step, such that shorter production times can be achieved after fermentation is completed.

In certain embodiments relating to water dispersible granule formulations, the preparation of water dispersible granules comprising a composition derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, via wet granulation enables the efficient preparation and recovery of granules of regular size and shape, and thus of similar dissolution and handling characteristics, among other advantages. Such regularity in particle size can be problematic to achieve with other formulation methods, such as dry compaction and fragmentation, which typically produces chips of irregular size and shape. In certain embodiments, the combination of wet granulation and lack of disintegrants in representative examples of WDG formulations comprising a composition derived from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, whether or not viable B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is present, provides an efficient and effective formulation for agricultural application and pest control.

In certain embodiments, the compositions described herein may be used in conjunction with other treatments such as cryo protectants, surfactants, detergents, soaps, dormant oils, polymers, and/or time-release or biodegradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation.

The compositions as described herein may also be used in consecutive or simultaneous application to a plant population or an environmental site singly or in combination with one or more additional agents, such as insecticides, pesticides, chemicals, fertilizers, or other compounds.

As discussed herein, compositions as described herein may be formulated as, for example, concentrates, solutions, sprays, aerosols, immersion baths, dips, emulsions, wettable powders, soluble powders, suspension concentrates, dusts, granules, water dispersible granules, microcapsules, pastes, gels and other formulation types by well-established procedures. These procedures will frequently include mixing and/or milling of the active components with agriculturally acceptable carrier substances, such as fillers, solvents, excipients, surfactants, suspending agents, speaders/stickers (adhesives), antifoaming agents, dispersants, wetting agents, drift reducing agents, auxiliaries and adjuvants.

In one embodiment solid carriers include but are not limited to mineral earths such as silicic acids, silica gels, silicates, talc, kaolin, attapulgus clay, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, aluminas calcium sulfate, magnesium sulfate, magnesium oxide, ground plastics, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, and ureas, and vegetable products such as grain meals, bark meal, wood meal, and nutshell meal, cellulosic powders and the like.

As solid carriers for granules, including for example the WDG formulations specifically contemplated herein, the following are suitable: crushed or fractionated natural rocks such as calcite, marble, pumice, sepiolite and dolomite; synthetic granules of inorganic or organic meals; granules of organic material such as sawdust, coconut shells, corn cobs, corn husks or tobacco stalks; kieselguhr, tricalcium phosphate, powdered cork, or absorbent carbon black; water soluble polymers, resins, waxes; or solid fertilizers. Such solid compositions may, if desired, contain one or more compatible wetting, dispersing, emulsifying or colouring agents which, when solid, may also serve as a diluent.

In various embodiments the carrier may also be liquid, for example, water; alcohols, particularly butanol or glycol, as well as their ethers or esters, particularly methylglycol acetate; ketones, particularly acetone, cyclohexanone, methylethyl ketone, methyl isobutyl ketone, or isophorone; petroleum fractions such as paraffinic or aromatic hydrocarbons, particularly xylenes or alkyl naphthalenes; mineral or vegetable oils; aliphatic chlorinated hydrocarbons, particularly trichloroethane or methylene ' chloride; aromatic chlorinated hydrocarbons, particularly chlorobenzenes; water-soluble or strongly polar solvents such as dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone; liquefied gases; or the like or a mixture thereof.

In one embodiment surfactants include nonionic surfactants, anionic surfactants, cationic surfactants and/or amphoteric surfactants and promote the ability of aggregates to remain in solution during spraying.

Spreaders/stickers promote the ability of the compositions as described herein to adhere to plant surfaces. Examples of surfactants, spreaders/ stickers include but are not limited to Tween and Triton (Rhom and Hass Company), Deep Fried™, Fortune®, Pulse, C. Daxoil®, Codacide oil®, D-C. Tate®, Supamet Oil, Bond®, Penetrant, Glowelt® and Freeway, Citowett®, Fortune Plus™, Fortune Plus Lite, Fruimec, Fruimec lite, alkali metal, alkaline earth metal and ammonium salts of aromatic sulfonic acids, e.g., ligninsulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid and dibutylnaphthalenesulfonic acid, and of fatty acids, alkyl and alkylaryl sulfonates, and alkyl, lauryl ether and fatty alcohol sulfates, and salts of sulfated hexadecanols, heptadecanols, and octadecanols, salts of fatty alcohol glycol ethers, condensation products of sulfonated naphthalene and naphthalene derivatives with formaldehyde, condensation products of naphthalene or naphthalenesulfonic acids with phenol and formaldehyde, polyoxyethylene octylphenol ethers, ethoxylated isooctylphenol, ethoxylated octylphenol and ethoxylated nonylphenol, alkylphenol polyglycol ethers, tributylphenyl polyglycol ethers, alkylaryl polyether alcohols, isotridecyl alcohol, fatty alcohol ethylene oxide condensates, ethoxylated castor oil, polyoxyethylene alkyl ethers, ethoxylated polyoxypropylene, lauryl alcohol polyglycol ether acetal, sorbitol esters, lignin-sulfite waste liquors and methyl cellulose. Where selected for inclusion, one or more agricultural surfactants, such as Tween are desirably included in the composition according to known protocols.

Wetting agents reduce surface tension of water in the composition and thus increase the surface area over which a given amount of the composition may be applied. Examples of wetting agents include but are not limited to salts of polyacrylic acids, salts of lignosulfonic acids, salts of phenolsulfonic or naphthalenesulfonic acids, polycondensates of ethylene oxide with fatty alcohols or fatty acids or fatty esters or fatty amines, substituted phenols (particularly alkylphenols or arylphenols), salts of sulfosuccinic acid esters, taurine derivatives (particularly alkyltaurates), phosphoric esters of alcohols or of polycondensates of ethylene oxide with phenols, esters of fatty acids with polyols, or sulfate, sulfonate or phosphate functional derivatives of the above compounds.

In one embodiment the preferred method of applying the composition as described herein is to spray a dilute or concentrated solution by handgun or commercial airblast.

As described above, the compositions as described herein may be used alone or in combination with one or more other agricultural agents, including pesticides, insecticides, acaracides, fungicides or bactericides (provided such fungicides or bactericides are not detrimental or toxic to any fungi or bacteria that are present in the composition), herbicides, antibiotics, antimicrobials, nemacides, rodenticides, entomopathogens, pheromones, attractants, plant growth regulators, plant hormones, insect growth regulators, chemosterilants, microbial pest control agents, repellents, viruses, phagostimulents, plant nutrients, plant fertilisers and biological controls. When used in combination with other agricultural agents the administration of the two or more agents or formulations may be separate, simultaneous or sequential. Specific examples of these agricultural agents are known to those skilled in the art, and many are readily commercially available.

Examples of plant nutrients include but are not limited to nitrogen, magnesium, calcium, boron, potassium, copper, iron, phosphorus, manganese, molybdenum, cobalt, boron, copper, silicon, selenium, nickel, aluminum, chromium and zinc.

Examples of antibiotics include but are not limited to oxytetracyline and streptomycin.

Examples of fungicides include but are not limited to the following classes of fungicides: carboxamides, benzimidazoles, triazoles, hydroxypyridines, dicarboxamides, phenylamides, thiadiazoles, carbamates, cyano-oximes, cinnamic acid derivatives, morpholines, imidazoles, beta- methoxy acrylates and pyridines/ pyrimidines.

Further examples of fungicides include but are not limited to natural fungicides, organic fungicides, sulphur-based fungicides, copper/calcium fungicides and elicitors of plant host defences.

Examples of natural fungicides include but are not limited to whole milk, whey, fatty acids or esterified fatty acids.

