Title of Invention

A METHOD OF PREPARING A COMPOSITION COMPRISING A COMBINATION OF A STRAIN OF BACILLUS THURINGIENSIS SUBSPECIES ISRAELENSIS AND A STRAIN OF BACILLUS SPHAERICUS, A COMPOSITION THEREOF AND A METHOD OF USE OF THE COMPOSITION.

Abstract The present invention discloses a method of preparing a composition comprising a combination of a strain of Bacillus thuringiensis subspecies israelensis and a strain of Bacillus sphaericus comprising the steps of fermenting the strains separately, concentrating each strain to the desired solids concentration or activity, combining the concentrated strains to form a slurry mixture and spray drying the slurry mixture to yield individual particles which contain toxins from both Bacillus thuringiensis subspecies israelensis and Bacillus sphaericus.
Full Text "A METHOD OF PREPARING A COMPOSITION COMPRISING A
COMBINATION OF A STRAIN OF BACILLUS THURINGIENSIS SUBSPECIES
ISRAELENSIS AND A STRAIN OF BACILLUS SPHAERICUS, A COMPOSITION THEREOF
AND A METHOD OF USE OF THE COMPOSITION"
Field of the Invention
The invention is directed to a method for controlling Dipteran larvae
or a method for inhibiting laryicidal_resisiance n Diptera by introducing a
larvicidally-effective amount of a combination of a strain of Bacillus
thuringiensis subspecies israeiensis 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. Patent No. 6,077,521. However, more recent
emphasis has been placed on the use of biopesticides. For example,
controlled-release formulations of at leasf one biological pesticidal
ingredient are disclosed in U.S. Patent No. 4,865,842; control of
mosquito larvae with a spore-forming Bacillus ONR-60A is disclosed in
U.S. Patent No. 4,166,112; novel Bacillus thuringiensis isolates with
activity against dipteran insect pests are disclosed in U.S. Patent Nos.
5,275,315 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. Patent No. 5,912,162 and a recombinantly derived
biopesticide active against Diptera including cyanobacteria transformed
with a plasmid containing a B. thuringiensis subsp. israeiensis
dipteracidal protein translationally fused to a strong, highly active native
cyanobacteria's regulatory gene sequence is disclosed in U.S. Patent
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
a/., 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 a/., 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.
Brief Summary of the Invention
The invention is directed to a composition comprising: a combination
of a strain of Bacillus thuringiensis subspecies israelensis and a strain of
Bacillus sphaericus. The strain of Bacillus thuringiensis subspecies
israelensis may be non-genetically modified, or the strain of Bacillus
sphaericus may be non-genetically modified, although a 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 may have from about 1:10 to about 10:1 weight ratio
of Bacillus thuringiensis subspecies israelensis to Bacillus sphaericus;
preferably from about 1:3 to about 3:1 weight ratio of Bacillus
thuringiensis subspecies israelensis to Bacillus sphaericus; more
preferably from about 1:2 to about2:1_weight ratio of Bacillus
thuringiensis subspecies israelensis to Bacillus sphaericus; and most
preferably a 1:1 ratio of Bacillus thuringiensis subspecies israelensis to
Bacillus sphaericus.
Additional components such as surface active agents, inert carriers,
preservatives, humectants, feeding stimulants, attractants, encapsulating
agents, binders, emulsifiers, dyes, U.V. protectants, buffers, drift control
agents, spray deposition aids, free-flow agents or combinations thereof
may also be utilized in conjunction with the combination in a larvicidal
composition.
The invention is also directed to a method of controlling Dipteran
larvae comprising the step of introducing a larviddally-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. 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 combination of a strain of Bacillus
thuringiensis subspecies israelensis and a strain of Bacillus sphaericus
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 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 subsp. 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.ti.) has been used
successfully in mosquito and blackfly control programs for many years.
B.ti. is utilized in clean to moderately clean organic breeding habitats,
and is most effective on Aedes species. A commercial formulation of
B.ti. is known by the trademark VECTOBAC, available from Valent
BioSciences Corp. Specific commerical formulations available from the
same supplier are VECTOBAC G, VECTOBAC CG, VECTOBAC 12AS
and VECTOBAC WDG. 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 chances
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. B.s. 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, SPHERIMOS AS and VECTOLEX
CG. Moreover, this strain was found to perform well in controlling
mosquitoes breeding in various habitats, especially ones with polluted
water.
B. s. is most effective on Culex species. The activity of B.s. is due to
a binary toxin, and repeated use can Jead 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.
B.t.i. and B.s. may be combined by mixing the powdered forms of
each of the individual strains, or by mixing the slurries of the fermentation
