USTE 20180084776A1MENTOR MAURORAIMA (19 ) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2018 / 0084776 A1 HURST et al. ( 43 ) Pub . Date : Mar . 29, 2018 (54 ) AGRICULTURAL COMPOSITION Publication Classification (51 ) Int. Ci. (71 ) Applicant: Agresearch Limited , Hamilton (NZ ) AOIN 25 / 12 (2006 .01 ) AOIN 25 /00 (2006 .01 ) (72 ) Inventors : Mark Robin Holmes HURST , AOIN 63 /04 ( 2006 . 01 ) (52 ) U . S . CI. Hamilton (NZ ); Jayanthi CPC ...... A01N 25 / 12 ( 2013 .01 ) ; AOIN 63 /04 SWAMINATHAN , Hamilton (NZ ) (2013 . 01 ) ; AOIN 25 /006 (2013 . 01 ) ( 73 ) Assignee : Agresearch Limited , Hamilton (NZ ) (57 ) ABSTRACT A granular composition that comprises one or more micro organisms, two or more plant powders and a biodegradable ( 21 ) Appl. No. : 15 /566 ,156 water - absorbent agent. Also provided is a method of pro ducing a granular composition by admixing a composition (22 ) PCT Filed: Apr. 13 , 2016 comprising two or more plant powders and a biodegradable water -absorbent agent, and ( i ) one or more lubricants to ( 86 ) PCT No. : PCT/ NZ2016 /050059 form a dough , and processing the dough to form granules , or ( ii ) coating a substrate with the dough to form the granular $ 371 (c )( 1 ) , composition . The microorganism can be located in the ( 2 ) Date : Oct. 12 , 2017 admixture , the dough or both the admixture and the dough . The granular composition is applied to soil to , for example , ( 30 ) Foreign Application Priority Data kill or control pests, maintain or increase plant or crop growth , maintain or increase pasture production , maintain or Apr. 13 , 2015 (NZ ) ...... 706932 increase plant, crop or pasture yield , deliver probiotics to Feb . 9 , 2016 (NZ ) ...... 716740 grazing animals or livestock , and /or bioremediate the soil. Patent Application Publication Mar . 29 , 2018 Sheet 1 of 6 US 2018 / 0084776 A1

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AGRICULTURAL COMPOSITION [ 0017 ] coating the dough with an exterior coating com prising one or more microorganisms to form the granular FIELD OF THE INVENTION composition . [0001 ] The present invention relates to an agricultural [0018 ] In a further aspect the invention relates to a method composition comprising one or more microorganisms, its of delivering one or more microorganisms to soil , compris methods of preparation and its application to soil . ing applying the granular composition of the invention on to soil . [0019 ] In a further aspect, the invention provides a method BACKGROUND TO THE INVENTION of controlling insect pests , the method comprising placing [ 0002 ] Microorganisms such as bacteria and fungi are insecticidal granular compositions in proximity to an insect useful alternatives to chemical agents for the improvement pest, the granular compositions being as claimed above . and / or maintenance of soil and plant health , including the [0020 ] Any one or more of the following embodiments control of pests . may relate to any of the aspects described herein or any [ 0003] The utility of microorganisms for application to combination thereof. soil is limited by the sensitivity of such microorganisms to [0021 ] In various embodiments the granular composition environmental conditions after application and during stor has age of compositions comprising microorganisms. [ 0022 ] a ) a bulk density of from about 0 . 2 g / cm² to [0004 ] Compositions that adequately protect the microor about 1 . 5 g / cm " , or ganism from environmental exposure are costly to manu [0023 ] b ) a mean diameter ( weight- based ) of from about facture . 1 mm to 5 mm , or 10005 ] It is an object of the present invention to provide an [0024 ] c ) a particle size distribution whereby at least agricultural composition comprising one or more microor 90 % of the granular composition have a diameter of ganisms, or to at least provide the public with a useful from 1 mm to 5 mm , or choice . 10025 ] d ) a particle size distribution whereby at least 90 % of the granular composition has a diameter within SUMMARY OF THE INVENTION 1 mm of the mean diameter , or [0006 ] In a first aspect, the invention provides a granular [0026 ] e ) a water activity of from about 0 . 2 to about composition comprising one or more microorganisms, two 0 . 95 , or or more plant powders and a biodegradable water- absorbent 100271 f ) a hardness ( the maximum load to crush ) of agent. from about 10 N to about 100 N , or [0007 ] In a further aspect, the invention provides a granu [0028 ] g ) a brittleness of from about 10 N to about 100 lar composition comprising one or more microorganisms N , or having a loading of less than 101° cells per gram of granular [0029 ] h ) a porosity of from about 1 to about 1000 , or composition , two or more plant powders and a biodegrad [0030 ] i) a swellability of from about 50 to about 300 % , able water- absorbent agent capable of absorbing at least or 10 % of its dry weight in water . [ 0031 ] j ) any combination of two or more of ( a ) to ( i ) [ 0008 ] In a further aspect, the invention provides a granu above . lar composition comprising one or more bacteria , two or [0032 ] In some embodiments the granular composition more plant powders and a biodegradable water- absorbent has a bacterial loading of less than about 1011 , 1010 , 10 % , 108 , agent capable of absorbing at least 10 % of its dry weight in 107, 106, 10 % or 104 cells per gram of granular composition , water. and useful ranges may be selected between any of these [0009 ] In a further aspect the invention relates to a method values . of producing a granular composition , the method comprising [0033 ] In various embodiments said two or more plant [0010 ] admixing a composition comprising one or more powders comprises a bran as at least one of said two or more microorganisms, two or more plant powders , a biodegrad plant powders . able water- absorbent agent capable of absorbing at least [0034 ] In various embodiments the granular composition 10 % of its dry weight in water, and one or more lubricants comprises a phagostimulant. to form a dough , and [0035 ] In various embodiments the granular composition [0011 ] processing the dough to form the granular compo comprises one or more polysaccharides . sition . [0036 ] In various embodiments the granular composition [0012 ] In a further aspect the invention relates to a method comprises one or more CO2- generating agents . of producing a granular composition , the method comprising 10037 ] In various embodiments the granular composition [0013 ] admixing a composition comprising one or more comprises at least about 1x104, 1x105 or 1x10 cfu / g after microorganisms, two or more plant powders and a biode storage at 4° C . for 14 days, and useful ranges may be gradable water - absorbent agent capable of absorbing at least selected between any of these values . 10 % of its dry weight in water , to form a dough , and [0038 ]. In various embodiments the granular composition [ 0014 ] coating a substrate with the dough to form the retains its integrity for at least about 48 hours after contact granular composition . ing about 0 . 1 , 0 . 2 , 0 . 3 , 0 . 4 , 0 . 5 , 0 . 6 , 0 . 7 , 0 . 8 , 0 . 9 or 1 ml of [0015 ] In a further aspect the invention relates to a method water per gram of granular composition . of producing a granular composition , the method comprising [0039 ] In various embodiments the microorganism is ( i ) [ 0016 ] admixing a composition comprising two or more one or more bacteria , ( ii ) one or more fungi, or ( iii ) one or plant powders , a biodegradable water -absorbent agent more bacteria and one or more fungi . capable of absorbing at least 10 % of its dry weight in water , 0040 ] In various embodiments the bacteria or fungi are and one or more lubricants to form a dough , and agriculturally and /or horticulturally useful, for example , the US 2018 / 0084776 A1 Mar . 29 , 2018 bacteria is pesticidal and/ or insecticidal , and /or supports water - absorbent polymer may comprise potato starch , corn plant growth and /or development, or any combination starch , sodium starch glycolate , cellulose , methylcellulose , thereof. peat, rice powder , sodium alginate , polyvinylpyrrolidone , [0041 ] In various embodiments the one or more bacteria sodium carboxymethyl cellulose , croscarmellose sodium or may comprise Serratia ( for example , Serratia entomophilia a combination of any two or more thereof. In a preferred or Serratia proteomaculans ) , Xanthamonas, Pseudomonas , embodiment the biodegradable water- absorbent agent com Rhizobium , Bifidobacterium , Lactobacillus, Streptococcus prises potato starch . ( Enterococcus ) , Yersinia ( for example , Yersinia ento 100531. In various embodiments the granular composition mophaga ), Pseudomonas, Bacillus, Pasteuria , Azobacter , may comprise about 0 .05 , 0 . 1 , 0 . 15 , 0 . 2 , 0 . 25 , 0 . 3 , 0 .35 , 0 . 4 , Enterobacter, Azospirillum , Cyanobacteria , Paecilomyces, 0 . 5 , 0 . 6 , 0 . 7 , 0 . 8 , 0 . 9 , 1 , 1 . 5 , 2 , 2 . 5 , 3 , 3 . 5 , 4 , 4 . 5 or about 5 % Streptomycetes , Chromobacterium , Rhanella , Burkholderia , by weight of the a biodegradable water - absorbent agent, and Paenibacillus, Collimonas, Sinorhizobium , Pantoea , useful ranges may be selected between any of these values . Lecanicillum , Erwinia , Pediococus, Leuconostoc, Aeromo [0054 ] In various embodiments the one or more saccha nas , Neptunomonas, Klebsiella , Ponchonia , Brevibacillus, rides may comprise a monosaccharide , a disaccharide, an Acinetobacter or a combination of any two or more thereof. oligosaccharide, a polysaccharide , or a sugar product , or a [ 0042 ] In various embodiments the one or more fungimay combination of any two or more thereof . In various embodi comprise Beauveria , Penicillium , Metarhizium , ments the one or more saccharides may comprise glucose , Trichoderma, Gliocladium , Coniothyrium , Verticillium , fructose , maltose , sucrose , lactose , trehalose , honey , such as Sclerotinia or a combination of any two or more thereof. clover honey, molasses , glucose syrup , corn syrup , for 10043 ] In various embodiments the bran may comprise example , high fructose corn syrup , golden syrup , treacle , wheat bran , rice bran , oat bran , barley bran , rye bran , cane sugar, refined sugar, raw sugar, brown sugar, white triticale bran , corn (maize ) bran , amaranth bran , millet bran , sugar, maltodextrin , cornflour, wheat flour, or a combination teff bran , quinoa bran , buckwheat bran , sorghum bran or a of any two or more thereof. combination of any two or more thereof. In a preferred [0055 ] In various embodiments the granular composition embodiment the bran comprises wheat bran . may comprise about 3 , 5 , 10 , 15 , 20 , 25, 30 , 35 , 40 , 45 or 10044 ] In various embodiments the granular composition about 50 % by weight of saccharides, and useful ranges may may comprise about 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 or about be selected between any of these values. 50 % by weight of bran , and useful ranges may be selected [0056 ] In various embodiments the granular composition between any of these values. may comprise about 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , or 90 % 10045 ] In various embodiments the plant powder may by weight of an inert bulking agent, and useful ranges may comprise wheat flour, rice powder , oat flour, barley flour, rye be selected between any of these values. In some embodi flour, triticale flour , corn (maize ) flour, amaranth flour, millet ments the inert bulking agent is gypsum or talc , such as a flour, teff flour, quinoa flour, buckwheat flour, sorghum flour clay. or a combination of any two or more thereof. In a preferred [0057 ] In various embodiments the one or more saccha embodiment the plant powder comprises rice powder or rides may be in the form of a powder or a liquid such as a maize flour. syrup , or a combination of a powder and a liquid . For [0046 ] In various embodiments the granular composition example , in one exemplary embodiment the one or more may comprise about 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55 , saccharides may comprise white sugar, and honey . In one 60 , 65 , 70 , 75 , 80 or about 85 % by weight of the one or more embodiment the one or more saccharides may comprise a plant powders , and useful ranges may be selected between powder and a liquid in a ratio of 200 : 1 , 175 : 1 , 150 : 1 , 125 : 1 , any of these values . 100 : 1 , 80 : 1 , 75 : 1 60 : 1 , 50 : 1 , 40 : 1 , 30 : 1 , 20 : 1 or 10 : 1 . [ 0047 ] In one embodiment the biodegradable water - absor [0058 ] In some embodiments the granular composition bent agent is capable of absorbing at least 10 % of its dry contains a lubricant. weight in water. [0059 ] In various embodiments the lubricant may be 10048 ] In various embodiments the biodegradable water selected from the group comprising a lipid such as a fat or absorbent agent is capable of absorbing at least 10 , 20 , 30 , oil . In various embodiments the lubricant may be selected 40 , 50 , 60 , 70 , 80 , 90 or 100 % water based on its dry weight, from the group comprising magnesium stearate , vegetable and useful ranges may be selected between any of these stearin , stearic acid , a mineral, a fatty acid ester, or an values . inorganic material, a polymer or a combination of any two [0049 ] In various embodiments the biodegradable water or more thereof. In other embodiments the mineral may absorbent agent is capable of absorbing at least 100 , 150 , comprise a talc or silica . In a preferred embodiment the 200 , 300 , 400 , 500 , 600 , 700 , 800 , 900 or 1 , 000 % water lubricant is magnesium stearate . based on its dry weight, and useful ranges may be selected 10060 ] In various embodiments the granular composition between any of these values. may comprise about 0 . 05 , 0 . 1 , 0 . 15 , 0 . 2 , 0 . 25 , 0 .3 , 0 .35 , 0 . 4 , [0050 ] In some embodiments the biodegradable water 0 . 5 , 0 . 6 , 0 . 7 , 0 . 8 , 0 . 9 or about 1 % by weight of the lubricant, absorbent agent is a polymer . and useful ranges may be selected between any of these [0051 ] In one embodiment the biodegradable water- absor values . bent agent is selected from the group comprising starches, [0061 ] In various embodiments the granular composition cross - linked polymers , and peat. may additionally comprise one or more processed cereals [0052 ] In various embodiments the biodegradable water selected from the group comprising wheat, corn , rice , oats, absorbent agent is a polymer selected from the group barley, amaranth , millet, quinoa , sorghum , and buckwheat . comprising a cold - water soluble starch , a pre - gelatinised For example , in various embodiments the one or more starch , a cross - linked polymer, or a combination of any two processed cereals may comprise kibbled wheat, puffed or more thereof. In various embodiments the biodegradable wheat, ground corn ( corn grits ) , puffed rice , rolled oats , US 2018 / 0084776 A1 Mar . 29 , 2018 puffed oats , puffed barley, puffed amaranth , puffed millet , bran and the one or more saccharides in a weight ratio puffed quinoa , puffed sorghum , or puffed buckwheat. In a of from about 5 : 1 to about 1 : 2 by weight, and the one preferred embodiment the composition additionally com or more plant powders and the one or more saccharides prises kibbled wheat and ground corn . in a ratio of from about 3 : 1 to about 1 : 1 by weight, or [ 0062 ] In various embodiments the granular composition [0074 ] h ) the bran and the one or more plant powders in may comprise about 5 , 10 , 15, 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55 a ratio of from about 2 : 1 to about 1 : 2 by weight, the or about 60 % by weight of the one or more processed bran and the one or more saccharides in a weight ratio cereals , and useful ranges may be selected between any of of from about 3 : 1 to about 1 : 2 by weight , and the one these values . or more plant powders and the one or more saccharides [0063 ] In various embodiments the granular composition in a ratio of from about 3 : 1 to about 1 : 1 by weight. comprises the bran and the one or more other plant powders [ 0075 ] In one embodiment the granular composition may in a ratio of about 8 :1 , 7: 1, 6 : 1, 5: 1 , 4 : 1, 3 : 1, 2 :1 , 3 :2 , 1 : 1 , additionally comprise a biodegradeable polymer. In one 2 : 3 , 1 : 2 , 1 : 3 or about 1 : 4 by weight, and useful ranges may embodiment the biodegradable polymer may comprise a be selected between any of these values . polysaccharide . [0064 ] In various embodiments the granular composition [0076 ] In one embodiment the biodegradable polymer comprises the bran and the one or more saccharides in a ratio may comprise a gum , such as a plant gum or a gum produced of about 5 :1 , 4 : 1, 3: 1, 2 : 1, 3 : 2, 1: 1 , 2 :3 , 1: 2, 1 :3 , 1: 4 or about by a microorganism . In various embodiments the biodegrad 1 : 5 by weight, and useful ranges may be selected between able polymer comprises an exopolysaccharide produced by any of these values . Achromobacter, Acetobacter, Acinetobacter , Agrobacterium , [ 0065 ] In various embodiments the granular composition Alcaligenes , Aspergillus, Aureobasidium , Aureomonas, Azo comprises the one or more plant powders and the one or tobacter, Bacillus , Beijerinckia , Lactobacillus , Lentinus, more saccharides in a ratio of about 8 : 1 , 7 : 1 , 6 : 1 , 5 : 1 , 4 : 1 , Leuconostoc , Mucorales , Pantoea stewartii, Pseudomonas , 3 : 1 , 2 : 1 , 3 : 2 , 1 : 1 , 2 : 3 , 1 : 2 , 1 : 3 or about 1 : 4 by weight, and Rhizobium , Schizophylum , Sclerotium , Serratia , Sinorhizo useful ranges may be selected between any of these values . bium , Sphingomonas, Streptococcus, Xanthomonas , [ 0066 ]. In one embodiment the granular composition com Zooglea , or Zymomonas spp . In various embodiments the prises biodegradable polymer may be selected from the group [ 0067 ] a ) the bran and the one or more plant powders in comprising xanthan gum , agar, alginate , cassia , dammar , a ratio of from about 4 : 1 to about 1 : 3 by weight , the pectin , beta - glucan , glucomannan , mastic , chicle , psyllium , bran and the one or more saccharides in a weight ratio spruce gum , gellan gum , acacia gum , guar gum , locust bean of from about 5 : 1 to about 1 : 2 by weight, and the one gum , carrageenans, gum arabic , karaya gum , ghatti gum , or more plant powders and the one or more saccharides tragacanth gum , konjac gum , tara gum , pullulan or a com in a ratio of from about 5 : 1 to about 1 : 1 by weight, or bination of any two or more thereof. [0068 ] b ) the bran and the one or more plant powders in [0077 ] In another embodiment the biodegradable polymer a ratio of from about 4 : 1 to about 1 : 3 by weight, the may comprise a synthetic polysaccharide , for example a bran and the one or more saccharides in a ratio of from synthetic polymer of sucrose . In one embodiment the poly about 3 : 1 to about 1 : 1 by weight, and the one ormore saccharide may comprise Ficoll® polysaccharide. plant powders and the one or more saccharides in a 10078 ] In one embodiment the granular composition may ratio of from about 5 : 1 to about 1 : 1 by weight , or additionally comprise bile salts . 10069 ] c ) the bran and the one or more plant powders in 10079 ]. In one embodiment the granular composition may a ratio of from about 4 : 1 to about 1 : 3 by weight , the be in the form of a dough , for example , an extruded dough . bran and the one or more saccharides in a ratio of from [ 0080 ] In one embodiment the granular composition may about 5 : 1 to about 1 : 2 by weight, and the one or more comprise an exterior coating comprising the one or more plant powders and the one or more saccharides in a microorganisms. In one embodiment the exterior coating ratio of from about 3 : 1 to about 1 : 1 by weight, or may comprise a biodegradable polymer , a non -cytotoxic oil , [0070 ] d ) the bran and the one or more plant powders in or a biodegradable polymer and a non -cytotoxic oil . Suitable a ratio of from about 4 : 1 to about 1 : 3 by weight , the biodegradeable polymers and non - cytotoxic oils for use in bran and the one or more saccharides in a ratio of from the exterior coating may be selected from those described about 3 : 1 to about 1 : 1 by weight, and the one or more above . plant powders and the one or more saccharides in a [0081 ] In one embodiment the granular composition may ratio of from about 3 : 1 to about 1 : 1 by weight, or comprise an external protective coating . [0071 ] e ) the bran and the one or more plant powders in 10082 ] In various embodiments the granular composition a ratio of from about 2 : 1 to about 1 : 2 by weight, the may comprise about 0 . 05 , 0 . 1 , 0 . 15 , 0 . 2 , 0 . 25 , 0 . 3 , 0 . 35 , 0 . 4 , bran and the one or more saccharides in a weight ratio 0 . 5 , 0 . 6 , 0 . 7 , 0 . 8 , 0 . 9 or about 1 % by weight of the biode of from about 5 : 1 to about 1 : 2 by weight, and the one gradeable polymer, and useful ranges may be selected or more plant powders and the one or more saccharides between any of these values. in a ratio of from about 5 : 1 to about 1 : 1 by weight, or [0083 ] In one embodiment the granular composition com 10072 ]. f ) the bran and the one or more plant powders in prises a ratio of from about 2 : 1 to about 1 : 2 by weight, the [0084 ] a substrate , and bran and the one or more saccharides in a weight ratio [0085 ] a layer that at least partially coats the substrate , the of from about 3 : 1 to about 1: 2 by weight , and the one layer comprising one or more microorganisms, two or more or more plant powders and the one or more saccharides plant powders , a biodegradable water - absorbent agent, one in a ratio of from about 5 : 1 to about 1 : 1 by weight, or or more saccharides , and one or more lubricants . 10073 ] g ) the bran and the one or more plant powders in (0086 ] In one embodiment the substrate may comprise a a ratio of from about 2 : 1 to about 1 : 2 by weight, the carbonate mineral, a clay granule , a silicate mineral, or an US 2018 / 0084776 A1 Mar . 29 , 2018 aluminosilicate mineral. In various embodiments the sub 3 to about 30 , about 3 to about 20 , about 3 to about 10 , about strate may comprise marble , sand , zeolite , diatomaceous 3 to about 5 , about 4 to about 50 , about 4 to about 30 , about earth or perlite . In one embodiment the substrate comprises 4 to about 20 , about 4 to about 10 , about 4 to about 5 N , a seed . about 5 to about 50 N , about 5 to about 40 N , about 10 to [0087 ] In various embodiments the granular composition about 50 N , about 10 to about 40 N , about 10 to about 30 N , may comprise the layer and substrate in a ratio of about 8 : 1 , about 15 to about 50 N , about 15 to about 40 N , about 15 to 7 : 1 , 6 : 1 , 5 : 1 , 4 : 1 , 3 : 1 , 2 : 1 or about 1 : 1 by weight. about 30 N , about 20 to about 50 N , about 20 to about 40 N , [0088 ] In one embodiment the granular composition fur or about 20 to about 30 N . ther comprises a densifier. In one embodiment the densifier [0097 ] In various embodiments the composition may have comprises a clay. In various embodiments the densifier a mean diameter of about 0 . 1 , 0 . 2 , 0 . 3 , 0 . 4 , 0 . 5 , 0 .6 , 0 . 7 , 0 . 8 , comprises gypsum , bentonite , talc , sand or silica. 0 .9 , 1 , 2, 3, 4 , 5 , 6 , 7 , 8, 9 or 10 mm , and useful ranges may 10089 ] In one embodiment the granular composition com be selected between any of these values . prises a further active . In some embodiments the further 0098 ] In some embodiments the granular composition is active is selected from one or more of an insecticide and a formed by extrusion . probiotic . [0099 ] In some embodiments the granular composition is [0090 ] In some embodiments the granular composition formed by wet granulation . comprises a fungicide . [0100 ] In one embodiment the method comprises extrud 10091 ] In various embodiments the granular composition ing the dough to form an agricultural composition . may comprise up to about 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , 13 , 14 , 10101 ] In one embodiment the method may comprise 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 or about 25 % by weight sterilising one or more of the bran , one or more plant moisture and useful ranges may be selected between any of powders , a biodegradable water -absorbent agent, one or these values . more saccharides , one or more lubricants , or a combination [0092 ] In various embodiments the granular composition of any two or more thereof. In one embodiment the steril may have a water activity A , of less than about 0 . 2 , 0 . 3 , 0 . 4 , ising may be achieved using gamma irradiation . 0 .45 , 0 .46 , 0 . 47 , 0 .48 , 0 . 49 , 0 . 5 , 0 .51 , 0 . 52 , 0 .53 , 0 .54 , 0 . 55 , [0102 ] In one embodiment the method may comprise 0 . 56 , 0 .57 , 0 .58 , 0 . 59, 0 . 6 , 0 .65 , 0 .66 , 0 .67 , 0 .68 , 0 .69 , 0 . 7 , drying the agricultural composition . In one embodiment the 0 .75 , 0 . 8 , 0 . 9 , 0 .95 , 0 . 98 , or about 0 . 99 , and useful ranges agricultural composition may be dried until the composition may be selected between any of these values . In a preferred has a water activity of less than about 0 .2 , 0 .25 , 0 .3 , 0 .4 , 0 . 5 , embodiment the granular composition has a water activity of 0 . 6 , 0 . 7 , 0 . 75 , 0 . 8 , 0 . 9 , 0 . 95 , 0 . 98 , or about 0 .99 , and useful less than about 0 . 55 . ranges may be selected between any of these values . [0093 ] In various embodiments the granular composition 10103 ] In one embodiment the method comprises may comprise at least about 1011, 5x1010 , 1010 , 5x10°, [0104 ] extruding the dough to form an extruded material, 2x10° , 10 % , 5x108, 109, 5x107, 107, 5x10 " , 10 " , 5x10 % , 10 % and or 104, 103 or 102 colony forming units (cfu ) of the one or [0105 ] processing the extruded material into granules to more microorganisms per gram of the composition after form an agricultural composition . about 1, 2 , 3, 4 , 5 , 6 , 7 days, or 2 , 3 , 4, 5 , 6 , 7 , 8 , 9, 10 weeks [0106 ] In another embodiment the composition compris storage, or 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12, 15 , 18 , 24 months ing one or more microorganisms may comprise one or more storage of the composition at a temperature of about 4 , 8 , 10 , saccharides listed above . 15 , 16 , 20 , 25 or about 30° C . or at ambient temperature , and [0107 ] In various embodiments the granular composition useful ranges may be selected between any of these values . is applied to soil to [ 0094 ] In various embodiments the one or more microor [0108 ] a ) kill or control pests , such as insect pests , or ganismsmay retain at least about 0 . 1 , 0 . 2 , 0 . 5 , 0 .75 , 2 , 5 , 10 , [0109 ] b ) reduce the population of a pest, such as an 15 , 20 , 25 , 30 , 35 , 40 , 50 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 , insect pest, or 98 , 99 or 100 % viability after 1 , 2 , 3 , 4 , 5 , 6 , 7 days , or 2 , [0110 ] c ) maintain or increase plant or crop growth , or 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 weeks , or 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , [ 0111 ] d ) maintain or increase pasture production , or 15 , 18 , 24 months storage of the composition at a tempera [0112 ] e ) maintain or increase plant, crop or pasture ture of about 4 to about 20° C . yield , or [0095 ] In various embodiments the number of viable units [0113 ] f) deliver probiotics to grazing animals or live of the one or more microorganisms in the composition after stock , or bioremediate the soil, or about 1 , 2 , 3 , 4 , 5 , 6 , 7 days, or 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 weeks , 101140 g ) or 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 15 , 18 , 24 months storage [0115 ] h ) a combination of any two or more of a ) to g ). of the composition at a temperature of about 4 , 8 , 10 , 15 , 16 , [0116 ] In various embodiments the granular composition 20 , 25 or about 30° C . or at ambient temperature, is about 50 , of the invention is used to control pests , preferably insect 60 , 70 , 75 , 80 , 90 , 95 , 100 , 110 , 120 , 125 , 150 , 175 , 200 , pests . 225 , 250 , 275 , 300 , 350 , 400 , 450 , or about 500 % of the [0117 ] In various embodiments the granular composition amount in the freshly prepared composition , and useful of the invention is used to maintain or increase plant growth . ranges may be selected between any of these values . [0118 ] In various embodiments the granular composition [ 0096 ] In various embodiments , the composition may of the invention is used to maintain or increase pasture have a hardness ( the maximum load to crush ) of at least production . about 0 . 5 , 1 , 1 . 5 , 2 , 3 , 4 , 5 , 7 . 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 or [0119 ] In various embodiments the granular composition about 50 N , and useful ranges may be selected between any may be applied to substantially cover a crop field or pasture . of these values , for example , from about 1 to about 50 , 2 to 0120 ] In various embodiments the composition may be about 50 , about 2 to about 30 , about 2 to about 20 , about 2 applied to the soil at a rate of at least about 5 , 10 , 20 , 30 , 35 , to about 10 , about 2 to about 5 , about 3 to about 50 , about 40 , 45 , 50 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 , 100 , 110 , 120 , US 2018 / 0084776 A1 Mar . 29 , 2018