Examples of organic fungicides include but are not limited to any fungicide which passes an organic certification standard such as biocontrol agents, natural products, elicitors (some of may also be classed as natural products), and sulphur and copper fungicides (usually limited to restricted use). An example of a sulphur-based fungicide is Kumulus™ DF (BASF, Germany). An example of a copper fungicide is Kocide® 2000 DF (Griffin Corporation, USA).

Examples of elicitors include but are not limited to chitosan, Bion™, BAB A (DL-3- amino-n- butanoic acid, b-aminobutyric acid) and Milsana™ (Western Farm Service, Inc., USA).

In some embodiments non-organic fungicides may be employed. Examples of nonorganic fungicides include but are not limited to Bravo™ (for control of PM on cucurbits); Supershield™ (Yates, NZ) (for control of Botrytis and PM on roses); Topas® 200EW (for control of PM on grapes and cucurbits); Flint™ (for control of PM on apples and cucurbits); Amistar® WG (for control of rust and PM on cereals); and Captan™, Dithane™, Euparen™, Rovral™, Scala™, Shirlan™, Switch™ and Teldor™ (for control of Botrytis on grapes).

Examples of pesticides include but are not limited to azoxystrobin, bitertanol, carboxin, CU2O, cymoxanil, cyproconazole, cyprodinil, dichlofluamid, difenoconazole, diniconazole, epoxiconazole, fen piclon i I , fludioxonil, fluquiconazole, flusilazole, flutriafol, furalaxyl, guazatin, hexaconazole, hymexazol, imazalil, imibenconazole, ipconazole, kresoxim-methyl, mancozeb, metalaxyl, R- metalaxyl, metconazole, oxadixyl, pefurazoate, penconazole, pencycuron, prochloraz, propiconazole, pyroquilone, SSF-109, spiroxamin, tebuconazole, thiabendazole, tolifluamid, triazoxide, triadimefon, triadimenol, triflumizole, triticonazole and uniconazole.

An example of a biological control agent is the BotryZen™ biological control agent comprising Ulocladium oudemansii.

The compositions may also comprise a broad range of additives such as stablisers and penetrants used to enhance the activity of the composition, and so-called 'stressing' additives such as potassium chloride, glycerol, sodium chloride and glucose. Additives may also include compositions which assist in maintaining stability or, when one or more microbes are present in the composition, microorganism viability, for example, during long term storage, for example unrefined corn oil and so called invert emulsions.

As will be appreciated by those skilled in the art, it is important that any additives used are present in amounts that do not interfere with the effectiveness of the composition.

Application

In one example, compositions as described herein are applied directly to the plant or its surroundings. In one embodiment, a composition as contemplated herein is applied to the environment of the pest, typically on to plants to be protected, equipment, ground or air. For example, a composition as described herein is admixed with a solvent, for example water, and applied as described herein.

In one embodiment, a composition as described herein is applied directly to the pest for example, by spraying, dipping, dusting or the like. It will be appreciated that, in certain circumstances, application to a plant or its surroundings will have the potential to include at least some direct application to a pest, for example, a pest already present on the plant or its surroundings.

In one embodiment, for example of a method for controlling one or more plant pests, the method comprising applying to a plant or its surroundings a composition as described herein.

The concentration of composition, or of active component(s) comprising the compostion, for example of the extract from B. iaterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, which is used for environmental, systemic, topical, or foliar application will vary widely depending upon the nature of the particular formulation, means of application, environmental conditions, and degree of biocidal activity.

In certain embodiments, a typical application rate of active agent, for example of the extract from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is from about O.lg/hectare to 10,000g/hectare. Commonly, the application rate is from about lOg/hectare to 5,000g/hectare, or 50 to 1500g/hectare. In various embodiments, the composition is admixed with water to a final concentration of active agent, for example of the extract from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, of about 0.5gm/L to about 10 gm/L prior to application, for example to a final concentration of about 5 gm/L.

In certain embodiments, a typical application rate of active agent, for example of the extract from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is from about 0.1 mL/hectare to 10,000 mL/hectare. Commonly, the application rate is from about 10 mL/hectare to 5,000 mL/hectare, or 50 to 1500 mL/hectare.

In various embodiments, the composition is admixed with water to a final concentration of active agent, for example of the extract from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, of about 0.5 mL/L to about 10 mL/L prior to application, for example to a final concentration of about 5 mL/L.

The composition will in various embodiments be administered to a particular plant or target area in one or more applications as needed, with a field application rate per hectare ranging on the order of from about 50 g/hectare to about 500 g/hectare of active ingredient, or alternatively, from about 500 g/hectare to about 1000 g/hectare may be utilized. In certain instances, it may even be desirable to apply the formulation to a target area at an application rate of from about 1000 g hectare to about 5000 g hectare or more of active component, for example of the extract from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946. In fact, all application rates in the range of from about 0.1 g of active agent per hectare to about 10,000 g/hectare are contemplated to be useful in the management, control, or killing, of target organisms using such formulations. As such, rates of about 100 g/hectare, about 200 g/hectare, about 300 g/hectare, about 400 g hectare, about 500 g/hectare, about 600 g/hectare, about 700 g/hectare, about 800 g/hectare, about 900 g/hectare, about 1 kg/hectare, about 1.1 kg/hectare, about 1.2 kg/hectare, about 1.3 kg/hectare, about 1.4 kg/hectare, about 1.5 kg/hectare, about 1.6 kg/hectare, about 1.7 kg/hectare, about 1.8 kg/hectare, about 1.9 kg/hectare, about 2.0 kg/hectare, about 2.5 kg/hectare, about 3.0 kg/hectare, about 3.5 kg/hectare, about 4.0 kg/hectare, about 4.5 kg/hectare, about 6.0 kg/hectare, about 7.0 kg/hectare, about 8.0 kg/hectare, about 8.5 kg/hectare, about 9.0 kg/hectare, and even up to and including about 10.0 kg/hectare or greater of active component may be utilized in certain agricultural, industrial, and domestic applications of the formulations described hereinabove.

Convenient and effective rates of application can be achieved by formulating the composition to deliver an effective amount of the active agent, for example of the extract from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, and applying said composition at a rate of about 1L per hectare. As discussed herein, such an application rate can be conveniently achieved by dissolution of the composition in a larger volume of agriculturally acceptable solvent, for example, water.

In various embodiments, the composition is admixed with water prior to application. In one embodiment, the composition is admixed with water and applied in at least about 100L water/Ha, in at least about 150L/Ha, in at least about 200L/Ha, in at least about 2501/Ha, in at least about 300L/Ha, in at least about 350L Ha, in at least about 400L/Ha, in at least about 450L/Ha, or in at least about 500L/Ha. Spraying, dusting, soil soaking, seed coating, foliar spraying, misting, aerosolizing and fumigation are all possible application techniques.

Generally, said application is by spraying.

Compositions formulated for other methods of application such as injection, rubbing or brushing, may also be used, as indeed may any known art method. Indirect applications of the composition to the plant surroundings or environment such as soil, water, or as seed coatings are possible.

As discussed above, the concentration at which the compositions are to be applied so as to be effective control compositions may vary depending on the end use, physiological condition of the plant; type (including plant species) or number of plants to be controlled; temperature, season, humidity, stage in the growing season and the age of plant; number and type of conventional treatments (including herbicides) being applied; and plant treatments (such as leaf plucking and pruning).

Other application techniques, including dusting, sprinkling, soil soaking, soil injection, seed coating, seedling coating, aerating, misting, atomizing, fumigating, aerosolizing, and the like, are also feasible and may be required under certain circumstances. These application procedures are also well- known to those of skill in the art.

Applications may be once only or repeated as required. Application at different times in plant life cycles, are also contemplated. For example, at harvest to prevent or minimise post harvest attack by pests.

Young seedlings are typically most susceptible to damage from competing plants and pests, such as insect pests. Therefore, application of the compositions as described herein to freshly planted- out crops, prior to emergence, is contemplated, as is application on emergence.

