CA2195969A1 - Mixtures of genetically modified insect viruses with chemical and biological insecticides for enhanced insect control - Google Patents

Abstract

Insecticidal compositions are described for use against insects comprising mixtures of genetically modified insect viruses with chemical and biological insecticides for enhanced insect control. The genetic modification of the virus comprises the insertion of a gene which expresses an insect controlling or modifying substance, for example, a toxin, a neuropeptide or a hormone, or an enzyme. The genetic modification of the virus also comprises a deletion in a gene.

Description

(~ ~u~p ~ 95969 ~ W096/03048 P~~

LL ~KES OF GENETICALLY LJ~I~l~ INSECT VIRUSES
WIT~ C~EMICAL AND T~T~TO~Tr~r INSECTICIDES
FOR F.I~T~ Tzn INSECT CONTROL

Field of the Invention This invention relates to insecticidal compositions for use against insects comprising mixtures of genetically ';f;e~ insect viruses with chemical and bio1ogicn1 inaecticides for ~nhDnr~d infiect control.

BackcTround o~ the Inve~tion Control of insect pests which infest commercially valuable crops has been the subject of a variety of approaches. ~h~m;r91 insecticides have been widely used; however, several concerns have been raised about their use. ~h~m;cn1 insecticides _ay affect b~n~f;r;D1 insect species in addition to target, non-b~n~f;r;n1 insect species. Insects tend to acquire resistance to such rh~m;r~1~, thereby requiring the dev~lc~ t of new chemicals. ~h~m;c may persist in the environment for periods of time a~ter their use.
In an effort to reduce the use of rh~m;r91 insecticides, insect-~per;f;r virusea are being ut; 1; ~od to attack insects in their larval stages.
Insect-sper;f;c viruses include both DNA and RNA
viruses. The DNA viruses include ~n~ ~ viruses (~EPV"), and Baculoviridae viruses, such as nuclear polyhedrosis viruses (nNPV"), grnn~1os;~ viruses (~GVn), and Baculovirinae non-ocrl~ baculoviruses (nNOB"), and the like. The RNA viruses include togaviruses, flaviviruses, picornaviruses, cytopls~m;c ~'?'~ 2t95969 ~
WO g6/03048 . ~ 1 1 polyhedrosis viruses ("CPV"), and the like. The Subfamily of double stranded DNA viruseA
~hA~--7Ovirin_e includes two genera, NPVs and GVs, which are particularly useful for bislog~c~l control because they produce oc~ inn bodies ("OBs") in their life cycle.
Examples of NPVs include Ly_antria dispar NPV (gypsy moth NPV), ~to~rArhA c~7ifnr";r~ MNPV, Syngrapha falcifera NPV (celery looper NPV), Spodopter_ litturalis NPV, Spodoptera frugiperda NPV, Spodoptera exigua NPV, ~eliothis ar_igera NPV, r- - LLa hrA~aiC~ NPV, Choristoneura f~m;f~r~"A NPV, Tri~hnpl-~;A ni NPV, ~elicoverpa zea NPV, etc.
r 1OA Of GVs include Cydia F -77A GV (codling moth GV), Pieris hr~ e GV, Trichoplusia ni GV, etc. r _lnn of NOBs are Orcytes rh i nnC~rsS NOB and ~eliothis zea NOB. r _1~A of ~n~ viruses include Melolontha ~elo~otha EPV, Amsacta moorei EPV, ~ocusta _igratoria EPV, r~-7Annp7us 8anguinipes EPV, Schistocerc~ greg~ri~ EPV, Aedes aegypt~ EPV, Ch;rono_us luridus EPV, etc.
Over 400 baculoviru8 isolate~ have been described as being present in invertekrates. The Autographa californica multiple nuclear polyhedrosis virus ("AcMNPV~) is the prototype virus of the Family Baculoviridae and has a wide hoat r nge. The AcNNPV
virus was originally ;~olated from Autogr~pha cA7ifnr";~ ~A. cal.), a lepidopteran noctuid (which ir, its adult stage is a nocturnal moth), cormonly known as the alfalfa looper. This virus infeota 12 Families and more than 30 species within the order of Lepidopterar, insects. It is not known to infect productively any species outside this order.
The life cycle of baculoviruses, as _lif;ed by AcMNPV, ~nal~A~ two stages. Each ,p~ ~rc 21 95969 W096/03048 ~ - -stage of the life cycle is represented by a 3pe~;~ic form of the virus: 3xtr~cel 1 nl ~r viral particles (nECV") which are nonoccluded, and oc~lu~ virus particles (~oVn). The extr~ nl~r and ocrlnA~d virus forms have the same genome, but exhibit different bio~ogio~l properties. The maturation of each of the two forma of the viru6 is directed by separate sets of viral genea, some of which are unique to each form.
In ita naturally occurring insect infectious form, multiple virions are found ~ in a paracrystalline protein matrix ~nown as an occlusion body ("OBn), which is also referred to as a polyhedron ;n~ n;~n body (~PIBn). The prot~in~r~ol~n vir~l ocrluRi~nn are referred to as polyhedra (polyhedron is the singular term). A polyhedrin protein, which has a molecular weight of 29 kD, is the major viral-encoded structural protein of the viral occl~o;~nn .
~Similarly, GVs produce OBs which are - _-ee~l primarily of granulin, r~ther than polyhedrin).
The viral oc~ i~n~ are an ; _ L~t part of the natural baculovirus life cycle, providing the means for horizontal (insect to insect) trAnnm;nsinn among susceptible insect species. In the environment, a susceptible insect (usually in the larval stage) ingests the viral ocrll~ni~nn from a ~nt~min~ted food source, such as a plant. The crystalline occlusions ~;nsori~te in the gut of the susceptible insects to release the infectious viral particles. These polyhedron derived viruses (nPDV") invade and replicate in the cells of the midgut tissue.
It is believed that virus particles enter the cell by endocytosis or fusion, and the viral DNA
is uncoated at the nuclear pore or in the nucleus.
Viral DNA replication is detected within six hours.

~' '' P ? ~ 2 W096/03048 ~ 9 5 9 6 9 1 ~ 3v3~5 By 10-12 hours po3t-infection ("p.i."), secondary infection spreads to other insect tissues by the budding of the extracellular virus ("ECV") from the surface of the cell. The ECV form of the virus is r~pnn~;hle for cell to cell spread of the virus within an individual infected insect, as well a6 transmitting infection in cell culture.
Late in the infection cycle (12 hours p.i.), polyhedrin protein can be detected in infected cells.
It is not until 18-24 hour~ p.i. that the polyhedrin protein assembles in the nucleus of the infected cell and virus particles become ~ ' in the prot~;n~r~n~ oc8lnninn~. Viral oor~ ;nn~ ac l~te to large numbers over 4-~ days as cells lyse. These polyhedra have no active role in the spread of infection in the larva. ECVs ~;~s~m;n~te within the infected larva, leading to the death of the larva.
When infected larvae die, m;ll;nn~ of polyhedra remain in the de _--;nJ tissue, while the ECVs are degradod. When other larvae are exposed to the polyhedra, for example, by ingestion of contaminated plants or other food material, the cycle is repeated.
In cum~ary, the ocrl~ d form of the virus is r~pnn~ihle for the initial infection of the insect through the gut, as well as the envi~, t~l stability of the virus. PDVs are ~n~n~; ~1 ly not infectious when administered by injection, but are highly infectious orally. The non-occluded form of the virus (i.e., ECV) is r~pnn~;hle for viral viremia and cell to cell infection in tissue culture. ECVs are highly infectious for cells in culture or internal insect tissues by injection, but ~s~nt;~11y not infectious by oral administration.
The~e insect viruses are not pathogenic to ' 2~95969 ~ W096l03048 ~ P~llr 50,~a vertebrates or plants. In addition, the baculoviruses generally have a narrow host range. Many strains are limited to one or a few insect species.
The use of baculoviruses as bioinsecticides holds great promise. One of the major ; ~ to their widespread use in agriculture is the time lag between initial infection of the insect and its death.
This lag can range from a few days to several weeks.
During this lag, the insect larva continues to feed, causing further damage to the plant. A number of researchers have attempted to ~veL. this drawback by inserting a heterologous gene into the viral genome, 80 ~8 to express an insect controlling or modifying substance, such as a toxin, nc L~_~tide and hormone or enzyme.
~owever, to date, such genetically ';f;ed insect viruses have not been u~ed in combination with ~h~; C il in8ecticides ag part of an integrated pest _ _~ t scheme. Combinations of wild-type insect viruses with ~ ;c~l insecticides have been reported, but their result~ were not optimum in view of the limitations of wild-type viru~es (R;h1;~rnphy entries 1-5). Researchers have al80 attempted to control insects with other biological control agents such as bacteria (e.g., Baclllu8 thuri"~;e"c;~), fungi, protozoans and nematodes, alone or in combination with insect viruses or ~he~; C~l insecticides, but they have also not provided optimum re~ults (2,3,5,6).
Lh~ler~Le, there is a need to develop combinations of chemical insecticides and genetically - 'ified insect viruses which will provide the benefits of both , t~ while reducing the amount of chemicals used and reducing the time of kill from that obtained with wild-type viruses through the use of genetically ~g;n~ed insect viruses.