130 , 140 , 150 , 160 , 170 , 180 , 190 or about 200 kg per purpose of providing a context for discussing the features of hectare, and useful ranges may be selected between any of the present invention . Unless stated otherwise , reference to these values . such sources of information is not to be construed , in any [ 0121 ] In various embodiments the composition may be jurisdiction , as an admission that such sources of informa applied to the soil at a rate of at least about 1x10°, 1x1010 , tion are prior art or form part of the common general 5x1010 , 1x1011 , 5x1011 , 1x1012 , 2 .5x1012 , 5x1012 , 7 .5x knowledge in the art . 1012 , 1x1013 , 2 .5x1013 , 5x1013 , 7 . 5x1013 , 1x1014 , 2 .5x1014 , [0132 ] The term “ comprising ” as used in this specification 5x1014 , 7 . 5x1014 , or about 1x1015 cfu of the one or more means “ consisting at least in part of" . When interpreting microorganisms per hectare and useful ranges may be statements in this specification which include that term , the selected between any of these values. features , prefaced by that term in each statement, all need to [ 0122 ] In one embodiment the granular composition may be present but other features can also be present. Related be applied when the soil is wet, or after irrigation or rainfall . terms such as “ comprise ” and “ comprised ” are to be inter [0123 ] In one embodiment application of the composition preted in the same manner . may reduce the population of a pest, for example Porina or black beetles, by at least about 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , BRIEF DESCRIPTION OF THE DRAWINGS 50 , 55 , 60 ,65 , 70 , 75 , 80 , 85 , 90 , 95 or 100 % in the 4 months [0133 ] The invention will now be described by way of following application , and useful ranges may be selected example only and with reference to the drawings in which : between any of these values . 10134 ] FIG . 1 is a graph showing the effect of water [ 0124 ] In one embodiment application of the composition activity on Yersinia entomophaga survival in standard baits leads to one or more of maintained or increased plant from four independent experiments .Mean CFUs by aw , and growth , maintained or increased pasture growth ,maintained their linear trends , estimated from GEE analysis . (Note : or increased plant yield , maintained or increased pasture Only four trend lines are visible because trend lines of yield , or a combination of any two or more thereof. aw = 0 .6172 and 0 .6452 overlap ) . water activity ( a ) of [0125 ] In various embodiments , application of the com 0 .5443 ( x ) , water activity ( a ) of 0 .5449 ( A ) , water activity position may increase pasture dry matter production by at ( a ) of 0 .6981 ( I ), water activity (aw ) of 0 .6452 ( O ) , water least about 5 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 150 , activity ( a ) of 0 .6172 ( © ) . 200 , 250 or 300 % , and useful ranges may be selected [0135 ] FIG . 2 shows the growth of the bacteria Serratia between any of these values . entomophila , over a duration of 96 hours after placement on [0126 ] In various embodiments the one or more microor dampened tissue paper at an initial loading of 1 .03x10° ( ), ganisms in the composition may retain at least about 0 . 1 , 0 . 2 , 1 . 59x10 ( ) and 7 . 33x104 ( A ) . 0 . 5 , 0 .75 , 2 , 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 50 , 55 , 60 , 65, 70 , [0136 ] FIG . 3 shows the growth of the bacteria Yersinia 75 , 80 , 85 , 90 , 95 , 98 , 99 or 100 % viability about 1 , 2 , 3 , 4 , entomophaga, over a duration of 96 hours after placement on 5 , 6 , 7 days , or 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 weeks, or 3 , 4 , 5 , 6 , dampened tissue paper at an initial loading of 1 .65x10 ' ( D ) , 7 , 8 , 9 , 10 , 11 , 12 , 15 , 18 , 24 months after application of the 3 .50x100 ( A ) , 1 .73x10 ( ) and 1 .72x10 ' ( O ) . composition to the soil , and useful ranges may be selected [0137 ] FIG . 4 shows the growth of the bacteria Serratia between any of these values. proteamaculans, over a duration of 96 hours after placement [0127 ] In various embodiments the number of viable units on dampened tissue paper at an initial loading of 7 . 33x108 of the one or more microorganisms in the composition ( ) , 8 . 90x10 ) and 2 .78x10 ' ( A ) . increases by at least about 1 . 2 , 1 . 25 , 1 . 5 , 1 . 75 , 2 , 2 . 5 , 3 , 3 . 5 , [0138 ] FIG . 5 shows baits viewed by scanning electron 4 , 4 .5 , 5 , 6 , 7 or 8 log increase about 1 , 2 , 3 , 4 , 5 , 6 , 7 days, microscopy : A ) extruded bait having high bacterial loading ; or 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 weeks, or 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , B ) extruded bait having low bacterial loading ; C ) wet 11 , 12 , 15 , 18 , 24 months after application of the composi granulated bait ; D ) blinding sand coated bait ; E ) extruded tion to the soil , and useful ranges may be selected between bait having cylindrical shape ; F ) extruded bait having star any of these values . shape; G ) extruded bait comprising 25 : 75 talc : bait dough ; 10128 ] In various embodiments the one or more microor ganisms are detectable in the soil for up to about 1 , 2 , 3 , 4 , H ) extruded bait comprising 50 :50 talc: bait dough . 5 , 6 , 7 , 8 , 9 , 10 weeks, or 3 , 4 , 5 , 6 , 9 or 12 months after 101391. FIG . 6 shows the proportion of porina larvae ( % ) application of the composition to the soil . that had consumed baits comprising Yersinia entomophaga [0129 ] It is intended that reference to a range of numbers ( YE ) or Metarhizium anisopliae var anisopliae (F178 ) Met( ) disclosed herein ( for example , 1 to 10 ) also incorporates after 24 (black bars ) , 48 ( light grey bars ) and 96 hours ( dark reference to all rational numbers within that range ( for grey bars ). Control baits contained no microorganisms. example , 1 , 1 . 1 , 2 , 3 , 3 . 9 , 4 , 5 , 6 , 6 . 5 , 7 , 8 , 9 and 10 ) and also 10140 ] FIG . 7 shows the mortality of porina larvae any range of rational numbers within that range ( for exposed to baits comprising A ) Yersinia entomophaga (YE ) example , 2 to 8 , 1 . 5 to 5 . 5 and 3 . 1 to 4 . 7 ) . or B ) Metarhizium anisopliae var anisopliae (F178 ) (Met ) [0130 ] To those skilled in the art to which the invention over 47 days . Control baits contained no microorganisms. relates, many changes in construction and widely differing [0141 ] FIG . 8 shows the mortality of black beetles embodiments and applications of the invention will suggest exposed to baits comprising Yersinia entomophaga ( YE ) themselves without departing from the scope of the inven over 15 days . tion as defined in the appended claims. The disclosures and the descriptions herein are purely illustrative and are not DETAILED DESCRIPTION OF THE intended to be in any sense limiting . INVENTION [0131 ] In this specification , where reference has been [ 0142 ] The present invention relates to a granular compo made to external sources of information , including patent sition , methods of preparing the granular composition , and specifications and other documents , this is generally for the application of the granular composition on to soil. The US 2018 / 0084776 A1 Mar . 29 , 2018