Repeated applications at the same or different times in a crop cycle are also contemplated. The compositions as described herein may be applied either earlier or later in the season. This may be over flowering or during fruiting, or immediately prior to harvest of the desired crop or plant, or after harvest to protect necrotic or senescing leaves, fruit, stems, machine harvested stalks and the like.

Application may be at a time before or after bud burst and before and after harvest. However, treatment preferably occurs between flowering and harvest. To increase efficacy, multiple applications (for example, 2 to 6 applications over the stages of flowering through fruiting) of the compositions as described herein is contemplated.

The compositions as described herein may also be formulated for preventative or prophylactic application to an area, and may in certain circumstances be applied to and around farm equipment, barns, domiciles, or agricultural or industrial facilities, and the like.

The compositions and methods described herein are applicable to any plant or its surroundings. Such plants include cereal, vegetable and arable crops, grasses, lawns, pastures, fruit trees and ornamental trees and plants.

Arable crops which may particularly benefit from use of the compositions and strain(s) of the invention include crucifers and brassicas. For example, cabbage, broccoli, cauliflower, brussel sprouts and bok choy.

Exemplary plants are in certain embodiments monocotyledonous or dicotyledonous plants such as alfalfa, barley, canola, corn, cotton, flax, kapok, peanut, potato, oat, rice, rye, sorghum, soybean, sugarbeet, sugarcane, sunflower, tobacco, tomato, wheat, turf grass, pasture grass, berry, fruit, legume, vegetable, ornamental plants, shrubs, cactuses, succulents, and trees. In further illustrative embodiments, the plant may be any plant, including plants selected from the order Solanales, including plants from the following families: Convolvulaceae, Hydroleaceae, Montiniaceae, Solanaceae, and Sphenocleaceae, and plants from the order Asparagales, including plants from the following families: Amaryllidaceae, Asparagaceae, Asteliaceae, Blandfordiaceae, Boryaceae, Doryanthaceae, Hypoxidaceae, Iridaceae, Ixioliriaceae, Lanariaceae, Orchidaceae, Tecophilaeaceae, Xanthorrhoeaceae, and Xeronemataceae.

The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.

EXAMPLES

Example 1: Assessment of insecticidal activity

This example describes a mortality trial to assess the insecticidal activity of various Brevibacillius laterosporus extracts against Diamondback moth ( Plutella xylostella, Lepidoptera: Plutellidae ).

Methods

Bioassay

Circular segments were cut from pak choy using the bottom plate of a petri dish as a template. Outer leaves of smaller pak choy (5-6 leaves) were used. After cutting, the leaves were soaked for ~ 60 min in filtered water with a small volume of peroxide solution to remove any bacteria that may otherwise cause rotting during the assay.

Leaf segments were placed on water agar in petri dishes, with the bottom of the leaf facing upwards. Segments were placed to ensure a seal was formed around the edge of the leaf segment to prevent larvae / insects from crawling underneath the leaf segment.

Diamondback moth larvae of 2^nd instar, from 2 mm to 4 mm in length, were gently placed on top of the leaf segment. 10 larvae were used per sample/plate, in accordance with experimental design.

Plates were sprayed within a Potters tower. Typically, 4 mL test and control samples were sprayed (at 5 psi to ensure good coverage) per plate as per experimental design.

After spraying, plates were transferred to a controlled climate environment (usually ~88 % humidity, ~ 24 °C, 8 hours dark), and observations were made in accordance with the experimental design. Typically, observations are made every 24 hours over the course of the trial.

Morbidity and mortality were recorded at each observation point, and photos were take to track eating habits and percentage of leaf damage, in addition to pathology where relevant.

Test samples

Test and control samples were prepared as follows:

Spinosad was used as a positive insecticide control, used as per the manufacturer's instructions. Water alone, and water with eNtomate™ surfactant (2.5 mL/L) were used as negative controls.

B. laterosporus NMI No. V12/001945 culture (not autoclaved, not filtered) comprising live cells together with eNtomate™ surfactant (2.5 mL/L) was used as a control.

Test samples comprised autoclaved unfiltered B. laterosporus NMI No. V12/001945 culture extract, autoclaved filtered B. laterosporus NMI No. V12/001945 culture extract, and filtered B. laterosporus NMI No. V12/001945 culture extract, each with eNtomate™ surfactant (2.5 mL/L). Filtered samples were prepared using a 3 kDa Amicon Ultra cutoff filter (Merck).

Samples comprising B. laterosporus NMI No. V12/001945 culture or extracts were diluted to 7.5 mL/L with water, and 4 mL of diluted sample was sprayed onto each sample pak choy leaf disc.

Mortality % was assessed once per day over the four day trial.

Results

As can be seen in Figure 1, the greatest mortality (90%) was observed with the autoclaved B. laterosporus NMI No. V12/001945 culture extracts, in which no viable bacteria were present. All samples either containing or derived from B. laterosporus NMI No. V12/001945 showed insecticidal activity against target lepidopterans above that of the negative controls.

The autoclaved filtered B. laterosporus NMI No. V12/001945 culture extract sample, comprising a sub 3 kDa fraction, showed comparable insecticidal activity to the unfiltered autoclaved extract. Notably, neither of these samples comprise viable B. laterosporus NMI No. V12/001945 cells.

Example 2: Assessment of insecticidal activity

This example describes a mortality trial to assess the insecticidal activity of various Brevibacillius laterosporus extracts against Diamondback moth ( Plutella xylostella, Lepldoptera: Plutellidae ).

The mortality bioassay was performed as described in Example 1. Test and control samples were prepared as follows:

Spinosad was used as a positive insecticide control, used as per the manufacturer's instructions.

Water alone, and water with eNtomate™ surfactant (2.5 mL/L) were used as negative controls.

B. laterosporus NMI No. V12/001945 culture (not autoclaved, not filtered) with eNtomate™ surfactant (2.5 mL/L) was used as a control (Not Auto. Control).

Test samples comprised:

- an above 3 kDa retentate of autoclaved B. laterosporus NMI No. V12/001945 culture extract

(Auto. Washed pellet),

- an above 3 kDa retentate of unautoclaved B. laterosporus NMI No. V12/001945 culture extract

(Not Auto. Washed pellet),

- a sub 3 kDa filtrate of autoclaved B. laterosporus NMI No. V12/001945 culture extract

(Autoclaved 3kDa),

- a sub 3 kDa filtrate of unautoclaved B. laterosporus NMI No. V12/001945 culture extract (Not

Autoclaved 3kDa).

All test samples were prepared with eNtomate™ surfactant (2.5 mL/L).

Filtered samples were prepared using a 3 kDa Amicon Ultra cutoff filter (Merck).

Mortality % was assessed once per day over the four day trial.

Results

As can be seen in Figure 2, samples comprising autoclaved B. laterosporus NMI No. V12/001945 culture extracts filtered through a 3 kDa cutoff filter, and thus comprising nothing larger than 3 kDa, showed greater than 90% mortality at 4 days. Again, all samples either containing or derived from B. laterosporus NMI No. V12/001945 showed insecticidal activity against target lepidopterans above that of the negative controls. The autoclaved filtered B. laterosporus NMI No. V12/001945 culture extract sample (Autoclaved 3kDa), comprising a sub 3 kDa fraction, showed rapid onset insecticidal activity, and greater than 90% insecticidal efficacy at 4 days. The filtered unautoclaved extract (Not Autoclaved 3kDa), comprising a sub 3 kDa fraction that had not been autoclaved, also showed greater than 80% insecticical efficacy at 4 days. Notably, neither of these samples comprise viable B. laterosporus NMI No. V12/001945 cells.

Example 3: Assessment of insecticidal activity and synergism

This example describes a mortality trial to assess the insecticidal activity of various Brevibacillius laterosporus extracts, with and without the synergist dillapiole, against Diamondback moth ( Plutella xylostella, Lepldoptera: Plutellidae).