broths of each strain, in the desired ratio, as illustrated by Examples 1-6
which follow. The ratio of B.t.i. to B.s. may be from about 10:1 to about
1:10; preferably from about 3:1 to about 1:3, more preferably from about
2:1 to about 1:2 and most preferably about 1:1.
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
carboxyiate, 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 suiphonate; 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
suiphonate 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-iinked 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.ti. 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 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 a surfactant. These compositions will be administered at the
rate of about 50 mg (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 pesticidal 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
longipaipis, Lutzomyia shannoni, Lycoriella mail, 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 mod, 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
fiouendana, 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., Cyciocephala borealis, Cyclocephala
immaculata, Macrodactylus subspinosus, Popillia japonica, Rhizotrogus
majalis, Alphitobius diapehnus, 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 combination larvicide of the present invention was formulated
as a mix of two commercially available strains: VECTOBAC CG, a
commercial granular formulation of B.t.i. with a label potency of 200
ITU/mg, and VECTOLEX CG, a commercial granular formulation of B.s.
with a label potency of 50 B.s. ITU/mg. Typically, the spray-dried technical
concentrate of each strain is first incorporated into a known amount of
vegetable oil binder. The amount of vegetable oil binder in the formula will
depend upon the amount of B.t.i. or B.s. spray technical concentrate in the
formula. The typical range may vary between 1% to 15% wt/wt.
depending upon the amount of B.t.i. or B.s. spray dried technical
concentrate, and the type, size and absorptive property of granular carrier
utilized in the formula. In this example, corn cob granules of the size
classification 10/14 Mesh were used. However, other size ranges such as
5/8 Mesh, 10/20 Mesh, 10/40 Mesh are also suitable. The slurry mixture
was sprayed onto the granular carrier while mixing in a suitable blender
and further blended until homogenous product was obtained.
Example 2 ....
A B.t.i. and B.s. granular formulation for enhanced and broad-
spectrum activity was prepared. Pre-formulated granular products of B.t.i.
and B.s., (VECTOBAC CG and VECTOLEX CG), were utilized in
developing the combination formulation. The resultant combination
granular product was targeted to contain B.t.i. at 100 ITU/mg and B.s. at
25 B.s. ITU/mg. The carrier utilized was 10/14 Mesh corn cob.
To prepare the formulation, 5 kg each of VECTOLEX CG and
VECTOBAC CG were charged into a blender and blended. The product
mix was then recovered.
Representative samples were taken for bioassay. Table 1 shows
the amount of the raw materials which were used to form the product
combination
* Commercial B.t.i. granular formulation with a label potency of 200
ITU/mg
** Commercial B.s. granular formulation with a label potency of 50
B.s. ITU/mg
Bioassay results for the B.t.i. and B.s. combination samples when
tested against fourth instar Aedes aegypti and third instar Culex
quinquefaciatus are shown in Table 2 below. ITU stands for international
toxicity units, which are based on a reference material of known specific
B.t.i. potency.
In a preferred formulation, the one to one weight ratio of B.t.i. to B.s.
has a potency of 100 ITU/mg for B.t.i. and 25 ITU/mg for B.s., which is
equivalent to a 4:1 ratio on a potency basis.
Example 3
The larvicidal combination product may also be formulated by
combining the required levels of both B.t.i. and B.s. technical powders in
the same binder liquid, and then impregnating or spraying onto the
granular carrier such as corn cob or any other suitable carriers.
To prepare the formulation, both spray dried technical concentrates
can be slurried in vegetable oil binder liquid and sprayed onto granular
carrier in a suitable blender and mixed until a homogenous product is
obtained. The theoretical components of the combination formulation
containing B.t.i. at 100 ITU/mg and B.s. at 25 B.s... ITU/mg are provided in
Table 3 below.
Example 4
The combined formulation may also be formed by pre-mixing
fermentation beers or slurry concentrates of Bti and Bs 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
formulation, the slurry concentrate 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 in Examples 2 and 3 or as wettable powders, water dispersible
granules, and aqueous or non-aqueous concentrates. These combined
powder concentrates may also be utilized in pellet and/or 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 Bti slurry concentrate was first
preserved with 0.12% wt/wt of potassium sorbate and 0.06% wt/wt of
methyl paraben. % solids in the preserved Bti slurry concentrate were
11.3% wt/wt. Similarly, a Bs slurry concentrate with 0.12% wt/wt of
potassium sorbate and 0.06% wt/wt of methyl paraben was preserved.
Per cent solids in the preserved Bs slurry concentrate had a mean %