granular composition comprises one or more microorgan - f0155 ] Various different types of substrates could be used . isms, two or more plant powders and a biodegradable For example , the substrate can be selected from a clay , a clay water- absorbent agent, for example, a cold water- soluble mineral, a seed , a pelletised grain , a granulate or an extruded stastarch . granule , a carbonate mineral, a silicate mineral , an alumi [0143 ] Thepresent invention also provides for the delivery nosilicate mineral , vermiculite , a fertiliser granule , zeolite , of beneficial microorganisms to soil in a composition that diatomaceous earth , perlite , sand or a combination of any supports the viability and or growth of the microorganism two or more thereof. during storage of the composition and following application 101561. If a seed , the seed substrate may be an angiosperm , of the composition to the soil . The granular compositions of vegetable , legume, cereal or conifer seed . For example , in the invention are suitable for delivery of a wide range of some embodiments the seed is a spinach , carrot, onion , microorganisms to improve soil quality and control pests soybean , lucerne , plantain , brassica , maize , rye grass , and disease in plants . canola or clover seed . In various embodiments the substrate [0144 ] The various embodiments of the composition and may comprise wheat, barley, bran ,maize , rye, rice , sorghum , methods of the invention have numerous advantages , includ millet, oats , forage brassica , canola or triticale , or a com ing but not limited to bination of any two or more thereof. [0145 ] attracts target insect pests encouraging ingestion 101571 The substrate may contain a fertiliser, for example , of the microorganism , a granule of urea, superphosphate , monoammonium phos [0146 ] improved structural integrity after application to phate (MAP ) , diammonium phosphate (DAP , single super soil and exposure to moisture , phosphate ( SSP ) , triple superphosphate ( TSP ) , calcium ammonium nitrate (CAN ) muriate of potash (MOP ) or a [0147 ] improved shelf life , nitrification inhibitor. [0148 ] growth of microorganism population in compo [0158 ] Examples of nitrification inhibitors included dicy sition after application to soil , andiamide (DCD ) , N - 2 , 5 - dichlorophenyl succinamic acid , [0149 ] improved pesticidal efficacy, 2 - chloro -6 - trichloromethyl pyridine (“ Nitrapyrin ” ), dicyan 10150 ) simple and cost- effective to manufacture , and /or diamide (“ DCD or “ DCDIN ”” ), zinc ethylene - bis - dithiocar [0151 ] suitable for broadcast application to soil. bamate , 2 , 4 , 6 -trichloroaniline , pentachlorophenol, thio urea , ammonium thiosulphate (ATS ), and methylpyrazole 1 . Granular Composition phosphates such as 3 , 4 -dimethypyrazole phosphate (DMPP ). [0152 ] As used herein the term “ granule ” and its deriva [0159 ] As explained above the substrate is coated by one tives ( i. e . granular ) includes prills , pellets, particles and or more layers . The one or more layers partially coats the grains . The granular composition has a mean particle size of substrate , or coats all of the substrate , or any amount in at least about 0 . 1 , 0 . 2 , 0 . 3 , 0 . 4 , 0 . 5 , 0 . 6 , 0 . 7 , 0 . 8 , 0 . 9 , 1 , 2 , between , for example a majority or substantially all of the 3 , 4 , 5 , 6 , 7, 8, 9 or 10 mm , and useful ranges may be substrate . selected from between any of these values, ( for example , [0160 ] The layers ( containing the microorganism and the about 0 . 1 to about 10 , about 0 . 1 to about 8 , about 0 . 1 to excipients ) comprise about 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , about 7 , about 0 . 1 to about 5 , about 0 . 1 to about 4 , about 0 . 1 55 , 60 , 65 , 70 or 75 % by weight of the substrate , and useful to about 3 , about 0 . 1 to about 2 , about 0 . 1 to about 1 , about ranges may be selected from between any of these values, 0 . 2 to about 10 , about 0 . 2 to about 8 , about 0 . 2 to about 6 , ( for example , 10 to about 75 , about 10 to about 60 , about 10 about 0 . 2 to about 2 , about 0 . 2 to about 0 . 8 , about 0 . 2 to to about 50 , about 10 to about 35 , about 10 to about 20 , about 0 . 5 , about 0 . 5 to about 10 , about 05 to about 7 , about about 15 to about 75 , about 15 to about 60 , about 15 to about 0 . 5 to about 4 , about 0 . 5 to about 1 , about 0 . 5 to about 0 . 8 , 50 , about 15 to about 35 , about 15 to about 20 , about 20 to about 0 . 7 to about 10 , about 0 . 7 to about 9 , about 0 . 7 to about 70 , about 20 to about 65 , about 20 to about 55 , about about 7 , about 0 . 7 to about 5 , about 0 . 7 to about 3 , about 0 . 7 20 to about 40 , about 20 to about 35 , about 20 to about 30 , to about 1 , about 0 . 8 to about 10 , about 0 . 8 to about 9 , about about 25 to about 70 , about 25 to about 60 , about 25 to about 0 . 8 to about 6 , about 0 . 8 to about 4 , about 0 . 8 to about 1 , 50 , about 25 to about 40 , about 30 to about 75 , about 35 to about 1 to about 10 , about 1 to about 8 , about 1 to about 7 , about 65 , about 35 to about 55 , about 35 to about 45 , about about 1 to about 5 , about 1 to about 3 , about 2 to about 10 , 40 to about 70 , about 40 to about 60 , about 40 to about 50 , about 2 to about 8 , about 2 to about 6 , about 3 to about 10 , about 45 to about 70 , about 45 to about 65 , about 45 to about about 3 to about 7 , about 3 to about 5 , about 5 to about 10 , 60 , about 55 to about 70 , about 60 to about 70 % by weight about 5 to about 8 , about 5 to about 7 , about 6 to about 10 , of the substrate ). about 6 to about 8 , about 7 to about 10 or about 7 to about [0161 ] The granular composition may comprise a binder 9 mm ) . to facilitate adherence of the coating to the substrate . In one embodiment the binder comprises a polymeric compound . 1. 1 the Granular Composition In various embodiments the binder comprises a polyhy [0153 ] The granular composition is formed of one or more droxyl compound , a copolymer of a C , - C , alkyl substituted microorganisms, such as bacteria and /or fungi, in combina with one or more lactams, a starch , a protein , a saccharide , tion with various excipients . or a combination of any two or more thereof. 10154 ] The granular composition can be formed from a 10162 ]. The binder can be selected from a polyvinyl alco substrate coated with one or more layers comprising a hol , or a cold water soluble starch or a pre - gelatinised homogenous mix of the one or more microorganisms and starch , or a combination thereof. excipients . The substrate is a material of sufficient density to 0163] In some embodiments the binder comprises sodium provide the composition with suitable ballistic properties to starch glycolate , maltodextrin , trehalose, rice powder, potato enable effective broadcast distribution of the composition . starch , kibbled wheat, corn grits , honey, cornflour , wheat US 2018 / 0084776 A1 Mar . 29 , 2018

flour, a polyvinyl pyrrolidone , hydroxymethylpropyl cellu health of pets or livestock or prevent disease in pets or lose and polyvinyl alcohols or a combination of any two or livestock . For example, to deliver bacterial therapeutics, more thereof. probiotics or low methane production microbes. [0164 ] In some embodiments of the present invention the [ 0169 ] Examples of suitable bacterial therapeutics include granular composition comprises, or consists of, a homoge the delivery of attenuated pathogenic strains such as neous mix of one or more microorganisms and excipients . Escherichia coli, Salmonella pullorum , Salmonella typhimu [0165 ] The granular composition may comprise an exter rium , Clostridium botulinum , Clostridium sporogenes and nal protective coating . For example , a talc , colloidal silicon Mycobacterium paratuberculosis . dioxide or stearic acid . The external protective layer can act [0170 ] Examples of suitable probiotics include Lactoba to protect and / or stabilise the granular composition . cillus species Bifidobacterium Species, Enterococcus spe 10166 ] In another embodiment the granular composition cies , Leuconostoc mesenteroides , Pediococcus acidllactici , comprises an outer layer comprising one or more microor Sporolactobacillus inulinus, Streptococcus thermophiles ganisms, a biodegradeable polymer and a non -cytotoxic oil . and the yeast Saccharomyces cerevisiae. For example , in one embodiment the granular composition [0171 ] Examples of suitable low methane production comprises a homogenous mix of one or more excipients , at microbes include, Fibrobacter spp ., spp ., Prevotella bryan least partially coated with a layer comprising one or more tii , and Sharpea azabuensis . microorganisms, a biodegradeable polymer and a non -cyto [0172 ] Microorganisms that infect plants ( e . g . to become endophytic and /or promote growth of the plant ) or animal toxic oil . pests or insects ( i .e . by killing or inhibiting reproduction or 1 . 2 Granular Composition Ingredients growth of the insect or pest ) may be suitable for use in the granular composition of the invention . [0167 ] Table 1 below sets out the functional excipients for [0173 ] The one or more microorganismsmay be selected use in the granular composition of the present invention . from the group comprising bacteria , yeast, fungi, spores , [0168 ] It will be appreciated that the granular composition fungal microsclerotia or a combination of any two or more of the invention may be used to deliver any microorganism thereof . If a bacteria , the bacteria may be selected from a that is agriculturally , horticulturally or veterinarily useful. gram positive bacteria , gram negative bacteria , or a combi For example, the granular composition of the invention may nation of gram positive and gram negative bacteria . be used to deliver one or more microorganisms that have [0174 ] In one embodiment the one or more microorgan pesticidal and / or insecticidal activity , that support plant isms comprise a probiotic to improve or support the gut growth , health or development and / or that promote the health of pets or livestock . TABLE 1 Functional agents for use in the present formulation

Functional Concentration Excipient Classification Examples Range Microorganism Single or multiple Bacteria ( e . g 1 x 106 - 1 x 1010 Yersinia cfu / g entomophaga ) , spores , fungi, yeast A biodegradable Starches , cross Potato starch , 0 .05 - 5 % by water- absorbent linked polymers , or polyvinylpyrollidone , weight agent peat. sodium alginate , Densifier Clays Gypsum , bentonite 1 - 20 % by weight Buffering agent Effervescent alginic acid , citric 1 - 20 % by agents acid , sodium weight bicarbonate or tartaric acid Bulking agent Plant powders , Rice powder , wheat 30 % -80 % by brans, processed bran , wheat ( e. g. weight cereals, starches , kibbled wheat) , corn clay ( e . g . corn grits ) , maltodextrin , gypsum Microorganism Biodegradable Xanthan gum , trace stabiliser polymers , e . g . Ficoll ? plant or bacterial gums, synthetic sucrose polymer Emulsifier Non -cytotoxic oils, Canola oil trace e. g . vegetable oils Phagostimulant Plant powders , rice powder , wheat 20 % - 85 % by brans, processed bran , wheat ( e . g . weight cereals , starches , kibbled wheat) , corn saccharides ( e . g . corn grits ) , maltodextrin , honey , sucrose US 2018 / 0084776 A1 Mar . 29 , 2018

TABLE 1 -continued Functional agents for use in the present formulation Functional Concentration Excipient Classification Examples Range Insecticide Carbamates , Carbaryl, 0 . 1 - 10 % or organophosphates , chlorpyrifos , < 1 % by weight pyrethroids , cypermethrin , neonicotinoids, imidacloprid , benzoyl phenyl diflubenzuron ureas Lubricant Lipids, fatty acid Magnesium 0 .05 - 1 % by esters , minerals , stearate , talc , silica weight inorganic materials , polymers CO2 - generating food acids, acid citric acid , tartaric 0 .01 - 5 % by agent anhydrides , acid , succinic weight carbonates anhydride , sodium bicarbonate , potassium carbonate

[0175 ] The microorganisms can be selected from bacteria about 0 . 1 to about 0 . 7 , about 0 . 2 to about 1 . 5 , about 0 . 2 to such as , for example , Serratia ( for example , Serratia ento about 1 .3 , about 0 . 2 to about 0 .8 , about 0 .3 to about 1 .5 , mophilia or Serratia proteomaculans ) , Xanthamonas , about 0 . 3 to about 1 , about 0 . 4 to about 1 . 5 , about 0 . 4 to Pseudomonas, Rhizobium , Bifidobacterium , Lactobacillus , about 1 . 4 , about 0 . 4 to about 1 , about 0 . 4 to about 8 , about Streptococcus (Enterococcus ) , Yersinia (for example , Yers 0 . 4 to about 7 , about 0 . 5 to about 1 . 5 , about 0 . 5 to about 1 . 2 , inia entomophaga ), Pseudomonas, Bacillus, Pasteuria , Azo about 0 . 5 to about 1 , about 0 . 5 to about 0 . 8 , about 0 . 5 to bacter, Enterobacter, Azospirillum , Cyanobacteria , Paecilo about 0 . 7 , about 0 . 5 to about 0 . 6 , about 0 . 6 to about 1 . 5 , myces, Streptomycetes, Chromobacterium , Rhanella , about 0 . 6 to about 1 . 2 , about 0 .6 to about 1 , about 0 . 7 to Burkholderia , Paenibacillus, Collimonas, Sinorhizobium , about 1 . 5 , about 0 . 7 to about 1 . 1 , about 0 . 8 to about 1 . 5 , Pantoea , Lecanicillum , Erwinia , Pediococus, Leuconostoc , about 0 . 8 to about 1 . 2 , about 0 . 9 to about 1 . 5 , about 1 to Aeromonas, Neptunomonas, Klebsiella , Ponchonia , Brevi about 1 . 5 , about 1 to about 1 . 1 , about 1 . 2 to about 1 . 5 g / m ) . bacillus, Acinetobacter or a combination of any two or more [0181 ] In one embodiment the densifier comprises a clay . thereof. In various embodiments the densifier comprises gypsum , [0176 ] The microorganisms can be selected from fungi bentonite , sand , silica or talc , or a combination thereof. such as , for example , Beauveria , Penicillium , Metarhizium , [0182 ] It is desirable that the granular composition main Trichoderma, Gliocladium , Coniothyrium , Verticillium , tains its structural integrity for a suitable period after appli Sclerotinia or a combination of any two or more thereof. cation to soil . For example , in an embodiment of the [0177 ] In one embodiment the microorganism is an invention in which the granular composition delivers one or Enterobacteriaceae species. In a specially contemplated more insecticidal microorganisms, the granular composition embodiment the microorganism is Yersinia entomophaga . retains its structural integrity for a period sufficient to allow 10178 ]. In one embodiment, in granular compositions com insect pests to ingest a sufficient amount of the granular prising gram negative bacteria , the agricultural composition composition to deliver an insecticidal quantity of the one or comprises bile salts . For example , in various embodiments more microorganisms. the composition comprises about 0 .02 % , 0 . 025 % , 0 .03 % , 0 . 04 % , 0 .05 % , 0 . 06 % , 0 .07 % or about 0 . 075 % by weight [0183 ] The inclusion of a biodegradable water - absorbent bile salts , and useful ranges may be selected from between agent in the granular composition provides for water absorp any of the values, for example from about 0 . 2 % to about tion and swelling of the granular composition while pre 0 . 75 % by weight. The inclusion of bile salts in the granular venting disintegration of the granular composition . composition inhibits the growth of some non - enteric micro [0184 ] The biodegradable water -absorbent agent is organism species , but does not affect the growth of gram selected from one or more of starches , cross - linked poly negative bacterial strains ( for example , Enterobacteriaceae mers, and peat. species ) . [0185 ] In various embodiments the water -absorbent agent [0179 ] In certain embodiments the granular composition is a polymer such as a starch or cross - linked polymer . comprises one or more densifiers . The inclusion of a densifer Examples of suitable starches and cross - linked polymers increases the density of the granular composition to enable includes cold -water soluble starch , a pre- gelatinised starch , effective broadcast distribution of the granular composition . a cross - linked polymer, or a combination of any two or more [0180 ] Suitable materials for use as densifiers in the granu thereof . In various embodiments the biodegradable water lar composition of the invention are materials having a bulk absorbent agent may comprise potato starch , peat, rice density of about 0 .2 , 0 .3 , 0 . 4 , 0 .5 , 0 .6 , 0 . 7 , 0 .8 , 0 . 9, 1, 1 . 1 , powder , corn starch , sodium starch glycolate , cellulose , 1 .2 , 1. 3 , 1. 4 or 1 . 5 g / m ", and useful ranges may be selected methylcellulose , sodium alginate , polyvinylpyrrolidone, between any of these values, ( for example , about 0 . 1 to sodium carboxymethyl cellulose, croscarmellose sodium , about 1 . 5 , about 0 . 1 to about 1 . 1 , about 0 . 1 to about 0 . 8 , alginic acid , citric acid , a combination of any two or more US 2018 / 0084776 A1 Mar . 29 , 2018 thereof. In a preferred embodiment the a biodegradable about 50 , about 35 to about 85 , about 35 to about 60 , about water- absorbent polymer agent comprises potato starch . 35 to about 55 , about 40 to about 85 , about 40 to about 60 , (0186 ] In various embodiments the granular composition about 45 to about 85 , or about 45 to about 60 % by weight) . may comprise about 0 . 05 , 0 . 1 , 0 . 2 , 0 . 3 , 0 . 4 , 0 . 5 , 0 . 6 , 0 . 7 , 0 . 8 , [0194 ] In various embodiments the granular composition 0 . 9, 1 , 2 , 3, 4 , or 5 % by weight of the a biodegradable comprises the bran and the one or more other plant powders water - absorbent agent, and useful ranges may be selected in a ratio of about 8 : 1 , 7 : 1 , 6 : 1 , 5 : 1 , 4 : 1 , 3 : 1 , 2 : 1 , 3 : 2 , 1 : 1 , between any of these values, ( for example , about 0 .05 to 2 : 3 , 1: 2 , 1 : 3 or about 1 :4 by weight , and useful ranges may about 5 , about 0 .05 to about 4 , about 0 .05 to about 1 , about be selected between any of these values ( for example, from 0 .05 to about 0 . 9 , about 0 . 1 to about 5 , about 0 . 1 to about 4 , 8 : 1 to about 1 : 4 , about 8 : 1 to about 1 : 2 , about 8 : 1 to about about 0 . 1 to about 2 , about 0 . 1 to about 0 . 6 , about 0 . 3 to 3 : 2 , about 8 : 1 to about 4 : 1 , about 7 : 1 to about 1 : 4 . 7 : 1 to about 5 , about 0 . 3 to about, about 0 . 3 to about 3 , about 0 . 3 about 1 : 2 , about 7 : 1 to about 1 : 1 , about 7 : 1 to about 3 : 1 , to about 1 , about 0 . 5 to about 5 , about 0 . 5 to about 3 , about about 7 : 1 to about 5 : 1 , about 6 : 1 to about 1 : 4 , about 6 : 1 to 0 .5 to about 1 , about 0 .6 to about 5 , about 0 .6 to about 3 , about 2 : 3 , about 6 : 1 to about 1 : 1 , about 6 : 1 to about 3 : 1 , about 0 . 6 to about 0 . 9 , about 0 . 8 to about 1 , about 0 . 8 to about 6 : 1 to about 4 : 1 , about 5 : 1 to about 1 : 4 , about 5 : 1 to about 5 , about 0 . 8 to about 3 , about 0 .8 to about 0 .9 , about about 2 : 3 , about 5 : 1 to about 3 : 1, about 4 : 1 to about 1: 4 , 1 to about 5 , about 1 to about 4 , about 1 to about 3 , about about 4 : 1 to about 1 : 2 , about 4 : 1 to about 2 : 1 , about 3 : 1 to 2 to about 5 , about 2 to about 4 or about 3 to about 5 % by about 1 : 4 , about 3 : 1 to about 1 : 2 , about 2 : 1 to about 1 : 4 , weight) . about 2 : 1 to about 1 : 3 , about 3 : 3 to about 1 : 4 , about 3 : 2 to [ 0187 ] In some embodiments , the granular composition about 1 : 2 , about 1 : 1 to about 1 : 4 , about 1 : 1 to about 1 : 2 , contains one ormore phagostimulants . Suitable phagostimu about 2 : 3 to about 1 : 4 , about 2 : 3 to about 1 : 3 , about 1 : 2 to lants are excipients that attract , or at least do not repel, target about 1 : 4 , about 1 : 2 to about 1 : 3 ) . pests , insects or animals . For example , in an embodiment of [0195 ] In various embodiments the granular composition the invention wherein the granular composition delivers one comprises the bran and the one or more saccharides in a ratio or more pesticidal microorganisms, it is desirable that the of about 5: 1 , 4 : 1, 3: 1, 2 : 1, 3 : 2 , 1: 1 , 2 :3 , 1: 2, 1 :3 , 1: 4 or about granular composition comprise excipients that are phago 1 :5 by weight, and useful ranges may be selected between stimulatory to the target pest or insect such that the target any of these values ( for example , about 5 : 1 to about 1 : 5 , pest or insect ingests a sufficient amount of the granular about 5 : 1 to about 1 : 5 , about 5 : 1 to about 1 : 2 , about 5 : 1 to composition to deliver a pesticidal quantity of the one or about 3 : 2 , about 5 : 1 to about 3 : 1 , about 4 : 1 to about 1 : 5 , more microorganisms. about 4 : 1 to about 1 : 4 , about 4 : 1 to about 1 : 2 , about 4 : 1 to [0188 ] It will be appreciated that suitable phagostimulants about 1 : 1 , about 4 : 1 to about 2 : 1 . 3 : 1 to about 1 : 5 , about 3 : 1 for use in the granular composition of the invention may be to about 1 : 3 , about 3 : 1 to about 2 : 3 , about 3 : 1 to about 1 : 1 , selected to achieve optimal attraction or phagostimulation of about 2 : 1 to about 1 : 5 , about 2 : 1 to about 1 : 3 , about 2 : 1 to the particular target pest, insect or animal to the granular about 2 : 3 , about 3 : 2 to about 1 : 5 , about 3 : 2 to about 1 : 2 , composition . about 1 : 1 to about 1 : 5 , about 1 : 1 to about 1 : 4 , about 1 : 1 to [0189 ] The phagostimulants are selected from the group about 1: 2, about 2 :3 to about 1 : 5, about 2 :3 to about 1 :3 or comprising one or more plant powders, one or more brans , about 1 : 3 to about 1 : 5 ) . one or more processed cereals , one or more starches , one or [0196 ] In various embodiments the granular composition more saccharides , and a combination of any two or more comprises the one or more plant powders and the one or thereof. [0190 ] In some embodiments the presence of a sugar, such more saccharides in a ratio of about 8 : 1 , 7 : 1 , 6 : 1 , 5 : 1 , 4 : 1 , as honey, is used as a phagostimulant. While not intended to 3 : 1 , 2 :1 , 3 : 2 , 1 :1 , 2 : 3, 1: 2 , 1: 3 or about 1 :4 by weight, and be limiting, phagostimulation may be provided by the glu useful ranges may be selected between any of these values. cose and fructose . [0197 ] The plant powders for use in the granular compo [ 0191] In some embodiments the granular composition sition include plant powders such as bran powders , flours , contains one or more bulking agents . It is desirable that the rice powders or any combination thereof. bulking agents promote the viability or growth of one or [0198 ] The bran is selected from wheat bran , rice bran , oat more microorganisms in the granular composition after bran , barley bran , rye bran , triticale bran , corn (maize ) bran , application of the granular composition to soil . amaranth bran , millet bran , teff bran , quinoa bran , buck 10192] The bulking agents suitable in the invention are wheat bran , sorghum bran or a combination of any two or selected from the group comprising brans , plant powders , more thereof. In a preferred embodiment the bran comprises saccharides, starches , processed cereals , clays , and a com wheat branul . bination of any two or more thereof. [01991 . The plant powder may contain wheat flour , rice [ 0193 ] In various embodiments the granular composition powder, oat flour, barley flour, rye flour, triticale flour, corn may comprise about 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55 , (maize ) flour, amaranth flour, millet flour , teff flour, quinoa 60 , 65 , 70 , 75 , 80 or about 85 % by weight of the one or more flour, buckwheat flour, sorghum flour or a combination of processed cereals , and useful ranges may be selected any two or more thereof. In a preferred embodiment the between any of these values ( for example , about 5 to about plant powder comprises rice powder or maize flour. 85 , 5 to about 60 , about 5 to about 40 , about 5 to about 30 , [ 0200 ] In various embodiments the processed cereals about 10 to about 85 , about 10 to about 60 , about 10 to about comprises wheat, corn , rice , oats, barley, amaranth , millet , 50 , about 10 to about 40 , about 15 to about 85 , about 15 to quinoa , sorghum , or buckwheat . For example , in various about 60 , about 15 to about 45 , about 20 to about 85 , about embodiments the one or more processed cereals may com 20 to about 60 , about 20 to about 55 , about 20 to about 40 , prise kibbled wheat, puffed wheat , ground corn ( corn grits ) , about 25 to about 85 , about 25 to about 60 , about 25 to about puffed rice , rolled oats , puffed oats , puffed barley , puffed 45 , about 30 to about 85 , about 30 to about 60 , about 30 to amaranth , puffed millet , puffed quinoa , puffed sorghum , or US 2018 / 0084776 A1 Mar . 29 , 2018 10 puffed buckwheat. In a preferred embodiment the compo [ 0208 ] In some embodiments the granular composition sition additionally comprises kibbled wheat and ground comprises one or more CO2- generating agents . It is desir corn . able that the CO2- generating agent acts to enhance the [ 0201 ) In some embodiments the granular composition attraction of the target insect pest to the granular composi contains a saccharide . The saccharides is selected from tion by generating CO2, an insect attractant, when the bait monosaccharides , a disaccharides , an oligosaccharides , a contacts water . Examples of suitable CO2- generating agents polysaccharides, or a sugar product , or a combination of any include food acids , acid anhydrides or carbonates or a two or more thereof. In various embodiments the one or combination of any two or more thereof . In various embodi more saccharides may comprise glucose , fructose , maltose , ments the CO2- generating agent comprises citric acid , tar sucrose, lactose , trehalose , honey , such as clover honey , taric acid , malic acid , succinic anhydride , sodium bicarbon molasses, glucose syrup , corn syrup , for example , high ate , sodium carbonate , potassium bicarbonate , potassium fructose corn syrup , golden syrup , treacle , cane sugar, carbonate, or a combination of any two or more thereof. refined sugar, raw sugar , brown sugar , white sugar, malto dextrin , cornflour , wheat flour , or a combination of any two 2 . Granular Composition Properties or more thereof. [0202 ] The saccharides may be in the form of a powder or [0209 ] As stated above , the granular composition is in the a liquid such as a syrup , or a combination of a powder and form of granules and may also be termed prills , pellets , a liquid . For example , in one exemplary embodiment the one particles , grains or the like . or more saccharides may comprise maltodextrin and honey . [0210 ] The granular composition can have a variety of [ 0203 ] In various embodiments the granular composition shapes. For example , the granular composition may be more contains a combination of at least two or more , at least three spherical when manufactured by granulation . The shape of ormore , at least four or more, at least five ormore , or at least the granular composition can also be elongate ( i . e . “ pellet six or more bulking agents or phagostimulants . shaped ” ) . For example , the granular composition may be [0204 ] In one embodiment the granular composition con pellet - shaped when formed by extrusion . It will be appre tains two or more brans , two or more plant powders and one ciated that the shape of the extruded granular composition or more saccharides . will relate to the shape of the extruder die . [0205 ] In one exemplary embodiment the granular com [0211 ] Themean diameter of the granular material is about position contains kibbled wheat, corn grits , wheat bran , rice 0 . 1 , 0 . 2 , 0 . 3 , 0 . 4 , 0 . 5 , 0 .6 , 0 . 7 , 0 . 8 , 0 . 9 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , powder, maltodextrin , and white sugar. In one embodiment 9 or 10 mm , and useful ranges may be selected between any the composition comprises kibbled wheat, corn grits , wheat of these values ( for example , about 0 . 1 to about 10 , about 0 . 1 bran , rice powder and maltodextrin in a ratio of 0 . 5 - 2 : 0 . 5 to about 8 , about 0 . 1 to about 5 , about 0 . 1 to about 3 , about 2 : 0 . 5 - 2 : 1 - 4 : 0 . 5 - 2 . In a preferred embodiment the composi 0 . 1 to about 1 , about 0 . 2 to about 10 , about 0 . 2 to about 9 , tion comprises kibbled wheat, corn grits , wheat bran , rice about 0 . 2 to about 7 , about 0 . 2 to about 30 . 2 to about 1 , powder and maltodextrin in a ratio of about 1 : 1 : 1 : 2 : 1 by about 0 . 2 to about 0 . 5 , about 0 . 3 to about 10 , about 0 . 3 to weight. about 9 , about 0 .3 to about 5 , about 0 .3 to about 1 , about 0 .3 [ 0206 ] The granular composition may also contain one or to about 0 . 8 , about 0 . 3 to about 0 . 6 , about 0 . 4 to about 10 , more buffering agents selected from the group comprising about 0 .4 to about 8 , about 0 .4 to about 6 , about 0 .4 to about alginic acid , citric acid , sodium bicarbonate, tartaric acid and 5 , about 0 . 4 to about 1 , about 0 . 4 to about 0 . 6 , about 0 . 5 to a combination of any two or more thereof. about 10 , about 0 . 5 to about 8 , about 0 . 5 to about 7 , about [ 0207] In various embodiments of the invention the granu 0 .5 to about 6 , about 0 . 5 to about 2 , about 0 . 5 to about 0 . 8 , lar composition contains an insecticide. It is desirable that about 0 . 6 to about 10 , about 0 . 6 to about 9 , about 0 .6 to the insecticide does not have a significant adverse effect on about 6 , about 0 .6 to about 4 , about 0 .6 to about 1 , about 0 .7 the viability or growth of one or more microorganisms in the to about 10 , about 0 .7 to about 8 , about 0 .7 to about 6 , about granular composition before or after application of the 0 . 7 to about 5 , about 0 . 7 to about 0 . 9 , about 0 . 8 to about 10 , granular composition to soil . Examples of suitable insecti about 0 . 8 to about 8, about 0 . 8 to about 7 , about 0 . 8 to about cides include carbamate , an organophosphate , a pyrethroid , 2 , about 0 . 9 to about 10 , about 0 . 9 to about 6 , about 0 . 9 to a neonicotinoid , or a benzoyl phenyl urea or a combination about 3 , about 1 to about 10 , about 1 to about 8 , about 1 to of any two or more thereof. In various embodiments the about 5 , about 2 to about 10 , about 2 to about 7 , about 2 to insecticide comprises carbaryl , furathiocarb , methmyl, about 5 , about 3 to about 10 , about 3 to about 6 , about 3 to oxamyl, pirimicarb , chlorpyrifos , diazinon , dichlorvos , about 4 , about 4 to about 10 , about 4 to about 8 , about 4 to dimethoate , fenitrothion , maldison , methamidophos, phor about 7 , about 5 to about 10 , about 5 to about 8 , about 5 to ate , terbufos , trichlorfon , cypermethrin , deltamethrin , fen about 7 , about 6 to about 10 , about 6 to about 8 , about 7 to valerate , cyhalothrin , fluvalinate , clothianidin , imidacloprid , about 10 mm ) . thiamethoxam , diflubenzuron , poncho , spinosad or a com [0212 ] Given that the granular material may have an bination of any two or more thereof. For example , in one irregular shape , it will be appreciated that the reference to a embodiment the granular composition comprises bacteria mean diameter is in respect to a notional circular shape . For and an insecticide . In one embodiment the granular compo example , if the granular composition was square or rectan sition comprises bacteria and an organophosphate insecti gular in shape , then the diameter would be the notional circle cide. In one embodiment the granular composition com that the vertices of the square or rectangle sat within . prises Yersinia entomophaga and an insecticide. In one [0213 ] As mentioned above , the granular compositions of embodiment the granular composition comprises Yersinia the present invention may include a substrate that is coated entomophaga and an organophosphate insecticide . In one with the composition containing the microorganism and exemplary embodiment the granular composition Yersinia excipients , ormay be formed solely from themicroorganism entomophaga and chlorpyrifos. and excipients . US 2018 / 0084776 A1 Mar . 29 , 2018 11