B. laterosporus NMI No. V12/001945 culture (not autoclaved, not filtered) with eNtomate™ surfactant (2.5 mL/L) was used as a control (Small Shake Flask).

Test samples comprised:

- B. laterosporus NMI No. V12/001945 culture (not autoclaved, not filtered) with dillapiole at

0.75% w/w (SSF + dillapiole 0.75%),

0.25% w/w (SSF + dillapiole 0.25%).

All test samples were prepared with eNtomate™ surfactant (2.5 mL/L).

Samples comprising B. laterosporus NMI No. V12/001945 culture were diluted to 7.5 mL/L with water, and 4 mL of diluted sample was sprayed onto each sample pak choy leaf disc.

Mortality % was assessed once per day over the four day trial.

Results

As can be seen in Figure 3, the mortality at day 4 with a sample comprising B. laterosporus NMI No. V12/001945 culture and dillapiole at 0.75% w/w was comparable to that observed with the positive control spinosad. Similarly, approximately 90% mortality was observed at day 4 with a sample comprising B. laterosporus NMI No. V12/001945 culture and dillapiole at 0.25% w/w. The presence of dillapiole at either concentration increased mortality beyond that observed with the sample comprising B. laterosporus NMI No. V12/001945 culture without dillapiole.

Notably, as shown in Figure 4, dillapiole at 0.25% w/w (with eNtomate™ surfactant at 2.5 mL/L) in water showed comparable mortality at day 4 to that observed with the water + eNtomate™ surfactant control. As such, the mortality observed with the dillapiole control is likely due to the presence of eNtomate™ surfactant, rather than the dillapiole itself.

This in turn strongly supports the conclusion that the increased insecticidal activity observed in samples comprising B. laterosporus NMI No. V12/001945 and dillapiole, as clearly seen in Figure 3, is the result of synergism between the dillapiole and components present in the B. laterosporus NMI No. V12/001945 culture and/or media. Example 4: Assessment of insecticidal activity and synergism

This example describes a mortality trial to assess the insecticidal activity of various Brevibacillius laterosporus extracts and potential synergists against Diamondback moth ( Plutella xylostella, Lepidoptera: Plutellidae).

B. laterosporus NMI No. V12/001945 culture (not autoclaved, not filtered) with eNtomate™ surfactant (2.5 mL/L) was used as a control (BL45 Control).

Test samples comprised:

- B. laterosporus NMI No. V12/001945 culture (not autoclaved, not filtered) with piperonyl butoxide (PBO) at 0.25% w/w (BL45 + PBO),

- B. laterosporus NMI No. V12/001945 culture (not autoclaved, not filtered) with parsley oil at

0.25% w/w (BL45 + Parsley Oil),

0.25% w/w (BL45 + Dillapiole).

All test samples were prepared with eNtomate™ surfactant (2.5 mL/L).

Mortality % was assessed once per day over the four day trial.

Results

As can be seen in Figure 5, observed mortality at days 3 and 4 with a sample comprising B. laterosporus NMI No. V12/001945 culture and dillapiole at 0.25% w/w was comparable to that observed with the positive control spinosad.

Observed mortality with sample comprising B. laterosporus NMI No. V12/001945 culture and either parsley oil, or piperonyl butoxide, was comparable to the culture extract control in the absence of these other agents.

Thus, the increased mortality in the presence of dillapiole was not observed in the sample comprising B. laterosporus NMI No. V12/001945 culture with the other added agents.

Example 5: Assessment of insecticidal activity of various formulations

This example describes a mortality trial to assess the insecticidal activity of various formulations of Brevibacillius laterosporus extracts against Diamondback moth ( Plutella xylostella, Lepidoptera: Plutellidae ).

Water alone, water with eNtomate™ surfactant (2.5 mL/L), water with eNtomate C™ surfactant (eNtomate™ + Trisol 70 + chemidet 24-3N, 2.5 mL/L), and a WDG formulation lacking B. laterosporus NMI No. V12/001945 (WDG Blank) were used as negative controls.

B. laterosporus NMI No. V12/001945 culture (not autoclaved, not filtered) with eNtomate™ surfactant (2.5 mL/L) was used as a control (BL45 Control). Test samples comprised:

- a WDG formulation comprising autoclaved B. laterosporus NMI No. V12/001945 culture (with eNtomate™ surfactant (2.5 mL/L), (BL45 WDG + Entomate),

- a WDG formulation comprising B. laterosporus NMI No. V12/001945 culture with eNtomate C™ surfactant (2.5 mL/L), (BL45 WDG + Entomate C),

Mortality % was assessed once per day over the four day trial.

Results

As can be seen in Figure 6, the mortality of ~80% was observed at day 4 for both the WDG formulation comprising B. laterosporus NMI No. V12/001945 with eNtomate C, and the B. laterosporus NMI No. V12/001945 culture control.

Accordingly, this data shows that a WDG formulation comprising B. laterosporus NMI No. V12/001945 which has comparable insecticidal efficacy to that of a fresh culture of B. laterosporus NMI No. V12/001945 can be prepared as described herein.

Example 6: Characterisation of insecticidal activity

This example presents a further characterisation of insecticidal activity from Brevibacillius laterosporus strain NMI No. V12/001945 (BI45).

The results presented herein confirm that the main insecticidal activity of BI45 was located within the culture supernatant.

As proteins generally denature at temperatures above 80°C and aggregate, with a concomitant loss of bioactivity, a sample of the culture supernatant was heated to get an indication of the molecular nature of the la rvicida I toxins present in the culture supernatant. To identify insecticidal toxins within the culture, for example whether the insecticidal activity comprised protein or a non- proteinaceous agent, or combinations thereof, proteins in the culture supernatant were precipitated with ammonium sulphate, concentrated and separatated by size exclusion chromatography.

The size exclusion fractions, original full strength culture, unwashed spores and unheated and heated culture supernatant were tested for activity against DBM larvae. Additionally, filtered culture supernatant derived from another sporulated culture of BI45 was included in the DBM biosassay. This additional sample (referred to below as FCS-D) was sent for further bioassay, and was also tested for DBM larvae activity.

Methods

General bioassay setup

Cabbage discs were cut out from a green cabbage leaf using a core borer with a 3 cm diameter. The leaf discs were washed in dH₂0 prior to treatment application. A 100 pi of treatment was spread onto both sides of each cabbage leaf disc and left to air dry on an angle in a sterile petri dish in a class 1 laminar flow cabinet. The air-dried leaf discs were put into sterile plastic containers (HuhTamaki, 30 ml volume) containing 3 cm diameter filter papers (Labserv, qualitative paper). The filter papers were hydrated with a 100 pi sterile Milli-Q water (MQW) before the leaf discs were added to the containers to prevent the cabbage discs from drying out too rapidly. Six in-house colony reared second to third instar diamondback moth, Plutella xylostella, caterpillars were added to each leaf disc with 3 blocks per treatment. The number of caterpillars per treatment was therefore 18 (N = 18). Bioassays were set up according to a randomised block design. DipelDF Bacillus thuringiensis subsp. kurstaki H-3a, 3b HDI (Nufarm, Valent BioSciences® Corporation) was used as a positive control at 32000 units/ml (1 mg/ml). Sterile MQW or mLB+ medium were used as negative controls. All treatments, including the controls, contained 0.5% Synoil adjuvant surfactant (Orion Agriscience). The bioassays were incubated at 23-25 °C with a 16:8 hour light dark cycle. Mortality rates were recorded every 24 hours after incubation for 4-9 days. The bioassay results were analysed by a One-Way ANOVA using Genstat version 16. Treatments with the constant values 0 or 100 were not included into the ANOVA to maintain variability in the statistical analysis. The least significant effect (LSE) was used to compare a constant valued treatment with a non-constant valued treatment.

