# Patent

## Title
Formulation and delivery of Bacillus thuringiensis subspecies Israelensis and Bacillus sphaericus in combination for broadspectrum activity and management of resistance to biological mosquito larvicides

## Abstract

A method for controlling Dipteran larvae or a method for inhibiting the development of larvicidal resistance, controlling resistant populations and reducing resistance levels in Diptera by introducing a larvicidally-effective amount of a combination of a strain of Bacillus thuringiensis subspecies israelensis and a strain of Bacillus sphaericus into an environment containing Dipteran larvae; and a composition of the combination are disclosed. Preferably both strains are non-genetically modified.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation in part of U.S. patent application Ser. No. 10/074,782, filed Feb. 13, 2002, now abandoned, which claims priority of U.S. Patent Application Ser. No. 60/269,5133i led on Feb. 16, 2001, now abandoned.


FIELD OF THE INVENTION
The invention is directed to a method for controlling Dipteran larvae or a method for inhibiting larvicidal resistance in Diptera by introducing a larvicidally-effective amount of a novel combination of a strain of Bacillus thuringiensis subspecies israelensis and a strain of Bacillus sphaericus into an environment containing Dipteran larvae; and a composition of the combination. Preferably, both strains are non-genetically modified.
BACKGROUND OF THE INVENTION
Mosquitoes and black flies are representative of the order Diptera which are pests that have plagued humans and animals for generations. Mosquitoes are the major vectors for a number of human and animal diseases, including malaria, yellow fever, viral encephalitis, dengue fever and filariasis.
Various chemical pesticides have been developed with the goal of controlling Diptera. For example, treatment of a water source with a water-soluble alcohol in water-miscible form for mosquito abatement is disclosed in U.S. Pat. No. 6,077,521. However, more recent emphasis has been placed on the use of biopesticides. For example, controlled-release formulations of at least one biological pesticidal ingredient are disclosed in U.S. Pat. No. 4,865,842; control of mosquito larvae with a spore-forming Bacillus ONR-60A is disclosed in U.S. Pat. No. 4,166,112; novel Bacillus thuringiensis isolates with activity against dipteran insect pests are disclosed in U.S. Pat. Nos. 5,275,815 and 5,847,079; a biologically pure culture of a Bacillus thuringiensis strain with activity against insect pests of the order Diptera is disclosed in U.S. Pat. No. 5,912,162 and a recombinantly derived biopesticide active against Diptera including cyanobacteria transformed with a plasmid containing a B. thuringiensis subsp. israelensis dipteracidal protein translationally fused to a strong, highly active native cyanobacteria's regulatory gene sequence is disclosed in U.S. Pat. No. 5,518,897.
Yet even these biopesticides have drawbacks; so the search for new biopesticides continues. One drawback of certain biopesticides is the potential build-up of pesticidal resistance.
Resistance is defined by differences in susceptibility that arise among populations of the same species exposed to a pesticide continuously over a period of time. These differences are identified by observing a statistical shift in the lethal dose (LD) either to kill 50% or 95% of the population (LD50 or LD95 respectively). Individual differences in susceptibility exist within each species, and pests that are substantially less susceptible may be present, generally at low frequencies, in at least some of the wild populations. In the presence of the pesticide, it is these substantially less susceptible pests that survive and reproduce. Since their ability to survive is a result of their genetic makeup, their resistant genetic makeup is then passed on to their offspring, resulting in shifts in the populations' susceptibility via pesticide-induced selection. Resistance to larvicides has been encountered among certain Dipteran species.
Specifically, the development of resistance in Culex quinquefasciatus to Bacillus sphaericus (B.s.) is noted by Rodcharoen et al., Journal of Economic Entomology, Vol. 87, No. 5, 1994, pp. 1133-1140. A method for overcoming this resistance, by combining B.s. with purified Cyt1A crystals isolated from Bacillus thuringiensis subsp. israelensis or by combining a recombinant B.t.i. with B.s., is disclosed by Wirth et al., Journal of Medical Entomology, Vol. 37, No. 3, 2000, pp. 401-407. However, improved but naturally derived or occurring biological larvicides and compositions to overcome Culex mosquito resistance to B.s. applications would be desirable. It is even more desirable if the biological Larvicide composition has the ability to effectively control broad spectrum mosquito species.
BRIEF SUMMARY OF THE INVENTION
The invention is directed to a novel method of combining a strain of Bacillus thuringiensis subspecies israelensis (ATCC Deposit Number SD-1276) and a strain of Bacillus sphaericus 2362 (ATCC Deposit Number, SD-1170) in which the individual strains are fermented in separate fermentation tanks, the activity of each strain is concentrated by a variety of methods including ultra filtration and centrifugation, and the slurry concentrates are combined in a suitable mix tank at pre-determined ratios to produce a combination slurry which may be spray dried to yield spray dried technical concentrate individual particles with toxins from both Bacillus thuringiensis subspecies israelensis and Bacillus sphaericus. This uniquely produced spray dried technical concentrate is further utilized to formulate wettable powders, granules, dry flowables or wettable granules or water dispersible granules, pellets, non-aqueous suspensions, briquettes, water soluble pouches and tablets which may be applied to mosquito habitats for enhanced and broad spectrum control of various mosquito species and also toward management of resistance that may inherently develop due to over use of biological larvicides such as Bacillus sphaericus. The strain of Bacillus thuringiensis subspecies israelensis may be genetically modified or non-genetically modified. The strain of Bacillus sphaericus may be genetically modified or non-genetically modified. The presently preferred combination includes a non-genetically modified strain of Bacillus thuringiensis subspecies israelensis and a non-genetically modified strain of Bacillus sphaericus. The combination slurry may have from about 1:10 to about 10:1 weight ratio and or potency ratio of Bacillus thuringiensis subspecies israelensis to Bacillus sphaericus; preferably from about 1:4 to about 4:1 weight ratio of Bacillus thuringiensis subspecies israelensis to Bacillus sphaericus; more preferably from about 1:2 to about 4:1 weight ratio of Bacillus thuringiensis subspecies israelensis to Bacillus sphaericus; and most preferably a 4:1 potency ratio of Bacillus thuringiensis subspecies israelensis to Bacillus sphaericus. 
Additional components such as surface active agents, inert carriers that may be natural, synthetic, organic or inorganic, natural and synthetic preservatives, humectants, feeding stimulants, attractants, encapsulating agents, binders, emulsifiers, dyes, sugars, sugar alcohols, starches, modified starches, dispersants, other polymers, vegetable and petroleum base oils, U.V. protectants, buffers, drift control agents, spray deposition aids, other stabilizers, free-flow agents or combinations thereof may also be utilized in conjunction with the combination in a larvicidal composition. The product form that may be used for delivery to mosquito habitats may be a technical powder, wettable powder, dust, pellet, briquette, tablet, impregnated granule (sand, corn cob, clay, synthetic), dry flowable or wettable granule or water dispersible granule or as non-aqueous or aqueous suspension concentrate. The biopotencies of thus combined product forms may range from 100 ITU/mg to 8000 ITU/mg for Bacillus thuringiensis subspecies israelensis and 25 Bs·ITU/mg to 3000 Bs·ITU/mg for Bacillus sphaericus. More preferably, biopotencies may range from 2000 ITU/mg to 6000 ITU/mg for Bacillus thuringiensis subspecies isralensis and 500 Bs·ITU/mg to 1500 Bs·ITU/mg for Bacillus sphaericus and most preferably the biopotencies may range from 1000 ITU/mg to 4000 ITU/mg for Bacillus thuringiensis subspecies israelensis and 250 Bs·ITU/mg to 1000 Bs·ITU/mg for Bacillus sphaericus. 
The invention is also directed to a composition and a method of controlling Dipteran larvae comprising the step of introducing a larvicidally-effective amount of a composition containing a strain of Bacillus thuringiensis subspecies israelensis and a strain of Bacillus sphaericus prepared by the process of the present invention into an environment containing Dipteran larvae. In this method, Dipteran may be a mosquito such as Culex pipiens, Culex quinquefasciatus, Aedes aegypti, Culex tarsalis, Culiseta incidens, Anopheles freeborni or a combination thereof.
The invention is additionally directed to a method for inhibiting larvicidal resistance in Diptera comprising the step of introducing a larvicidally-effective amount of a composition containing a strain of Bacillus thuringiensis subspecies israelensis and a strain of Bacillus sphaericus that is prepared by the process of the present invention into an environment containing Dipteran larvae. Preferably, the Diptera is Culex and larvicidal resistance is developed against Bacillus sphaericus. 


