Progress 10/01/99 to 09/30/04
Outputs
Elevated concentrations of carbon dioxide (CO2) prevented neonate larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte, from locating the roots of growing corn in behavioral bioassays conducted in soil tubs. When CO2 was pumped into one end of a soil tub, significantly more larvae were recovered from soil at the treated end than from soil around a growing corn plant at the opposite end of the tub. In controls with ambient air pumped into one end of a soil tub, significantly more larvae were recovered from the soil around the corn plant than from soil oil the treated side. Larvae were unable to locate the roots of corn seedlings when CO2 generating materials were mixed into the soil. CO2 concentrations in soil were measured by mass spectrometry with selected ion monitoring, at m/z, 44. Granules composed of baker's yeast, yeast nutrients, and all organic substrate were prepared as a CO2 source and were tested in larger soil tub bioassays.
Significantly fewer larvae were recovered from corn roots in the soil tubs with yeast granules than from corn roots in control soil tubs. The CO2-generating granules produced soil CO2 concentrations between 15.8 and 18.5 mmol/mol (compared with 1.7-2.6 mmol/mol in control tubs), and this was sufficient to prevent larvae from locating corn roots. in geld trials, organic and inorganic CO2 generating treatments resulted in root ratings that were significantly lower than for the control plants.
Impacts
Western corn rootworms are among the most damaging pests in corn, responsible for more than $1 billion annually in the United States in combined damage and control costs. Volatile attractants can be used to monitor adult populations to assess damage potential, and can also be combined with pesticides to minimize the amounts required for control.
Publications
- Bernklau EJ, Fromm EA, Bjostad LB. Disruption of host location of western corn rootworm larvae (Coleoptera : Chrysomelidae) with carbon dioxide. Journal of Economic Entomology 97 (2): 330-339 APR 2004
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Progress 01/01/03 to 12/31/03
Outputs
We found in field trapping tests in cornfields that three-day-old germinating corn is highly attractive to adults of the western corn rootworm (WCR), Diabrotica virgifera virgifera. Field catches with germinating corn showed a female-biased sex ratio of 9:1, and a 3-fold increase in trap catches over the positive control p-methoxycinnamaldehyde (PMC), a synthetic attractant widely used for WCR adult monitoring. Activated carbon collection of germinating corn followed by GC/MS analysis showed that geosmin and a series of sequiterpenes, copaene, caryophyllene, humulene, longifolene, and b-ylangene were the major volatile components of germinating corn headspace. However, no field activity was found with these compounds. Steam distillation of germinating corn generated a clear, strong-smelling distillate that was attractive to adult rootworms in the field for approximately 24 hours. Trap catches with distillate were half those of PMC. The most field-active collection was
the aqueous residue remaining from the steam distillation. The residue had a yellow color and a subtle pungent odor. GC/MS analysis of dichloromethane extracts of the residue indicated a series of phenolics and two benzoxazines. C-18 fractions of the residue were analysed by GC/MS. The MeOH fractions predominantly contained phenolics, and the water fraction contained a series of small polar molecules and oxygen heterocycles. A number of the phenolics present were of immediate interest because of structural similarities with PMC. Ferulic acid, p-coumaric acid, syringaldehyde and coniferyl aldehyde all possess a cinnamyl or cinnamate group that is para to an oxy-substituent, in these respects resemble PMC. In PMC the oxy-substituent is a methoxy group, but in the phenolics identified from corn they are hydroxyl groups. Three blends of phenolics plus indole were field tested. Ferulic acid:p-coumaric acid:indole, syringaldehyde:indole and coniferyl aldehyde:indole were statistically
equivalent with PMC and PMC:indole and half that of residue. These results strongly suggest that PMC may mimic the activity of one of the phenolic compounds identified from germinating corn.
Impacts
Western corn rootworms are among the most damaging pests in corn, responsible for more than $1 billion annually in the United States in combined damage and control costs. Volatile attractants can be used to monitor adult populations to assess damage potential, and can also be combined with pesticides to minimize the amounts required for control.