Size exclusion chromatography of BI45 culture supernatant ammonium sulphate precipitate

Ammonium sulphate precipitation

A 100 ml of culture supernatant filtered through a 0.2 pm filter (Millpore) was collected from a six-day-old sporulated culture of BI45. A magnetic stirrer was used to stir the culture supernatant slowly on ice while ammonium sulphate was gradually added until it reached a concentration of 85%, causing the proteins to precipitate. The precipitate was collected by centrifugation at 10,000 x g for 20 minutes at 4°C. The pellet was resuspended in 20 ml resuspension buffer (20 mM Tris-HCI and 150 mM NaCI; pH 7.5). Subsequently, the suspension was washed three times in resuspension buffer by centrifugation at 8000 x g for 15 minutes at 4°C, using a Vivaspin 20, 5000 molecular weight cut off (MWCO) concentrator column (GE Healthcare). After the third wash, the concentrate was concentrated down to 5 ml using the Vivaspin 20, 5000 MWCO column, and stored at 4°C until further use.

Size exclusion chromatography

The 5 ml desalted protein suspension, as described above, was concentrated to 1 ml by centrifugation at 5800 x g for 10 minutes at 15°C using a 10,000 MWCO concentrator column (Millipore). The concentrate was resuspended in 5 ml TBS column buffer (25 mM Tris-HCI and 150 mM NaCI; pH 7.4) and concentrated down to 1 ml using the 10,000 MWCO concentrator column. The sample was subsequently injected into a Sephacryl S200 High Resolution (GE Health care Life Sciences) column (1.5 x 42 cm) for the separation of the proteins present in the sample. The size exclusion chromatography ran for 2.5 hours at a speed of 1 ml/minute operated by a Bio-Rad Biologic LP (low Pressure) chromatography system. Fractions of 1 ml were collected by a fraction collector (BioFracTM, BioRad) and stored at 4°C until further use. Fractions were analysed by SDS-PAGE,

Native PAGE and Bradford measurements. A DBM bioassay was conducted with the protein fractions of interest as described above. Sterile MQW was used as a negative control.

The collection of crude culture fractions of BI45

A volume of 50 ml of a six day old sporulated culture of BI45, cultured in mLB+, was centrifuged at 10,000 x g for 15 minutes at 4°C to separate the spores from the culture supernatant. Prior to centrifugation, 5-10 ml of full strength culture was kept in storage at 4°C or -20°C to be tested in DBM insect bioassays. The spore pellet was washed thrice in 50 ml of sterile MQW by centrifugation at 10,000 x g for 15 minutes at 4°C. The washed spores were resuspended in 50 ml sterile MQW and stored at 4°C or -20°C until further use. The culture supernatant was centrifuged six-seven times under the same conditions as above to remove as many particles as possible. Subsequently, the culture supernatant was filtered through a 0.8 pm/0.2 pm vacuum filter. The filtered culture supernatant was kept at 4°C or -20°C until further use. A part of the culture supernatant was heated at 65°C and/or 95°C in a water bath prior to being tested for toxicity against DBM caterpillars. The samples were bioassayed against DBM as described above. Bioassay treatments consisted of the full strength culture, the washed spore suspension, the culture supernatant kept at 4°C, heated culture supernatant at 65°C and/or heated at 95°C. Sterile MQW was used as a negative control.

Results

The size exclusion chromatography yielded two distinct peaks (Figure 7). Protein analysis of the peak fractions showed a poor separation with a generally high number of bands in each fraction (Figures 8 and 9). Fractions with similar protein profiles were pooled to test for activity in a DBM larvae bioassay. The DBM bioassay results reveal that the size exclusion fractions did not have any significant lethal activity (Table 1).

Table 1. Cumulative mortality (%) at day 4 of the DBM bioassay with BI45 size exclusion chromatography fractions.

Treatment Protein Cumulative concentration Mortality (%) LSD^a

(mg/ml)

Day 4 5% 10%

1. Dipel Bt subsp. kurstaki NM^b (100.0)

2. Negative control (sterile MQW) 20.0 NS NS

3. BI45 full strength culture 5.5 60.0 NS *

4. BI45 unwashed spore suspension 3.6 80.0

5. BI45 supernatant 4°C 7.0 60.0 NS *

6. BI45 supernatant 65°C 6.3 20.0 NS NS

7. BI45 supernatant ammonium sulphate 8.5 66.7 precipitate

8 Size exclusion fractions 26-36 6.9 6.7 NS NS

9. Size exclusion fractions 37-39 4.7 (0.0)

10. Size exclusion fractions 40-42 3.9 (0.0)

11. Size exclusion fractions 43-47 3.9 26.7 NS NS

12. Size exlusion fractions 48-67 0.1 26.7 NS NS

13. FCS-D ND^C (100.0)

14. FCS-D, lyophilised ND (100.0)

LSD 5% 43.11

LS effect 5% (LSD 5%/V2) 30.48 Overall P-value 0.021

LSD 10% 35.51

LS effect 10% (LSD/V2) 25.11 Overall P-value 0.021

Overall level of significance * * * ^a I The LSD was used to compare the treatments included in the ANOVA with the negative control. ^b|

NM = not measured. ^c| ND = non detectable.

NS = non significant; * = somewhat significant = 5% (P < 0.05); ** = significant = 1% (P < 0.01);

*** = highly significant = 0.1% (P < 0.001).

General ANOVA of the cumulative mortality (%) at day 4 of the diamondback moth bioassay with BI45 size exclusion chromatography fractions. Treatments that are constant, 0 or 100, have been omitted from the ANOVA (means are in brackets). To compare two un-bracketed means, the LSD is used. To compare a bracketed mean with an un-bracketed mean, the LS Effect is used.

The unwashed spores and ammonium sulphate precipitate had a significantly higher cumulative mortality than the other BI45 treatments when the LSD was 5% (P = 0.021). The full strength culture and the unheated culture supernatant, unwashed spores and ammonium sulphate precipitate had a significantly higher cumulative mortality when the LSD was 10% (P = 0.021). The heated culture supernatant lost a lot of activity and had no significant mortality, indicating that at least some insecticidal compounds in the culture supernatant were heat sensitive and most likely of proteinaceous nature.

The FCS-D samples were not included in the ANOVA because of their constant values on day four, where the mortality was 100%, but the results could be interpreted using the LS Effect. The FCS-D samples had a significantly higher mortality than the other treatments, except for the unwashed spores and Dipel, with both 5% and 10% LS Effect levels (Table 1, P = 0.021). This was unexpected, given the low number of protein bands observable in these samples by SDS-PAGE (Figure 10) and the fact that no proteins could be detected by Bradford measurement.

The results of this experiment showed a loss of activity in certain culture supernatant samples when heated at 65°C, suggesting that certain insecticidal compounds within the supernatant were likely of proteinaceous nature. However, the size exclusion fractions comprising protein did not display any significant activity.

Addtionally, the FCS-D samples were highly toxic and had an undetectable amount of protein. This suggested that at least some of the high insecticidal activity observed with the FCS-D samples was likely not caused by proteins, but rather a non-proteinaceous agent.

Example 7: Field trial of insecticidal activity

This example describes a large scale field trial to assess the insecticidal activity of various formulations of Brevibacillius laterosporus extracts against Diamondback moth ( Plutella xylostella, Lepldoptera: Plutellidae).

The trial site was a greenhouse of Xuejin Vegetable farm at Tongbo village, Jiangshan Town, Xinluo District. Non-heading Chinese cabbage, planted at 6,000 plants per Mu (Chinese unit of area,

= 0.0667 hectare) in sandy, medium fertile soil, was at the rosette stage. A great number of diamondback moth larvae was found evenly distributed in the greenhouse, with peak numbers of 1^st- 3^rd instar larvae.