DETAILED DESCRIPTION OF THE INVENTION
The invention is directed to a method for controlling Dipteran larvae or a method for inhibiting larvicidal resistance in Diptera by introducing a larvicidally-effective amount of a novel combination of a strain of Bacillus thuringiensis subspecies israelensis and a strain of Bacillus sphaericus into an environment containing Dipteran larvae; and a composition of the combination. Preferably both strains are non-genetically modified. A detailed discussion of the composition, and the methods utilizing the composition follows.
The Larvicidal Compositions
Biopesticides are a class of naturally occurring pesticides frequently derived from unicellular or multicellular organisms which have developed natural defenses against other organisms. The group of microorganisms pathogenic to insects is varied and diverse. The gram-positive soil bacterium Bacillus thuringiensis subsp. israelensis is one of many B. thuringiensis strains able to produce insecticidal proteins. These proteins, expressed during the sporulation cycle of the bacterium, assemble into parasporal crystalline inclusion bodies. The parasporal crystal produced by B. thuringiensis subspecies israelensis is toxic when ingested by the larvae of Diptera, including mosquitoes and black flies. Upon ingestion, crystal proteins are solubilized in the larval midgut and disrupt the epithelium of the larval midgut region. Swelling and/or lysis of the epithelial cells is followed by larval death from starvation.
Bacillus thuringienesis subspecies israelensis (B.t.i.) has been used successfully in mosquito and blackfly control programs for many years. B.t.i. is utilized in clean to moderately clean organic breeding habitats, and is most effective on Aedes species. Commercial formulations of B.t.i. available under the trademark VECTOBAC® are available from Valent BioSciences Corporation. Specific commercial formulations available from the same supplier are VECTOBAC® G (5/8 Mesh corncob based granular formulation with a label claim of 200 ITU/mg), VECTOBAC® CG (1014 Mesh corncob based granular formulation with a label claim of 200 ITU/mg), VECTOBAC® 12AS (Aqueous suspension formulation with a label claim of 1200 ITU/mg) and VECTOBAC® or DF (Water dispersible granule or dry flowable formulation with a ibel claim of 3000 ITU/mg). B.t.i. is effective against a broad range of mosquito species, offers low mammalian toxicity and is easy to apply. B.t.i. also has a very low susceptibility to the development of resistance, because its larvacidal activity is based on multiple toxins. The probability that individual mosquitoes within a treated population will not be susceptible to all toxins is extremely small.
Bacillus sphaericus (B.s.) is a rod-shaped, aerobic, spore-forming bacterium found commonly in soil and other substrates. To date, at least 16 strains have been found to show mosquitocidal properties of various degrees. Several strains such as 1593M, 2362 and 2297 exhibit high toxicity to mosquito larvae. Bacillus sphaericus strain 2362, (VECTOLEX®, available from Valent BioSciences Corp.) has been utilized in many countries successfully. Specific commercial formulations of B.s. available from the same source are VECTOLEX® WDG (Water dispersible granular formulation with a label claim of 650 Bs. ITU/mg) and VECTOLEX® CG 1014 Mesh corncob granular formulation with a label claim of 50 Bs. ITU/mg). Moreover, this strain was found to perform well in controlling mosquitoes breeding in various habitats, especially ones with polluted water.
Bacillus sphaericus is most effective on Culex species. The activity of B.s. is due to a binary toxin, and repeated use of this toxin in the same habitats has been reported to lead to development of resistance.
However, various levels of resistance to B.s. by mosquito larvae have been observed in Culex pipiens and Culex quinquefasciatus. 
We have now found that a combination of B.t.i. and B.s. is an effective larvicidal formulation. Non-genetically modified components are utilized, which are desirable if the larvicide is to be utilized in an environment connected with production or harvesting of food sources such as crops, cattle or swine. Non-genetically modified B.t.i. or B.s. may be defined as strains which occur naturally, and are not strains resulting from recombinant DNA techniques.
A novel method of combining B.t.i and B.s is illustrated in Example 1 which follows. The ratio of B.t.i. to B.s. may be from about 10:1 to about 1:10; preferably from about 4:1 to about 1:4, more preferably from about 4:1 to about 1:2 and most preferably about 4:1 B.t.i:B.s either on a solids or ITU basis.
The compositions disclosed above may also include additional components such as a surface active agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, a drift control agent, a spray deposition aid, an encapsulating agent, a binder, an emulsifier, a dye, a U.V. protectant, a buffer, a free-flow agent, or any other component which stabilizes the active ingredient, facilitates product handling and application for the particular target pests, Diptera.