solids of 10.1% wt/wt. Slurry mixtures prepared and their rations on solids
basis are given in Table 4
The compositions as shown in Table 4 were combined and spray
dried utilizing a Niro spray. 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 5.
Table 5 Potency values of Bti + Bs. spray dried technical powders as
affected by their ratios on solids basis. Mean Bti spray dried technical
powder potency = 7474 ITU/mg. MeanBs spray dried technical powder
potency = 3030 Bs. ITU/mg (All assays are average of initial and 2 month
5 °C stored samples )
Biopotency data presented in Table 5 reveal an interesting but very
synergistic increase in actual potency of both Bti and Bs over theoretical
potencies, which are, based on actual potencies of 100% of either Bti or
Bs spray dried technical powders. The best combination for increased
activity on both Aedes and Culex appeared to be when Bti and Bs slurry
concentrates are combined at 1 part of Bti to 2 parts of Bs on solids basis.
For enhancing the Bs potency, the best combination was when two parts
of Bti solids was combined with 1 part of Bs solids. In this combination, Bs
potency showed 47% increase over theoretical potency. By combining the
Bti and Bs slurry concentrates prior to spray drying, Bti potency on
average showed an increase of 35% over mean theoretical mean potency
while Bs 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 Bti and Bs toxins and spores. In other words, these
are not physical mixtures as revealed in Example 2 or 3. 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 5
The combination larvicidal formulation may also be prepared in
liquid form by adding, at the desired level, both VECTOBAC WDG (3000
ITU/mg) and VECTOLEX WDG (650 ITU/mg) to water in the spray tank
and mixing until a homogeneous dispersion is obtained. The suspension
so formed may be delivered to the target habitat by various application
methods. A liquid formulation is ideal for spray operations.
Example 6
A liquid formulation may be prepared from liquid formulations of
each individual strain. VECTOBAC 12AS and SPHERIMOS AS (aqueous
suspension product forms marketed by Valent BioSciences Corp.) may
also be mixed in water in the spray tank and applied by various spray
application equipment. Formulating B.t.i. and B.s. as one aqueous
formulation with preservatives, stabilizers, surfactants, dispersants,
diluents is yet another preferred method for delivering both toxins to the
mosquito habitats.
Example 7
The change in susceptibility of Culex quinquefasciatus laboratory
colonies known to be B. s. resistant, in response to selection with a
mixture of B.t.i. and B.s. was 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 susceptible larvae collected from a waste water lagoon of a dairy in
the western United States. Field collected larvae were subjected to
selection at LC90 every generation for forty generations. At the 40th
generation, the colony shwed a 54.4 and 14.2 fold resistance at LC50 and
LC90 levels respectively. This colony was used for the subsequent tests.
A resistant colony is one which 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 explore the concept of resistance more fully.
A susceptible colony is one which is effectively killed by a particular
pesticide. For example, in Culex quinquifasciatus, 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.
Stock suspensions were prepared by mixing 0.2 g of B.t.i. or B.s. in
20 ml of distilled water to make a 1% suspension; mixtures were made by
combining the stock suspensions in the desired ratio. Suspensions were
subsequently diluted as required for test treatments.
The parental colony and the tenth generation 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 either larvicide alone,
as well as the mixture. Five to seven different concentrations in the range
of 0.0001-0.1 ppm 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 were 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.
Colonies treated individually at the LC80 level with B.s. (Bacillus
sphaericus strain 2362, ABG-6184, VECTOLEX, available from Valent
BioSciences Corp.) or B.t.i. (Bacillus thuringiensis subsp. israelensis
(VECTOBAC available from Valent BioSciences Corp.) were compared to
a colony treated with a 1:2 weight ratio of B.s. (VECTOLEX) to B.t.i.
(VECTOBAC) for five generations at the LC50 level, followed by treatment
with a 1:1 weight ratio of B.s. (VECTOLEX) to B.t.i. (VECTOBAC) for five
more generations at the LCso level.
Following the first five generations of these treatments, lower mean
B.s. LC50 values were obtained for the colonies subjected to selection by
the 1:2 combination than for either colony selected by the individual
components, indicating an increased susceptibility and consequently
decreased resistance as illustrated by Table 6. Mean B.s. LC50 values
continued to decline under selection with the 1:1 combination during the
subsequent five generations.
Table 6
This table shows data from the original selection study through F25. One
key change in the selection was that the B.t.i., selected colony was
switched to B.s. selection after F10 to assess stability of susceptibility in
the colony.
Table 6 Change in B.s. Susceptibility in ppm of a B.s. Resistant Colony in