Each treatment had 4 replicates of 50 square metre trial plots randomly arranged throughout the greenhouse.

Test and control samples were prepared as follows:

34% Spinosad-methoxyfenozide (Dow AgroSciences, U.S.), diluted x 1000, was applied in accordance with the manufacturer's instructions, was used as a positive control. Water was used as a negative control.

B. laterosporus NMI No. V12/001945 culture extract, with 1.5 x 10⁹/rmL average spore count (not autoclaved, not filtered) with eNtomate C™ surfactant (2.5 mL/L), diluted to 0.5 mL/L.

Test and control samples were sprayed with an electric sprayer at 60 kg/Mu (0.0667 hectare), with both the front and back sides of the plants evenly sprayed. The spraying was repeated at 7 days.

The quantity of larvae was investigated before spraying, and at 3, 7, and 10 days after spraying. In each trial area, 10 plants were selected for quantification of infestation.

Results

As can be seen in Table 2, the B. laterosporus NMI No. V12/001945 culture extract achieved greater than 90 % control efficiency at 10 days post spraying, comparable to that of the spinosad positive control. Rapid control was achieved, with greater than 50 % control efficiency achieved in as little as 3 days post sprarying. No harmful effect on the crops themselves was observed.

Table 2. Control efficiency in greenhouse field trial

No. live = number of live DBM larvae; %Redn = Reduction rate %; Contol% = control efficiency %

Example 8: Field trial of insecticidal activity

This example describes a large scale field trial to assess the insecticidal activity of various formulations of Brevibacillius laterosporus against Fall Armyworm (FAW, Spodoptera frugiperda).

The main objectives were to:

Bioassay maize young leaf spray (with different concentrations on L2, L3, L4 and L5 larval stages)

Bioassay artificial diet spray to optimise concentration for subsequent trials, and

Bioassay whole plant spray using the optimal concentration.

Methods

Experiments on different FAW larval stages (L2, L3, L4 and L5, IITA-Benin) were used to establish optimized concentrations of B. laterosporus NMI No. V12/001945.

For the first experiment, only the larval stage L3 was used. The following concentrations, in a completely random block, were used:

Control: 0 spores/ml

5 x 10⁷ spores/ml

10^s spores/ml

- 1.5 x 10⁸/ml

Four additional experiments were then set up: L2, L3 (resumed), L4 and L5. Another biopesticide, botanical (TopBio) was used for comparison. Topbio is a neem oil base product manufactured in Benin. It is used at a dose of 2L/ha in vegetable crops and now testing against FAW.

The following Concentrations were used:

Control : 0 spores/ml

10⁵ spores/ml

10⁶ spores/ml

10⁷ spores/ml

5 x 10⁷ spores/ml

10^s spores/ml

- 10⁹/ml

- Topbio 0.12%

Young, pre-germinated maize leaves were washed in 10% bleach. Samples comprising approximately 10 g leaves were each soaked in a solution of 20 ml of the test samples (above) for several minutes. FAW ( Spodoptera frugiperda) larvae of 5, 7, 9, 11 days (L2, L3, L4, L5), fasted for 6 hours were introduced individually into aerated boxes. 20 larvae per sample were used, with three repetitions.

The young leaves soaked with each of the test solutions were then introduced into the boxes (one sheet per box). Mortality monitored daily up to fifteen days.

Data analysis

Mean Survival Times (TMS) were calculated, using a survival Kaplan Meier analysis (IBM SPSS Statistics 21, 1989-2012). Log-Rank test was used to compare pairwise the mean survival times of the larvae. Differences in larval mortality rates were subjected to analysis of variance (ANOVA). When the F values are statistically significant, the means were compared using the Student-Newman-Keuls (SNK) test at the 5% probability level.

The LC50 is defined as the concentration of an agent (chemical or biological) required to produce the death of half of the organisms tested at a given time after application (Maddox, 1982).

The analysis and modelling of time-concentration-mortality data was performed using the cox- regression model (IBM SPSS Statistics 21, 1989-2012). Only data from product concentrations were used for this analysis.

Results and discussion

Larval mortality

Mean mortality in L3 larvae ranged from 15% (control) to 98% (10^s spores/ml 8i 1.5 x 10^s spores/ml). No significant difference was observed between the two highest concentrations at the 5% threshold ( F_3r8 = 128.067, P <0.000) (Figure 11).

Lethal concentration 50 (LC50); Experiment 1

Lethal concentrations were calculated based on the time taken to kill 50% of the treated larval population. Table 3 shows the LC50 values for 3, 5 and 7 days.

It should be noted that the concentrations increase with the number of days. Thus, it took 5.63 x 10⁹ spores/ml to kill 50% L3 larvae in 3 days, while it took 5.43 x 10⁶ spores / ml to kill 50% L3 larvae in 7 days.

Table 3: LC50 of L3, Experiment 1

Mean survival times (MST)

The survival of third instar larvae was significantly more affected by the higher concentrations used, 10^s spores/ml and 1.5 xlO⁸ spores/ml, but the latter had similar TMS (Table 4)

Table 4: MST of L3 lavae as a function of concentration

In table 4 above, means followed by the same letter are not significantly different from one another (analysis of variance followed by the SNK test at the 5% threshold).

Series of experiments on L2, L3, L4 and L5 Larval mortality

In general, the observed mean mortality rate increased with larval age, with later larval stages exhibiting higher mortality. Without wishing to be bound by any theory, applicants infer this may be a result of increased consumption of bacteria over time.

As can be seen in Table 5 below, the concentrations 5 x 10⁷ spores/ml, 10^s spores/ml, 10⁹ spores/ml showed the highest mortality rates, with 10⁹ spores/ml significantly higher regardless of the larval stage (Table 5).

In general, the mortality observed with the control agent Topbio was comparable to that observed with low concentrations of B. laterosporus NMI No. V12/001945. B. laterosporus NMI No. V12/001945 samples at 10⁹ spores/ml and 10^s spores/ml exhibited mean mortality rates significantly higher than those observed with Topbio, apart from with L5 larvae (Table 5).

Table 5: Mortality rate by stage and concentration of Brevibacillus laterosporus (% ± SE)

In table 5 above, means in the same column followed by the same letter are not significantly different from one another (analysis of variance followed by the SNK test at the 5% threshold).

Lethal concentration 50 (LC50); Series of experiments

The concentrations of the B. laterosporus NMI No. V12/001945 product required to kill 50% of the larvae in 3, 5, 7 days of the different larval stages used are recorded in Table 6. Concentrations necessary to kill 50% of the larval population in 3 days were very high. They ranged from 2.26 x 10¹⁷ spores/ml (L2) to 6.88 x 10^s spores/ml (L5). The lowest concentrations were those required to kill the larval population in 5 and 7 days (see Figure 12 - 15). This supports the high mortality rates observed among older larvae.

Table 6: LC50 values, after inoculation of the larvae of S. frugiperda to different concentrations of EBJ2

Mean survival time (days ±SE)

In general, as observed with the mortality rates and LC50, mean larval survival times decreased with the age of the larval stages. The older the larvae, the more vulnerable they are (Table 7). 10⁹ spores/ml has the least average survival time followed by 10^s spores/ml.

Table 7: Mean Survival Time (TMS) (days ± SE) of S. frugiperda larvae L2, L3, L4 and L5 inoculated with spores of Brevibacillus laterosporus

Conclusion

These data show that B. laterosporus NMI No. V12/001945 has significant insecticidal activity against FAW larvae, particularly at higher but readily achievable in the field concentrations such as 10^s and 10⁹ spores/ml.

Example 9: Assessment of insecticidal activity

Spinosad was used as a positive insecticide control, used as per the manufacturer's instructions. Water alone (Water), and water with eNtomate™ surfactant (2.5 mL/L, Entomate) were used as negative controls.