Suitable surface-active agents include anionic compounds such as a carboxylate, for example, a metal carboxylate of a long chain fatty acid; a N-acylsarcosinate; mono or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulphate such as sodium dodecyl sulphate, sodium octadecyl sulphate or sodium acetyl sulphate; ethoxylated fatty alcohol sulphates; ethoxylated alkylphenol sulphates; lignin sulphonates; petroleum sulphonates; alkyl aryl sulphonates such as alkyl-benzene sulphonates or lower alkylnaphthalene sulphonates, e.g., butyl-naphthalene sulphonate; salts or sulphonated naphthalene-formaldehyde condensates; salts of sulphonated phenol-formaldehyde condensates; or more complex sulphonates such as the amide sulphonates, e.g., the sulphonated condensation product of oleic acid and N-methyltaurine or the dialkyl sulphosuccinates, e.g., the sodium sulphonate or dioctyl succinate.
Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g., polyoxythylene sorbitan fatty acids esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7 diol, or ethoxylated acetylenic glycols.
Examples of a cationic surface-active agent include, for instance, an aliphatic mono-, di-, or polyamide as an acetate, naphthenate or oleate; an oxygen-containing amine such as an amine oxide of polyoxethylene alkylamine; an amid-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.
Examples of inert materials include inorganic minerals such as kaolin, mica, gypsum, fertilizer, sand, phyllosilicates, carbonates, sulphate, or phosphates; organic materials such as sugars, starches, or cyclodextrins; or botanical materials such as wood products, cork, powdered corncobs, rice hulls, peanut hulls, and walnut shells.
The formulation may also contain added drift control agents or spray deposition aids to control droplet size and to facilitate aerial application. Examples of suitable compounds for these purposes include polyvinylalcohol polymer solutions, polyamide copolymer solutions, polymerized acrylic acid derivatives and blends thereof, vegetable oils and blends thereof, petroleum oils and blends thereof, as well as natural and synthetic polymers.
In the formulations, more than one of the additional components described above may advantageously be utilized.
The compositions of the present invention can be applied as a liquid, an aqueous suspension, an emulsifiable suspension, or a solid by conventional application techniques for each. Solid formulations are presently preferred. In general, the application rate of the larvicidally-effective combination of the present invention will deliver a quantity of pesticide sufficient to control the population of a target pest.
Solid compositions may be formed by spray drying the B.t.i. and B.s slurries separately and combining the powder or by combining the slurries and spray drying the combined slurry to form a powder.
The composition of the present invention can be in a suitable product form for direct application or as a concentrate or primary composition which requires dilution with a suitable quantity of water or other diluent before application. The pesticidal concentration will vary depending upon the nature of the particular formulation, specifically, whether it is a concentrate or to be used directly. The composition may contain from about 1 to 98% by weight of a solid or liquid inert carrier, and 0.1 to 50% by weight of other suitable components such as a surfactant, a dispersant, a suspending agent, a free-flow agent, a stabilizing agent, UV protecting agent, a polymer or combination thereof. These compositions will be administered at the rate of about 50 g (liquid or dry) to 20 kg or more per hectare.
The Methods
The combination of the present invention can be treated prior to formulation to prolong the insecticidal activity when applied to the environment of a target pest as long as the pretreatment is not deleterious to the combination. Such treatment can be by chemical and/or physical means as long as the treatment does not deleteriously affect the properties of the composition(s). Examples of chemical reagents include, but are not limited to, halogenating agents; aldehydes such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride; alcohols, such as isopropanol and ethanol; and histological fixatives, such as Bouin's fixative and Helly's fixatives.
The compositions of the invention can be applied directly to the environment to be treated. Ponds, lakes, streams, rivers, still water, and other areas subject to infestation by dipteran pests are examples of environments needing such treatment. The composition can be applied by spraying, dusting, sprinkling, and broadcasting, among others.