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W096/03048 ~ ~ 2 1 9 5 9 6 9 ~ f ~

SummarY of the Invention It i8 an object of this invention to provide insecticidal compositions for use against lepidopteran insects comprising mixtures of g~n~~ lly modified insect viruses with ~hnmic~l and biological insecticides for ~nh~n~ insect control. The genetic ';fic~tion of the virus comprises the insertion of a gene which ~ .asea an insect controlling or modifying sub6tance, for example, a toxin, a neuropeptide or a hormone, or an enzyme. The genetic ';fi~t;nn of the virus al~o compriaes a ~1etinn in a gene.
Thi6 invention provides insecticidal lS compoaitions comprising:
(a) an effective amount of a ~h~mi~1 insecticide selected from the class of ~h~m;c~1~ consiRting of pyrethroidR, arylpyrroles, diacylhydrnzines and for~m;~in~; and (b) an effective amount of a genetically ~'f;~ Autograph~ ~7ifnrn;ce nuclear polyhedrosis virus ("Ac-~7PV") which ~nnt~;n~ either: (i) an inserted gene which ~ aaes Au~cL~us ~ustralis insect toxin ("AaIT"), or (ii) a deletion in the gene ~nro~;ng ecdysteroid UDP-glucosyl transferase (~EGT") of Ac-~7PV, wherein said compositions are used against lepidopteran in6ect~, with the proviso that when the insects are ~eliothis zea insects and the ~h~m;c91 insecticide is a forr-m;~;n~, the genetically modified AcMNPV cnn~n;n~ an inserted gene which eA~Lesses AaIT.
In one -'; t, this invention provides ~ Q-~ ~(~
W096/03048 _ 7 _ P~

insecticidal compositions for use against Heliothis virescens insecta comprising:
(a) an effective amount o~ a ~h~m;c~l insecticide selected from the class of ~h~m;c~l~ consisting of pyrethroids and arylpyrroles; and (b) an effective amount of a genetically modified AcMNPV which cont~;n~ either:
(i) an inserted gene which expresaes AaIT, or (ii) a deletion in the gene ~n~o~;n~ EGT of AcNNPV.
In another : '-'; t.~ this invention provides insecticidal compositions for use against Neliothis zea insects comprising:
(a) an effective amount of a ~bam; ~Al insecticide selected from the class of rh~m;c~lA consi~ting of arylpyrroles and diacylhydrazines; and (b) an effective amount of a genetically modified AcMNPV which c~n~9;nA either:
(i) _n inserted gene which expresses AaIT, or (ii) a deletion in the gene ~n~o~;n~ EGT of AcMNPV.
In still another ~ t, this invention provides insecticidal compositions for use against Neliothis zea insects comprising:
(a) an effective amount of a ~h~m;r~l insecticide selected fron the class of ~h~m; ~Al ~ con8isting of f r ' ~; n~8;
and (b) an effective anount of a genetically _odified AcMNPV which c~nt~;n~ _n inserted gene which e~L eS~C~ AaIT.
This invention further provides a method for the control of lepidopteran insects which comprise~

~ ~5q69 W096/03048 ~ rj~ r~ a admini~tering to said in~ect~ or to a crop where ~aid insects feed the insecticidal compositions described above.

B~ief DescriPtion of the Fi~ure~

Figure 1 i8 a graphical depiction of the data presented in Table 13 below, that is, percent mortality at 1, 4 and 10 days for the first three treatments set forth in Table 13, with the exception that the ~'Untreated check" data in Table 13 is not depicted in Figure 1.
Figure 2 is a graphical depiction of the data presented in Table 14 below, that is, percent mortality at 1, 4 and 10 days for the first three tr~ 'n set forth in Table 14, with the exception that the "Untreated check" data in Table 14 is not depicted in Figure 2. nAcNNPV AaIT-ins." in Table 14 is the ~ame as "rNPV" in Flgure 2.
Det~iled Descri~tion of the Invention Insects ~uch a8 Lepidoptera undergo a well-characterized se~uence of events during their devDl~ t from egg to adult insect. After hatching of the egg, the insect larva enters a period of extensive feeding. During this time, it molts several times to allow for c~nt;n~d growth. Stages between s~c~;ve molts are referred to as instars. At the end of the larval growth period, the larva pupates and emerges as the adult in~ect. It is the goal of this invention to enhance the control of pestiferous in~ects during the larval stages. Lepidopteran f~; 1; ~A which are known to be ; _ L~lt pests of crops include Noctuidae, Noto~n~; ~A~ Arctiidae, ~ pJ~
W096l03048 ~ ~ 2 ~ 9 59 69 P~

Pyralidae, Plutellidae, Pieridae and Geometridae.
Two criteria are utilized to determine whether ~n insecticidal compoaition provides effective control of insect pests. One is the number of larvae killed over a period of time. This is referred to as "% mortality". Another is the speed of kill. Even if the % mortality over the final time period is not ; _ ~v~d, if more larvae are killed in the early stages of the time period, this i8 b~n~f;C;nl, in that there is less feeding time and thus less damage to the crop. Thus, if either the % mortality or the speed of kill is ; _ ~ved, the composition tested can be said to be an ; _ ~v~ t. over existing compositions.
A combination of a genetically ';f;~
insect virus with a rh~; c91 or biological insecticide i~ said to be ~synergistic~ if the mortality of the combination is greater t_an the sum of the single : ~t~ applied individually; "additive" if the mortality of the - ;n-t;~n is equal to the sum of the single tn applied individually; "sub-additive" if the mortality of the combination is greater than either of the single _ ~q applied individually, but less t A n the sum of the single _ ~ ~n applied individually; and "nnt~g~nintiC" if the mortality of the combination is less than either of the single - _ ta applied i~dividuallY.
Benefits are obtained when the combinations nre either synergistic or additive. Even when the combination is additive, by reducing the dose of either or both of the - _ tn compared to the dose when applied individually, there is a savings in cost, ns well envi., t~l benefitg guch as reduction in the amount of ~h9~; c91 insecticide which reduces about persi~tence and dev~ t of resistance.
The insecticidal composition ig b~n~f;~

