Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/40—Viruses, e.g. bacteriophages
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology

Definitions

• Control of insect pests by chemical means has long been a useful method to protect crops from damage caused by insect attack and infestation. More recently, methods to control insect crop damage have been introduced which are specific to the target insect and avoid environmental and ecological compromise associated with traditional pesticide usage.
• One of these methods employs a gene- tically modified crop which produces insect-specific toxins, e.g., the Cry toxin from Bacillus thuringiensis .
• insect-specific toxins e.g., the Cry toxin from Bacillus thuringiensis .
• the B. thuringiensis-Cry-toxin-expressing crop may exhibit varying degrees of protection from an array of lepidopteran pest species.
• Cry ⁇ A(c) expressing cotton varieties are highly resi- stant to tobacco budworm, Heliothis virescens, but only moderately resistant to cotton bollworm Helicoverpa zea (J.H. Benedict et al.,1996, Journal of Economic Entomology, Vol. 89 (1), p. 230) .
• NPV nucleopolyhedrosis virus
• rNPV recombinant nucleopolyhedrosis virus
• NPV and rNPV may vary in the level of virulence/potency against various insect species, depending upon the host range of the viral vectoring agent and the potency of the toxin encoded by the inserted gene.
• the insect species Helicoverpa zea is highly susceptible to the NPV and rNPV designated HzNPV and HzAalT, respectively, but only moderately susceptible to the Au- tographa californica NPV (AcNPV) or its rNVP, AcAalT (Treacy et al., 1999, Proceedings Beltwide Cotton Conf . , pp. 1076 - 1083).
• AuNPV Au- tographa californica NPV
• AcAalT AcAalT
• the present invention provides a method for synergistic insect control which comprises applying to the locus of a transgenic crop a synergistically effective amount of a recombinant insect virus containing a vector which is highly virulent to said in- sect.
• a recombinant insect virus which contains a vector which is highly virulent to the target insect species to a transgenic crop, preferably a transgenic crop which has been genetically altered to produce an insect toxin (insecticide) demonstrates a significant synergistic effect (i.e. the resultant insect control is much greater than that which could be predicted from the insect control of the virulent recombinant insect virus when used alone or from the insect control of the transgenic crop when used alone) .
• This synergistic effect enables a commercially useful level of insect con- trol via a non-chemical biological means.
• the synergistic insect control method of the invention allows for effective resistance management compatable with sustainable agriculture practices which are environmentally and ecologically sound.
• the application of a synergistically effective amount of a recombinant insect virus preferably a recombinant nucleopolyhedrosis virus (rNPV) , containing a vector which is highly virulent to the target insect species to a transgenic crop variety, preferably a transgenic crop which is genetically altered to produce an insect toxin, provides synergistic control of the insect pest. That is, the application of the virulent recombinant insect virus to the transgenic crop results in a combination of insecticidal components which produces a greater insecticidal effect than that which would be expectedfrom the individual insecticidal compondnts employed individually (synergistic effect) .
• rNPV nucleopolyhedrosis virus
• Recombinant insect viruses containing a highly virulent vector which are suitable for use in the method of invention include rNPVs such as HzNPV, HzAalT, EGTdel, or a combination thereof.
• Transgenic crops which produce an insect toxin suitable for use in the method of invention include Bt expressing lines of maize and cotton (BTK lines), such as NuCotn 33BTM, a transgenic cotton variety derived from Deltapine DP5415TM by the BollgardTM transformation event, or transgenic maize varieties such as those which express the MON 810TM transformation event (YieldGardTM, Monsanto Co. ) .
• BTK lines Bt expressing lines of maize and cotton
• NuCotn 33BTM a transgenic cotton variety derived from Deltapine DP5415TM by the BollgardTM transformation event
• transgenic maize varieties such as those which express the MON 810TM transformation event (YieldGardTM, Monsanto Co. ) .
• the virulent recombinant insect virus may be applied in the form of a formulated composition, such as a wetta- ble powder, to the locus, foliage or stems, preferably the foliage, of a transgenic crop, particularly a transgenic crop which has been genetically altered to produce an insect toxin.
• a formulated composition such as a wetta- ble powder
• foliage or stems preferably the foliage
• transgenic crop particularly a transgenic crop which has been genetically altered to produce an insect toxin.
• a pre- ferred formulation is that described in co-pending U.S. patent application Serial No. 09/094,279, filed June 9, 1998, incorporated herein by reference thereto.
• the synergistically effective amount of the virulent recombinant insect virus may vary according to prevailing conditions such as the degree of insect resistance of the transgenic crop, the application timing, the weather conditions, the mode of applica- tion, the density of the insect population, the target crop species, the target insect species, and the like.
• synergistic insect control may be obtained when the virulent recombinant insect virus is applied to the transgenic crop at rates of lxlO 10 occlusion bodies per hectare (OB/ha) to lxlO 13 OB/ha, pre- feralbly 5x10 ! ° OB/ha to 12X10 1 ! OB/ha.
• synergism for two-way insecticidal combinations is determined by the Colby method (Colby, S.R., Weeds, 1967 (15), pp.20-22), i.e. the expected (or predicted) results (percentage of insects eliminated) of the combination is calculated by taking the sum of the results for each insecticide component applied alone and subtracting the product of these two results divided by 100. This is illustrated mathematically below, wherein a two-way combination is composed of component X plus component Y.
• the percent insect control (no external insecticide applied) exhibited by a transgenic crop of this invention relative to a closely related control crop could be represented by X; and the percent control of a recombinant insect virus of the invention when used an the control crop could be represented by Y.
• the foregoing Colby formula can be used to calcu- late the expected percent control for the combination of the virus and the transgenic crop. If the observed results (actual percent control.) of the combination of the transgenic crop treat®d with the -virus is greater than the calculated expected results, then the combination is synergistic.
• Plants are grown from seed in 3.8-liter plastic pots which are filled with commercial potting soil.
• conventional Deltapine DP5415 cotton is included in the study. Viral applications to cotton are initiated about 1.5 months after the cotton planting date. Potted plants are sprayed in an enclosed chamber which is equipped with an overhead, rotary hydraulic boom. The boom is fitted with three hollow cone nozzles (TX3, Spraying Systems, Wheaton, IL) ; one nozzle is mounted to apply spray directly over plants and two nozzles are mounted an drop tubes angled at about 45° to spray sides of plants.
• the sprayer is calibrated to deliver 189 liters/ha at 3.5 kg/cm 2 ; compressed air is used as the spray propellant.
• the formulated rNPV insecticide is suspended in dechlorinated water, along with the gustatory stimulant, CoaxTM (CCT Corp., Carlsbad, CA) , at 3.5 L/ha. Plants are sprayed three times at 7 -day intervals. Potted cotton plants are arranged in a completely randomized design with four replica ⁇ tions an table-tops which are flooded with water to a depth of about 2 cm to prevent larval migration between plants . Two plants per treatment are given replicate doses, with replicate subsam- ples taken from separate tests. Environmental parameters for the greenhouse during the course of the study are programmed for an average daily low temperature of about 27°C and an average daily high of about 32°C.
• the plants are infested with laboratory-reared, neonate H. zea at about 1 hr after each spray session. With the use of a small paint brush, larvae are placed an leaves and squares throughout the upper portion of each cotton plant. A total of 30 freshly hatched larvae are placed an each plant following each of the three spray sessions. Artificial placement of larvae an plants is designed to approximate natural distribution of eggs and small larvae of this pest species an cotton (Farrar & Bradley, 1985, Environ . Entomol . ) . Efficacy of treatments applied to cotton is determined 7 days after the third application session by recording numbers of damaged and non-damaged squares per plant. Signi- ficant differences among treatments in injury to cotton by H. zea are determined by analysis of variance (ANOVA, SAS Institute, 1989) . Treatment means are separated by Duncan's multiple range test (DMRT; SAS Institute, 1989).
• foliar application of a virulent recombinant insect virus (HzAalT) to a transgenic crop (NuCotn33) at a rate of 12X10 11 OB/ha reduces the insect damage by 4.2-fold as compared to the insect damage to the untreated transgenic crop
• a virulent recombinant insect virus HzAalT
• DP5415 susceptible crop