[0214 ] For the granular compositions that do not include a [0221 ] It is desirable that when the granular composition substrate , they have a bulk density of about 0 .2 g /cm3 to is exposed to moisture it rapidly takes up water and expands about 1 .5 g/ cm ", and useful ranges may be selected between in size ( swell) . any of these values , ( for example, about 0 .6 g / cm to about f0222 ] Swellability is measured by sorption assay . The 1 .5 g / cm , from about 0 . 7 g /cm3 to about 1 .5 g /cm ”, from granular composition is exposed to moisture and the amount about 0 .8 g / cm3 to about 1 . 5 g / cm ", from about 0 . 9 g / cm² to of water uptake and volume displacement is measured using about 1. 5 g / cm " , from about 1 g / cm to about 1 .5 g/ cm ”, macrophotography. The swellability of the granular compo from about 1. 1 g /cm to about 1 .5 g /cm ", from about 1. 2 sition is from about 50 , 75 , 100 , 125 , 150 , 175 , 200 , 225 , g / cm² to about 1 . 5 g / cm " , or from about 1 . 3 g /cm² to about 250 , 275 or 300 % and useful ranges may be selected from 1. 5 g/ cm ). between any of these values , ( for example , about 50 to about [0215 ] For the granular compositions that do include a 300 , about 50 to about 250 , about 50 to about 225 , about 50 substrate , they have a bulk density of about 0 . 2 , 0 .5 , 0 .75 , 1 , to about 200 , about 50 to about 125 , about 50 to about 100 , 1 .25 , 1 . 5 , 1 . 75 or at least about 2 cm " , and useful ranges may about 75 to about 300 , about 75 to about 275 , about 75 to be selected from between any of these values, ( for example , about 200 , about 75 to about 150 , about 75 to about 100 , from about 0 . 2 to about 1. 5 , about 0 . 5 to about 1 .4 , about 0 .5 about 100 to about 300 , about 100 to about 250 , about 100 to about 1 . 3 , about 0 . 5 to about 1 . 2 , about 0 . 5 to about 1 . 1 , to about 225 , about 100 to about 175 , about 150 to about about 0 . 5 to about 1 , about 0 . 5 to about 0 . 9 or about 0 . 5 to 300 , about 150 to about 275 , about 150 to about 250 , about 150 to about 200 , about 175 to about 300 , about 175 to about about 0 . 8 g / cm ) . 275 , about 175 to about 250 , about 175 to about 200 , about [0216 ] The density of the granular composition may be 200 to about 300 , about 200 to about 275 , about 200 to about determined using standard methods known in the art. For 250 , about 250 to about 300 % ) . example , bulk density may be measured as “ freely settled ” [0223 ] In various embodiments the granular composition or “ poured ” density, or “ tapped ” density . may have a water activity of less than about 0. 2 , 0 .25 , 0 .3 , [ 0217 ] As discussed above, it is desirable that the granular 0 .35 , 0 .4 , 0 .45 , 0 .5 , 0 .55 , 0 .6 , 0 .65 , 0 .7 , 0 .75 , 0 . 8, 0 .85 , 0 . 9, composition substantially retains its structural integrity after 0 .95 , 0 . 99 , and useful ranges may be selected between any application to the soil and do not readily disintegrate . of these values , ( for example , about 0 . 2 to about 0 . 99 , about [0218 ] The brittleness of the granular composition is 0 . 2 to about 0 . 90 , about 0 . 2 to about 0 . 6 , about 0 . 2 to about determined by measuring the amount of force required to 0 .5 , about 0 . 3 to about 0 . 9 , about 0 .3 to about 0 .8 , about 0 . 3 cause the granular composition to disintegrate . Brittleness to about 0 .6 , about 0 . 35 to about 0 . 99, about 0 . 35 to about may be measured using an Instron instrument. The brittle 0 . 85 , about 0 . 35 to about 0 . 7 , about 0 . 35 to about 0 . 6 , about ness of the granular composition is about 10 , 20 , 30 , 40 , 50 , 0 .4 to about 0 .95 , about 0 .4 to about 0 . 90 , about 0 . 4 to about 60 , 70 , 80 , 90 or 100 N and useful ranges may be selected 0 .6 , about 0 . 55 to about 0 . 99 , about 0 .55 to about 0 .85 , about from between any of these values , ( for example , about 10 to 0 . 55 to about 0 . 6 , about 0 . 6 to about 0 . 99 , about 0 . 6 to about about 100 , about 10 to about 80 , about 10 to about 6 , 0 10 to 0 . 95 , about 0 . 6 to about 0 .85 , about 0 . 7 to about 0 . 95 , about about 50 , about 10 to about 40 , about 10 to about 30 , about 0 . 7 to about 0 . 8 , about 0 . 8 to about 0 .99 ) . In a preferred 10 to about 20 , about 20 to about 100 , about 20 to about 90 , embodiment the granular composition has a water activity of about 20 to about 70 , about 20 to about 60 , about 20 to about less than about 0 . 55 . 50 , about 20 to about 40 , about 30 to about 100 , about 30 to [0224 ] It will be appreciated by those skilled in the art that about 80 , about 30 to about 60 , about 30 to about 50 , about the optimal water activity for the granular composition may 30 to about 40 , about 40 to about 100 , about 40 to about 80 , vary. about 40 to about 60 , about 40 to about 50 , about 50 to about 0225 ]. In the instance of Yersinia entomophaga a water 100 , about 50 to about 80 , about 50 to about 60 , about 60 to activity of Aw : 0 .54 to 0 . 55 is desired . With respect to about 100 or about 60 to about 80 N ) . Yersinia entomophaga , a range up to 0 .69 had a faster [0219 ] The granular composition of the invention is suf decline in viable cell numbers . Water activity ( AW ) is mea ficiently porous wherein such that following absorption of sured with a dew point meter (Decon devices ) . water, there are adequate spaces within the structure of the [0226 ] The growth of undesirable fungi in the granular granular composition for the one or more microorganisms to composition of the invention may be limited at higher pH . In various embodiments the composition has a pH of at least survive and grow . about pH 6 , pH 6 . 5 , pH 7 , pH , 7 . 5 , pH 8 , pH 8 . 5 , pH 9 , pH [ 0220 ] The porosity of the granular composition may be 9 . 5 or about pH 10 , and useful ranges may be selected from measured using scanning electron microscopy (SEM ) and by between any of these values , for example , from about pH 6 determining the water activity of the granular composition . to about pH 10 or about pH 7 to about pH 9 . The porosity of the granular composition is about 1 , 100 , [0227 ] The growth of undesirable bacteria in the granular 200 , 300 , 400 , 500 , 600 , 700 , 800 , 900 or 1000 um , and composition may be limited at an acidic pH . The growth of useful ranges may be selected from between any of these undesirable fungi in the granular composition may be lim values , ( for example , about 1 to about 1000 , about, to about ited by the addition of a fungicidal agent. 900 , about 1 to about 800 , about 1 to about 600 , about 1 to about 400 , about 200 to about 1000 , about 200 to about 800 , about 200 to about 600 , about 200 to about 500 , about 300 3 . Granular Composition Manufacture to about 1000 , about 300 to about 700 , about 300 to about [0228 ] In various embodiments the composition of the 600 , about 300 to about 500 , about 500 to about 1000 , about present invention is formed by extrusion . For example , the 500 to about 800 , about 500 to about 700 , about 600 to about granular composition of the present invention is formed by 1000 , about 600 to about 800 , about 700 to about 1000 , admixing a composition comprising one or more microor about 700 to about 800 or about 800 to about 1000 um ) . ganisms, two or more plant powders , a biodegradable water US 2018 / 0084776 A1 Mar . 29 , 2018 absorbent agent, and one or more lubricants to form a dough , f0239 ] In some embodiments the granular composition is and then processing the dough to form the granular com selected for a size of 0 . 1 , 0 . 5 , 1 . 0 , 1 . 5 , 2 .0 , 2 . 5 , 3 . 0 , 3 . 5 , 4 . 5 position . or 5 mm selecting for a bait size between 1 and 2 . 8 mm , and [0229 ] In various embodiments the lubricants are selected useful ranges may be selected from between any of these from lipids, fatty acid esters , minerals , inorganic materials values ( for example , 0 . 1 to about 5 , about 0 . 1 to about 4 , and polymers . Examples of specific lubricants include mag about 0 . 1 to about 3 . 5 , about 0 . 1 to about 2 . 5 , about 0 . 1 to nesium stearate , talc and silica . In various embodiments the about 1 . 5 , about 0 . 1 to about 0 . 5 , about 0 . 5 to about 5 , about lubricant is present at about 0 . 05 , 0 . 10 , 0 .15 , 0 .20 , 0 .25 , 0 . 5 to about 4 . 5 , about 0 . 5 to about 3 , about 0 . 5 to about 2 , 0 .30 . 0 . 35 . 0 . 40 . 0 . 45 , 0 .50 , 0 .55 , 0 .60 . 0 .65 , 0 .70 , 0 .75 , 0 . 80 , about 0 . 5 to about 1 , about 1 to about 5 , about 1 to about 4 , 0 .85 , 0. 90 , 0 . 95 or 1 .00 % by weight, and useful ranges may about 1 to about 2 , about 2 to about 5 , about 2 to about 4 . 5 , be selected from between any of these values ( for example , about 2 to about 3 . 5 , about 3 to about 5 , about 3 to about 4 0 .05 to about 1 , about 0 . 05 to about 0 . 90 , about 0 .05 to about mm ) . 0 .65 , about 0 . 05 to about 0 . 50 , about 0 .05 to about 0 .40 , [0240 ) It will be appreciated that various granulation tech about 0 .05 to about 0 . 30 , about 0 .10 to about 1 , about 0 . 10 niques can be used to make the composition of the present to about 0 . 85 , about 0 . 10 to about 0 . 75 , about 0 . 10 to about invention . For example , the use of wet granulation . 0 .65 , about 0 . 10 to about 0 . 40 , about 0 .25 to about 1 . 00 , [0241 ] Granulation occurs through the agglomeration or about 0 . 25 to about 0 . 75 , about 0 . 25 to about 0 .65 , about cohesiveness of particles to form larger particles . It will be 0 .25 to about 0 . 35 , about 0 .30 to about 1 .00 , about 0 .30 to appreciated that the use of wet granulation is useful in the about 0 .60 , about 0 . 30 to about 0 . 50 , about 0 . 45 to about preparation of a substrate -containing granular composition . 1 .00 , about 0 .45 to about 0 . 80 , about 0 . 45 to about 0 .70 , In such manufacture the granulation process is used to build about 0 . 45 to about 0 .60 , about 0 .65 to about 1 .00 , about 0 .65 to about 0 . 90 , about 0 .65 to about 0 .75 , about 0 .70 to up material about the substrate . about 1 . 00 , about 0 .70 to about 0 . 90 , about 0 .70 to about [0242 ] Granules are formed using a fluidised powder bed 0 .80 , about 0 .85 to about 1 .00 or about 0 . 85 to about 0 .95 % through the addition of a liquid carrier to the fluidised by weight) . powder bed . The powder particles are wetted by the liquid [0230 ] The ingredients and excipients are typically mixed , carrier and aggregate to form wet granules . In various for example in a suitable mixing vessel such as a blender. embodiments the granulation process will include a binder [0231 ] In some embodiments the mixed ingredients and /or to maintain the granules once dry . In some embodiments the excipients are reduced in particle size . For example , in some binder is selected from sodium starch glycolate , maltodex embodiments the ingredients and / or excipients are reduced trin , trehalose , rice powder , potato starch , kibbled wheat , to a particle size of about 10 , 100 , 200 , 300 , 400 , 500 , 600 , corn grits , honey, cornflour, wheat flour, a polyvinyl pyr 700 , 800 or 900 um . Preferably the excipients and /or ingre rolidone , hydroxymethylpropyl cellulose and polyvinyl dients are ground to a particle size of less than about 850 um . alcohols or a combination of any two or more thereof. 0232 ] The particle size can then be size selected such as [0243 ] In various embodiments the manufacture of the through the use of a sieve . In some embodiments the sieve granular composition of the present invention is formed by has a pore opening of about 50 , 100 , 200 , 300 , 400 , 500 , 600 , admixing a composition comprising one or more microor 700, 800 , 900, 1000 , 1100 or 1200 um . Preferably the pore ganisms, two or more plant powders , a biodegradable water opening is about 850 um . absorbent agent to form a dough , and then coating a sub [ 0233 ] In some embodiments the mixed composition is strate with the dough to form the granular composition . then combined with the lubricant and the biodegradable [0244 ] In other embodiments of the invention the granular water - absorbent agent. composition is formed by admixing a composition compris [0234 ] The microorganism can then be added to the dry ing two or more plant powders, a biodegradable water mixture to prepare a dough . The microorganism can be absorbent agent, and one or more lubricants to form a dough , added using a mixer for example . and then coating the dough with an exterior coating com [ 0235 ] As a specific example to demonstrate this method , prising one or more microorganisms to form the granular a cell suspension of Yersinia entomophaga ( in a 0 .05 M composition . trehalose solution with 0 .18 % by weight premium clover [0245 ] In other embodiments of the invention the granular honey ) can be added to the dry mixture to prepare a dough composition is formed by admixing a composition compris using a cake mixer at speed “ 2 ” for 5 - 10 min . This provides ing one or more microorganisms, two or more plant pow an effective dough for preparing the composition of the ders, a biodegradable water- absorbent agent, and one or invention . more lubricants to form a dough , and then coating the dough [0236 ] The dough is then extruded through a die , for with an exterior coating comprising one or more microor example, using a hand extruder or mechanical extruder . ganisms to form the granular composition . Suitable extruders would be known and would be operated [0246 ] In various embodiments one or more of the granu in a typical manner . For example , at a pressure of 2 bar. lar composition excipients are heat treated or gamma irra [ 0237] The extruded dough can then be dried . Various diated to kill or reduce the amount of undesirable microor drying methods can be used . For example , the dough could ganisms in the granular composition . be dried in a laminar flow at 20° C . under ambient humidity . 102471 In one embodiment the method may comprise During or after drying the dough can then be broken into drying the agricultural composition . In one embodiment the small pieces to form the granular composition . agricultural composition may be dried until the composition [ 0238 ] In some embodiments the granular compositions has a water activity of less than about 0 . 2 , 0 .25 , 0 . 3 , 0 . 4 , 0 . 5 , can be further size selected to remove any undesirably fine 0 .6 , 0 . 7 , 0 .75 , 0 . 8 , 0 . 9 , 0 .95 , 0 . 98 , or about 0 . 99 , and useful or large particles . ranges may be selected between any of these values . US 2018 / 0084776 A1 Mar . 29 , 2018