The compositions of the present invention may be effective against insect pests of the order Diptera, e.g., Aedes sp., Andes vittatus, Anastrepha ludens, Anastrepha suspensa, Anopheles sp., Armigeres subalbatus, Calliphora stygian, Calliphora vicina, Ceratitis capitata, Chironomus tentans, Chrysomya rufifacies, Cochliomyia macellaria, Culex sp., Culiseta sp., Coquillettidia sp., Deino cerities sp., Dacus oleae, Delia antiqua, Delia platura, Delia radicum, Drosophila melanogaster, Eupeodes corollas, Glossina austeni, Glossina brevipalpis, Glossina fuscipes, Glossina moristans centralis, Glossina morsitans morsitans, Glossina morsitans submorsitans, Glossina pallidipes, Glossina palpalis gambiensis, Glossina palpalis palpalis, Glossina tachinoides, Haemagogus equines, Haematobia irritans, Hypoderma bovis, Hypoderma lineatum, Leucopis ninae, Lucilia cuprina, Lucilia sericata, Lutzomyia longlpaipis, Lutzomyia shannoni, Lycoriella mali, Mansonia sp., Mayetiola destructor, Musca autumnalis, Musca domestica, Neobellieria sp., Nephrotoma suturalis, Ochlerotatus sp., Ophyra aenescens, Orthopodomyia sp., Phaenicia sericata, Phlebotomus sp., Phormia regina, Psorophora sp., Sabethes cyaneus, Sarcophaga bullata, Scatophaga stercoraria, Stomoxys calcitrans, Toxorhynchites amboinensis, Tripteroides bambusa, Uranotaneia sp. and Wyeomyia sp. However, the composition of the invention may also be effective against insect pests of the order Lepidoptera, e.g., Achroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp., Cochylis hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus sibericus, Desmia funeralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria, Eoreuma loftini, Ephestia elutella, Erannis tiliaria, Estigmene acrea, Eulia salubricola, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisinia americana, Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantria cunea, Keiferia lycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra configurata, Manduca quinquemaculata, Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis, Paleacritia vernata, Papilio cresphontes, Pectinophora gossypiella, Phryganidia californica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynota flouendana, Platynota sultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella, Pontia protodice, Pseudaletia unipuncta, Pseudoplusia includens, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella, Spilonota ocellana, Spodoptera sp., Thaurnstopoea pityocampa, Tineloa bisselliella, Trichoplusia ni, Udea rubigalis, Xylomyges curialis, Yponomeuta padella; Coleoptera, e.g., Leptinotarsa sp., Acanthoscelides obtectus, Callosobruchus chinensis, Epilachna varivestis, Pyrrhalta luteola, Cylas formicarius elegantulus, Listronotus oregonensis, Sitophilus sp., Cyclocephala borealis, Cyclocephala immaculata, Macrodactylus subspinosus, Popillia japonica, Rhizotrogus majalis, Alphitobius diaperinus, Palorus ratzeburgi, Tenebrio molitor, Tenebrio obscurus, Tribolium castaneum, Tribolium confusum, Tribolius destructor, Acari, e.g., Oligonychus pratensis, Panonychus ulmi, Testranychus urticae; Hymenoptera, e.g., Iridomyrmex humilis, Solenopsis invicta; Isoptera, e.g., Reticulitermes hesperus, Reticulitermes flavipes, Coptotermes formosanus, Zootermopsis angusticollis, Neotermes connexus, Incisitermes minor, Incisitermes immigrans; Siphonaptera, e.g., Ceratophyllus gallinae, niger, Nosopsyllus fasciatus, Leptopsylla segnis, Ctenocephalides canis, Ctenocephalides felis, Echicnophaga gallinacea, Pulex irritans, Xenopsylla cheopis, Xenopsylla vexabilis, Tunga penetrans; and Tylenchida, e.g., Melodidogyne incognita, Pratylenchus penetrans. 
In a specific embodiment, the compositions of the invention are active against insect pests of the sub order Nematocera of the order Diptera. Nematocera include the families Culicidae, Simulidae, Chironomidae, Psychodidae, Sciaridae, Phoridae and Mycetophilidae.
The ability of combination of the present invention to inhibit larvicidal resistance is described in detail hereinafter in the Examples. These Examples are presented to describe preferred embodiments and utilities of the invention and are not meant to limit the invention unless otherwise stated in the claims appended hereto.
Example 1
The most preferred and novel method of combining two biological actives is by pre-mixing fermentation beers or slurry concentrates of B.t.i and B.s at the desired solids or potency level and spray drying the slurry mixture to produce a combined technical spray dried powder concentrate. In such a combination formulation, the slurry concentrates may contain preservatives, stabilizers, surfactants, dispersants and other binders. The spray-dried technical concentrate or powder may then be utilized in formulating a granular product as wettable powders, water dispersible granules, and aqueous or non-aqueous suspension concentrates. These combined powder concentrates may also be utilized in pellet, tablet, and briquette formulations. A spray drying experiment was performed combining Bti and Bs fermentation slurry concentrates at various ratios based on % solids level in each of the slurry concentrates. A B.t.i slurry concentrate was first preserved with 0.12% wt/wt of potassium sorbate and 0.06% wt/wt of methyl paraben. Percent solids in the preserved B.t.i slurry concentrate were 11.3% wt/wt. Similarly, a B.s slurry concentrate was preserved with 0.12% wt/wt of potassium sorbate and 0.06% wt/wt of methyl paraben. Percent solids in the preserved B.s slurry concentrate had a mean % solids of 10.1% wt/wt. Slurry mixtures prepared and their ratios on solids basis are given in Table 1.