~096/03~4~ P ~,~S P~ J~

lf it provides snh~nre~ control of either or both permissive and semi-pGrm;~ive insecta. A pGrm;~sive insect is generally lO0-l,000 fold more susceptible to an insect virus or rhGm;eA1 insecticide than a semi-S p~rm;R~ive insect. For example, the tobacco budworm (~. virescens) is psrm;~sive to AcNNPV, whereas the cotton bollworm (A. zea) is Gsemi-pGrm;nsive to AcNNPV.
An ~nr;11~ry benefit of this invention is that the combination of the rhG~;r91 insecticide and insect virus is that more types of insects can be targeted than through the individual _ _ ~nts alone.
Both rhsm;rs1 insecticides and insect viruses have sper;f;c host ranges. The combinations may expand the host range because of the presence of both , However, this effect is not due to any interaction between the insecticidal A lnrge number of classes of insecticidal rh~m;c91~ are ~t;~ to control insect pests. A
summary of a number of these classes and a description of their mode of action will now be set forth.
Pyrethroids are __ '- which bind to a sodium ion channel protein, which sllh~ uGn~ly causes a change in the action potential across the axonal membrane. In turn, this disrupts proper functioning of the insect nervous system. r _ 1~ of pyrethroids include cypermethrin (~-cyano-3-phen~yL~ yl-cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcycl~ Pr-rh~ylate; FMC Corp.), ~K~ . . ~ I N (3-phL..~yL_~zyl-cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcycloprop~nGrsnh~Yylate;
Coulston International Corp.), fenvalerate (~-cyano-3-ph_l~AyL_~lzyl-2-(4-chlo~he~yl)-3-methylbutyrate) and cyhalothrin (~-cyano-3-pho.-~yL nzyl-3-(2-chloro-3,3,3-trifluoro-prop-l-enyl)-dimethylcyclopror-nGrsrhr~ylate).

~:~ i p ~
wos6/0304s ~ r~.,.J., sw~s For~-m;~;nen are c~ ~n which have several postulated mode~ of action, ;nn~ ;ng binding to octop-m;ne ~a neurnh. A/neurotransmitter) receptor and acting as an agonist, en~-- t of cAMP
production and induction of behavioral changea, or in_bition of mixed function or ~m;nP oxidasea.
Examples of fnr~-mi~;ne~ include Amitraz (N'-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino]methyl]-N-methylmeth~n;m;~Am;~e; NOR-AM, Schering AG) and chlordimeform (N'-(4-chloro-O-tolyl)-N,N-dimethylfnrr-m;~;ne).
Arylpyrroles are mitochondrial toxins which exert their lethal effects by ~nro~pl;ng oxidative pho~h~Lylation. r ,len of arylpyrroles include 4-lS bromo-2-(p-chlorophenyl)-l-(ethoxymethyl)-5-(trifluoromethyl)-pyrrole-3-carbonitrile (U.S. Patent Number 5,310,938) and c _ IR described in U.S.
Patent Number 5,010,098.
Diacylhydrazines are non-steroidal insect growth regulants, whose prim ry mode of action is as an e_dy G-.C agonist. r _l~n of diacylhydrazines include dibenzoyl-t-butylhydrazine (whose preparation i5 described in U.S. Patent Number 5,300,688) and MIMIC~ (3,5-dimethylhen~ojc ~cid l-(l,l-dimethylethyl)-2-(4-ethylbenzoyl) hydrazide; Rohm &
Haas Co . ) .
Cyclodiene~ bind to a receptor subunit of the GA73A complex. An example of a cyrlo~;ene is en~ln8nl~ n (6,7,8,9,10,10-he~ hloro-1,5,5,6,9,9-hexahydro-6,9-methano-2,4,3-b~n~Q~;~Y~thiepin 3-oxide;
Hoechst).
Carbamates act as inhibitors of rhnl;nenterase. Examples of carbamates include ~h; o~; C~ rh (dimethyl-N,N-(thiobis(methylimino)carbonyloxy)-~ 95~69 W096l03048 ~ >i ~ r~

bi~(e~hon;m;~nthioate); Rhone-Poulenc) and methomyl (S-methyl N-[(methylnArh ~l)oxy] thioacetimidate).
Org-nnFhnsrh-tes act as inhibitors of ~hnl;n~#terase. r _1P~ of ,JL~ hn~hotes include profenofos (0-4-bromo-2-chlor~hc~yl O-ethyl S-propyl phosphorothioate Ciba-Geigy), malathion (O,O-dimethyl pho~hoL~dithioate o_ diethyl mercaptosuccinate), sulprophos (O-ethyl 0-~4-(methylthio)phenyl] S-propyl phosphorodithioate and dimethoate (O,O-dimethyl (S-methylc~ ' ~lmethyl)-phoO~huLodithioate.
Pyrazole~ are inhibitors of mitochondrial respiration by acting ~pe~;f;c~lly at Complex I of the electron transport system. r _l~# of pyrazoles include tebufenpyrad (N-(4-t-butylbenzyl)-4-chloro-3-ethyl-1-methylpyrazole-5-c~rh~Y~m;~; Mitsubishi Rasei, American Cyanamid Company) and __ '-described in p--hl;Qh~d ~uropean Patent Application Number 289,879.
Nitrog~-n;~;n~ prevent binding of acetylrhnl;n~ to certain acetyl~hnl;n~ receptors in the postsynaptic ~ ~c; by binding to the receptors themselves, these ~ _ '~ disrupt nC~LL- ---- QQinn.
r _le~ of nitrog~l-n;~;n~Q include ;m;~--loprid (1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-2-;m;~o~ ;n;m;n~; Bayer) and its derivatives.
Milbemycins first ~ind to a site in the GA3A
receptor/chloride ion channel complex, and then induce paralysis and death in insects by inhibiting signal tron~ ;nn at the ne 'Ll QC'll or junction. An example of a _ilbemycin is abamectin (mixture of av~ Lins cnn~o;n;ns ~80~ ~v~ - Lin Bla and c20 ~ve ~ Lin Blb; Merck, 8harp & Dohme).
3enzoylphenylureas are insect growth regulators which interfere with chitin synthesis, thereby disrupting the process of cuticle formation ~ W096l03048 2 1 9 5969 during insect molting. An example of a benzoylphenylurea is diflubenzuron (1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl) urea; 'Jniroyal Chemical Co., Inc.).
~i~;n~hydrazoneg are inhibitors of mitochondrial respiration by inhibiting electron transport at Complex II. An example of an n-nir;n~hydrazone is Lyd~ Lhylnon (tetrahydro-5,5-dimethyl-2(lH)-pyr;m;~;n~nO [3,-[4-(trifluoromethyl)phenyl]-1-[2-[4-(trifluoromethyl)phenyl]ethenyl]-2-propenylidene;
American Cyanamid Company).
It will be ~ud.z~Lood by persons skilled in the art that additional examples of the foregoing classes of ~ho?n; c~l ~ are known and available either from co~mercial suppliers or described in the patent and scientific literature.
In aac~.d~.cc with this invention, the insecticidal composition compri~es an insecticidal rho?n;~l (or biolog;c~l insecticide as described below) and a genetically ~;f;ed insect virus.
In one . ' '; t of this invention, the genetic ';f;c2tion of the inaect virus conpriaes the insertion of a gene which expresses an insect controlling or mcdifying uuL~L~e at any suitable location within the viral genome. The subatance, for example, is a toxin, a neuropeptide or a hormone, or an enzyme. A subatance thus expressed onh~n~oQ the bioinsecticidal effect of the virus.
Such toxins include the insect-specific toxin AaIT from the ~corpion And-~.L~us australis (7), a toxin from the straw itch mite species }~yemotes tritici (8), ~ci777-C thuringiensis toxins (9,10), and a toxin isolated from spider venom (11). Examples of such neuropeptides or lJ e- include eclosi~n ~ ~ 9596 9 W096l03048 ~ ~,'C,~a hormone (12), prothoracicotropic hormone (PTTH), p~k;nPtiC hormone, diuretic hormone and proctolin (13). An example of auch enzymea is juvenile hormone eateraae (J~E) (14).
Thia invention ia ~ _1;f;~ with a genetically medi_ied AcMNPV which cnntA;n~ an inserted gene which expreaaea AaIT. The starting point _or the genetic 'if;r~tion is the wild-type atrain of AcNNPV
de~ignated E2 (ATCC V~-1344). The toxin inaerted into thia viral atrain ia AaIT, which i8 ~,~d~ced by the venom of the North African scorpion Anll ~L~ ~8 auatralis ~ector. The toxin is 70 amino acids in length and binds to aodium rh~nnP1~ in inaects and cauaes contractile paralysis at the n~ to microgram range in insect larvae. Becauae AaIT doea not bind to 1;~n 80dium rh9nn~1~, AnIT is a candidate for uae aa a bio~n~Pcticide to protect cropa ~ecause it can be aafely ingested by humans.
The region ~L,c~ of the coding region of the AaIT gene ;nr1n~ a nignal sPr~lPnre which directr the secretion of AaIT _rom the cell. Sp~C;fic911y, the signal s~r,~Pnre directs the toxin through the ~e_,~toLy pathway to the cell surface where it is secreted from the cell. During trnnsport, enzymea cleave the aignal ae~~nre, leaving the mature A IT.
It haa been found that heterologous aignal aequencea are uaeful in the expreaaion and secretion of insect toxins, such as AaIT (15). A preferred heterologoua signal se~nre is the cuticle signal aequence of Dro8o~hi7A 7An~gA~ter (_or an exoakeletal protein), which aecretea a large guantity of associated mature proteina.
In turn, a codon optimized DNA aequence Pnro~ng the cuticle signal ae~Pnre and AaIT ia used.
The degeneracy of the genetic code permita variationa