Response to selection with B.t.i., B.s. and mixtures
Example 8
The bioassay procedure described in Example 7 was utilized in
order to assess the susceptibility of B.s. resistant and susceptible Culex
quinquefasciatus colonies to B.t.i., B.s. a 2:1 mixture of B.t.i. to B.s.
Mean LC50 values were determined for each treatment. A lower
mean LC50 value indicates that the particular treatment can be used
effectively at a lower concentration, which indicates that the organisms are
more susceptible to the treatment.
The results are provided in Table 7 below. As expected, the B.s.
treatment gave the highest mean LC50 for the B.s. resistant colony.
However, the 2:1 ratio mixture shows an improved result over the result for
B.t.i. and B.s. alone, both in susceptible and resistant colonies, indicating a
higher susceptibility and possible synergism when the components are
combined.
Example 9
The efficacy of a 2:1 mixture of B.t.i. to B.s. on Culex
quinquefasciatus colonies determined to be susceptible to B.s. was tested
as follows.
A mixed susceptible colony of Culex quinquefasciatus was
established from a combination of egg rafts collected from a site in the
western United States. The collected egg rafts were individually
transferred to 230 ml waxed paper cups each holding 200 ml tap water
and 0.5 g rabbit pellets as larval diet. The larvae were hatched out. Then
pupae were removed into cups with water and placed in screen cages,
where the adults emerged. The adults were provided with 10% sucrose
solution, and on day five after emergence, females were allowed to feed
on restrained chicks. On day 5 subsequent to this blood feeding,
oviposition cups were introduced into the cages to collect eggs. To
maintain the colony in the laboratory, 4-5 egg rafts were placed in an
enamel pan containing 2 liters of tap water and 2 g. of rabbit pellets as
larval diet.
The sample preparation conditions, bioassay method, and LC50
determination were the same as those described in Example 7. The
results are provided in Table 8 below. The results show that over time, the
test colony which was B.s. susceptible, becomes less susceptible by the
fifth generation in response to treatment with B.s. alone, since the LC50
value increases. In contrast, the 2:1 mixture does not show the same
tendency to the same extent. This fact is indicated by the data which,
while showing an increase in LC50 over the parental strain, also show less
of an increase than for the treatment with B.s. alone. Therefore, use of the
2:1 B.t.i. and B.s. mix slowed the resistance over time.
Table 8
Table 8 also shows data from the original selection study through F20.
One key change in the selection was that the mixture-selected colony was
switched from a 2:1 B.t.i./B.s. (VectoBac WDGA/ectoLex WDG) to a 1:1
mixture selection after F5.
Table 8 Change in susceptibility of a B.s. Susceptible Colony in Response
to B.t.i and B.s. mixtures.
Example 10
The following experiment was performed to demonstrate the utility
of the mixture for controlling mosquitos of varying species. In this
example, the effectiveness of the mixture and each individual larvicide
were determined on a mixed population of Culex quinquefasciatus and
Aedes aegypti.
Twenty plastic rearing tubs were placed outside in a midwestern
United States location. The tubs were filled with deionized water and
enriched with 2.4 g of ground guinea pig chow. The tubs were then
infested with 100 third instar Culex quinquefasciatus and 100 third instar
Aedes aegypti. One hour after the infestation, and just prior to treatment,
tubs were sampled and the number of larvae in a test sample from each
tub were counted, to obtain a control value.
Test larvicides included VECTOLEX CG (50 B.s. ITU/mg on corn
cob granules), VECTOBAC CG (200 ITU/mg on corn cob granules) and a
1:1 mixture of VECTOLEX CG and VECTOBAC CG, as described in
Example 2. Each tub was treated with an appropriate amount of one of
the three test larvicides, equivalent to a single treatment rate of 5, 10 or 20
lbs/acre.
On the fifth and twelfth day after treatment, 100 third instar Culex
quinquefasciatus and 100 third instar Aedes aegypti were added to each
tub.
Larvae were sampled from the tubs on the second day, the seventh
day and the fourteenth day after treatment to determine how many were
still alive. The numbers were obtained and compared to the number of
larvae alive prior to treatment. 100% reduction indicates that all larvae
were killed. A positive number for percent reduction indicates that the
larvicide does kill. The results are provided in Table 9 below, indicating
that the 1:1 mixture of B.t.i. to B. s. can control Culex quinquefasciatus and
Aedes aegypti larvae, even over a period of several days, at each
application rate tested.
Example 11
Another experiment was performed to demonstrate the utility of the
mixture for killing mosquitos of varying species. In this example, the
effectiveness of the mixture and each individual larvicide were determined
on Culex tarsalis.
The test was performed on mosquitoes in a waste water pond of a
waste water treatment facility in the western United States. The pond was
extremely polluted, and standard tests showed the presence of Culex
tarsalis. Tall reeds covered 80% of the water surface. The edge of the