TABLE 1







B.t.i and B.s slurry mixtures evaluated









Ratio of B.t.i to B.s on solids basis














1:0
1:1
3:1
1:3
2:1
1:2


Material
A
B
C
D
E
F





B.t.i
8.85 Kg
4.42 Kg
6.64 Kg
2.21 Kg
5.89 Kg
2.95 Kg


preserved


slurry


concentrate


B.s preserved
—
5.10 Kg
2.55 Kg
7.65 Kg
3.41 Kg
6.80 Kg


slurry


concentrate


De-ionized
1.15 Kg
0.48 Kg
0.81 Kg
0.14 Kg
0.70 Kg
0.25 Kg


water


Total
10.0 Kg
10.0 Kg
10.0 Kg
10.0 Kg
10.0 Kg
10.0 Kg









The compositions as shown in Table 1 were combined and spray dried utilizing a Niro spray dryer. Inlet temperature ranged between 180° C. and 190° C. and outlet temperature during drying ranged between 68° C. to 81° C. The technical powders were sieved through 100-mesh standard sieve and samples were bioassayed against L4 Aedes aegypti and L3 Culex quinquefasciatus. Average potency data is represented in Table 2.







TABLE 2







Potency values of B.t.i + B.s. spray dried technical powders as affected by their ratios


on solids basis. Mean B.t.i spray dried technical powder potency = 7474 ITU/mg. Mean


B.s spray dried technical powder potency = 3030 Bs. ITU/mg (All assays are average


of initial and 2 month 5° C. stored samples)
