Publications
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A. 2003. Patent Application 2730-65-PZA (South Africa), Serial No. 2001/3584
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A., Morton, H. V. 2003. Patent Application 2730-65-CIP-PZA (South Africa), Serial No. 2002/3664
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Progress 01/01/02 to 12/31/02
Outputs
We have isolated and and completed the chemical identifications of feeding stimulants from corn roots for western corn rootworm larvae. Corn root extracts were made by using a laboratory press at 10,000 pounds per square inch to squeeze the juices from the roots of 3-day-old germinating corn seeds. We previously showed that the complete set of feeding stimulants includes both water-soluble and organo-soluble components. The water-soluble components were identified by gas-liquid chromatography and mass spectrometry (GC-MS) of derivatives prepared by reaction with hydroxylamine hydrochloride in pyridine to generate Schiff bases from compounds with keto groups, followed by reaction with bis(trimethylsilyl)trifluoroacetamide (with 1% dimethylchlorosilane as acid catalyst) to generate trimethylsilyl derivatives. A series of derivatives was identified, including saccharides, amino acids, small organic acids, inorganic compounds, and polar glycerolipids. The organo-soluble
components were analyzed by direct GC-MS analysis of organic extracts and also by derivatization of glycerolipids with acid methanolysis to generate methyl esters, followed by GC-MS of the derivatives. The feeding activity of synthetic mixtures was compared with the feeding activity of corn root extracts. Candidate feeding stimulant blends were tested by application to disks of filter paper, and the extracts elicited strong feeding from larvae, with three distinct behavioral elements that included grasping movements with the mandibles on the filter paper, a downward posture of the head, and back and forth movements of the head normally seen when a larva is tunneling into a corn root. These behaviors are not always easy to see, and observations every few minutes were required. A second bioassay technique was also used, based on addition of the fluorescent dye D-282 ("UV blue water soluble dye", Day-Glo Color Corporation, Cleveland, Ohio) to the corn root extracts at a concentration of
1 mg dye per 1 ml solution. When larvae fed on a filter paper disk wetted with a root extract containing the fluorescent dye, their gut contents fluoresced brightly under ultraviolet light after only 10 minutes of feeding. Larvae acquired a permanent record as they fed, becoming more and more brightly visible during a one-hour bioassay, but larvae that merely remained on the paper without feeding did not fluoresce. Corn root extracts elicited strong feeding from nearly 100% of the larvae tested, and a synthetic mixture was developed that also elicited strong feeding from nearly 100% of the larvae tested. In contrast, poor feeding was observed with subsets of the synthetic mixture that lacked either the water-soluble components or the organo-soluble components. Our identification of rootworm feeding stimulants complements our development of CO2-generating materials for rootworm attraction that can similarly be used for the development of new pest control approaches based on behavioral
manipulation.
Impacts
Western corn rootworms are among the most damaging pests in corn, responsible for more than $1 billion annually in the United States in combined damage and control costs. Feeding stimulants can be used to interfere with host selection damage, and can also be combined with pesticides to minimize the amounts required for control.
Publications
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A., Morton, H. V. 2002. Patent Application 2730-65-CIP-PBR (Brazil), Serial No. PI 0015510-1
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A., Morton, H. V. 2002. Patent Application 2730-65-CIP-PJP (Japan), Serial No. 2001-534229
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A., Morton, H. V. 2002. Patent Application 2730-65-CIP-PZA (South Africa), Serial No. 2002/3664
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A., Morton, H. V. 2002. Patent Application 2730-65-CIP-PEP (Europe), Serial No. 00937570.0
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A. 2002. Patent Application 2730-65-PJP (Japan), Serial No. 2000-580439
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A. 2002. Patent Application 2730-65-PZA (South Africa), Serial No. 2001/3584
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A. 2002. Patent Application 2730-65-PUS (United States), Serial No. 09/831,094
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A., Morton, H. V. 2002. Patent Application 2730-65-CIP (Continuation-In-Part), Serial No. 09/573,795
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A., Morton, H. V. 2002. Patent Application 2730-65-CIP-PCT (International), Serial No. PCT/US00/13477
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A., Morton, H. V. 2002. Patent Application 2730-65-CIP-PAU (Australia), Serial No. 52719/00
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A. 2002. Patent Application 2730-65-PEP (Europe), Serial No. 99961587.5