2 1 9 5 9 ~ 9 W096/03048 ~ u~ 5 of the nucleotide sequence, while still producing a polypeptide having the ; ~ont; ral amino acid sequence a8 the polypeptide encoded by the native DNA Eequ~nre.
The procedure known as codon optimization provides one with a means of ~et7isn;ng such an altered DNA sequence to reflect the codon frequency utilized by the host insect. In this _'; t, codon use tahles for Drosophila - - 7an~g~ter are u~ o~ to generate a codon optimized DNA s~ nre ~nro~ing the cuticle signal sequence and AaIT.
An additional means to improve AaIT
expression is the use of the AcMNPV DA26 ~'early"
promoter. This promoter is inserted upstream of the codon optimized DNA onro~;n, the cuticle signal sequence and AaIT.
Samples of a genetically modified AcMNPV E2 strain crn~a;n;ng the DA26 promoter and the codon optimized DNA ~nro~;n~ the cuticle signal sequence and AaIT are constructed in accordance with the procedures set forth in co-pending, commonly-Arai,nod 7Jnited States Patent ~ppl;cat;~n Serial Number 08/070,164, which is hereby incu~ ted by reference. Samples of the resulting viral construct, designated AC1001, have been deposited with the American Type Culture Collection and have been Ar~ gno~ ATCC accD~si~n nu~ber VR-2404. Other constructs using wild-type AaIT
DNA sequences, other heterologous signal sequences and other promoters can be generated by persons skilled in the art using conventional tPrhn;~
T ~ t of insect viral p_lCo ~uce in controlling insects by genetic ~;f;rae;rn of the insect virus also takes the form of a deletion in a gene. An example is a deletion in the gene ~nro~;nrJ
ecdysteroid UDP-glucosyl transfera~e ("EGT"). Miller et al. have reported the construction of such EGT

p ~ S 2 1 9 5 9 6 9 W096/03048 '' r~

Dtrains of inRect viruces ~16). In particular, Miller deRcribed the construction of an AcMNPV EGT- atrain.
Expression of the e~t gene causes the production of EGT. EGT inactivates insect molting ~ -- (ecdy_one), which prevents the insect larva from molting or pupating. When the e~t gene is inactivated, _uch as by generating an EGT- strain, molting and pupation of the larva infected with the insect viru6 can proceed. In turn, this continued development of the in6ect results in such b~nef;ci~
crop protection results as reduced feeding, reduced growth and more rapid death. This is because the EGT
in6ect virus fail_ to block larval molts and pupation, along with the cessation of feeding in preparation for these molting events. r~nR~ n~ly, the EGT- infected insect6 are much more prone to die earlier than wild-type (EGT') infected insects when they attempt to molt while they are in an infected state. Thus, infecting insects with EGT- ~trainc i6 more effective than infectin~ incect6 with wild-type viru6 in termc of LT~o valuea (the time it t~kec one-half of a group of in~ectc to die after being infected with a virus).
The Ç~t gene i8 inactivated by 8ub8tituti~g in its place or incerting within it another gene 6uch as the nonviral marker gene for ~-galactosida6e. Any DNA sequence can be used to disrupt the e~t gene as long as it disrupts expression of the e~t coding _equence. Alternatively, all or part of the e~t gene can be removed from the genome by deleting or mutating an appropriate coding 6egment. In addition, the regulatory part of the genome that controls e~t gene expre86ion can be altered or removed. The result of these ';f;cstions is undeLe~L~6-ion of the eqt gene. Deletions inactivating the eqt gene can also be produced by 8erial virus pas8age in insect8 or insect ~!,'p~@ ~
~ W096~03048 2 ~ 9 ~ 9 6 9 ~ S~S

cell culture. All of these insertions, deletionA or - mutations are achieved using convent;~n-l means. The re~ulting deleted insect viruses have the advantage that they contain no foreign DNA and differ from wild-type virusea only in that they lack a ~unctional e~t gene.
Niller ~ _l;f;~d an AcMNPV EGT- virus by the ~ ';n~nt ~signrt~ vEGTDEL, in which a portion of the ect geme was deleted. Miller obtained vEGTDEL
by cotransfecting into SF cells a plasmid pEGTDEL
(which is the product of cleavage of a plasmid c~nt~;n;ng the çqt gene with ~coRI and XbaI 80 as to excise part of the gene) and DNA from the virus vEGTZ
(which ~~nt~;nR the lacZ gene inserted in frame with the preceding ect coding 8r~ n). ~ l~go~
~ e~ ' n~t;nn results in the r~rlrc - of the eat-lacZ fusion gene in vEGTZ with the deleted e~t gene from pEGTDEL, yielding the le ~n~nt virus vEGTDEh which ia EGT-.
Z0 Miller used a atrain of AcNNPV ~rn;~n~ted ~1, which is a clonal isolate of the oris;n~lly ;rolAt~ wild-type strain (ATCC VR-1345). Nore recently, a strain of AcMNPV ~n~n~t~ V8 has been ~ ted and characterized. Samples of this V8 strain have been deposited with the ~ n Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., and have been A~R;gn~d ATCC aec~ n nu~ber ~R 2463. The techn;1U~ described in Miller for constructing an Ll EGT- strain are readily rrpl; ~-hle to constructing a V8 EGT- strain.
_ Conv~nt;~n~l f lrt;~n te~hnol~gy ~nown to persons skilled in the art is used to prepare the compo~itions o~ this invention. The compoaitions are in the form of wettable powders, granulea, suspensions, ~ nr, solutions, so7~lt;rn~ for SUBSTITUTE SHEET (RULE 26) _ _ _ _ _ _ _ .. , . , .. ,, . ,, .. , . ,,,,, . . _ _ W096/03048 ~ ~~ 5'~ 5 aerosols, baita and other conv~nt;~n~l insecticide preparations.
The compositions frequently include an inactive carrier, which aan be a liquid such as water, alcohol, hydroc-rhrn~ or other organic solvents, or a mineral, animal or vegetahle oil, or a powder such as talc, clay, silicate or kieselguhr.
The insecticidal compositions of this invention are applied using conv~n~ n~l techniques known to persons skilled in the art. These include exposing the insect pests to the compositions by inhalation (through spraying or dusting crops where said insects feed), ingestion or direct contact.
The insecticidal compositions are administered in several ways. The virus and rh~m;
are administered at the same time, either in one dosage form or simultaneously with two dosage forms.
If two dosage forms are used, they are packaged separately and then admixed, if n~c~ y in the presence of a diluent, to generate the final composition. Alternatively, one of the virus or rh~ic~l can be administered first to stress the insect, follr ' by the other ~ _ t.
The insecticidal compo8itions of thi8 invention are ndministered ~t dosages in the range of 2.4X10~-2.4X101' PIBs/hectare of genetically modified virus with 0.001-1.0 kg/hectare of rh~;r~l insecticide. These dosages L~yL~ralt dosage ranges e8t~hl;rh~d in the art for each ~ _ t individually, a~ well as reductions made po8~ible by the combination insecticidal compositions o~ thi8 invention.
The rrnr~ntrations of each of the active _ _ ~n~r of the compositions needed to produce optimum insec~;r;~lly effective compositions for -~ P.~'~iPt~ 2~95969 5 W096/03048 - F~