Actual





Ratio


Potency


of B.t.i


expressed

Actual
Actual Potency


to B.s
Theoretical
Actual
as % of
Theoretical
B.s
expressed as %


on
B.t.i
B.t.i
theoretical
B.s
Potency
of theoretical


solids
Potency*
Potency
potency in
Potency+
(Bs.
potency in


basis
(ITU/mg)
(ITU/mg)
Column 2.
(Bs. ITU/mg)
ITU/mg)
Column 5
















1:1
3737
5174
138%
1515
1642
108%


3:1
5606
6122
109%
758
1088
144%


1:3
1869
4769
255%
2273
2655
117%


2:1
4983
5499
110%
1009
1479
147%


1:2
2489
3738
150%
2020
2503
124%


Mean
3737
5060

1515
1873









Biopotency data presented in Table 2 reveal an interesting but very synergistic increase in actual potency of both B.t.i and B.s over theoretical potencies, which are, based on actual potencies of 100% of either B.t.i or B.s spray dried technical powders. The best combination for increased activity on both Aedes and Culex appeared to be when B.t.i and B.s slurry concentrates are combined at 1 part of B.t.i to 2 parts of B.s on solids basis. For enhancing the B.s potency, the best combination was when two parts of B.t.i solids was combined with 1 part of B.s solids. In this combination, B.s potency showed 47% increase over theoretical potency. By combining the B.t.i and B.s slurry concentrates prior to spray drying, B.t.i potency on average showed an increase of 35% over mean theoretical mean potency while B.s potency showed an increase of 24% over theoretical mean potency. There appeared to be significant advantage in combining the slurry concentrates prior to spray drying and further formulating these powders as granules, wettable powders, water dispersible granules, or pellet formulations. The best possible explanation for these enhanced potency values appeared to be due to the fact that each spray dried particle carries both B.t.i and B.s toxins and spores. Thus, these novel formulation approaches are likely to not only result in broad-spectrum activity but also will minimize the potential for build up of resistance. In other words, resistance management can also be achieved yet by another novel formulation approach.
Example 2
Example of Changes 1N Susceptibility of Bacillus sphaericus Susceptible Culex pipiens quinquefasciatus Colonies Upon Selection with Bacillus sphaericus and two B.t.i: B.s. Technical Dry Formulations Derived by Combining Both Fermentation Slurry Formulations and Spray Drying
This experiment was conducted using Culex pipiens quinquefasciatus colonies established from susceptible field populations collected in Thailand. The field mosquitoes were collected from an area known to have a high potential for development of resistance to Bacillus sphaericus. The colonies were subjected to selection each generation at the LC80 level with the following materials.

    
    
        1. Untreated Control—(no selection)
        2. B.s technical powder
        3. 1:1 ITU ratio B.t.i: B.s Technical Spray Dried Formulation (VBC-60033)
        4. 4:1 ITU ratio B.t.i: B.s Technical Spray Dried Formulation (VBC-60034)
    
    


The colonies were assayed for susceptibility to Bacillus sphaericus prior to the selections and each fifth generation for twenty-five generations. The colonies were bioassayed by placing 20 late third or early fourth instar larvae in a 116 ml waxed paper cup containing 100 ml distilled water. One drop of larval diet (2 g of ground up rabbit pellets in 20 ml distilled water) was added per cup. The cups were treated with a range of concentrations of each formulation. Five to seven different concentrations were utilized in each bioassay to yield mortalities. Each concentration was replicated four to five times in each test. The treated larvae were held at 82-85° F. To determine LC50 values, the number of dead larvae was counted at regular intervals from the time of treatment with the test larvicide. Once all larvae died, the concentration wherein 50% had been killed could be determined.
The following table shows the changes in LC90 values in each of the selected colonies over 25 generations. Susceptibility of B.s selected colonies decreased, while colonies selected with VBC-60033 and VBC-60034 (Formulations derived by combining fermentation slurry concentrates and spray drying) maintained stable susceptibilities relative to the unselected colony.







TABLE 3







LC90 VALUES OF EACH FIFTH GENERATION AFTER SELECTION














F-0
F-5
F-10
F-15
F-20
F-25



LC90
LC90
LC90
LC90
LC90
LC90


TREATMENTS
ppm
ppm
ppm
ppm
ppm
ppm
















Unselected
0.013
0.022
0.03
0.022
0.061
0.034


B.s @ LC80
0.013
0.075
0.149
2.332
1.256
1.551


VBC - 60033 @ LC80
0.013
0.074
0.022
0.049
0.048
0.047


VBC - 60034 @ LC80
0.013
0.007
0.024
0.03
0.021
0.022









Example 3
Example of Changes 1N Susceptibility of Bacillus sphaericus Resistant Culex pipiens quinquefasciatus Colonies Upon Selection with Two B.t.i:B.s Technical Dry Formulations Derived by Combining Both B.T. and B.S Fermentation Slurry Formulations and Spray Drying
A resistant colony is one that demonstrates a significant decrease in susceptibility to a particular pesticide over that expected for wild type insects. Generally, a five-fold or more decrease in susceptibility indicates resistance. Rodcharoen et al., Journal of Economic Entomology, Vol. 87, No. 5, 1994, pp. 1133-1140 explores the concept of resistance more fully.
A susceptible colony is one that is effectively killed by a particular pesticide. For example, in Culex quinquefasciatus, if a particular pesticide, Bacillus sphaericus, has a measured LC50 value of less than 0.1 ppm, the colony is characterized as susceptible to that pesticide.
The change in susceptibility of Culex quinquefasciatus laboratory colonies known to be B.s. resistant, in response to selections with VBC-60033 and VBC-60034 (Technical dry formulations derived by combining fermentation slurry concentrates of both B.t.i and B.s and spray drying) were determined in the laboratory in the following manner. Selection refers to treatment at less than LC100 level.
A colony of Culex quinquefasciatus resistant to B.s. was started from highly resistant larvae collected from a water body in Wat Pikul, Bang Yai District, Nonthaburi Province, Thailand. Field collected larvae were subjected to selection at LC80 every generation for twenty generations. At the 20th generation, the colony showed a 47,000 and 1,153,846 fold resistance at LC50 and LC90 levels respectively. This colony was used for subsequent tests.
The experiment was conducted using sub-colonies established from the highly B.s. resistant colonies. The colonies were subjected to selection each generation at the LC80 level with the following materials.