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A. 2002. Patent Application 2730-65-PROV (Provisional), Serial No. 60/107,285
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A. 2002. Patent Application 2730-65-PCT (International), Serial No. PCT/US99/26074
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A. 2002. Patent Application 2730-65-PAU (Australia), Serial No. 18134/00
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A. 2002. Patent Application 2730-65-PBR (Brazil), Serial No. PI 9915306-8
- Bjostad, L.B., Bernklau, E.J., Fromm, E.A. 2002. Patent Application 2730-65-AR (Argentina), Serial No. P990105629
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Progress 01/01/01 to 12/31/01
Outputs
Western corn rootworm larvae (Diabrotica virgifera virgifera) feed selectively on the roots of corn and a few other grasses, but the chemical cues responsible for this selective feeding behavior are not known. Laboratory feeding bioassays with neonate larvae were conducted with root extracts made by using a laboratory press at 10,000 pounds per square inch to squeeze the juices from the roots of 3-day-old germinating corn seeds. The root extract was applied to disks of filter paper, and elicited strong feeding from larvae, with three distinct behavioral elements that included grasping movements with the mandibles on the filter paper, a downward posture of the head, and back and forth movements of the head normally seen when a larva is tunneling into a corn root. These behaviors are not always easy to see, and observations every few minutes are required. An improved bioassay technique was developed using the fluorescent dye D-282 ("UV blue water soluble dye", Day-Glo
Color Corporation, Cleveland, Ohio), added to the corn root extracts at a concentration of 1 mg dye per 1 ml solution. When larvae fed on a filter paper disk wetted with a root extract containing the fluorescent dye, their gut contents fluoresced brightly under ultraviolet light after only 10 minutes of feeding. Larvae acquired a permanent record as they fed, becoming more and more brightly visible during a one-hour bioassay, but larvae that merely remained on the paper without feeding did not fluoresce. Only a single observation was therefore necessary at the end of the bioassay to determine the feeding stimulant activity of a candidate material. Both bioassays were used to study the nature of the chemical cues involved in feeding behavior. Aqueous root extracts elicited strong feeding behavior from nearly 100% of the larvae. An aqueous root extract was extracted with hexane, and aliquots of both portions were applied to filter paper disks. When the disks were dried and then wetted
with water, the aqueous portion elicited feeding from 50% of the larvae and the hexane portion did not elicit feeding at all. Bioassays were also performed on the recombined extract by applying an aliquot of the hexane portion to a filter paper disk, drying it, then applying an aliquot of the aqueous portion, drying it again. Disks with the recombined materials were wet with water, and nearly 100% of the larvae fed on these disks containing both portions, indicating that corn roots contain both water-soluble and organosoluble compounds that elicit feeding by WCR larvae. Khapra beetle (Trogoderma granarium) is a serious grain pest not known to occur in the United States, and possible introductions are monitored each year with infochemical traps in grain facilities throughout Colorado. Although Khapra beetle was not found, the dermestid beetle species Trogoderma teukton was detected for the first time in Colorado. This species previously has been reported in the U. S. only from Iowa,
Minnesota, North Dakota, and Washington. The occurrence of T. glabrum, T. granarium, T. inclusum, T. simplex, and T. variabile was also determined.
Impacts
Western corn rootworms are among the most damaging pests in corn, responsible for more than $1 billion annually in the United States in combined damage and control costs. Feeding stimulants can be used to interfere with host selection damage, and can also be combined with pesticides to minimize the amounts required for control. Khapra beetle trap data is useful in export enhancement.
Publications
- Bishop, R.J., Bernklau, E.J., Bjostad, L.B. 2001. A new state record for Trogoderma teukton (Coleoptera : Dermestidae) in Colorado with notes on other Trogoderma species. Entomological News 112 (4): 281-284.
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Progress 01/01/00 to 12/31/00
Outputs
Laboratory bioassays were conducted with neonicotinoid insecticides in combination with CO2-generating formulations for the control of western corn rootworm larvae. Our laboratory has previously shown that CO2 attracts western corn rootworm larvae, which follow CO2 gradients to locate corn roots underground. In laboratory tests, CO2-generating granules containing small amounts of neonicotinoid insecticides were added to tubs of soil with a corn plant at one end and neonate western corn rootworm larvae at the other end. Neonicotinoid granules with CO2-generating materials added to them significantly reduced the number of larvae attacking corn plants compared to granules containing neonicotinoids alone.