plant protection depend on the type of organism, rh~m;~rl and insect virus ~if;r~tion used and the formulation o~ the composition. These concentrations are readily det~m; n~d by a person skilled in the art.
As an alternative to rh~;C~l insecticides, biological control agenta are - i n~ with insect viruaes. Biological control agents include bacteria such as Rart77ua thuringiensis, available, for example from Abbott Laboratories as X3NTARIW and DIPLLW 2X.
Other bi~lr,rj;c~l control agents include protozoans such as Nosema polyvora, ~. grandis and Br~con mellitor ~5). Still other biological control agents include entomopathogenic fungi (5) and nematodes.
Nematodes are administered in a liquid formulation or dispersed in a gel where they are in dormant stage until ready for use.
In order that this invention may be better understood, the foll~ ~ng ~ a are set forth. The , lea are for the purpose of illustration only and are not to be construed as limiting the scope of the invention.

~ le~
3xam~1e 1 Bio~ssay T~rhn; r.~l~

The bioassay technique used in these examples is the diet overlay method. The bioassays ~re carried out as follows. The insects u~ed are E.
virescens (tobacco budworm) and H. zea (cotton bollworm). The larvae are reared on a soybean/wheat germ agar-based diet (Stoneville diet), adapted from the ~SDA Insectary Labs, Stoneville, MS. 3ach colony i8 kept at 28~~ under constant fluo t~cG~t light. All bioassays are conduoted on Stoneville diet with second W096l03048 ~;~?;~P S 2 ~ 9 5 9 6 9 ~u~7~ -3~5 instar larvae (~. virescens four days old nd ~. zea three days old).
Bioassay trays (C-D International, Ino., Pitman, NJ) each contain 32 separate arenas. Each 4X4 cm arena contains 5 ml of Stoneville diet. Clear vented adhesive tops (C-D International, Inc.) enclose the insect in the arena f~l1 n~ treatment and infestation. The clear tops allow for easy scoring.
For log/PROBIT~ (~RO Group, Inc.) analysis, serial dilutions are made from viral stock solutions in acetone:double distilled water prior to each experiment. The dilutions are made in log in~ ~ tR
from lX10~ to lX101 PIBs/ml, ~PrPn~;ng upon the apecies tested. Viral stocks are concentrated, when n~c~A~-ry, by centrifugation. Terhn;c~l grade insecticides are pl~aLad in a variety of concentrations, measured in parts per million ("ppmn) based on weight of insecticide to volume of diluent.
To the surface of the artificial diet (which had hardened) is added by pipettins 0.4 ml of an acetone:water (60:40) solution of one of the follc -'nJr: viral solut;~n, rhP~;r~l e~l~lt;nn, viral plus rhPm;c~l solution or untreated solut;nn. For viral sol~t;~nA, the ~;l--t~rnA range from lX10' to lX101 PIBs/ml, in 10-fold dilutions, ~p~n~;ng upon the insect species tested. The rhP~;c~l nrpl;r?t;~nR
range from 1000 ppm to 0.1 ppm, ~p~n~;n~ upon the rh~;r-l studied and the insect species tested. Each ~;lllt;rn is teated with 32 larvae and repeated with 3-4 replicates. The applications are evenly distributed by rotation of the tray and solutions are allowed to dry in a fume hood. Once dried, one larvae is added to each test arena and allowed to feed for a period of 8 to 10 days. N. vire~cens are fed for 8 days; ~. zea for 12 days. Bioassay trays are kept at 28~C in ~ ; 2 1 9 5 9 6 9 W096/03048 ~ r5~5 c~n~;n~l~u~ fluorescent light th-~uyLvut the study period. Readings are taken once a day to observe early onset time of in~ection. At each reading, a larva i8 ~noi ~ned dead if it exhibits no movement even after shaking the diet tray or if the body becomes l;~.;f;~A. ~h~m;r~l and viral LC20 and LCso values (concentration at which 20% or 50% mortality is obscrv~d) are calculated, based on 3-4 replicates.
Statistica are computed u~ing the SAS log/PROBIT~
program, mortality versus dose, at 8 or 10 days post-treatment. Once these PROBIT'~ values are calculated, tests are conducted with the r~ ' c~l o alone at the predicted LC~o and LCso doses, the viruses alone at the LC,o and LCso doses, and all poo~hle ~h~m;c~l/virus permutations, using the same diet overlay method.
nLC,oll is the dose which i8 predicted to cause 20%
mortality of the larvae by application of the product, while ~LCso~ is the dose which is predicted to cause 50~ mortality of the larvae by application of the product.
The ~no~n~nation of PIBs/ml is indicated in the ensuing Tables, for example, as "5E4n, which is 5Xl01, where "E" means ~ t. The term "DAT" in the Tables stands for day(s) after treatment. In these Tables, AcNNPV "AaIT insertedn is the g~n~t;~Ally modified E2 strain ~nn~A;ntng the DA26 promoter with the codon optimized DNA ~n~o~;ng the cuticle signal se~uence and Aa~T.
rnh~n~ in~ect control is obtained from the compositions cnnt~in;ng a combination of genetically ~;f;ed insect virus and oh~m;C~l insecticide when either (or both) increased mortality or ; _ ~v~d speed of kill results.
Examples 2-5 present the results of experiments with ~elicoverpa zea; r _le~ 6-8 present W096/03048 r p ~ p ~ ~ 2 1 9 5 9 6 9 ~ S5~ ~ -the results of experiments with Heliothis virescens.

ExamPle 2 Co~bination of the F~ '~;n~, Amitraz With GeneticallY Modified Insect Viruaes In the firat experiment, the for~-m;~;ne, Amitraz i~ tested in combination with the insect virus AcMNPV, which i~ ge~etically ';f;ed to either contain AaIT or be EGT-. The resulta are presented in Tables 1 and 2.

? ~ ~ c ~ ~" ! '~
WO 96/03048 - 23 _ 2 1 9 5 9 6 9 1 ~1/U~,3,~

4~
O
L E~ ~ ~ o Ul ,, ~ . O
-- O
~~ 11 H _I ~ L~
r ~ m r ~ 1 4 V

~ ~ ~ U
~ U~ N U) O nJ
m r , r o W
O V

L
n~ m ~ -' tl .- H
;, L P' ~. ~
U~ , ~
v nl U .
o ~ nl , n o m k ~, u ~ P' ~,. ~ ' o H
H H ~
p ~1 . ~ U
n p r ~ , r~ . L ~ L ~¢ o ~"

~ 1 95~69 W096/03048 ? ~ p ~ 24 - r~ 3~3 ~ ~ ~ N ~D Co~
r~
r 1,~ ~
o _l ; V

r~ r r~ ~ ~ ~ U
E Ir~ N N Cl p O p~ _ O
., 'q~

O V
~a --P~
~ .
C r ~I r L ~ ~ V U O
O

V.~ r, o o JJ ~ ,~ ~ r ,a ~ ~ . . .