[0248 ] In one embodiment the method comprises viability of the microorganisms in the granular composition [0249 ] extruding the dough to form an extruded mate is maintained or not reduced to non - viable levels during rial, and storage of the granular composition for prolonged periods. [ 0250 ] processing the extruded material into granules to [0258 ] For example , the granular composition retains its form an agricultural composition . shelf storage stability when stored at 20° C . for up to 6 , 7 , [ 0251] In another embodiment the composition compris 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 or 18 months, and useful ing one or more microorganismsmay comprise one or more ranges may be selected between any of these values, ( for saccharides listed above . In one exemplary embodiment the example from about 6 to about 18 , about 6 to about 17 , about composition comprising one or more microorganisms com 6 to about 15 , about 6 to about 12 , about 6 to about 8 , about prises trehalose and honey . 7 to about 18 , about 7 to about 17 , about 7 to about 15 , about 10252 ] In another embodiment the composition compris 7 to about 12 , about 7 to about 10 , about 8 to about 18 , about ing one or more microorganisms or the exterior coating may 8 to about 16 , about 8 to about 14 , about 8 to about 12 , about comprise a biodegradable polymer, a non - cytotoxic oil, or a 10 to about 18 , about 10 to about 17 , about 10 to about 15 , biodegradable polymer and a non -cytotoxic oil. about 10 to about 13 , about 10 to about 11 , about 11 to about 18 , about 11 to about 15 , about 12 to about 18 , about 12 to 4 . Use about 14 , about 13 to about 18 or about 13 to about 14 [ 0253] The granular composition of the present invention months) . can be applied to plants , pastures, fields or any area to be 10259 ) After storage of microorganism - containing prod treated . The granular material can be applied to the surface ucts it is important that they retain a level of microorganisms of the land , onto plants or drilled into the soil. In some such that they retain the viability of the product when used . embodiments the granular composition of the present inven In relation to the granular composition of the present inven tion can be added to animal feed . The granular composition tion , it retains at least about 1x10² , 1x10°, 1x104, 1x10 % , contain one or more microorganisms that are supported in 5x105 , 1x106 , 5x106, 1x107, 5x107 , 1x108, 5x108 , 1x10° , the composition such that upon delivery to plants , pastures 2x10° , 1x10° , 5x1010 , or 1x1011 colony forming units and fields, upon contact with water the one or more micro ( cfu 's ) of the one or more microorganisms per gram of the organism multiplies within the granular composition . The composition after storage . Therefore , after storage the microorganism ' s growth within the granular composition is granular compositions of the present invention retain at least supported by the ingredients in the granular composition about 0 . 1 , 0 . 2 , 0 . 5 , 0 .75 , 2 , 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 50 , such as the particular plant powders and /or the sugars. 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 , 98 , 99 or 100 % viability . Through this method the granular compositions can deliver The storage is for about 1 , 2 , 3 , 4 , 5 , 6 , 7 days, or 2 , 3 , 4 , 5 , an effective amount of the microorganism to the area to be 6 , 7 , 8 , 9 , 10 weeks storage , or 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , treated without the requirement of providing a high micro 15 , 18 , 24 months storage of the composition at a tempera organism loading in the granular composition since the ture of about 4 , 8 , 10 , 15 , 16 , 20° C . or at ambient microorganism can multiple in the granular composition temperature , and useful ranges may be selected between any upon delivery to the site of action . This has the advantage of of these values. lowering the costs of the granular composition given it is not [ 0260 ] As described above, the granular composition of required to carry a high loading of the microorganism . the present invention can be utilised to [0254 ] The granular composition may be spread by spray [ 0261] a ) kill or control pests , such as insect pests or ing ( land or aerial) , blowing , spinning or pneumatic appli other invertebrate pests , b ) reduce the population of a cation the granular composition such that the granular pest , such as an insect pest, composition cover 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55 , [0262 ] c ) maintain or increase plant or crop growth , 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 or 100 % of the plants , field , [0263 ] d ) maintain or increase pasture production , pasture or area to be treated and useful ranges may be (02641 e ) maintain or increase plant, crop or pasture selected between any of these values, (for example , 5 to yield , or about 100 , about 5 to about 95 , about 5 to about 90 , about [ 0265 ] f ) bio - remediate contaminated land , or 5 to about 70 , about 5 to about 6 , 0 5 to about 40 , about 10 [0266 ] g ) a combination of any two or more of a) to f) . to about 100 , about 10 to about 90 , about 10 to about 70 , [0267 ] In relation to increasing pasture dry matter produc about 10 to about 60 , about 20 to about 100 , about 20 to tion , the granular composition of the present invention when about 90 , about 20 to about 70 , about 20 to about 60 , about used on pasture will advantageously increase pasture dry 30 to about 100 , about 30 to about 70 , about 30 to about 50 , matter production by at least about 5 , 10 , 20 , 30 , 40 , 50 , 60 , about 40 to about 100 , about 40 to about 80 , about 40 to 70 , 80 , 90 , 100 , 150 , 200 , 250 or 300 % and useful ranges about 70 , about 40 to about 60 , about 50 to about 100 , about may be selected between any of these values , ( for example , 50 to about 80 , about 50 to about 70 , about 60 to about 100 , from about 5 to about 300 , about 5 to about 200 , about 5 to about 60 to about 90 , about 70 to about 100 % ) . about 150 , about 5 to about 100 , about 5 to about 80 , about [0255 ] The granular compositions of the present invention 5 to about 70 , about 5 to about 50 , about 5 to about 30 , about can be spread on a pasture using spreaders carried by trucks, 10 to about 300 , about 10 to about 200 , about 10 to about tractors , planes or any other vehicle . To obtain good spread 100 , about 10 to about 60 , about 20 to about 300 , about 20 ability the granular compositions have a bulk density that to about 250 , about 20 to about 150 , about 20 to about 80 , assist spreadability . about 20 to about 50 , about 30 to about 300 , about 30 to [ 0256 ] In one embodiment the granular composition may about 250 , about 30 to about 200 , about 30 to about 150 , be applied when the soil is wet , or after irrigation or rainfall. about 30 to about 100 , about 40 to about 300 , about 40 to [0257 ] An advantage of the present invention is that the about 200 , about 40 to about 100 , about 50 to about 300 , granular composition of the present invention have an about 50 to about 150 , about 50 to about 100 , about 60 to improved shelf life . The applicants have found that the about 300 , about 60 to about 250 , about 60 to about 100 , US 2018 / 0084776 A1 Mar . 29 , 2018 14 about 80 to about 300 , about 80 to about 100 , about 100 to [ 0274 ] When a granular composition of the present inven about 300 , about 100 to about 250 , about 100 to about 200 tion is applied to soil the granular composition is applied at or about 150 to about 300 % ) . a rate of at least about 5 , 10 , 20 , 30 , 35 , 40 , 45 , 50 , 55 , 60 , [0268 ] As mentioned the granular composition of the 65, 70 , 75 , 80 , 85 , 90 , 95, 100 , 110 , 120 , 130 , 140 , 150 , 160 , present invention can be used to kill or reduce the population 170 , 180 , 190 or about 200 kg per hectare , and useful ranges of insect pests or invertebrate pests such as grubs , beetles , may be selected between any of these values , ( for example, caterpillars , weevils , snails , slugs, crickets , termites, or ants . about from 5 to about 200 , about 5 to about 170 , about 5 to In various embodiments the insect pest is beetle larvae , such as grass grubs , chaffers , Torito (Diloboderus abderus ), Cor about 160 , about 5 to about 120 , about 5 to about 90 , about bie grubs (Oncopera intricate) , Winter Corbie grubs (Onco 5 to about 80 , about 5 to about 70 , about 5 to about 30 , about pera rufobrunnea ), Tasmania grass grubs ( Acrossidius tas 10 to about 200 , about 10 to about 180 , about 10 to about maniae ) , weevils such as clover root weevils ( Sitona 140 , about 10 to about 100 , about 10 to about 90 , about 10 lepidus ), adults beetles such as black beetles. Leipdopterans to about 80 , about 10 to about 70 , about 10 to about 50 , such as Cuncunilla negra , Dalaca pallens , True armyworm about 30 to about 200 , about 30 to about 180 , about 30 to (Mythimna unipuncta ) , African armyworm ( Spodoptera about 170 , about 30 to about 150 , about 30 to about 140 , exempta ), greasy cutworm ( Agrotis ipsilon ) , Cosmopolitan about 30 to about 110 , about 30 to about 90 , about 30 to armyworm (Mythimna separate ), corn rootworms (Dia about 80 , about 30 to about 70 , about 45 to about 200 , about brotica ), wire worms ( Elateridae ) , other insects pests such 45 to about 180 , about 45 to about 150 , about 45 to about as locust ( locusta migratoria ) or a combination of any two 110 , about 45 to about 100 , about 45 to about 90 , about 45 or more thereof. to about 80 , about 45 to about 70 , about 50 to about 200 , [ 0269 ] In one particularly contemplated embodiment, about 50 to about 160 , about 50 to about 120 , about 50 to application of the granular composition may kill or reduce about 100 , about 50 to about 80 , about 60 to about 200 , the population of Porina , such as Wiseana spp . including about 60 to about 170 , about 60 to about 160 , about 60 to Wiseana cervinata , Wiseana copularis , Wiseana fuliginea , about 140 , about 60 to about 120 , about 60 to about 100 , Wiseana jocosa , Wiseana mimica , Wiseana umbraculata or about 60 to about 90 , about 70 to about 200 , about 70 to Wiseana signata . In another particularly contemplated about 180 , about 70 to about 160 , about 70 to about 140 , embodiment the granular composition may kill or reduce the about 70 to about 120 , about 70 to about 110 , about 80 to population of black beetles (Heteronychus arator ) . about 200 , about 80 to about 190 , about 80 to about 150 , [0270 ] For example, application of the granular composi about 80 to about 120 , about 90 to about 200 , about 90 to tion may reduce the population of a pest, for example Porina about 170 , about 90 to about 150 , about 90 to about 150 , or black beetles , by at least about 10 , 15 , 20 , 25 , 30 , 35 , 40 , about 110 to about 200 , about 110 to about 150 , about 120 45 , 50 , 55, 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 or 100 % in the four to about 200 , about 120 to about 160 , about 130 to about months following application , and useful ranges may be 200 , about 130 to about 180 or about 130 to about 170 kg selected between any of these values, (for example , from per hectare ) . about 10 to about 100 , about 10 to about 90 , about 10 to about 75 , about 10 to about 60 , about 10 to about 50 , about [0275 ] In various embodiments the one or more microor 10 to about 40 , about 20 to about 100 , about 20 to about 90 , ganisms in the composition may retain at least about 0 . 1 , 0 . 2 , 20 to about 75 , 20 to about 60 , 20 to about 50 , 20 to about 0 . 5 , 0 . 75 , 2 , 5 , 10 , 15, 20 , 25 , 30 , 35 , 40 , 50 , 55 , 60 , 65 , 70 , 40 , 40 to about 100 , 40 to about 90 , 40 to about 75 , 40 to 75 , 80 , 85 , 90 , 95 , 98 , 99 or 100 % viability about 1 , 2 , 3 , 4 , about 60 , 40 to about 50 , 50 to about 100 , 50 to about 90 , 5 , 6 , 7 days , or 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 weeks, or 3 , 4 , 5 , 6 , 50 to about 75 , 50 to about 60 , 60 to about 100 , 60 to about 7 , 8 , 9 , 10 , 11, 12 , 15 , 18 , 24 months after application of the 90 , 60 to about 75 , 75 to about 100 , 75 to about 90 , or from composition to the soil, and useful ranges may be selected about 75 to about 80 % ) . between any of these values for example , from about 40 to [ 0271] The granular composition of the present invention about 100 % , about 60 to about 100 % , about 70 to about can be used to deliver a wide variety of organisms, and may 100 % or from about 80 to about 100 % . be used in combination to deliver other compounds such as probiotics . [0276 ] In various embodiments the number of viable units 10272 ] As one example , the granular composition of the of the one or more microorganisms in the composition present invention may be used for bioremediation of soil . increases by at least about 1 . 2 , 1 . 25 , 1 . 5 , 1 .75 , 2 , 2 . 5 , 3 , 3 . 5 , For example , the granular composition of the present inven 4 , 4 . 5 , 5 , 6 , 7 or 8 log increase about 1 , 2 , 3 , 4 , 5 , 6 , 7 days , tion can be applied to affected soil to release the microor or 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 weeks, or 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , ganism for colonisation of the affected soil . 11 , 12 , 15 , 18 , 24 months after application of the composi [ 0273] When the granular composition of the present tion to the soil, and useful ranges may be selected between invention is used for application to plants , pasture or a field , any of these values , ( for example from about 1 . 2 fold to the granular composition is applied at a rate of at least about about 5 fold , about 1 . 5 fold to about 4 fold , about 2 fold to 1x10°, 1x1010 , 5x1010, 1x1011 , 5x1011 , 1x1012 , 2. 5x1012 , about 4 fold or about 2 fold to about 3 fold ) . 5x1012 , 7 .5x1012 , 1x1013, 2. 5x1013 , 5x1013 , 7. 5x1013 , 1x1014 , 2 . 5x1014 , 5x1014 , 7 .5x1014 , or about 1x1015 cfu of [0277 ] In various embodiments the one or more microor the one or more microorganisms per hectare and useful ganisms are detectable in the soil for up to about 1, 2 , 3 , 4 , ranges may be selected between any of these values , ( for 5 , 6 , 7 , 8 , 9 , 10 weeks , or 3 , 4 , 5 , 6 , 9 or 12 months after example , from about 1x109 to about 1x1015 , about 1x101° to application of the composition to the soil. about 1x1014 , about 1x1011 to about 1x1014 , 1x1012 to about [0278 ] The invention consists in the foregoing and also 1x1014 , about 1x1013 to about 1x1015 , or about 1x1013 to envisages constructions of which the following gives about 1x1014 cfu per hectare ) . examples only and in no way limit the scope thereof. US 2018 / 0084776 A1 Mar . 29 , 2018 15