Impacts
Neonicotinoid insecticides are among the newer classes of environmentally benign materials that are encouraged by the Food Quality Protection Act, with better differential toxicity and residue properties than older classes of insecticides. Western corn rootworms are among the most damaging insect pests in corn, responsible for more than $1 billion annually in the United States in combined damage and control costs.
Publications
- Bjostad LB, Bernklau EJ, Fromm EA, Morton V. Filed 26 June 2000. Method and Device for Attracting Insects. PCT Patent Application No. PCT/US00/13477.
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Progress 01/01/99 to 12/31/99
Outputs
Previous work in our laboratory has shown that CO2 is the only volatile compound from corn roots that attracts western corn rootworm larvae. This suggested to us the possibility that CO2-generating formulations applied to the soil may cause behavioral disorientation and prevent larvae from locating corn plants, providing a new method for pest control. Yeast-nutrient granules were prepared as a CO2-generating formulation. In laboratory tests, the granules were mixed into tubs of soil containing a corn plant at one end and a group of newly-hatched larvae at the other end. Significantly fewer larvae located corn plants in soil tubs containing the CO2-generating granules. Previous work in our laboratory also showed that CO2 is attractive to the termite species Reticulitermes flavipes and Reticulitermes tibialis, in glass T-tube bioassays using defined concentrations of CO2 delivered with a syringe pump. We have now shown that Reticulitermes virginicus is also attracted to
CO2. All 3 species are optimally attracted to 0.5-1.0 % CO2.
Impacts
Western corn rootworms are the most damaging insect pests in corn ($1 billion annually, and we are developing less toxic means for the control of these pests by manipulating their behavior with CO2, a non-toxic compound. This is consistent with the goals of the Food Quality Protection Act. We have now demonstrated that CO2-emitting traps are effective in attracting and catching termites in the field. This will be useful in developing improved baiting technology for termites, the most successful and environmentally benign new technique for termite control that is on the market.
Publications
- Bjostad LB, Bernklau EJ, Fromm EA. Filed 19 November 1998. Method and Device for Attracting Insects. U. S. Patent Provisional Application 60\107,285.
- Bjostad LB, Bernklau EJ, Fromm EA. Filed 4 November 1999. Method and Device for Attracting Insects. Patent Cooperation Treaty Application PCT/US99/26074.
- Bjostad LB, Bernklau EJ, Fromm EA. Filed 5 November 1999. Method and Device for Attracting Insects. Argentina Patent Application P990105629.
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Progress 01/01/98 to 12/31/98
Outputs
In strong contrast to earlier published results, we now conclude that the attraction of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae to corn roots is due to CO2 alone, and that no other volatile chemical cues are involved in attracting the larvae. Choice test behavioral bioassays were conducted in the laboratory, with volatile corn compounds on 1 side of the bioassay apparatus and with different concentrations of CO2 on the other side (mass spectrometry was used to measure CO2 concentrations on both sides of the apparatus). Larvae were strongly attracted to volatile compounds from corn when ambient air was present on the other side of the bioassay. However, larvae chose equally between the 2 sides of the bioassay when volatile compounds from corn were present on 1 side and an equivalent concentration of CO2 was present on the other side. When given a choice between corn volatiles and a higher concentration of CO2, the larvae chose the CO2 side
significantly more often. In an experiment conducted both with diapausing and non-diapausing strains, the headspace from germinating corn seeds was collected and continuously injected into 1 side of the bioassay apparatus, and a defined concentration of CO2 was continuously injected into the other side. We tested the possibility that compounds of limited volatility may be involved in larval attraction by preparing glass beads coated directly with volatiles produced by germinating corn seeds, and also by testing soil that was removed from corn roots. All these experiments indicated that compounds other than CO2 were not involved in larval attraction. In other experiments, the soil atmosphere surrounding the roots of growing corn plants was not as attractive as an equivalent concentration of CO2 alone, and the headspace from feeding-damaged corn roots was not as attractive as an equivalent concentration of CO2 alone, indicating that weak repellents were present in these treatments
together with the strong attractant CO2. Tests with solvent extracts and cryogenic extracts of germinating corn seeds in conjunction with CO2 also indicated the presence of weak repellents in corn for the larvae.