Test samples comprised:

- B. laterosporus NMI No. V12/001946 cells only prepared from centrifuged cell culture from which the culture supernatant has been removed (Pellet Recon);

- a culture supernatant prepared from B. laterosporus NMI No. V12/001946 cell culture

(Supernatant);

- a sub 3 kDa filtrate of autoclaved B. laterosporus NMI No. V12/001946 culture extract (<

3kDa),

- an autoclaved B. laterosporus NMI No. V12/001946 whole cell culture (Autoclaved WC); and

- a B. laterosporus NMI No. V12/001946 whole cell culture (Whole Culture).

All test samples were prepared with eNtomate™ surfactant (2.5 mL/L).

Samples comprising B. laterosporus NMI No. V12/001946 culture or extracts were diluted to 1 x 10^s spores/mL with water, and 4 mL of diluted sample was sprayed onto each sample pak choy leaf disc.

Mortality % was assessed once per day over the four day trial.

Results

As can be seen in Figure 16, all samples either containing or derived from B. laterosporus NMI No. V12/001946 showed insecticidal activity against target lepidopterans above that of the negative controls.

Furthermore, all samples comprising components of the culture supernatant from B. laterosporus NMI No. V12/001946 cell culture had at least comparable, if not superior, insecticidal activity than B. laterosporus NMI No. V12/001946 cells alone: compare the mortality observed with each of Whole Culture, Autoclaved WC, < 3kDa, and Supernatant mortality with that observed with Pellet Recon.

The autoclaved B. laterosporus NMI No. V12/001946 whole culture sample (Autoclaved WC) showed a rapid onset of insecticidal activity with over 60% mortality observed at day 1, and approximately 80% insecticidal efficacy at 4 days - close to the 90% efficacy observed with whole cell culture in which living B. laterosporus NMI No. V12/001946 was present.

These data establish that substantial insecticidal efficacy can be achieved using culture extracts from living B. laterosporus NMI No. V12/001946, including sub 3 kDa fraction from such cultures, and indeed substantial efficacy can be achieved without the need for viable B. laterosporus NMI No. V12/001946 cells being present.

Example 10: Assessment of insecticidal activity

Test samples comprised:

- B. laterosporus NMI No. V12/001944 cells only prepared from centrifuged cell culture from which the culture supernatant has been removed (Pellet Recon);

- a culture supernatant prepared from B. laterosporus NMI No. V12/001944 cell culture

(Supernatant);

- a sub 3 kDa filtrate of autoclaved B. laterosporus NMI No. V12/001944 culture extract (<

3kDa),

- an autoclaved B. laterosporus NMI No. V12/001944 whole cell culture (Autoclaved WC); and

- a B. laterosporus NMI No. V12/001944 whole cell culture (Whole Culture).

All test samples were prepared with eNtomate™ surfactant (2.5 mL/L).

Samples comprising B. laterosporus NMI No. V12/001944 culture or extracts were diluted to 1 x 10^s spores/mL with water, and 4 mL of diluted sample was sprayed onto each sample pak choy leaf disc.

Mortality % was assessed once per day over the four day trial.

Results

As can be seen in Figure 17, all samples either containing or derived from B. laterosporus NMI No. V12/001944 showed insecticidal activity against target lepidopterans above that of the negative controls.

Furthermore, all samples comprising components of the culture supernatant from B. laterosporus NMI No. V12/001944 cell culture had at least comparable, if not superior, insecticidal activity than B. laterosporus NMI No. V12/001944 cells alone: compare the mortality observed with each of Whole Culture, Autoclaved WC, < 3kDa, and Supernatant mortality with that observed with Pellet Recon.

Both the cell-free supernatant (Supernatant) sample and the autoclaved B. laterosporus NMI No. V12/001944 whole culture sample (Autoclaved WC) showed a rapid onset of insecticidal activity, with over 50% insecticidal efficacy at day 1 - close to the 70% efficacy observed at day 1 with whole cell culture in which living B. laterosporus NMI No. V12/001944 and culture media was present.

These data establish that substantial insecticidal efficacy can be achieved using culture extracts from living B. laterosporus NMI No. V12/001944, including a sub 3 kDa fraction from such cultures, and indeed substantial efficacy can be achieved without the need for viable B. laterosporus NMI No. V12/001944 cells being present.

Example 11: Assessment of insecticidal activity

This example describes a mortality trial to assess the insecticidal activity of various Brevibacillius laterosporus extracts against Diamondback moth ( Plutella xylostella, Lepldoptera: Plutellidae ) after long term storage.

Spinosad was used as a positive insecticide control, used as per the manufacturer's instructions. A test sample (Soluble concentrate) comprising B. laterosporus NMI No. V12/001945 culture was formulated as shown in Table 8 below. This was stored at 54 °C for 2 weeks (equivalent to storage at room temperature for 2 years) prior to testing.

Table 8: Soluble concentrate formulation

Freshly harvested cell cultures of B. laterosporus NMI No. V12/001945 grown in a large bioreactor (EBJ2 Big Reactor), and in a mini bioreactor (EBJ2 Mini Reactor) were also tested.

Water alone, and a composition comprising the soluble concentrate formulation set out in Table 8 but lacking B. laterosporus NMI No. V12/001945 (Excipients of soluble concentrate) were used as negative controls.

Samples comprising B. laterosporus NMI No. V12/001945 were diluted to 1 x 10^s spores/mL with water, and 4 mL of diluted sample was sprayed onto each sample pak choy leaf disc.

Mortality % was assessed once per day over the four day trial.

Results

As can be seen in Figure 18, rapid insect mortality approaching 100% was observed on day 1 with the freshly cultured B. laterosporus NMI No. V12/001945, comparable to the positive control Spinosad. The observed toxicity of freshly cultured samples comprising B. laterosporus NMI No. V12/001945 exhibited both contact action associated with secreted metabolites and/or the >3 kDa fraction, and toxicity associated with viable B. laterosporus NMI No. V12/001945 cells.

Approximately 50% mortality was observed on day 1 with the Soluble concentrate sample stored at 54 °C emulating long term storage. Ultimately, approximately 80% insect mortality was observed with this sample at day 4, indicating that formulations of B. laterosporus NMI No. V12/001945 are able to be prepared that maintain insecticidal activity after long term storage.

This data shows that formulations comprising B. laterosporus NMI No. V12/001945 having significant, biologically relevant and commercially useful insecticial efficacy prepared as described herein can support such activity even after long term storage. Example 12: Assessment of insecticidal activity

This example describes a mortality trial to assess the insecticidal activity of various Brevibacillius laterosporus extracts against Diamondback moth ( Plutella xylostella, Lepidoptera: Plutellidae ) after long term storage.

The mortality bioassay was performed as described in Example 1. Test and control samples were as described above in Example 11, except in this experiment the Soluble concentrate samples comprising B. laterosporus NMI No. V12/001945 (see Table 8 above) were stored for 2 months at room temperature (Soluble concentrate Shelf) or at 4 °C (Soluble concentrate Fridge).

Spinosad was used as a positive insecticide control, used as per the manufacturer's instructions. Freshly harvested cell cultures of B. laterosporus NMI No. V12/001945 (EBJ2 Mix) and a 7 day old culture (Day 7) were also tested.

Water alone, and a composition comprising the soluble concentrate formulation set out in Table 8 but lacking B. laterosporus NMI No. V12/001945 (Soluble concentrate excipients) were used as negative controls.

Mortality % was assessed once per day over the four day trial.

Results

As can be seen in Figure 19, rapid insect mortality approaching 60% was observed on day 1 with both of the Soluble concentrate samples that had undergone 2 months' storage, with comparable activity between the sample stored at room temperature and that stored at 4 °C. Ultimately, approximately 80% insect mortality was observed with these samples at day 3, indicating that formulations of B. laterosporus NMI No. V12/001945 are able to be prepared that maintain insecticidal activity after long term storage, even at room temperature.