EXAMPLES assumed a negative binomial distribution for all counts [ 02791. A number of examples were conducted to examine through log link function . The a " values were used as a the stability and manufacture of the baits , as well as the covariate in the GEE analysis — i . e . the model was : bait ' s effectiveness in the field , including methods of deter Log CFU change= a + bxaw mining its effectiveness . [ 0280 ] Described below is some of the methods common Bait Water Activity Versus Viable Count Change to the examples, such as the growth of Yersinia ento mophaga : the bacteria used to examine the efficacy of the [0288 ] Yersinia entomophaga numbers CFUS that were baits . measured during storage periods up to 90 days were ana lysed using a GEE approach in statistical software SAS 1 . Porina Preference Assay version 9 . 3 . The GEE analysis took account of correlation [0281 ] Porina larvae were reared from eggs . between the duplicated ( or triplicated ) counts at each sam 0282] For the experimental set up , a click clack container pling time, and assumed group -specific negative binomial ( W 150 mmxL 220 mmxH 60 mm ) was filled approximately distributions through log link function . Five groups were two - thirds with Hauraki gold Peat Moss and gently shaken determined based on water activity values at To . The CFU so that the surface was level . Tap water was then added to change in each group over the period was modelled as a the peat and mixed until the peat was moist . The containers linear trend - i. e. decrease at a constant rate ( b ) on the loge were left overnight to equilibrate . scale: [ 0283] The following morning six porina larva ~ 30 mm in log CFU = a ; - b ;xDays length were placed on top of the peat and allowed to burrow . [0289 ] Then , these decreasing rates were compared After the porina larvae had entered the peat profile , four small piles of baits that were visually equal in size were between the five water activity groups in the GEE analysis , placed on the peat surface . Each sample was tested at least to statistically test the relationship between the CFU change three independent times with one sample per container but and awo in a randomised design . Data from visual assessments by photography were recorded on the basis of the amount of Example 1 Stability sample that had been disturbed , as determined by photo graphing on a 24 - hour basis over a 72 hour duration . 1 . Method [0290 ] The baits were stored at 4 and 20° ambient humid 2 . Bioassay of Bait Variants Against Porina Larvae ity and the samples were assessed as close as possible to 1 [ 0284 ] Individual bait weighing approximately 0 . 02 g was hour, 1 day , 28 days , 60 days and 90 days after production , added to the top of fertilizer - free potting mix placed to a allowing the stability trend of Yersinia entomophaga to be depth of approximately 45 mm in a standard specimen defined . container (measuring 45 mm in diameter by 60 mm in [0291 ] Previous stability trials have found the bait to be height) that contained one porina larva approximately 25 - 35 stable at 4° C . ( Table 3 ) , so samples stored at 4° C . were only mm in length . The containers were then left at 15° C . for 7 assessed when required . days in a dark incubator ( Contherm ) and monitored daily for the presence of live or dead larvae . To ensure the burrows [0292 ] Attributes such as hardness, ability to " process” , that the porina larvae had formed throughout this time e . g . extrusion properties , and water content were assessed . period were not damaged , larvae were assessed visually by [0293 ] The following composition was tested for stability looking for signs of movement through the sides of the at 4 and 20° C . transparent specimen container . TABLE 2 3 . Statistical Analysis Composition of bait stability tested at 4 and 20° C . [ 0285 ] In all experiments statistically analysed , cells at Amount time = 0 are at a value of 100 % . Ingredient Function ( % w / w ) Yersinia entomophaga Active ingredient 38 . 37 % Fluidised Bed Drier Experiment Water Activity suspension with live bacteria Kibbled wheat Porina 10 . 12 % [0286 ] Because each drying time in each replicated experi phagostimulant ment resulted in two or three different water activity values Corn grits Porina 10 . 12 % at T . (referred to as am, hereafter ) and viable bacterial cell phagostimulant count (CFU ) was measured at rather different intervals in San Bran ® plant powder Porina 10 . 12 % each experiment, this analysis compared CFU change values phagostimulant Rice powder Bait stabiliser 20 . 24 % from T , to Toe day between different water activity values . Maltodextrin Bait stabilizer 10 . 12 % Each CFU change value was calculated as: Porina phagostimulant CFU change (cfu / g )= mean CFU at 70 - CFU at 728 Xanthan gum Bacteria stabiliser 0 .23 % day Canola oil Bacteria stabiliser 0 .23 % Magnesium stearate Lubricant 0 . 13 % [ 0287 ] These CFU change values were analysed using a Premium clover honey Porina 0 . 18 % generalised estimating equations (GEE ) approach in statis phagostimulant tical software SAS version 9 . 3 . The GEE analysis took account of correlation between the duplicated counts , and US 2018 / 0084776 A1 Mar . 29 , 2018

[ 0294 ] The bait was manufactured such that the bacteria TABLE 4 were incorporated both on the surface of the bait ( by Ingredients , its function , percentage ( W / w or v / w ) in spraying ) and distributed throughout the bait . the undried dough and amount required to prepare 1 kg 2 . Results of the bait dough for extrusion and coating a core Extruded bait Coated marble [0295 ] The stability of the composition of Table 2 is ( “ Standard " ) substrate * shown below in Table 3 . Ingredient % Amount % Amount TABLE 3 Yersinia entomophaga suspension 35 . 30 550 mL 25 . 18 170 mL Kibbled wheat 10 . 70 166 . 7 g 12. 35 83 . 35 ?g Shelf life stability of the composition of Table 2 . Corn grits 10 . 70 166 . 7 g 12 . 35 83 . 35 g San Bran ® plant powder 10 .70 166 . 7 g 12 . 35 83 . 35 g Time Viable cell counts (cfu / g ) Rice powder 21. 40 333 . 3 24 .70 166 .7 g Maltodextrin 10 . 70 166 . 7 12 . 35 83. 35 g (days ) 4° C . 20° C . Paselli BC (Potato starch ) 0 .30 5 . 2 g 0 . 39 2 . 6 ? Magnesium stearate 0 . 13 2 . 0 g 0 . 15 1 . 0 g - 10 1010 10 x 1010 Premium clover honey 0 . 18 2 . 7 g 0 . 20 1 .38 g ? 10 x 1010 9 x 1010 ? 10 x 1010 7 x 1010 * 50 : 50 substrate to build up ratio ? 10 x 1010 5 x 1010 ?10 x 1010 3 x 1010 ?10 x 1010 2 x 1010 [0301 ] Moisture content of the formulated baits was deter ? 10 x 1010 2 x 1010 mined using an oven -drying method and expressed as per ? 10 x 1010 2 x 1010 centage based on wet weight. One g of bait was withdrawn 9 x 1010 from an individual bag and dried at 80° C . for 24 h . After 24 9 x 1010 h , the dry weight of the sample was recorded and the moisture content calculated as % increase in weight. [0302 ] Water activity was measured using an Aqualab Water Activity Meter Series 3TE (Decagon Devices ) . Example 2 — Ingredients /Manufacturing Variables 10303 ] Bait components varied in its ability to take up water ( Table 5 ) . Corn grit increased in weight by 48 % 1 . 1 Assessment of Formulation Binders through water uptake , kibbled wheat by 91 % and bran by 129 % . [0296 ] To determine the effect of potential binders or anti - desiccants in place of Paselli starch , 2 % Acacia gum or 1 % or 5 % oil was included in the dry standard bait ingre TABLE 5 dient. Water uptake values of the standard bait and its ingredients . 02971. The baits prepared with either acacia gum ( 9 . 93x 10 ' ) or 1 % oil (6 .73x104 ) or 5 % oil ( 7 .55x104 ) were not Water stable in storage with a significantly reduced shelf life Replicate samples activity Initial Soaked Swelling average apparent by day 28 compared with the standard bait (3 .01x ( 1 and 2 ) ( aw ) weight weight ( % ) % 10 ' ) . Teabag 1 * 0 . 15 0 . 309 Teabag 2 * 0 . 15 0 . 291 Kibbled wheat 1 4 . 003 7 .736 93 . 0 1 . 2 Assessment of Water Activity in “ Standard ” Bait Kibbled wheat 2 0 .4607 4 . 822 9 . 136 89 . 1 91 . 1 [ 0298 ] From preliminary assessments of data accrued Corn grit 1 4 . 784 7 . 104 46 . 8 Corn grit 2 0 .5103 5 . 276 7 . 93 48 . 8 47 . 8 throughout the project it was observed that baits of a low Rice powder 1 3 . 54 6 . 102 71. 2 water activity had higher rates of survival than stored Rice powder 2 0 . 5662 2 . 355 4 . 01 68 . 3 69 . 7 standard baits with a higher water activity . For all experi San Bran ® 1 plant 4 . 25 9 .618 127 . 3 ments in this study standard bait was used as an internal powder control and water activity was always measured as a pro San Bran ® 2 plant 0 . 3571 4 . 003 9 . 15 129 . 7 128 . 5 duction variable . This enabled a detailed analysis comparing powder Standard bait 3 . 841 5 .874 51. 0 Yersinia entomophaga survival against water activity . mixture 1 10299 ] From the GEE analysis it is evident that the rate of Standard bait 0 .47496 .0499 .433 54 . 8 52 . 9 decrease ( over the course of each experiment ) in viable cell mixture A2 numbers was significantly greater as water activity at T . Paselli BC (Potato 0 .4958 increased . As shown in FIG . 1 , there was significantly less starch ) * * survival at ay , of 0 .698 , and greater survival at the lower a Maltodextrin * * 0. 4679 of 0 .544 . * The teabag is the bag that contains the formulation . 1 . 3 Water Holding Capacity * * not tested due to the fine nature of component. [0304 ] Based on the information presented in Table 5 , two 10300 ] To determine the ability of various bait components bait ingredients were identified that differed in water uptake : to absorb water , bags were independently filled with each rice ( ~ 70 % ) and bran ( ~ 129 % ) . Although corn grits had a component ( as shown in Table 4 ) and the filled teabag lower water sorption ( ~ 48 % ) than rice , this was a property immersed in water under stirring for 1 h to allow for of its smooth and waxy (hydrophobic surface ), limiting its maximum uptake ofwater . The weight of the material before ability to absorb moisture . (“ dry weight” ) and after water uptake (“ wet weight” ) was recorded , and corrected for the dry and wet weight of the tea 1 .4 Modified Bait Variants bag . The moisture uptake ( swelling ) was calculated as % [ 0305 ] Based on the results of bait physiochemical prop increase in weight. erties , two new variants were developed by either removing US 2018 / 0084776 A1 Mar . 29 , 2018 17 the bait component to be tested or replacing a component such as San Bran® plant powder with a different form of TABLE 6 - continued bran (wheat bran ) that had different properties (i . e . no Details of variation to the standard bait variants , the sugar ) . Using the standard bait formulation ( see B of Table rational and its attributes noted during production 10 ) , a bait was made without bran and another without rice powder — in these instances the remaining bulk was made Bait manufacture Bait through the addition of extra rice or bran , respectively , to Rationale attributes palatability make the final volume. The omission of various components cost and enhance resulted in different physical attributes of the bait , such as its stability in ability to be extruded and broken post -extrusion . storage Replacement of Cheaper source Dough consistency YES [0306 ] The properties tested are listed in Table 6 . Of note , San Bran® plant of bran not uniform and heat -treated bait also assessed at this time, was found to be powder with hard to extrude . brittle , while the omission of rice led to a bait that was easy wheat bran The baits became more brittle to extrude but was slow to take up moisture . The alteration Freeze dried Alternative Nil effect ND of bait components also changed the baits ' appearance standard extruded production relative to the standard bait . bait method Addition of Determine if Nil effect ND TABLE 6 acacia addition of a 22 gum into bacterial polysaccharide Details of variation to the standard bait variants , the concentrate enhances survival rational and its attributes noted during production of cells during production and Bait manufacture Bait storage Rationale attributes palatability Addition of oil Reduce potential Nil effect NDND to the dry desiccation Standard bait Reference bait Reduce cost YES standard stress to Heat treated Reduce microbial Rapid uptake of ND bait mixture Yersinia competitors bacteria concentrate , entomophaga the dough hard to extrude IND = not determined ; YES = palatable to Porina ; NO = not palatable to Porina . pH 8 Reduce microbial Nil effect ND competitors , the [ 0307 ] Along with the standard bait , two baits “ standard pH is not minus San Bran® plant powder " or standard minus rice conducive to powder ” were placed in a long term stability trial and fungal growth assessed periodically for surviving Yersinia entomophaga . Bile salts Reduce microbial Nil effect ND competitors ; bile Initial data revealed that the standard bait minus rice powder salts are not had higher viable cell numbers at day 60 relative to the conducive to the standard bait and significantly greater than the historic bait . growth of non The standard bait without San Bran® plant powder exhib enterobacteria ited reduced survival of Yersinia entomophaga . Based on Bacterial load Determine effect Nil effect - reduced 3ND of Yersinia load leads to greater this information no further replicates of this bait were entomophaga survival and undertaken . density dependant economical 10308 ]. In other instances , such as bait packaging or competition replacement of San - Bran® plant powder with wheat bran , a Packaging Determine effect greater porosity N 3D favourable storage trend was observed from where further of gas exchange leads to greater bait batches were produced and placed in a second or third through the use of survival different assessment allowing for robust statistical analysis . packaging . 10309 ) Baits where wheat bran was used in place of San Replacement of Determine the Rapid uptake of 3NO Bran® plant powder, resulted in greater than 10 - fold higher plant powder impact of San bacteria concentrate Yersinia entomophaga survival over 90 days relative to the Bran ® with rice Bran ® plant and the bait became standard bait ( San Bran® plant powder ) . This was validated powder powder on brittle post drying absorption and by statistical LMM analysis of the data . drying Replacement of Determine the Wet 3NO 1 . 5 Packaging Materials rice powder with impact of San San Bran ® plant Bran® plant [0310 ] Physical processes such as gas and moisture trans powder powder on fer can occur through the package containing the bait , which absorption and could affect Yersinia entomophaga survival in the stored drying product. To determine if this might be the case , standard Replacement of Determine Nil effect 3NDND baits were packaged in materials that differed in its gas rice powder with influence of barley flour different starch transfer abilities ( Table 7 ) . types on dough [0311 ] Each of the formulations was independently pack quality, reduce aged ( 15 g /bag ) in either TGT bags ( pore size 120 um , cost and enhance 10x10 cm ) , gas transferable GT( ) bags (pore size 80 um stability in thick , 10x10 cm ) , mesh bags ( pore size 280 um , 10x10 cm ) storage or foil bags ( pore size < 1 um , 10x10 cm ) . The mesh bags are Replacement of Determine Nil effect rice powder with influence of ND made from commercially sourced screen silk ; there is cur maize flour different starch rently no information on either the water or oxygen trans types on dough mission rate . quality , reduce 0312 ] The bags were then stored at 4 and 20° C . and ambient humidity . Viable cell counts, water activity and US 2018 / 0084776 A1 Mar . 29 , 2018 18 moisture content were assessed after drying of the bait ( 1 day ) and after 28 , 60 and 90 days of storage. At each TABLE 8 -continued sampling time, one bag was destructively sampled . Viable cells numbers (cfu / g ) and water activity values from extruded standard baits stored at either 10 or 20° C . TABLE 7 Temp After 28 After 60 Storage bags and its rate of permeability Bait formulation (°C . ) aw Initial days days 30 h dried 10 0 .47 6 . 0 x 10° 1 x 10 % 6 . 2 x 108 Water vapour Oxygen 30 h dried 20 0 . 47 6 . 0 x 109 3 x 107 2 .5 x 105 Bag transmission rate transmission rate Pore size Foil 0 . 4 g /m² / 24 h 0 . 3 cc /m² / 24 h < 1 um 1 . 7 Yersinia entomophaga Bait Loading GT 5 g /m² / 24 h 6000 to 8000 80 um cc/ m² / 24 h 10320 ) To determine if bacterial loading influences the ?G? 2 . 5 g /m² / 24 h 625 cc/ m² / 24 h 120 um survival dynamics of Yersinia entomophaga in a bait , a Mesh bag * 280 um series of standard baits were prepared containing different densities of Yersinia entomophaga , ranging from 10 % , 107 , 108, 101° cells per gram of the bait . [ 0313] Each % survival value was calculated at each [0321 ] After the 28 -day shelf storage period , a higher post- storage time as: initial bait loading led to a greater rate of cell death . LMM % Survival ( % ) = CFU at each post -storage time/ analysis of the 28 day storage data found the log . % mean CFU at 70x100 Survival = 3 .7171 - 0 .6620xlog10 initial loading was a statis 0314 ] The % survival values were analysed using a linear tically significant ( P = 0 .024 ) relationship . mixed modelling ( LMM ) approach in statistical software SAS version 9 . 3 . In the LMM analysis , correlation between TABLE 9 the duplicated (or triplicated ) measurements at each sam pling time was modelled as a random effect . In the instance Mean % surviving cells of bait packaging the LMM also included the effects of two Standard bait Mean % surviving factors : packaging (Package ) and post- storage measuring (per g of bait ) cells time (Day ) , and its interaction (Package * Day ) as fixed effects . loading of 1010 0 . 05 loading of 108 0 . 14 [0315 ] Bait stored in the GTbag over 60 days appeared to loading of 107 0 . 24 have higher numbers of surviving Yersinia entomophaga loading of 105 1 . 05 than when the bacterium was stored in either a TGT or a foil packaged bag . [ 0316 . To validate this, the experiment was repeated and a " mesh ” bag was included as an additional treatment. Example 3 — Manufacture of Baits Although at day 60 there appeared to be a consistent trend of improved survival in the more porous bag types [0322 ] Two different types of baits were tested : extruded (MESH > GT> TGT> FOIL ) , this was not statistically signifi baits and baits formed with a coated marble core . The baits cant. with a coated marble core comprised a 50 : 50 substrate to build up ( coating ) ratio . 1. 6 Effect of Storage Temperature on Survival of Yersinia in [0323 ] The key ingredients used in the baits and the Baits amount of the key ingredient are shown in Table 4 . To [0317 ] The initial aim of the project was to achieve determine the ability of various bait components to absorb 3 -month stability at 20° C . where the bacteria count does not water , bags were independently filled with each component go below ~ 6x10° / g bait . It should be noted that commercial (as shown in Table 4 ) and the filled teabag immersed in food cool stores are typically maintained in a temperature water under stirring for 1 h to allow for maximum uptake of range of between 6 - 7° C . water. The weight of the material before (" dry weight” ) and [0318 ] In one experiment the viability of Yersinia ento after water uptake ( “ wet weight" ) was recorded , and cor mophaga , in a standard bait in TGT packaging stored at rected for the dry and wet weight of the tea bag . The either 20° C . or 10° C ., was assessed after 28 and 60 days . moisture uptake (swelling ) was calculated as % increase in [ 0319 ] When stored at 10° C . there was less than one log weight . drop in numbers of Yersinia entomophaga , in comparison 103241. To determine the ability of various bait components with a large decrease in numbers in bait stored at 20° C . This to absorb water, bags were independently filled with each indicates that a change in temperature has a significant effect component (as shown in Table 4 ) and the filled teabag on the survival of Yersinia entomophaga within the bait and immersed in water under stirring for 1 h to allow for that there may be a yet to be determined threshold tempera maximum uptake of water. The weight of the material before ture between 10 - 20° C . , above which the rate of survival of ( " dry weight” ) and after water uptake (“ wet weight" ) was recorded , and corrected for the dry and wet weight of the tea the bacteria in the bait rapidly decreases. bag . The moisture uptake (swelling ) was calculated as % TABLE 8 increase in weight. Viable cells numbers (cfu / g ) and water activity values from 1 . Method extruded standard baits stored at either 10 or 20° C . Temp After 28 After 60 1 . 1 Extruded Bait Bait formulation (°C . ) w Initial days days [0325 ] The ingredients used to prepare the standard 24 h dried 10 0 .60 7 . 6 x 10° 2 x 10° 5 . 4 x 108 extruded bait and their amounts are listed in Table 4 . 24 h dried 20 0 .60 7 . 6 x 10° 2 x 107 9 . 7 x 105 (0326 ). The excipients were ground in a blender, sieved ( 850 um ), and mixed together with magnesium stearate and US 2018 / 0084776 A1 Mar . 29 , 2018