Impacts
(N/A)
Publications
- Bernklau, E.J., and Bjostad, L.B. 1998. Behavioral responses of first-instar western corn rootworm to carbon dioxide in a glass bead bioassay. J. Econ. Entomol. 91:444-456.
- Bjostad, L.B. 1998. Electrophysiological methods. Chapter 9. Methods in Chemical Ecology. J.G. Millar and K.F. Haynes, eds. ISBN 0-412-08071-0. Kluwer Academic Publishers, Norwell, Massachusetts.
- Bernklau, E.J., and Bjostad, LB. 1998. Re-investigation of host location by western corn rootworm larvae: CO2 is the only volatile attractant. J. Econ. Entomol. 91: 1331-1340.
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Progress 01/01/97 to 12/31/97
Outputs
Attraction of western corn rootworm larvae to CO2: Laboratory behavioral bioassays showed that larvae were attracted to a broad range of CO2 concentrations from 1.125 mmol/mol up to 100 mmol/mol, with optimal attraction to 2.51-4.20 mmol/mol CO2. The concentration of CO2 in soil near growing corn roots was 4.36+0.31 mmol/mol, which was consistent with the behavioral optimum determined for the larvae. The concentration of CO2 in soil that contained no corn was 1.38+0.03 mmol/ mol, and the concentration in ambient air was 0.94+0.01 mmol/mol. WCR larvae were highly competent in recognizing shallow gradients of CO2, and in laboratory choice tests, significantly more larvae were attracted to 1.125 mmol/mol CO2 than to 1.000 mmol/mol, a concentration difference of only 12.5%. Repellents for western corn rootworm larvae: Volatile compounds isolated from corn were repellent to neonate western corn rootworm larvae inbehavioral bioassays. Diethyl ether extracts of germinating
corn seeds were repellent to larvae when the attractive compound CO2 was present on the treatment side of a choice test and the control contained only ambient air. The repellent activity was recovered from a silica gel separation in the 100% hexane fraction and in the 50:50 hexane:ether fraction, indicating that more than one compound is involved in the effect. Gas liquid chromatography was conducted in conjunction with mass spectrometry to characterize the compounds in these two fractions.
Impacts
(N/A)
Publications
- Bernklau, E.J. 1997. Infochemicals and host location by western corn rootworm larvae. M.S. Thesis. Colorado State University.
- Jewett DK, and Bjostad LB. 1997. Structure-activity study with haloalkane attractants of western corn rootworm (Coleoptera: Chrysomelidae) larvae using a behavioral bioassay. J. Entomol. Sci. 32: 91-105.
- Hibbard, B.E., T.L. Randolph, E.J. Bernklau, E.M. Abou-Fakhr & L.B. Bjostad. 1997. Electroantennogram-active components of maize silk for adults of the western corn rootworm (Coleoptera: Chrysomelidae). Environ. Entomol. 26: 285-296.
- Hibbard, B.E., T.L. Randolph, E.J. Bernklau, & L.B. Bjostad. 1997. Electroantennogram-active components in buffalo gourd root powder for western corn rootworm adults. Environ. Entomol. 26: 1136-1142.
- Hibbard, B.E., Jewett, D.K., Bjostad, L.B. 1997. Pentanoic acid attracts Olcella parvis (Adams)(Diptera: Chloropidae) in Colorado corn fields. J. Kansas Entomol. Soc. 70: 67-69.
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Progress 01/01/96 to 12/30/96
Outputs
A new behavioral bioassay was developed for feeding stimulants from corn roots for western corn rootworm larvae. Aqueous extracts of corn roots placed on small disks of filter paper elicited feeding from nearly 100% of the larvae. Preliminary chemical identification of the compounds in aqueous corn extracts indicates that the feeding stimulants are likely to include monosaccharides, amino acids, small organic acids, and inorganic salts.
Impacts
(N/A)
Publications
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