This data shows that formulations comprising B. laterosporus NMI No. V12/001945 having significant, biologically relevant and commercially useful insecticial efficacy prepared as described herein can support such activity even after long term storage.

Publications

Baxter, S.W., Badenes-Perez, F.R., Morrison, A., Vogel, H., Crickmore, N., Kain, W., Wang, P., Heckel, D.G. and Jiggins, C.D. (2011) Parallel evolution of Bacillus thuringiensis toxin resistance in Lepidoptera. Genetics October, 189, 675-679.

Favret, M.E. and Yousten, A. A. (1985) Insecticidal activity of Bacillus laterosporus. J. Invertebrate Pathology 48,195-203.

Tabashnik, B.E., Cushing, N.L., Finson, N., Johnson, M.W. (1990) Field Development of Resistance to Bacillus thuringiensis in Diamondback Moth (Lepidoptera: Plutellidae). Journal of Economic Entomology, 83, 1671- 1676.

Tabashnik, B.E., Liu, Y., Malvar, T., Heckel, D.G., Masson, L. and Ferre, J. (1998) Insect resistance to Bacillus thuringiensis: uniform or diverse? Phil. Trans. R. Soc. Land. B 29 October 353, 1751-1756.

As used in this specification, the words "comprise", "comprises", "comprising", and similar words, are not to be interpreted in an exclusive or exhaustive sense. In other words, they are intended to mean "including, but not limited to". When interpreting each statement in this specification that includes the term "comprise", "comprises", or "comprising", features other than that or those prefaced by the term may also be present. The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.

Where in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.

It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the present invention.

The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.

Aspects of the invention have been described by way of example only, and it should be appreciated that variations, modifications and additions may be made without departing from the scope of the invention, for example when present the invention as defined in the indicative claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.

Claims

1. A composition comprising a cellular extract obtained from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, and/or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is or has been grown, together with one or more agriculturally-acceptable carriers.

2. The composition as claimed in claim 1, comprising a cellular extract obtained from B. laterosporus NMI No. V12/001945 and/or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown.

3. The composition as claimed in claim 1 or claim 2, wherein the cellular extract or the composition is substantially free of viable B. laterosporus NMI No. V12/001944 cells, is substantially free of viable B. laterosporus NMI No. V12/001945 cells, is substantially free of viable B. laterosporus NMI No. V12/001946 cells, or is substantially free of viable B. laterosporus cells.

4. The composition as claimed in any one of claims 1 to 3, wherein the cellular extract or the composition is substantially free of protein.

5. The composition as claimed in claim 4 wherein the cellular extract or the composition is substantially free of native protein, is substantially free of functional protein, and/or is substantially free of undenatured protein.

6. The composition as claimed in any preceding claim, wherein the extract or composition comprises a composition enriched in or to which has been added a sub-3 kDa fraction from B. laterosporus NMI No. V12/001945 or a culture thereof.

7. The composition as claimed in any preceding claim, wherein the composition comprises: a) from about 1% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001945; or b) from about 1% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; or c) both a) and b) above.

8. The composition as claimed in any preceding claim, wherein the extract or composition comprises a composition enriched in or to which has been added a sub-3 kDa fraction from B. laterosporus NMI No. V12/001946 or a culture thereof.

9. The composition as claimed in any preceding claim, wherein the composition comprises: a) from about 1% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001946; or b) from about 1% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; or c) both a) and b) above.

10. The composition as claimed in any preceding claim, wherein the extract or composition comprises a composition enriched in or to which has been added a sub-3 kDa fraction from B. laterosporus NMI No. V12/001944 or a culture thereof.

11. The composition as claimed in any preceding claim, wherein the composition comprises: a) from about 1% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001944; or b) from about 1% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; or c) both a) and b) above.

12. The composition as claimed in any preceding claim, wherein the extract or composition is substantially non-proteinaceous.

13. The composition as claimed in any preceding claim, wherein the extract or composition is one to which has been added an undenatured proteinaceous extract or fraction from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946.

14. The composition as claimed in any preceding claim, wherein the extract or composition is one which is enriched in undenatured protein from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946.

15. An insecticidal composition formulated as a water dispersible granule (WDG) comprising one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001945; b) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001945; c) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001945 is or has been grown; d) from about 1% to about 50% w/w one or more wetting agent; e) from about 1% to about 50% w/w one or more dispersant; f) from about 2% to about 50% w/w one or more humectant or agent to control water activity; g) from about 0% to about 50% w/w one or more protectants; h) from about 0% to about 50% w/w one or more nutrients or mixture thereof; i) from about 5% to about 80% w/w one or more filler; j) from about 0% to about 20% w/w one or more antioxidant or UV radiation protectant; k) from about 1% to about 50% w/w one or more binding agent;

16. An insecticidal composition formulated as a water dispersible granule (WDG) comprising one or more of the following: a) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001946; b) from about 2% to about 80% w/w viable B. laterosporus NMI No. V12/001944; c) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001946; d) from about 2% to about 80% w/w cellular extract obtained from B. laterosporus NMI No. V12/001944; e) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001946 is or has been grown; f) from about 2% to about 80% w/w of a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944 is or has been grown; g) from about 1% to about 50% w/w one or more wetting agent; h) from about 1% to about 50% w/w one or more dispersant; i) from about 2% to about 50% w/w one or more humectant or agent to control water activity; j) from about 0% to about 50% w/w one or more protectants; k) from about 0% to about 50% w/w one or more nutrients or mixture thereof;

L) from about 5% to about 80% w/w one or more filler; m) from about 0% to about 20% w/w one or more antioxidant or UV radiation protectant; n) from about 1% to about 50% w/w one or more binding agent; o) from about 0% to about 50% one or more disintegrating agent; p) from about 0% to about 10% w/w water; q) any combination of two or more of any of a) to p) above.

17. The composition as claimed in any preceding claim wherein the composition comprises one or more of piperonyl butoxide, safrole, apiole, parsley oil, and dillapiole.

18. The composition as claimed in any preceding claim wherein the composition is a synergistic composition comprising dillapiole.

19. A method of treating or protecting a plant or its surroundings, and/or plant derived materials, from pest infestation wherein the method comprises applying to the plant or its environment a composition comprising an effective amount of a cellular extract of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946.

20. A method of treating or protecting a plant or its surroundings, and/or plant derived materials, from pest infestation wherein the method comprises applying to the plant or its environment a composition as claimed in any preceding claim.

21. A method of controlling and/or preventing a pest infestation characterised by the step of applying a composition comprising an effective amount of a cellular extract of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, to a surface.

22. A method for controlling one or more insect pests, comprising the step of applying to a plant or its surroundings or a locus at which insect pests are present a composition as claimed in any preceding claim.

23. A method of controlling a pest or pest population, the method comprising contacting the pest or the pest population with a pesticidally-effective amount of a composition as claimed in any preceding claim.

24. A method of preparing a pesticidal or insecticidal composition, the method comprising a) optionally growing a culture of B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946; b) providing a cellular extract obtained from B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, and/or a composition comprising or derived from media in which B. laterosporus NMI No. V12/001944, and/or B. laterosporus NMI No. V12/001945, and/or B. laterosporus NMI No. V12/001946, is or has been grown; c) admixing the cellular extract and/or composition with one or more agriculturally- acceptable carriers; to provide the insecticidal composition.

25. The method as claimed in any preceding claim, wherein the composition is a composition as claimed in any one of claims 1 to 18.

26. The method as claimed in any preceding claim, wherein the composition is formulated as a water dispersible granule.

27. The method as claimed in claim 26, wherein the water dispersible granule comprises a WDG formulation as described herein.