Paselli BC (Potato starch ) . Yersinia entomophaga cell sus 2 . Results pension ( in a 0 .05 M trehalose solution with 2 . 7 g premium clover honey ) was added to the dry mixture to prepare a dough using a cake mixer at speed “ 2 ” for 5 - 10 min . 2 . 1 Marble Chip Coated Bait Development [ 0327] The dough was extruded through a 3 mm die using 0338 ]. The resultant PVP baits were smooth in appearance either a hand extruder or mechanical extruder ( operated at 2 and felt hard . The use of Paselli BC (Potato starch ) produced bar pressure ) onto clean plastic trays . The extruded bait a bait of good consistency and was therefore pursued as a dough was dried for 24 h in the laminar flow (with air " on " ) binder for core coating . Of note , approximately 60 % of the at 20° C . under ambient humidity . weight of a 1 g marble chip coated bait was the marble chip core , with the remaining 40 % comprised of the standard bait [0328 ] Around 3 - 4 h from the start of the drying process the bait material was broken into small pieces . At the end of material applied as the coating . the 24 h period , the dried baits were sieved to remove any fine or large particles selecting for a bait size between 1 and 2 . 2 Fluidised Bed Drier Experiment 2 .8 mm . (0339 ] Tests showed solid cores coated with the standard bait matrix formed discrete free - flowing coated baits that 1 .2 Marble Chip Coated Bait were suitable for fluidised bed drying experiments . [0340 ) Similar to the assessment of the water activity of [0329 ] An alternative bait production strategy of coating extruded standard bait , marble chip coated bait of a lower the standard bait material to a solid dense core (marble chip ) water activity has a higher rate of survival. This observation was assessed . The resultant bait was assessed in palatability combined with that of all other standard extruded bait data assays and under controlled conditions using a fluidised bed where aw was assessed . Water activity at TO was plotted drier . against Yersinia entomophaga viable cell numbers change [ 0330 ] A technology was developed where the standard after 28 days in storage . Cell viability significantly bait material is coated onto a solid inert core material. The decreased as water activity increased according to the equa chosen core was marble chip , as it is a chemically inert and tion : Loge CFU change = 17 . 9065 + 5 . 1106xaw ( P < 0 .0001 ) . non - porous material that should not influence the dynamics [0341 ] Water activity has a significance influence on the of the applied bait and is unlikely to influence Yersinia survival of Yersinia entomophaga under storage at 20° C . entomophaga survival dynamics in the bait matrix . [0331 ] Given the new production process and the 2 .3 Bait Absorption Assay unknown ability of a bait mix to bind to the core material, three formulation binders were independently assessed . [0342 ] In this study 1 gram of bait containing Yersinia These were entomophaga , Serratia entomophila or Serratia pro teamaculans was placed , at various cell densities , onto moist [0332 ] 2 % w / w polyvinyl pyrrolidone (PVP ), and paper towels . The samples were destructively assessed and [0333 ] 0. 3 % , Paselli BC (Potato starch ). Yersinia entomophaga , Serratia entomophila or Serratia [0334 ] Marble chips coated with the bait matrix were proteamaculans enumerated as outlined above . prepared in a Cimbria CC2 laboratory scale seed coating 10343 ] As shown in FIGS . 2 - 4 the number of bacteria machine . Screened 1 - 2 mm marble chips (500 g ) were added within the bait increased within 20 - 48 hours . FIG . 2 dem to the drum and coated with a bait mixture ( Table 3 ) using onstrates approximately 7 -log increase from a loading of concentrated Yersinia entomophaga live cells suspended in 7 . 33x104 cells/ g . FIG . 3 demonstrates an increase approxi mately 7 - log increase from a loading of 1 . 73x10 cells / g . 0 . 05M trehalose ( 170 mL ) to bind the coating to the core. FIG . 4 demonstrates approximately 4 - log increase from a [ 0335 ] In order to determine optimal moisture contents for loading of 8 .9x10 ' cells / g . storage stability it was necessary to establish a repeatable drying process. A fluidised bed drier allows the rapid assess ment of the effects of drying time on the survival dynamics Example 4 — Effectiveness of the Bait in the Field of Yersinia entomophaga in the bait. Heated air is forced up through a porous matrix on which the material to be dried is 1 . Method positioned . The movement of air has the force to uplift the material being dried resulting in a suspended tumbling [0344 ] The effectiveness of baits containing Yersina ento motion leading to the rapid drying of the test bait material. mophaga was tested against porina (Wiseana spp ) . A fluidised bed drier maintains a constant programmed temperature and humidity can be measured within the cham [0345 ] As shown in Table 10 , the baits tested in the ber. preference assay were [ 0336 ] The marble chip coated baits were transferred to a [0346 ] a ) bait 38 % Yersinia custom -built fluidised bed dryer . The drying bed was 190 [0347 ] b ) standard 35 % Yersinia mm in diameter and approximately 30 mm in depth . The [0348 ] c ) standard (minus rice powder , replaced with marble chip coated baits were dried using air at a flow rate San Bran® plant powder ) of approximately 1 m / s . Although the inlet air was unheated , the heat produced by the machine resulted in the heating of [0349 ] d ) standard (San Bran® plant powder replaced inlet air to approximately 35° C . Marble chip coated baits with wheat bran ) were dried for up to 120 minutes with samples collected at [0350 ] e) standard (minus rice powder ) 10 , 30 , 60 and 120 minutes for assessment of water activity [ 0351 ] f ) standard (minus San Bran® plant powder . and viability ( CFU / g ) . replaced with rice powder ) [ 0337 ] The water activity ( outlined below ) of the resultant [0352 ] g ) marble chip coated with the standard bait product at the set time points was determined . material and a clover leaf as a control. US 2018 / 0084776 A1 Mar . 29 , 2018

TABLE 10 Examples 5 - 10 [0357 ] Methods common to Examples 5 - 10 are described Composition of the baits tested below . Ingredient/ Percentage w / w or w / v 1 . Manufacture and Composition of Baits excipient Function A [0358 ] Baits tested in Examples 5 - 10 were manufactured Function B C D F as follows . Yersinia active 38 35 35 35 35 [03591 . The ingredients used in baits prepared by extru entomophaga sion , wet granulation and coating on a core of blinding sand (MH96 ) live and their amounts are listed in Table 12 . bacteria Kibbled wheat timulant 10 1111 1111 11 11 Corn grits Phagostimulant 10 11 1111 11 11 TABLE 12 San bran Phagostimulant 10 11 32 11 0 Rice powder Phagostimulant 20 210 21 32 Ingredients , percentage ( w / w or v / w ) in the undried dough Maltodextrin Phagostimulant 10 11 11 11 11 and amount required to prepare 1 kg of the bait dough for Paselli BC ( Potato stabiliser 0 .3 0. 3 0 .3 0 .3 extrusion , wet granulation or coating a blinding sand core starch ) Extruded and wet Coated blinding sand Magnesium stearate lubricant 0. 3 0 . 3 0 . 3 0 . 3 granulated baits substrate * Xanthan gum stabiliser 0 . 12 0 . 13 0 . 13 0 . 13 0 . 13 Canola oil emulsifier 0 . 23 Ingredient % Amount % Amount Premium clover phagostimulant 0 .23 0. 18 0 . 18 0 .18 0 . 18 honey Bacterial or spore suspension 35 . 30 550 mL 25 . 18 170 mL Kibbled wheat 10 . 70 166 . 7 g 12. 35 83. 35 g Corn grits 10 . 70 166 . 7 g 12 . 35 83 . 35 Fine wheat bran 10 . 70 166 . 7 g 12. 35 83 . 35 g [ 0353] A surface coated bait was used as a comparison in Rice powder 21. 40 333. 3 g 24 . 70 166 .7 09 all but one replicate , to determine if porina had a greater Milled Chelsea white sugar 10 . 70 166 . 7 12 . 35 83 . 35 Paselli BC (Potato starch ) 0 . 30 5 .2 g 0 .39 2 . 6 g feeding preference for that bait relative to the other baits . Magnesium stearate 0 . 13 2 .0 g The coated marble chip bait was also used . Premium clover honey 0 . 18 2. 7 g 0 . 20 1 . 38 g [ 0354 ] The remaining baits assessed had shown improved * 50 :50 substrate to build up ratio shelf life characteristics in previous experiments . These were a standard bait with wheat -bran in place of San Bran® plant powder and a standard bait that contained no rice 1. 1 Preparation of Bacterial and Spore Suspensions powder . [0360 ] For each granule production run , the bacteria ( Yers inia entomophaga , Serratia entomophila or Serratia pro [ 0355 ] Where rice powder or san bran was omitted (Group teamaculans ) were produced by fermentation or batch cul C and Group F ) , the baits swelled and were not touched by ture . Cells were collected by centrifugation (6300 g , 16° C . , the larvae. These baits were therefore considered not prac 10 min ) and the pellet was re - suspended with 0 . 05 M tical to use . In general the other baits that were assessed in trehalose , making the total volume 25 % of the original the assay were frequently disturbed , indicative of interest by volume. the larvae . [0361 ] The fungus Metarhizium anisopliae var anisopliae ( F178 ) was grown on solid substrate ( rice ) and wet har [ 0356 ] There was significant Porina mortality in treat vested by washing the rice grains with 0 .01 % Triton - X 100 ments containing Yersinia entomophaga baits over the 7 - day resulting in a spore suspension of 3x108/ ml for either duration while control treatments showed negligible mor incorporation into the granule or coating on zeolite granule . tality . The marble chip coated with the standard bait material Total spore loadings were determined by dissolving the had reduced efficacy with a delayed 28 % mortality at day 7 granules in 0 . 01 % Triton - X 100 and performing a haemo ( Table 11 ). It should be noted that the bacterial loading of cytometer count of total spores per gram of granule , and a these marble coated baits was low with 1 .81x10° CFU / g and viable spore count determined through dilution plating onto this likely reduced efficacy of this treatment. potato dextrose agar (PDA ) . TABLE 11 1 .2 Extruded Bait Ability of bait variants to cause mortality against Porina larvae . [ 0362] Extruded baits were prepared as described in Values are number of healthy ( H ) or dead ( D ) larvae ( out of a Example 3 . total of 14 per treatment) after introduction of the bait . 1 .3 Wet Granulated Bait Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 [0363 ] Bacterial cell suspension was added to the dry mixture to prepare a dough using a cake mixer at speed “ 2 ” Control 14 H 14 H 14 H 14 H 14H 1D 13H 1 ) standard 1D 13H 4D 10H 8D 6H 10D 4H 13D 1H 13D 1H for 5 - 10 min . 5 .86 x 10° [0364 ] The dough was extruded in a wet granulator (FSG CFU / g TG 2000 ) using 3 . 15 mm mesh sieve at 50 rpm speed . 2 ) with bran 14H 4D 10H 7D 7H 9D 5H 12D 2H 12D 2H [ 0365 ] The extruded baits were dried , broken up and 7 .59 x 10° sieved as for the extruded baits described in Example 3 . CFU / g 3 ) marble 14H 14H 1D 13H 3D 11H 4D 10H 4D 10H 1 . 4 Blinding Sand Coated Bait 1 . 81 x 106 [0366 ] Blinding sand coated with the bait matrix was CFU /g prepared in a Cimbria CC2 laboratory scale seed coating machine. Screened 1 - 2 mm blinding sand (500 g ) was added to the drum and coated with a bait mixture ( Table 12 ) using US 2018 / 0084776 A1 Mar . 29 , 2018 concentrated bacterial cells suspended in 0 .05M trehalose [0375 ) The samples were studied in a Jeol JSM 7000F ( 170 mL ) to bind the coating to the core . Scanning Electron Microscope operating at 10 kV . 2 . Enumeration of Bacteria and Fungi 7 . Assessment of Brittleness and Complete Crushing Force [0367 ] Ten - fold serial dilutions of bacterial liquid cultures were prepared in Special M (SpecM ) buffer ( 0 . 1 % Tween 10376 ) Brittleness ( point of fracture ) and complete crush 80 ; 2 mM tetra - sodium pyrophosphate ). The resultant dilu ing force were measured using an Instron 4204 . tions were then plated onto selective agar. Plates were [0377 ] The force was measured using the load cell 100 incubated at 30° C . for 48 h and colony counts were recorded as CFU mL - 1. Newton / 10 kg, foot area 11134 .57 mm or 11 .35 cm ? , [0368 ] To enumerate Yersinia entomophaga , Serratia compression speed 10 mm /min , force applied 5 kg and the entomophila or Serratia proteamaculans in granules , one gauge length 5 mm . gram of the granule formulation was weighed out and added [ 0378 ] Five baits for each formulation were measured . to 9 g SpecM buffer . The granules were dispersed in the buffer using a Homogenizer (Polytron ) at 11000 rpm for 8 . Assessment of Bulk Density 30 -60 seconds until the samples were completely dispersed . Ten - fold serial dilutions were prepared in SpecM buffer and [0379 ] Bulk density of the baits was measured in triplicate 100 UL aliquots of the relevant dilutions were plated on to ( n = 3 ) by measuring the mass of the known volume of bait selective agar . Plates were incubated at 30° C . for 48 h and and expressed in grams per cubic centimetre ( g / cm ) derived counts recorded as CFU per gram ( g ) of granule . [ 0369 Spore viability of Metarhizium anisopliae var from the formula : anisopliae (F178 ) was determined via an overnight germi Bulk density = mass ( g ) /( volume (mL ) nation test on PDA where viable spores germinate and can be enumerated . 9. Porina Bioassay 2 . 1 Absorption Assay [0380 ] Individual porina larvae were set up in 100 ml [ 0370 ] One gram of extruded baits containing a pre plastic lidded tubes ( 20 per treatment) filled with a 60 : 40 determined amount of microorganism were placed onto mix of soil and peat moistened to 20 % , and allowed to moist paper towels . Containers were left with their lids burrow overnight before treatments were applied . Two baits raised allowing air flow at room temperature ( approximately ( equivalent to 0 .060 g ) were placed on the soil surface of 22° C . ) for the duration of the experiment. At 0 , 24 , 48 , 72 each tube and the lid replaced . Tubes were checked after 24 , and 96 h post placement on moist filter paper, the samples 72 and 96 hours for signs of granule consumption . Four days in triplicates ( one random sample from each of the three post bait application the larvae were fed with cut ryegrass, independent containers ) were independently destructively then again at day seven , and at seven day intervals thereafter. assessed and the amount of microorganism enumerated . 3 . Assessment of Moisture Content 10 . Black Beetle Bioassay [ 0371] Pre -weighed baits were dried at 80° C . for 24 h . [0381 ] Black beetle adults were confined individually in The dry weight of the sample was recorded and the moisture 12 -well trays and fed either carrot cylinders ( 2 . 5 mm thick content calculated according to the formula : with a diameter of 7 mm , replaced every 2 - 3 days ) or 0 . 02 g of a Y. entomophaga bait . Sample size was 30 beetles per Moisture content ( % ) = 100x (wet weight- dry weight )/ treatment with a 50 :50 male : female ratio . Beetles were kept wet weight at 22° C . for the duration of the bioassay . 4 . Assessment of Water Activity [ 0382 ] Moistened Y . entomophaga extruded granules received a single drop of water to increase moisture before 10372] Water activity was measured using an Aqualab being added to the trays . Water Activity Meter Series 3TE (Decagon Devices ). [0383 ] Beetle mortality was assessed at 1 , 7 and 14 days 5 . Assessment of Swellability post initiation of the experiment. [0373 ] Tea bags were independently filled with 0 .5 g of each bait immersed in water under stirring for 1 h to allow Example 5 — Bacterial Growth and Survival in the for maximum uptake of water . The weight of the material Bait before (“ dry weight” ) and after water uptake ( “ wet weight " ) was recorded in triplicate , and corrected for the dry and wet 1 . Method weight of the tea bag and average results calculated (n = 3 replicates ). The moisture uptake (swelling ) was calculated 0384 ] Bacterial survival and growth in extruded baits as % increase in weight using the formula : comprising Yersinia entomophaga , Serratia entomophila or Moisture uptake = 100x ( (soaked weight ingredient Serratia proteamaculans at two different initial bacterial soaked weight tea bag ) - initial weight ingredi loadings was determined by absorption assay . ent - initial weight tea bag ) ) / ( initial weight ingre dient - initia 2 . Results 6 . Assessment of Porosity [0385 ] The results are shown in Table 13 . [ 0374 ] Samples were mounted on to aluminium stubs with [0386 ] Irrespective of the initial bacterial loading , the conductive carbon tape and sputter coated from a gold / number of viable bacteria in the extruded baits increased to palladium source to impart conductivity to the samples . a loading of about 1010 - 10 cfu / g over 96 hours . US 2018 / 0084776 A1 Mar . 29 , 2018

TABLE 13 Viable cell counts for Yersinia entomophaga , Serratia entomophila and Serratia proteamaculans incorporated into extruded baits . Viable cell count ( CFU / g + SD ) after Bacteria 0h 24 h 48 h 72 h 96 h Yersinia 1 . 73 + 1 . 47 + 2 . 19 + 6 . 40 + 1 . 33 + entomophaga 0 . 15 x 103 0 . 12 x 105 0 . 19 x 108 1 . 30 x 10° 0 .32 x 1010 3 .50 1 . 95 £ 9 .03 2 .40 – 1 .75 + 0 .52 x 105 2. 19 x 105 1. 69 x 108 0 . 35 x 1010 0. 09 x 1010 Serratia 7 .33 £ 3 . 96 8 . 35 7 .27 + 1 .72 + entomophila 2 .08 x 104 0 . 35 x 106 0 .64 x 109 1 . 96 x 1010 0 . 15 x 1011 1 . 59 = 1 .02 + 1 .11 + 2 .00 2 . 13 + 0 . 11 x 106 0 .23 x 10° 0 .15 x 1011 0 .26 x 1011 0. 32 x 1011 Serratia 8 . 90 – 1 . 07 + 3 . 85 + 1 . 00 + 1 .78 + proteamaculans 1. 14 x 105 0. 81 x 10° 0 .48 x 1010 0 .23 x 1011 0. 25 x 1011 2 . 78 + 3 . 17 + 1 . 00 1. 83 4 . 13 = 0 .28 x 107 0. 38 x 106 0 .00 x 10° 0. 65 x 1010 0. 85 x 1010