    
    
        1. 1:1 ITU ratio B.t.i:B.s or VBC-60033 (Technical dry formulation derived by combining B.t.i and B.s fermentation slurry formulations and spray drying)
        2. 4:1 ITU ratio B.t.i:B.s or VBC60034 (Technical dry formulation derived by combining B.t.i and Bsph fermentation slurry formulations and spray drying)
    
    


The colonies were assayed for susceptibility to B.s prior to the selections and at each fifth generation for twenty-five generations of selection. Bioassay methods described in Example 2 were also used in this experiment.
The following table shows the changes in LC90 values in each of the selected colonies over 25 generations. LC90 of the 4:1 B.t.i:B.s or VBC-60034 (Technical dry formulation derived by combining B.t.i and B.s fermentation slurry formulations and spray drying) selected colony was 157 times lower than the unselected colony after 25 generations of selection.







TABLE 4







LC90 VALUES OF EACH FIFTH GENERATION FOLLOWING


SELECTIONS














F-0
F-5
F-10
F-15
F-20
F-25


TREAT-
LC90
LC90
LC90
LC90
LC90
LC90


MENTS
ppm
ppm
ppm
ppm
ppm
ppm
















Unselected
267231
56565
19851
20451
17021
19331


VBC -
15000
20860
10989
1334
1304
353


60033 @


LC80


VBC -
15000
29642
531
206
107
123


60034 @


LC80









Example 4
Example of Effectiveness OF B.t.i:B.s. (VBC-60033 and VBC-60034) for Control OF B.s Susceptible Mosquito Larvae.
Tests were carried out in controlled small plots infested with mosquito larvae. Plastic wading pools infested with larvae via natural oviposition were used. Species present included Culex pipiens and Culiseta incidens. Plots were filled with water, then enriched with  ̃100 g of rabbit chow each. Yard waste was added to provide substrate, and the plots were left to infest for two weeks. Plots were infested with roughly equivalent numbers of L1-L3 larvae at the initiation of the test.
The following treatments were made:

    
    
        1) UTC
        2) VectoBac® CG (200 ITU B.t.i)
        3) VectoLex® CG (50 Bs. ITU)
        4) VBC-60035 (4:1 ITU B.t.i:B.s granules using VBC-60034)
        5) VBC-60037 (1:1 ITU B.t.i.B.s granules using VBC-60033)
    
    


Rates: All materials were applied at 2.55 kg/ha (200 gm/plot)
Test Design:

    
    
        Replication—5 replicates
        Randomization—Modified RCB based on initial populations.
        Total plots used=25
        Plot size=0.785 sq meters
    
    


Sampling: All plots were sampled by dipping prior to treating the plots. Post treatment samples were taken on days 3, 7, 14, 21 and 28 following treatment. Four dips were taken per plot. Time was allowed between dips for larvae to re-surface in each plot. This was accomplished by taking the first dip from all the plots, then returning to the first plot for the second dip, taking the second dip from all plots and continuing in a like fashion for all four dips. Total number of L1-L2, L3-L4 and pupae collected in each plot were counted and recorded.
Results
Populations of L3-L4 larvae were initially reduced by all treatments. Reductions achieved by ABG-6185, VBC-60035 and VBC-60037 were similar throughout the study, and were significantly lower than the UTC on days 3 to 21 (p=0.05). Reductions achieved by ABG-6138S were statistically lower than UTC on days 3-14, but higher than the other three treatments on days 7 to 28 (p=0.05). L3-L4 Population trends are shown in the following table. Both VBC-60035 and VBC-60037 formulations provided high levels of initial and residual control.