pond was divided into six plots ranging from 0.1 to 0.2 acres in size, for the
purposes of the test.
Just prior to treatment, each test plot was sampled and the number
of larvae in a test sample from each plot were counted, to obtain a control
value.
Test larvicides were the formulations described in Example 10, and
then each plot was treated with an appropriate amount of one of the three
test larvicides, equivalent to a single treatment rate of 5 or 10 lbs/acre.
Larvae were sampled from the test plots on the second day, the
seventh day and the fourteenth day after treatment, to determine how
many were still alive. The numbers were obtained and compared to the
number of larvae alive prior to treatment. 100% reduction indicates that all
larvae were killed. A positive number for percent reduction indicates that
the larvicide does kill. The results are provided in Table 10 below,
indicating that the 1:1 mixture of B.t.i. to B. s. can control Culex tarsalis
larvae.
Example 12
Another experiment was performed to demonstrate the utility of the
mixture for killing mosquitos of varying species. In this example, the
effectiveness of the mixture and each individual larvicide were determined
on Culexpipiens and Culiseta incidens outdoors in a roadside ditch.
Two sections of a roadside ditch separated by a driveway in the
western United States were the site of the study. The ditches were
vegetated with grasses and aquatic plants and received seepage runoff
from.a septic system as well as rainfall. A drainage swale with similar
hydrology and vegetation was selected as the untreated control. Each site
was populated with Culex pipiens and Culiseta incidens at the time of
treatment.
Just prior to treatment, each test plot was sampled and the number
of larvae in a test sample from each plot were counted, to obtain a control
value.
Test larvicides were of the formulations described in described in
Example 10, and then two of the test sites were treated with an
appropriate amount of one of the two test larvicides, equivalent to a single
treatment rate of 20 lbs/acre. The remaining test site was untreated, to
serve as the control.
Larvae were sampled from the test plots on the fourth day and the
seventh day after treatment. The numbers were obtained and compared
to the number of larvae prior to treatment. 100% reduction indicates that
all larvae were killed. A positive number for percent reduction indicates
that the larvicide does kill. The results are provided in Table 11 below,
indicating that the 1:1 mixture of B.t.i. to B. s. can control Culex pipiens
and Culiseta incidens larvae.
Example 13
The efficacy of a 2:1 mixture B.s. (VECTOLEX) to B.t.i.
(VECTOBAC) was determined in the field in the following manner.
The test field was a rice field in the western United States,
measuring 156 acres. Levees were used a buffer zones between test
plots. At the time of the treatment, Anopheles freeborni larvae were
present at a density of 0.5-3.0 per dip, according to a standard dip test.
Each individual larvicide, and the larvicidal combination were applied at a
rate of 12 lbs/acre by airplane, which applied the granules at a speed of 85
miles per hour and with a swath width of 60 feet.
Larval counts were performed on days 2, 6 and 15 post-treatment,
and measured against the larval count of an untreated control test plot.
The results are shown in Table 12 below, illustrating that a 2:1 mix
effectively kills larvae, as a positive number for percent control of larvae
indicates that the larvicide does kill.
Example 14
The following studies demonstrate the susceptibility of various B.s.
susceptible and non-susceptible mosquitoes to mixtures. They support
the claims of mixtures as a method of controlling mosquitoes, and
controlling B.s. resistant mosquitoes. Three of the studies also support the
method of mixing technical powders prior to formulation of granules.
Efficacy of a 1:1 mixture of B.t.i. and B.s. Formulations for Control of B.s.
Resistant Culex quinquefasciatus Field Populations Compared to Each
Formulation Separately.
Materials and Methods
A highly resistant population of Culex quinquefasciatus was
identified in Wat Pikul, Bang Yai District, Nonthaburi Province, Thailand.
This population was treated with various doses of VectoBac WDG (3000
Bti ITU) and VectoLex WDG (650 Bs ITU) and a 1:1 mixture of the two
between January and September of 2001. Following each treatment,
population changes were assessed overtime by dipping, and percent
control of late instar larvae and pupae was calculated for post treatment
days.
Results
Doses of VectoLex WDG as high as 200 mg/m2 resulted in little or
no control of this population. VectoBac WDG was found to provide control
at doses as low as 20 mg/m2. A 1:1 mix of the two products was found to
be more effective than either product alone at a dose of 20mg/m2.
Table 13
Percent reduction of a B.s. resistant Culex quinquefasciatus field
population after treatment with B.s., B.t.i., and a mixture of B.t.i and B.s.
Efficacy of Two B.t.i./B.s. Combination Formulations For Control of Culex
pipiens and Culiseta incidens Compared to Standard B.s. Formulation in
Artificial Plots
Materials and Methods
Twenty eight artificial test plots were set up using wading pools in