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• 2000
  • 2000-12-21 EP EP00991987A patent/EP1244360A2/de not_active Withdrawn
  • 2000-12-21 JP JP2001551300A patent/JP2003519638A/ja not_active Withdrawn
  • 2000-12-21 CA CA002396562A patent/CA2396562A1/en not_active Abandoned
  • 2000-12-21 BR BR0016924-2A patent/BR0016924A/pt not_active Application Discontinuation
  • 2000-12-21 CZ CZ20022301A patent/CZ20022301A3/cs unknown
  • 2000-12-21 IL IL15037900A patent/IL150379A0/xx unknown
  • 2000-12-21 AU AU37270/01A patent/AU3727001A/en not_active Abandoned
  • 2000-12-21 PL PL00357639A patent/PL357639A1/xx not_active Application Discontinuation
  • 2000-12-21 CN CN00818240A patent/CN1420725A/zh active Pending
  • 2000-12-21 WO PCT/EP2000/013094 patent/WO2001050865A2/en not_active Ceased
  • 2000-12-21 KR KR1020027008779A patent/KR20020065923A/ko not_active Withdrawn
  • 2000-12-21 SK SK967-2002A patent/SK9672002A3/sk unknown
  • 2000-12-21 HU HU0203815A patent/HUP0203815A2/hu unknown
  • 2000-12-30 EG EG20001602A patent/EG22209A/xx active
• 2001
  • 2001-01-05 AR ARP010100058A patent/AR026806A1/es unknown

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