O . ~ , O
~i , ~ X

r~ ~ p ~ P~ ~ 2 1 9 5 9 5 9 W0 96/03048 . ~ JL,S, J5.~5 The cnn~ inn~ are as follows: Amitraz ~t 100 ppm synergizes the bioactivity of AcMNPV "AaIT
inserted" against H. zea larvae. The synergism of the afure t;nn~d virus is somewhat dose-~r~n~nt, since co_bination~ of this L. ~ inAnt virus plus Amitraz at 1000 ppm produce additive, rather than synergi~tic, effects on X. zea.
In contr~ct, Amitraz ha~ no ~ign;f;c~nt e_fect on the bioactivity of AoMNPV "EGT deleted~
against ~. zea larvae. There is a numerical trend suggesting ~. zea lG~v.~e to the forr~m;~;nG/"EGT
deleted" co_bination is slightly less than additive.

r 1 ~ 3 Combination of an Arylpyrrole With Geneticallv Mn~; f;ed Insect Viruse~

In the next experiment, the arylpyrrole 4-bromo-2-(p-chlo- v~h_.-yl) -l- (ethoxymethyl)-5-(trifluoromethyl)-pyrrole-3-carbonltrile is tested in combination with the insect virus AcNNPV, which is genetically modified to either contain AaIT or be EGT-.
The results are u~ e_ t~ in Tables 3 and 4.

~l~p ~p "~ ~1 9~969 WO 96/03048 ~ 26 - F~

O
V V

E~ C o V
H nl V
. = 0~o _ ~I Cl 01 N CDa) U
N
r~ ~ ;) .
D), ' ~ ~G .
o " ~ o 8 .
~ V
N . C) O ~ r C ~--I r .

C~

~ ~ d' U') _ I r V ~ V

o 4 v S4 L4 U
~C r V . V E-H ~ H 1 ~
~ . r,, ~ N
r ~ j C) ~
H r - r 5!; r U ,~ 41 2U '' ~ ~

' , ! O, ~ P ' ~ ~
WO 96/03048 - 27 _ 2 1 9 5 9 6 9 r~l,u~o~

~, - 0 ~
,i V
. r ,~ ~¢ . V
r1 0 .LI

W r~ ~
~ C U ' ' ri o~ N O ~ ~1 r ~
m Id O ~ a ~ h .
~ 4 ~
N ~ V
~ Id r W ~ U V
41 m Y~ 4 O
V r~ r; ; ; r ~4 h ~G . V
H V I E~
_ r ~ r ~ ~

5 h ~ ~
It L U

W096/03048 '~ ~ p ~ P.r ~ 2 t 9 5 9 6 9 ~ , 3~

The rnnrln~; nn~ are as follows: The arylpyrrole 4-bromo-2-(p-chlorophenyl)-1-(ethoxymethyl)-5-(trifluoromethyl)-pyrrole-3-carbonitrile significantly hastens the speed-of-kill properties o~ AcMNPV "AaIT inserted" against ~. zea larvae [i.e., based on data taken at three days posttreatment]. ~owever, at five and eight days posttreatment, ~. zea response to this aryl-pyrrole/L~ ~inAnt virus co~bination is additive (or slightly less than additive).
The arylpyrrole has no statistically sign;f;r~nt e~iect on the mean mortality of AcMNPV-V8 "EGT deleted" against second-instar ~. 8ea. ~owever, there iB a numerical trend (at 3 DAT) suggesting the arylpyrrole slightly hastens speed-of-kill properties of "EGT deleted" against ~. zea larvae.

1~ 4 Corbination of a Diacylhydrazine With GeneticallY Mn~; f;ed Insect Viruses In the next experiment, the diacylhydrazine dibenzoyl-t-butylhydrazine ia te8ted in ',nAt;nn with the in8ect virus AcMNPV, which i8 genetically ';f;ed to either contain AaIT or be EGT-. The resulta are presented in Tables 5 and 6.

P ~ 2~9~969 W0 96103048 - Z9 - P~ 5 N N 0 .-O ~1 _ O

V
V
E ,~ V v V
O r I ~ V
v r ~V ~ .
. ~ ..
r r~ ' O.

O
r _ -I ~r r.

, ; . ,~lj3 ~ ) V

~V '~ O O ~
N N
O V V
V

H ~ H .¢
. ~ p ' ' 1~1 . .P~ o N
r ~p _ ~ ~,, ~') ;, ;, Lr) ,~ ~ ,e ~ ~ ~

2 ~ 9 5 9 6 9 W0 96/03048 ~ - 30 ~ JU3~ J~S

O _ a ¢ ~ 0 0 '' ~m v a Ul o ~ ~ v h u~
O ~ --I V
., E-l S
~ U

3 ~

., ~ . .
_ ~ ,a _ ; s '~ ' fi v ~ . o -o - --I ~ _ .
. -.
_ ~ ' ,~ , , .~
.
fi ~ fi v S_ oO IG o O ,~ m N N
O V V
U' ' r ' ' -9 , ~ L
. ' ) ~ 9 ~ ~ ' ' ~
r ~ ~ ;', O

~ ~. . o ~ fJ~, p j~
~ Wo96/03D48 ' ~ ~m~ a The c~n~l n#iAn~ are as follow~: The diacylhydrazine, dibenzoyl-t-butylhydrazine ~igniiicantly hastens the ~peed-oi-kill properties of AcNNPV AaIT inserted" again~t X. zea larvae [i.e., ba~ed on data collected at 3 DAT].
The diacylhydrazine al80 ~i~n;f;c~ntly ha~ten~ the speed-of-kill properties of Ac~NPV "EGT
deleted" against ~. zea laryae [i.e., based on data collected at 3 DAT].
Exam~le 5 Combination of a Benzoylphenylurea With Genetic~lly M~; fied Insect Viru~e8 In the next ~Yp~; t, the benzoylphenylurea ~;fll~h~n~uron i~ te~ted in combination with the insect viru~ AcMNPV, which is genetically modi~ied to either contain AaIT or be EGT-.
The result~ are pre~ented in Tahles 7 and 8.

W096/03048 - 32 - ~ 1 9 59 69 ~ 3i~

r~ ~
L ~ ' ~
- ~

E~ C
rt '¢ N ~ 'N m J
wO rJ ~i N N . C
dP .q .
rJ ~ o~ r ~ C

~0 ~ , ' _~ r~ _ o h . ~ C

_' ' r ~ ; r r . h a ~
r~ d~W
W ~ ~
r, Vl ~ ~ rJ O

, O
O N N
U r~ r~ ' ' ' r~ . -- H
r~ ' C
t' p~ C N
C ~ 3~j . ,¢ .C 40~ o 'J'~ I,, 2 1 9 5 9 6 9 W096/03048 -- 33 - r~V~ .. J~525 W
,~ o P~
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P. ~3 N 1'7 ~ ~ g