Example 6 - Assessment of Baits Comprising an TABLE 15 Inert Bulking Agent Normalised viable cell counts for Yersinia entomophaga 1 . Method incorporated into extruded baits comprising talc . [0387 ] Extruded baits were prepared comprising the bait Bait dough of Table 12 and talc , an inert bulking agent, in a composition weight ratio of 25 :75 , 50 : 50 or 75 : 25 . Baits comprising (Ratio Normalised viable cell count after 100 % bait dough were prepared as a control. talc :dough ) 0h 24 h 48 h 72 h 96 h [ 0388 ] Growth of Yersinia entomophaga in the baits was assessed by absorption assay. 25 :75 1 . 00 138 .06 - 9297. 05 15002 .97 – 19260 .65 + 112 . 15 4747 .41 3463. 53 10602 . 96 50 :50 1 . 00 322 .71 – 7569 .43 12418 .50 16507 .57 + 2 . Results 220 .67 2514 . 50 4474 .25 6 290 . 99 [ 0389 ] The results are shown in Table 14 . The results 75 :25 1 . 00 22 .09 59 .33 65 .68 + 63. 97 + normalised to the initial cell loading are shown in Table 15 . 4 .60 4 .55 10 . 49 4 . 10 [ 0390 ) Irrespective of the initial bacterial loading , the 100 % dough 1 . 00 159 . 46 + 7810 . 09 + 12261. 38 + 12499 . 30 + number of viable bacteria in the extruded baits increased to 43 .24 2660 .57 3774 .47 2575 .20 a loading of about 1040 - 1011 cfu / g over 96 hours . The Example 7 - Physical Characteristics of Granules addition of an inert bulking agent in the form of talc did not 1 . Method inhibit the growth of Yersinia entomophaga in the baits [0392 ] Extruded , wet granulated and blinding sand coated when used at a ratio of 25 : 75 or 50 :50 . baits comprising Yersinia entomophaga , some comprising [ 0391 ] The 75 : 25 bait had a sloppy texture . The lower talc in varying amounts as an inert bulking agent, were growth rate of this bait may reflect the high initial cell prepared . loading and that many of the bait 's nutritional ingredients [0393 ] Viable cell count, water activity , moisture content, may have been utilised by the bacteria at the time of density , brittleness and swellability , compressive strength manufacture . and porosity of the baits were measured . TABLE 14 Viable cell counts for Yersinia entomophaga incorporated into extruded baits comprising talc . Bait composition (Ratio Viable cell count (CFU / g + SD ) after talc :dough ) 0h 24 h 48 h 72 h 96 h 25 : 75 4 . 25 + 4 .56 + 3 . 33 5 .98 + 6 . 82 1 .65 x 106 2 . 26 x 108 0 . 38 x 1010 1. 49 x 1010 1 .65 x 1010 50 :50 3 .23 € 8 . 98 + 2 .18 3 . 54 + 4 . 74 + 1 . 20 x 106 3 .45 x 108 0 .08 x 1010 0 . 12 x 1010 0 .76 x 1010 75 : 25 3 .89 8 .45 + 2 . 28 + 2 . 50 = 2 .46 0 .42 x 108 1 .38 x 109 0 .06 x 1010 0 . 17 x 1010 0 . 96 x 1010 100 % dough 8 .02 1 . 28 6 . 18 + 9 . 65 + 9 . 92 + 0 .49 x 106 0 .43 x 109 2 .09 x 1010 2 .62 x 1010 1 .74 x 1010 US 2018 / 0084776 A1 Mar . 29 , 2018 23

2 . Results [0394 ] FIG . 5 shows images of the baits captured by a scanning electron microscope . [0395 ] The physical characteristics of the baits are pre sented in Table 16 . [ 0396 ] All baits had a porosity of 0 .05 - 50 um . TABLE 16 Cell loading , water activity ( aw ) , moisture content, density , brittlenessness , sweswellability , porosity and force for complete crushing of baits . Cell Force for loading Moisture Density complete ( cfug) = a + content (( g/ cm3cm )) 1= Brittleness Swellability crushing SD SD ( % ) + SD SD ( n ) + SD ( % ) SD (N ) - SD Standard baits Extruded 7 . 7 + 1 . 3 x 0 .567 10 . 1 + 0 . 54 + 26 . 9 + 4 .08 113 . 3 + 9 . 9 29 . 9 + 108 0 .001 0 . 10 0 . 007 2 .65 Wet 7 . 7 + 0 . 588 – 10 . 6 + 0 .52 + 26 . 1 + 6 .67 122 . 7 + 49 . 7 33. 4 + granulated 12 . 0 x 0 .0001 0 . 22 0 .013 12 . 01 108 Blinding 1 . 7 + 0 . 2 x 0 .697 + 7 . 4 + 0 .27 0 .91 + 14 . 3 + 6 . 92 86 . 0 + 15 . 6 23. 9 + sand 108 0 .001 0 .007 9 . 13 coated Granule size / shape Extruded , 7 . 7 + 1 . 3 x 0 .567 – 10 . 1 + 0 .54 26 . 9 + 4 .08 113 . 3 + 9 . 9 29 . 9 + cylindrical 108 0 . 001 0 . 10 0 .007 2 .65 1 - 2 . 8 mm Extruded , 8 . 2 + 1 . 5 0 .671 + Not 0 . 57 + Not 110 . 7 + 32 . 5 NA star 10 % 0 . 001 determined * 0 .01 determineda shaped , 10 mm Extruded baits comprising talc 25 talc : 1 . 7 + 1 . 6 x 0 .636 8 . 8 + 0 .14 0 .58 + 10 .2 - 0 .88 118 .0 = 22 . 3 22 . 1 + 75 dough 106 0 . 0008 0 . 002 0 . 89 50 talc : 1 . 4 + 1 . 2 x 0 .465 4 . 3 0 . 63 + 10 .2 + 0 .88 122 .0 + 32. 1 17 . 5 + 50 dough 105 0 .0003 0 .036 2 . 45 75 talc : 2 . 4 = 0 . 3 x 0 .528 10 . 5 0 .63 + 10 .2 + 0 .54 86 . 7 + 40. 4 – 25 dough 107 0 .0001 0 . 017 Standard 2 . 5 = 3 . 0 x 0 .647 † 12 . 0 + 0 .57 + 21 . 6 £ 3 . 92 150 . 7 + 17 . 5 26 . 7 + extruded 106 0 . 0004 0 .07 0 . 005 7 . 97 granule ( no talc ) due to shape of the extrusion the brittleness of the granule was unable to be determined . " extrudability of the granules were not smooth and continuous. Example 8 Assessment of Water Activity in Baits TABLE 17 - continued Dried Under Controlled Conditions 1 . Method Yersinia entomophaga cell counts (CFU / g ), water activity [ 0397 ] Extruded baits were prepared . The baits were dried in a food dehydrator ( placed in a room with a constant (aw ) and moisture content ( % ) of extruded baits post drying . temperature of 20° C . ) and sampled at 2 , 4 , 6 , 24 , 30 and 48 h post drying and assessed for cell viability , water activity Drying Cell counts Water activity Moisture and moisture content. Temperature and relative humidity time ( h ) ( CFU / g ) + SD ( a . ) + SD content ( % ) monitored inside the food dehydrator using an ibutton data logger , during the experiment period . 2 . 3 + 0 .895 + 0 .0000 24 . 1 2 . Results 0. 07 x 1010 [0398 ] The results are shown in Table 17 . o 1 . 8 + 0 .748 + 0 . 0000 14 . 0

m 0 .07 x 1010 TABLE 17 6 . 3 = 0 . 351 + 0 . 0021 6 . 6

Yersinia entomophaga cell counts ( CFU / g ) , water activity i 0 .14 x 109 (2 . ) and moisture content ( % ) of extruded baits post drying . 5 . 7 0 . 329 + 0 .0021 6 . 4 Drying Cell counts Water activity Moisture 0 . 21 x 10° time ( h ) (CFU / g ) SD (aw ) – SD content ( % ) sono 7 . 1 + 0 .439 + 0 .0000 7 . 8 2 . 1 + 0 . 954 + 0 . 0007 35 . 6 1 . 2 x 10° 3 . 5 x 1010 US 2018 / 0084776 A1 Mar . 29 , 2018 24

Example 9 - Assessment of Baits Comprising anisopliae ( F178 ) zeolite granules were not initially con Fungi sumed although they were pulled into the burrows after 72 hours . 10399 ) Extruded baits were prepared comprising 1 . 5x108 [0408 ] Baits comprising Metarhizium anisopliae (F178 ) CFU / g ( high rate ) or 2 . 10x10 CFU / g (low rate ) of the provided greater than 60 % efficacy at day 47 . fungus Metarhizium anisopliae var anisopliae ( F178 ) . 10409 ). The results demonstrate that baits prepared by all [ 0400 ] The viability of spores in the baits was tested by three manufacturing methods are an effective delivery sys placing individual granules on 1. 5 % water agar at 22° C . and tem for the delivery ofmicroorganisms (bacteria or fungi ) to assessing spore germination , growth of mycelium and sub a target insect host. sequent sporulation . 10401 ] The high loading rate bait sporulated within 4 days. 2 . 2 Black Beetle Bioassay Hyphae and fungal spores were observed in the low loading [0410 ] The Yersinia entomophaga baits were tested . The rate bait after 7 - 10 days . results are shown in FIG . 8 . 10402 ] This example demonstrates the utility of the bait as [0411 ] This example demonstrates that baits prepared by a delivery vehicle for fungal spores . three different manufacturing methods are effective for the delivery of microorganisms (bacteria or fungi) to a target Example 10 — Effectiveness of Baits Comprising insect host . Fungi or Bacteria Against Insects 1 . A granular composition comprising one or more micro organisms, two or more plant powders and a biodegradable water - absorbent agent capable of absorbing at least 10 % of 1 . Method its dry weight in water. [0403 ] Extruded , wet granulated and blinding sand -coated 2 . A composition of claim 1 wherein the one or more baits comprising Yersinia entomophaga or Metarhizium microorganisms are fungi. anisopliae (F178 ) were prepared . The baits tested are set out 3 . A composition of claim 1 wherein the one or more in Table 18 . Baits comprising no microorganisms were microorganisms are bacteria . prepared as a control. 4 . A composition of claim 2 or 3 wherein the fungi are [0404 ] A prior art bait that is an existing standard in the selected from Beauveria , Penicillium , Metarhizium , field was also prepared . The prior art bait comprised zeolite Trichoderma, Gliocladium , Coniothyrium , Verticillium , granules coated in a coating comprising Metarhizium Sclerotinia or a combination of any two or more thereof, anisopliae (F178 ) . and /or the bacteria are selected from Serratia , Xanthamonas, Pseudomonas, Rhizobium , Bifidobacterium , Lactobacillus, [04051 . The insecticidal effectiveness of the baits was Streptococcus, Yersinia , Pseudomonas, Bacillus, Pasteuria , tested against porina (Wiseana spp ) and black beetle . Azobacter, Enterobacter, Azospirillum , Cyanobacteria , Paecilomyces, Streptomycetes , Chromobacterium , TABLE 18 Rhanella , Burkholderia , Paenibacillus, Collimonas , Sinorhizobium , Pantoea , Lecanicillum , Erwinia , Pedioco Baits for bioassay testing cus, Leuconostoc , Aeromonas, Neptunomonas, Klebsiella , Bacteria / fungi Formulation Ponchonia , Brevibacillus, Acinetobacter or a combination of any two or more thereof. Yersinia entomophaga Ye Extruded granules : extruded bait formulation 5 . A composition of claim 3 or 4 having a bacterial loading ( Ye ) Ye Wet Granulation : wet granulated bait formulation of less than about 1011 to about 104 cells per gram of Ye Blinding sand : coated blinding sand bait granular composition . formulation 6 . A composition of claim 1 or 2 having a loading of one Ye Control: Extruded bait formulation , no or more microorganisms less than 10 + to about 104 cells per microorganism gram of granular composition . Metarhzium anisopliae Ma Zeolite Granule : Granules comprising 24 . 5 % 7 . A composition of any one of claims 1 to 6 having ( F178 ) (Ma ) Ma spore suspension , 0 . 3 % xanthan gum a ) a bulk density of from about 0 . 2 g / cm to about 1 . 5 ( stabiliser ) , 63 % zeolite ( carrier) , 2 . 4 % bentonite ( filler ), 9 . 8 % talc ( filler) with a theoretical g / cm ”, or loading of ~ 10 ' spores / g granules b ) a mean diameter (weight - based ) of from about 1 mm to Ma granular formulation Low : extruded bait 5 mm , or formulation with a theoretical loading of ~ 104 c ) a particle size distribution whereby at least 90 % of the spores/ g granules granular composition has a diameter of from 1 mm to Ma granular formulation High : extruded bait 5 mm , or formulation with a theoretical loading of - 107 spores/ g granules d ) a particle size distribution whereby at least 90 % of the Ma Control: extruded bait formulation , no granular composition have a diameter within 1 mm of microorganism . the mean diameter , or e ) a water activity of from about 0 . 2 to about 0 . 95 , or f ) a hardness of from about 10 N to about 100 N , or g ) a brittleness of from about 10 N to about 100 N , or 2 . Results h ) a porosity of from about 1 to about 1000 , or i ) a swellability of from about 20 to about 300 % , or 2 . 1 Porina Bioassay j ) any combination of two or more of ( a ) to (i ) above. 8 . A composition of any one of claims 1 to 7 comprising [0406 ] The Metarhizium anisopliae (F178 ) and Yersinia a ) about 0 . 05 to about 5 % by weight of a biodegradable entomophaga baits were tested . The results are shown in water - absorbent agent, or FIGS. 6 and 7 . b ) a phagostimulant, or 10407 ] Test baits and the no microorganism control baits c ) one or more polysaccharides , or were consumed almost immediately , with the larvae drag d ) at least about 5 to about 50 % by weight plant powders , ging the baits into their burrows . The Metarhizium or US 2018 / 0084776 A1 Mar . 29 , 2018 25

e ) a lubricant, or A ) one or more lubricants to form a dough , and processing f ) a substrate , or the dough to form granules , or g ) a densifier, or B ) coating a substrate with the dough to form the granular h ) one or more processed cereals as the two or more plant composition , powders , or wherein i) an active agent selected from one or more of an i ) the admixture comprises one or more microorganisms, insecticide and a probiotic , or or j ) any combination of two or more of ( a ) to ( i ) above . ii ) the dough is coated with an exterior coating comprising 9 . A composition of any one of claims 1 to 8 having one or more microorganisms, or a ) a mean diameter of about 0 .5 to about 10 mm , or iii ) both ( i ) and ( ii ) . b ) a moisture content of about 5 to about 25 % by weight, 22 . A method of claim 21 wherein the dough is an or extruded dough . c ) a water activity ( a ) of less than about 0 .55 to about 23 . A method of claim 22 wherein the granular compo 0 . 95 , or sition is formed by wet granulation . d ) any combination of two or more of ( a ) to ( c ) above . 24 . A method of any one of claims 21 to 23 wherein the 10 . A composition of any one of claims 1 to 9 wherein the granular composition is dried to a water activity (aw ) of less biodegradable water - absorbent agent is capable of absorbing than about 0 .2 to about 0 . 95 . at least 10 to about 100 % water based on its dry weight, or 25 . The use of the granular composition of any one of about 100 to about 1000 % water based on its dry weight. claims 1 to 21 on soil to 11 . A composition of any one of claims 1 to 10 wherein a ) kill or control pests , such as insect pests, or the biodegradable water - absorbent agent is selected from the b ) reduce the population of a pest , such as an insect pest , group comprising starches , cross -linked polymers , and peat. or 12 . A composition of any one of claims 1 to 10 wherein c ) maintain or increase plant or crop growth , or the biodegradable water - absorbent agent is a polymer. d ) maintain or increase pasture production , or 13 . A composition of any one of claims 1 to 12 comprising e ) maintain or increase plant, crop or pasture yield , or at least about 1x100 cfu / g after storage at 4° C . for 14 days . f ) deliver probiotics to grazing animals or livestock , or 14 . A composition of any one of claims 1 to 13 when used g ) bioremediate the soil, or retains its integrity for at least about 48 hours after contact h ) a combination of any two or more of a ) to g ) . ing about 0 . 1 to about 1 . 0 mL of water per gram of granular 26 . The use of a composition of claim 25 wherein the composition . granular composition is applied to soil 15 . A composition of any one of claims 1 to 14 wherein a ) at a rate of at least about 5 to about 200 kg per hectare , the two or more plant powders comprises a bran as at least or one of said two or more plant powders . b ) at a rate of at least about 1x10° to about 1x101s cfu of 16 . A composition of claim 15 comprising , in addition to the one or more microorganisms per hectare , or bran , a plant powder selected from the group comprising c ) to target a pest wherein the application of the granular wheat flour, rice powder, oat flour , barley flour, rye flour, composition to soil reduces the population of the tar triticale flour, corn (maize ) flour, amaranth flour , millet flour, geted pest by about 10 to about 100 % in the 4 months teff flour, quinoa flour, buckwheat flour and sorghum flour or following application , or a combination of any two or more thereof. d ) and retains about 0 . 1 to about 100 % viability from 1 17 . A composition of claim 15 or 16 comprising a ratio of day to about 24 months after application , or bran to the one or more other plant powders of about 8 : 1 to e ) and wherein the one or more microorganisms are about 1: 4 by weight. detectable in the soil for up to about 1 to about 10 18 . A composition of any one of claims 15 to 17 com weeks or about 3 to about 12 months after application prising a ratio of bran to the one or more saccharides of of the granular composition to soil , or about 5 : 1 to about 1 : 5 by weight . f ) a combination of any two or more of a ) to e ). 19 . A composition of any one of claims 15 to 18 com 27 . A method of delivering one or more microorganisms prising a ratio of the one or more plant powders to the one to soil , comprising applying the granular composition of any or more saccharides of about 8 : 1 to about 1 : 4 by weight . one of claims 1 to 20 to soil . 20 . A composition of any one of claims 1 to 19 comprising 28 . A method of delivering one or more microorganisms about 1x10° to about 1x10 ' colony forming units ( cfu ' s ) of to animal feed , comprising applying the granular composi the one or more microorganisms per gram of the composi tion of any one of claims 1 to 20 to soil. tion after about 1 day to about 24 months after storage when 29 . A method of controlling insect pests , the method stored at a temperature of about 4 to about 20° C . comprising placing insecticidal granular compositions in 21 . A method of producing a granular composition com proximity to an insect pest, the granular compositions being prising admixing a composition comprising two or more as claimed in any one of claims 1 to 20 . plant powders and a biodegradable water - absorbent agent 30 . A granular composition as herein described in any one capable of absorbing at least 30 % of its dry weight in water, or more of the examples herein . and * * * *