the parking area of Multonomah County Mosquito Control District's facility
at 5235 N. Columbia Blvd, Portland, OR on July 27, 2001. The pools were
set out in four rows with seven pools per row. Each row was designated
as a test series. The pools were filled to a depth of approximately 8" with
tap water from the District's water supply. This depth was. maintained
throughout the study. Each pool had an approximate surface area of .785
M2. Each pool was enriched with straw and rabbit chow (100gr.) to
provide habitat and food for the mosquito larvae.
The pools were left to season the hay/rabbit chow mix, and to allow
natural populations of the local mosquitoes to become established.
Artificial stocking of the test pools was done after the local mosquito
populations failed to produce adequate populations in the pools for the
study. After this initial stocking, populations of Culex pipiens and Culiseta
incidens maintained themselves by natural re-infestation.
Three formulations, designated ABG6185, VBC60015 and
VBC60019 were compared in the study. ABG-6185 consisted of B.s.
technical powder formulated onto corncob and had a potency of 50 B.s.
ITU. VBC-60015 and VBC-60019 were combinations of B.t.i. and B.s.
technical powders formulated onto corncob and had theoretical potencies
of 200B.t.i./50B.s. ITU and 100B.U./50B.S. ITU respectively.
Formulations were tested at application rates of 2.5kg/ha and 5kg/ha
compared to untreated controls. Four replications were done for each
application rate and UTC in a random pattern throughout the test series.
Sampling was done by taking 5 dips per pool using standard
mosquito dippers and concentrating the larval catch with fine mesh
strainers. Composite samples were preserved in alcohol for counting and
species identification. Larval counts were recorded as L1-L2, L3-L4 and
pupae.
Pretreatment counts, and the test product applications were done
on August 20, 2001. The initial post- treatment larval counts were done on
August 23, 2001 approximately 64 hours after the treatment. Follow-up
counts were done on August 27, August 31 and a final count on
September 6, 2001.
Control success was determined by calculating mean numbers of
3rd and 4th stage larvae, and pupae in the pre and post-treatment counts
from the four replicates of each test. Percent control was calculated by
and applying Mulla's formula to the overall population means for each
treatment.
Results
Following treatment, mean populations in all treated plots declined
relative to the untreated control plots, and were significantly lower (P=.05
Student-Newman-Keuls) than the UTC at 7 days post treatment. There
were numerical, but not statistically significant differences between
individual treatments. Initial reductions (3 days post treatment) were
highest overall for the combination formulations, and similar control was
seen from the formulation throughout the duration of the study. Percent
reductions from treatments at 2.5 kg/ha are shown in Table 14.
Table 12 Corrected percent reduction of L3-L4 Culex and Culesita larvae
in artificial plots following application of treatments at 2.5 kg/ha.*
Efficacy of Two B.t.i./B.s. Combination Formulations for control of Culex
tarsalis and Culex pipiens Compared to Standard B.s. Formulation in
Small Field Plots
Materials and Methods
The test site was a marsh where a small stream entered the
Yakima River near Yakima, Washington. Natural populations of Culex
tarsalis and Culex pipiens were present. Water was essentially stagnant.
Depth was 6-12 inches and remained constant throughout the experiment.
Water temperature ranged between 72 and 77 throughout the experiment.
Vegetation was primarily grass with scattered broadleaf weeds covering
80% of the surface, plant height was 6-15 inches in height. Cattle
occasionally grazed in the site, but were not present during the test.
Organic matter was very high at the site. Predator populations were
generally low in the plots.
The test was a randomized complete block experiment with three,
1000 square foot plots per treatment. Plots were sampled immediately
prior to the application on 7-19-01 and again 48 hours, 7 and 14 days after
the application. Twenty dips with a standard mosquito dipper were made
in each plot. Larvae instar 1-2, larvae instar 3-4 and pupae were counted
in each dip.
Three formulations, designated ABG6185, VBC60015 and
VBC60019 were compared in the study. ABG-6185 consisted of B.s.
technical powder formulated onto corncob and had a potency of 50 B.s.
ITU. VBC-60015 and VBC-60019 were combinations of B.t.i. and B.s.
technical powders formulated onto corncob and had theoretical potencies
of 200B.t.i./5QB.s. ITU and 100B.t.i../50B.S. ITU respectively.
Formulations were tested at an application rate of 5 lb/acre and compared
to untreated controls. Data were analyzed with Analysis of Variance.
Percent reductions were calculated with Mulia's formula and based on
large larvae and pupae only. Overall population means for each treatment
were used in this calculation.
Results
At two days after treatment, all three formulations provided
significant although not outstanding control. The rate of 5 lbs/A may have
been rather low for this site. Control at 7 and 14 days was not significant
largely because the larval population in replicate two of the UTC had very