4~ P H H NC _ ri V _ d~~ ~ .
U
C ~

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n~ .
." . o ~0.
~ 4 ,~ C C

--I ~r r r r-~ . _ V ~ V

SV r , " a ~ 40 111 "
O N N
V . V V ' "
U

H ~ ~ r CO W p c . . ' P ' ~ C ~;
r~ U ,~ 41 C ., .. r~

~ ~; P ~ ?195969 ~
W096/03048 F~ '0,~

The c~nrl~ n~ are as follows: The benzoylphenylurea, ~;fll-h ,v,,~, does not improve ~ctivity of AcMNPV-E2 "AaIT inserted" against H. zea larvae; further, N. zea reaponse to this combination is less than additive.
The benzoylphenylurea also does not improve activity of AcNNPV-E2 "EGT deleted" againat ~. zea larvae; further, ~. zea reaponse to this combination ia less than additive.
Exam~le 6 Combination of a Pyrethroid With Wild-TYDe or GeneticallY Modified Insect Viru~e3 In the next experiment (in second instar ~.
virescens), the pyrethroid, ~y~- -thrin ia tested in combination with the insect virus AcMNPV, which is either wild-type or genetically f;e~ to either contain AaIT or be EGT-. The results are presented in Table~ 9-14.
Table 9 depicts the combination of ~y~ thrin with the wild-type E2 8train of AcNNPV.
The combination Ut; 1 i ~ a do8age equivalent to the predicted LC~o of each _ ~ t used alone.
~ABLE 9 MEAN % NORTALITY AT
lDAT 4DAT lODAT
Cypermethrin (0.5 ppm) ll 19 19 AcMNPV "wild-type" (400 PIB8/ml) 0 13 27 Cypermethrin (0.5 ppm) I
AcMNPV "wild-type" (400 PIBs/ml) 5 31 41 ~ntreated Check O O O

t ,l p -t . C 2 1 9 5 9 6 9 WO 96/03048 F~ 11 In,: JSJ2 ~ - 3~ --Synergism is not ob#erved with the combination co~pared to the individual _ --ts, as was also reported by Aspirot (1).
Table 10 depictE the combination of cypermethrin with the V8 EGT strain of AcMNPV. The combination utilizes a do-age equivalent to the predicted LC~o of each _ t used alone.

MEAN % ~rTTy AT
lDAT 4DAT lODAT

Cypermethrin (0.5 ppm) 11 19 19 AcMNPV "EGT del." (775 PIBs/ml) 0 2 3 Cypermethrin (0.5 ppm) +
AcNNPV "EGT del." (775 PIBs/ml) 23 27 34 Untreated Check O O O

Synergism is GLE__v~d with the co~bination o _- ad to the individual c _ ~ tn. This synergism contrasts with the lack of synergism observed with the combination of cypermethrin and the wild-type viruD.
Table 11 depict- the co~bination of ~y~ thrin with the E2 "AaIT inn-erted" strain of AcNNPV. The combination ut;l; 7~- a dosage eguivalent to the predicted LC~o of each ~ t used alone.

MEAN ~ MORTALITY AT
lDAT 4DAT lODAT

Cypermethrin (0.5 ppm) 11 19 l9 AcMNPV ~AaIT ins." (1000 PIBs/ml) 0 6 22 _ _ y ~ ~ :
1 95q~q W096/03048 ~ P~

Cypermethrin (0.5 ppm) +
AcMNPV "AaIT ins.~ (1000 PIBs/ml) 22 38 44 Untreated Check 0 0 0 Synergi~m is obee~v~d with the combination compared to the individual ~ t~ at 1 and 4 DAT.
This earlier speed of kill is superior to that o served with the combination of ~y~ 'h~in and the wild-ty-pe virus.
Table 12 depicts the combination of cypermethrin with the E2 wild-type strain of Ac~NPV.
The combination llt; 1; ~~~ a dosage equivalent to the predicted hCso of each ~ _ t used alone.

MEAN % ITTTY AT
lDAT 4DAT lODAT

Cypermethrin (1 ppm) 39 58 58 AcMNPV "wild-type" (1200 PIBs/ml) 0 25 53 Cypermethrin (1 ppm) I
Ac~NPV "wild-type" (1200 PIB8/ml) 48 77 91 ~ntreated Check 0 0 0 Except for one DAT, synlergiam is not observed with the combination compared to the individual ~
Table 13 depicts the combination of ~y~ -thrin with the V8 EGT- 8train of AcNNPV. The combination utilizes a dosage equivalent to the predicted LC20 for ~y~ -thrin and the predicted LC50 for the AcMNPV V8 EGT- strain.

~ 2t95969 WO 96/03048 P~ J~J~a ~ 37 -MEAN % ,rTTy AT
lDAT 4DAT 10DAT

Cypermethrin (0.5 ppm) 11 19 19 AcMNPV "EGT del." (1100 PIB~/ml) 0 0 8 Cypermethrin (0.5 ppm) +
AcMWPV "EGT del." (1100 PIBs/ml) 34 47 50 Vntreated Check 0 0 0 The results of Table 13 are also depicted grnph; rnl ly in Figure 1. Synergigm is obse.v.d with the combination compared to the individual ~
This synergism contrasts with the lack of synergism oL~e v~d with the combination of cypermethrin and the wild-type virus, even though a smaller dose of cypermethrin is used with the geuetically ';f;~d viru~.
Table 14 depicts the comoination o~
cypermethrin with the E2 "AaIT inserted" strain of AcMNPV. The combination nt; 1; ~'F a do~age equivalent to the predicted LCso of e~ch t used alone.

MEAW % MORTALITY AT
lDAT 4DAT 10DAT

Cypermethrin (1 ppm) 31 31 31 AcMNPV "AaIT ins. n (5000 PIBs/ml)0 6 16 Cypermethrin (l ppm) +
AcMNPV "AaIT ins." (5000 PIBs/ml)25 63 72 Vntreated Check 0 0 0 ~ ~ 9~6~

p W096/03048 .cl/~,rus~
.~

The results of Table 14 are also depicted gr~ph;r~l1y in Figure 2. Synergism is observed with the combination compared to the individual ,- _ ts at 4 and 10 DAT. This synergism contrasts with the

5 lack of synergism oL~~Lved with the combination of cypermethrin and the wild-type virus.
Thus, the ~in~t;nn of cypermethrin with either the virus g~n~t;~slly ~;f;~ to contain AaIT
or be EGT is superior to the combination of ~y~ -thrin and the wild-type virua. These results are not prr~;ctshle in view of the prior data using only oombinationa of the wild-type virus with pyrethroids ~1).

ExamDle 7 ~ ~;n-t;~n of a Diacylhydrazine With Wild-TYDe or Geneticallv Modified In~ect Viruse8 In the next experiment (in third instar ~.
virescens~, the diacylhydrazine, dibenzoyl-t-butylhydrazine is tested in combinAtion with the in~ect viru~ AcNNPV, which i9 either wild-type or genetically ';f;ed to be EGT- (L1 strain). The results are presented in Tables 15-16. The combinations utilize lower dosages than those used in Example 6.
Table 15 depicts the combination of the diacylhydrazine with the wild-type Ll strain of AcMNPV.

: . , . ' A
P~P'~ 2~95969 W096l03048 P~ g~

TAB~E 15 -Treatment/Dose Percent Mortality lDAT 4DAT lODAT

AcMNPV "wild-type" (lE2 PIBs/ml) 00 25 Diacylhydrazine (lOOppm) O 0 31 AcMNPV "wild-type" (lE2 PIBs/ml) +
Diacylhydrazine (lOOppm) O 19 69 Acetone:Water Check Q O O

6ynergism is oh~erv~d with the combination at 4 and 10 DAT.
Table 16 depicts the combination o~ the diacylhydrazine with the genetically ~;fied EGT~
strain) of AcMNPV.

Treatment/Dose Percent Mortality lDAT 4DAT lODAT

Re, ' in~nt (lE3 PIBs/ml) 0 6 88 Diacylhydrazine (lOOppm) O 0 31 R~ ' ;n~n~ (lE3 PIBs/ml) +
Diacylhydra~ine (100 ppm) 0 13 100 Acetone:Water Check O O O

Observations indicate thi~ re~ponse is slightly dif_erent than additive with the combination at 4 DAT.