TABLE 5







POPULATION TRENDS IN PLOTS


LARGE LARVAE/4 DIPS














DAY
DAY
DAY
DAY
DAY
DAY



0
3
7
14
21
28
















UTC
312
735
345
376
326
278


ABG - 6138S
293
43
76
118
182
249


ABG - 6185
317
6
1
1
27
103


VBC - 60035
262
1
0
7
52
93


VBC - 60037
254
10
1
28
11
56









Example 5
Example of Effectiveness of B.t.i: B.s Formulations for Control of Bacillus sphaericus Tolerant Mosquito Larvae
Efficacy studies of Vectobac® CG (B.t.i @200 ITU/mg) and VBC-60035 at an application rate of 1.25 lb/acre were conducted in small field plots against colony reared Ochlerotatus taeniorhynchus 2nd/3rd instar larvae. This mosquito species is tolerant to B.s exposure. The formulations were added to the plots following flooding and larval introduction.
Approximately 1000 2nd/3rd instar Oc. taeniorhynchus larvae were reared in the laboratory and added to each plot at the time of flooding. The water surface area in the plots measured approximately 8 ft2 with a depth of approximately 6′′ and a salinity of 3-5 ppt (Floore et al. 1998). Emergent grasses were present in all the plots. The water temperature (not yet available) was recorded using an Onset HOBO ProSeries Temperature data recorder and the salinity measured with a Salinity Refractometer (SR-1). The pH was recorded with Oakton pH Tester 2 (not yet available). The study was initiated Jun. 19, 2003. Rainfall during the study was 3.5′′.
The randomized study consisted of 4 replications of the treatments of the formulations and untreated control plots. The application rates were based on the plot surface area and weighed on a Mettler electronic balance and applied by hand. Larvae were fed daily approximately 100 ml of larval food (3 parts liver powder: 2 parts brewer's yeast).
The following treatments were made:

    
    
        1) UTC
        2) VectoBac® (B.t.i. @ 200 ITU/mg)
        3) VBC-60035 (4:1 of B.t.i:B.s with 200 ITU/mg+50 Bs·ITU/mg on Corncob granules)
    
    


Rates: Materials were applied at 1.25 lb/a
Sampling: All plots were sampled by dipping the plots. Post treatment samples were taken at 24 and 48 hours following treatment. Eight dips were taken per plot. Numbers of larvae collected by dipping were counted and recorded.
Results
Mean larval numbers sampled in plots treated with VBC-60035 formulation were lower than in the plots treated with B.t.i and significantly lower than in the UTC at both 24 and 48 hours following treatment. Corrected percent reductions in the plots treated with the VBC-60035 were 71% and 92% at 24 and 48 hours respectively. Like values in the B.t.i treated plot were 65% and 81%.







TABLE 6







MEAN NUMBER OF LARVAE SAMPLED IN PLOTS AT 24


AND 48 HOURS AFTER TREATMENTS











TREATMENT
24 HRS
48 HRS







UTC
12.75 c
41.25 c



VectoBac ®
 4.5 ab
 8 ab



CG



VBC - 60035
 3.75 a
 3.25 a










The present invention is illustrated by way of the foregoing description and examples. The foregoing description is intended as a non-limiting illustration, since many variations will become apparent to those skilled in the art in view thereof. It is intended that all such variations within the scope and spirit of the appended claims be embraced thereby.
Changes can be made in the composition, operation and arrangement of the method of the present invention described herein without departing from the concept and scope of the invention as defined in the following claims:

## Claims

1. A method of preparing a composition comprising a combination of a strain of Bacillus thuringiensis subspecies israelensis that produces toxins and a strain of Bacillus sphaericus that produces toxins comprising the steps of separately culturing the strains, concentrating each strain, combining the concentrated strains to form a slurry mixture and spray drying the slurry mixture to yield individual particles which contain toxins from Bacillus thuringiensis subspecies israelensis and toxins from Bacillus sphaericus that have larvicidal and resistance reduction activity.

2. A composition comprising a combination of a strain of Bacillus thuringiensis subspecies israelensis that produces toxins and a strain of Bacillus sphaericus that produces toxins, where the strains were separately cultured, concentrated individually, where the concentrated strains were combined to form a slurry mixture, where the slurry mixture was spray dried, and where the spray dried slurry mixture yielded individual particles which contained toxins from Bacillus thuringiensis subspecies israelensis and Bacillus sphaericus that have larvicidal and resistance reduction activity.

3. A method of controlling Dipteran larvae comprising introducing a larvicidally-effective amount of a product of claim 2 into an environment containing Dipteran larvae.

4. The method of claim 1, wherein the particles are used in various mosquito larvicide product forms selected from the group consisting of granules, powders, wettable granules or dry flowables, tablets, briquets, pellets and liquid suspensions.

5. The method of claim 1, wherein the particles are mixed with at least one member selected from the group consisting of other pesticides, adjuvants, oils, pre-formed granules and extruded pellets.

6. The composition of claim 2, wherein said composition has from about 1:4 to about 4:1 weight ratio of Bacillus thuringiensis subspecies israelensis to Bacillus sphaericus.

7. The composition of claim 2, wherein said composition has about 4:1 weight ratio of Bacillus thuringiensis subspecies israelensis to Bacillus sphaericus.

8. The composition of claim 2, wherein said composition has about 2:1 weight ratio of Bacillus thuringiensis subspecies israelensis to Bacillus sphaericus.

9. The composition of claim 2, wherein said composition has from about 1:10 to about 10:1 weight ratio of Bacillus thuringiensis subspecies israelensis to Bacillus sphaericus.

10. The composition of claim 2, wherein said composition has from about 1:2 to about 4:1 weight ratio of Bacillus thuringiensis subspecies israelensis to Bacillus sphaericus.