low population. Nonetheless, percentage control was higher in all the
treated plots than in untreated plots on day 7 and 14. In comparing the
formulations, there did not appear to be any difference between treatments
at day 2 but both the VBC formulations were better than ABG-6185 at day
7. Corrected percent reductions calculated using overall population
means of L3-pupae are shown in Table 15.
Table 15 Corrected percent reduction of L3-L4 Culex larvae and pupae in
small field plots following application of treatments at 5 lb/acre.*
Efficacy of Four B.t.i./B.s. Combination Formulations for Control of
Ochlerotatus taeniorhynchus Compared to Standard B.t.i. Formulation in
Artificial Plots
Materials and Methods
Twenty-four artificial test plots located at the John A. Mulrenan, Sr.,
Public Health Entomology Research and Education Center, in Panama
City, Florida were utilized in this study. The plots were filled to a depth of
approximately 6" with 3-5 ppt saline water. This depth was maintained
throughout the study. Each plot had an approximate surface area of 8 ft2.
Emergent grasses and a sandy soil substrate were present in the plots.
Water temperature averaged 75 degrees Fahrenheit during the study.
Artificial infestation of the test plots was done prior to initial
treatment and every other day following the treatments. Approximately
1000 third instar Ochlerotatus taeniorhynchus larvae were added to each
plot on each infestation day.
Five formulations, designated ABG6138s, VBC60015, VBC60016,
VBC60018 and VBC60019 were compared in the study. ABG-6138s
consisted of B.t.i.. technical powder formulated onto corncob and had a
potency of 200 B.t.i. ITU. VBC-60015, VBC60016, VBC60018 and VBC-
60019 were combinations of B.t.i. and B.s. technical powders formulated
onto corncob and had theoretical potencies of 200B.ti./50B.s. ITU,
100B.t.i./25S.s. ITU, 200B.t.i./25 B.s. ITU, and 100B.t.i./50B.S. ITU
respectively. Formulations were tested at application rates of 2.8kg/ha
compared to untreated controls. Four replications were done for each
application rate and UTC in a random pattern throughout the test series.
Sampling was done by taking 8 dips per plot, using standard
mosquito dippers. Numbers of larvae collected from each plot were
recorded. Initial sampling was done 1 day following initial infestation and
treatment, and repeated on days 2, 3, 5, 7, 10, and 13 following treatment.
Control success was determined by comparing number of larvae
collected in treated plots to numbers collected in the UTC plots. Percent
mortality was calculated for each treatment using the following formula.
% mortality = (# larvae in control - # of larvae in treatment)/ # larvae in
control
Results
One day after treatment, populations in all treated plots were
significantly lower than in the UTC plots. Mean percent reductions on day
one ranged from 79.7% for VBC60015 to 98.5 % for VBC60018. There
were no significant differences between the treatments on this day. The
materials continued to show efficacy through day 7 of the study, after
which time percent control declined rapidly. Three of the combination
treatments, VBC60015, VBC60018 and VBC60019, provided significantly
higher overall percent reductions through the course of the 13-day study
(LSMEANSSS multiple comparison test p=0.05).. Percent reductions
through day 7 from treatments at 2.8 kg/ha are shown in Table 16.
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:
WE CLAIM :
1. A method of preparing a composition comprising a combination of a strain of Bacillus
thuringiensis subspecies israelensis and a strain of Bacillus sphaericus comprising the steps of:
a) preparing fermentation slurries of each strain separately to produce fermentation
slurries;
b) concentrating said fermentation slurries at the desired solids or potency level to
produce concentrated fermentation slurries/ powders;
c) combining by mixing said concentrated fermentation slurries/ powders in the desired
ratio of 1:3 to 3:1 to produce a combined concentrated slurry mixture/ powder;
and
d) spray drying the concentrated slurry mixture to produce a combined technical spray
dried powder concentrate.
2. The method as claimed in claim 1, wherein the powder concentrate is used in various
larvicide product forms selected from the group consisting of powders, granules, wettable powders,
water dispersible granules, pellets, briquettes, aqueous suspensions, emulsifiable suspensions, and
aqueous or non-aqueous concentrates.
3. The method as claimed in claim 1, which involves the step of adding to said composition a
component selected from the group consisting of a surface active agent, an inert carrier, a
preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an
emulsifier, a dye, a U.V. protectant, a buffer, a drift control agent, a spray deposition aid, a free-flow
agent and combinations thereof.
4. A composition produced by the method as claimed in claim 1, wherein the ratio of Bacillus
thuringiensis subspecies israelensis to Bacillus sphaericus is 3:1 to 1:3.
5. A method of controlling Dipteran larvae comprising introducing a larvicidally-effective
amount of a composition produced by the method as claimed in claim 1 into an environment
containing Dipteran larvae.