?'~'''JP~ ~195969 wo 96/03048 . ' ~ s Exam~le 8 Combination of an Arylpyrrole With Wild-TYPe or Genetically M~;f;ed Insect Viruses In the next experiment ~in 6econd instar N.
virescen6) ~ the arylpyrrole, 4-bromo-2-(p-chlo~h~.-yl)-l-~ethoxy~methyl)-5-~tri_luoromethyl)-pyrrole-3-carbonitrile is tested in combination with the insect virus AcMNPV, which i6 either wild-type or genetically ';f;~ to either contain AaIT or be V8 3GT-. The results are pre~ented in Tables 17-l9.
Table 17 depicts the combination of the arylpyrrole with the wild-type E2 strain of AcM-Npv.
The combination ut;l;~ a dosage equivalent to the predicted LC~o ~or each - ' used alone.

TAB~E 17 ME~UN % MO~TALITY AT
lDAT 4DAT lODAT

Arylpyrrole (1 ppm) 3 8 13 AcMNPV "wild-type" ~400 PIB6/ml) 0 0 6 Arylpyrrole ~1 ppm) +
AcMNPV "wild-type" ~400 P B s/ml) 2 19 41 ~ntreated ChecX 0 0 0 Synergism is obselv~d with the combination at 4 and 10 DAT.
Table 18 depicts the combination of the arylpyrrole with the g~n~ lly ';f;ed EGT (V8 strain) of AcMNPV.

~ ' p ~
W096l03048 2 1 9 5 9 6 9 ~ L,~.05~5 MEAN % MORTALITY AT
lDAT 4DAT 10DAT

Arylpyrrole (2 ppm) 20 52 84 AcMNPV "EGT del. n (1100 PIBs/ml) 0 0 3 Arylpyrrole (2ppm) +
AcMNPV "EGT del." (1100 PIBs/ml) 33 50 72 Untreated Check 0 0 0 Results indicate that, with the combination at 1 DAT, an ; , ~v~d earlier speed of kill is observed compared to the combination of the arylpyrrole and the wild-type virus.
Table 19 depicts the combination o~ the arylpyrrole with the g~n~t;cAlly modi~ied AaIT
in6erted E2 strain o~ AcMNPV.

MEAN ~ MORTALITY AT
lDAT 4DAT 10DAT

Arylpyrrole (2 ppm) 20 52 84 AcMNPV "AaIT ins." (1000 PIBs/ml) 0 3 22 Arylpyrrole (2ppm) +
AcMNPV "AaIT ins." (1000 PIBs/ml)39 69 78 Untreated Check 0 0 0 Synergism iB observed with the combination at 1 and 4 DAT, indicating an ; ~ ~v~d earlier speed oi kill compared to the - ~ ;nAt;~n of the arylpyrrole and the wild-type virus.
Thus, on an overall basi~, the combination i~ .P~o, ~ 5~69 of the arylpyrrole 4-bromo-2-(p-chloropher,yl)-1-(ethoxymethyl)-5-(trifluoromethyl)-pyrrole-3-carboritrile with either the viru~ genetically modi~ied to contair, AaIT or be EGT- ia superior to the co~bination of the arylpyrrole and the wild-type virus.

~ ~t~ . 2 ~ ~ 5 9 6 ~
W096/03048 P~ 3''~

Biblioc~raPhY

1. Aapirot, J., et al., United States Patent Number 4,668,511.
2. Mohamed, A.}., et al., Environ. ~ =
EntomoloqY, 12, 478-481 (1983).
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EntomoloqY, 12, 1403-1405 (1983).
4. Velichkova-K~zhllkhArova, M., et al., Ra~teniçv'l~n; ~llk;, 25, 80-86 (1988).
5. Jagues, R.P., et al., ~Compatability of Pathogens with Other Methods of Pest Control and with Different Crops", Chapter 38, pages 695-715.

6. Geervliet, J.B.F., et al., Med. Fac.
J~n~h~ll~w. Riikauniv. Gent, 5Ç, 305-311 (1991).

7. Zlotkin, E., et al., Toxico~, 9, 1-8 (1971).

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MicrobioloaY, 56, 2764-2770 (1990).

10. Federici, B.A., In Vitro, 28, 50A ~_ (1992).

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12. Eldridge, R., et al., Insect Bio 21, 341-351 (1992).

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16. Miller, B.K.., et al., International Patent Application Number WO 91/00014.

Claims (16)

What is claimed is:

1. An insecticidal composition comprising:
(a) an effective amount of a chemical insecticide selected from the class of chemicals consisting of pyrethroids, arylpyrroles, diacylhydrazines and formamidines; and (b) an effective amount of a genetically modified Autographa californica nuclear polyhedrosis virus ("AcMNPV") which contains either: (i) an inserted gene which expresses Androctonus australis insect toxin ("AaIT"), or (ii) a deletion in the gene encoding ecdyateroid UDP-glucosyl transferase ("EGT") of AcMNPV, wherein said composition is used against lepidopteran insects, with the proviso that when the insects are Heliothis zea insects and the chemical insecticide is a formamidine, the genetically modified AcMNPV
contains an inserted gene which expresses AaIT.

2. The insecticidal composition of Claim 1 wherein the composition comprises:
(a) an effective amount of a chemical insecticide selected from the class of chemicals consisting of pyrethroids and arylpyrroles; and (b) an effective amount of a genetically modified AcMNPV which contains either:
(i) an inserted gene which expresses AaIT, or (ii) a deletion in the gene encoding EGT of AcMNPV, wherein said composition is used against Heliothis virescens insects.

3. The insecticidal composition of Claim 1 wherein the composition comprises:
(a) an effective amount of a chemical insecticide selected from the class of chemicals consisting of arylpyrroles and diacylhydrazines; and (b) an effective amount of a genetically modified AcMNPV which contains either:
(i) an inserted gene which expresses AaIT, or (ii) a deletion in the gene encoding EGT of AcMNPV, wherein said composition is used against Heliothis zea insects.

4. The insecticidal composition of Claim 1 wherein the composition comprises:
(a) an effective amount of a chemical insecticide selected from the class of chemicals consisting of formamidines;
and (b) an effective amount of a genetically modified AcMNPV which contains an inserted gene which expresses AaIT, wherein said composition is used against Heliothis zea insects.

5. The insecticidal composition of Claim 2 wherein the chemical insecticide is selected from the class of chemicals consisting of pyrethroids.

6. The insecticidal composition of Claim 5 wherein the pyrethroid is .alpha.-cyano-3-phenoxybenzyl-cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate.

7. The insecticidal composition of Claim 2 or Claim 3 wherein the chemical insecticide is selected from the class of chemicals consisting of arylpyrroles.

8. The insecticidal composition of Claim 7 wherein the arylpyrrole is 4-bromo-2-(p-chlorophenyl)-l-(ethoxymethyl)-5-(trifluoromethyl)-pyrrole-3-carbonitrile.

9. The insecticidal composition of Claim 3 wherein the chemical insecticide is selected from the class of chemicals consisting of diacylhydrazines.

10. The insecticidal composition of Claim 9 wherein the diacylhydrazine is dibenzoyl-t-butylhydrazine.

11. The insecticidal composition of Claim 4 wherein the formamidine is N'-(2,4-dimethylphenyl)-N-[(2,4-dimethylphenyl)imino]methyl]-N-methylmethanimidamide.

12. The insecticidal composition of any of Claim 2, Claim 3 or Claim 4 wherein the effective amount of the chemical insecticide is 0.001-1.0 kg/hectare.

13. The insecticidal composition of any of Claim 2, Claim 3 or Claim 4 wherein the genetically modified AcMNPV contains an inserted gene which expresses AaIT.

14. The insecticidal composition of any of Claim 2, Claim 3 or Claim 4 wherein the genetically modified AcMNPV contains a deletion in the gene encoding EGT of AcMNPV.

15. The insecticidal composition of any of Claim 2, Claim 3 or Claim 4 wherein the effective amount of the genetically modified AcMNPV is 2.4 X 10 8-2.4 X 10 12 polyhedron inclusion bodies ("PIBs")/hectare.

16. A method for the control of lepidopteran insects which comprises administering to said insects or to a crop where said insects feed the insecticidal composition of Claim 1.