The present invention discloses a method of preparing a composition comprising a
combination of a strain of Bacillus thuringiensis subspecies israelensis and a strain of
Bacillus sphaericus comprising the steps of fermenting the strains separately,
concentrating each strain to the desired solids concentration or activity, combining the
concentrated strains to form a slurry mixture and spray drying the slurry mixture to yield
individual particles which contain toxins from both Bacillus thuringiensis subspecies
israelensis and Bacillus sphaericus.

Documents:

973-kolnp-2003-abstract.pdf

973-kolnp-2003-assignment.pdf

973-kolnp-2003-claims.pdf

973-kolnp-2003-correspondence.pdf

973-kolnp-2003-description (complete).pdf

973-kolnp-2003-examination report.pdf

973-kolnp-2003-form 1.pdf

973-kolnp-2003-form 18.pdf

973-kolnp-2003-form 3.pdf

973-kolnp-2003-form 5.pdf

973-KOLNP-2003-FORM-27.pdf

973-kolnp-2003-gpa.pdf

973-kolnp-2003-granted-abstract.pdf

973-kolnp-2003-granted-assignment.pdf

973-kolnp-2003-granted-claims.pdf

973-kolnp-2003-granted-correspondence.pdf

973-kolnp-2003-granted-description (complete).pdf

973-kolnp-2003-granted-examination report.pdf

973-kolnp-2003-granted-form 1.pdf

973-kolnp-2003-granted-form 18.pdf

973-kolnp-2003-granted-form 3.pdf

973-kolnp-2003-granted-form 5.pdf

973-kolnp-2003-granted-gpa.pdf

973-kolnp-2003-granted-reply to examination report.pdf

973-kolnp-2003-granted-specification.pdf

973-kolnp-2003-reply to examination report.pdf

973-kolnp-2003-specification.pdf


Patent Number 235094
Indian Patent Application Number 973/KOLNP/2003
PG Journal Number 26/2009
Publication Date 26-Jun-2009
Grant Date 24-Jun-2009
Date of Filing 30-Jul-2003
Name of Patentee VALENT BIOSCIENCES, CORP.
Applicant Address 870 TECHNOLOGY WAY, LIBERTYVILLE, IL 60048
Inventors:
# Inventor's Name Inventor's Address
1 DECHANT PETER 16443 SOUTHEAST MEADOWLAND, PORTLAND, OR 97236
2 DEVISETTY BALA N 1561 BUNESCU LANE, BUFFALO GROVE, IL 60089
PCT International Classification Number A01N 63/00
PCT International Application Number PCT/US2002/04162
PCT International Filing date 2002-02-13
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/269,513 2001-02-16 U.S.A.