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United States Department of Agriculture

Agricultural Research Service

Research Project: BIORATIONAL TECHNOLOGIES FOR MANAGEMENT OF CHRYSOMELID BEETLE PESTS OF AGRICULTURAL CROPS
2008 Annual Report


1a.Objectives (from AD-416)
Investigate, develop and evaluate the use of predators, parasitoids, entomopathogens, feeding stimulants or deterrents, cultural controls, host resistance, and other environmentally -friendly tactics in IPM programs for Colorado potato beetle, corn rootworm and other chrysomelid pests. Develop methods for improved management of gypsy moths in non-forest and newly infested forested areas of the United States, particularly mating disruption techniques.


1b.Approach (from AD-416)
Characterize and develop new tactics to manage chrysomelid leaf beeetles, particularly Colorado potato beetle and corn rootworms, and in labratory, semi-field and field experiments, evaluate various methods of deployment, such as primary toxicants or pathogens, habitat augmentation, conservation biocontrol, baits or other attracticidal formulations, or incorporation in crop or trap plants. Studies will encompass biology, host specificity, behavior, non-target effects and environmental impact of biological control agents and other tactics. Studies will be conducted in cooperation with U.S. Forest Service "slow the spread" national Gypsy Moth Project and partners to disrupt gypsy moth mating.


3.Progress Report
This project contributed to the understanding of biocontrol of key crop pests, especially Colorado potato beetle, and its natural enemies, plant hosts and microbial symbionts. IIBBL scientists documented pest and natural enemy response to cover-crop treatments in potato and cucurbit crop systems, as well as intraguild predation, and determined diel periodicity of predation (activity cycle over day and night) for Colorado potato beetle predators. Scientists determined important factors affecting measurement of Colorado potato beetle egg predation by quantitative PCR for the key predator Coleomegilla maculata, and are expanding this work to include other foods such as pollen, aphids, and yeast, to gain insight into omnivory, which is often important for generalist predators. Molecular methods for identifying predators have been improved greatly by IIBBL contributions, including matching of immature and adult stages, and the development of non-destructive techniques for DNA fingerprinting of arthropod specimens. Regarding crop hosts and resistance, researchers at IIBBL have cloned the regulatory region of an infestation-induced gene from poplar, linked it to a marker gene and transformed Arabidopsis with this transgene, with the result that the marker gene was expressed upon infestation. The project also demonstrated that bacteria living in the digestive tract of the Colorado potato beetle appears to be composed mainly of potato endophytes (microbes that live inside of plants). Most of the enteric bacteria inhibited the growth of a entomopathogenic bacterium, Photorhabdus temperata, and several also inhibited the entomopathogenic fungus Beauveria. This shows that these bacteria may serve an important role in defending the pest from pathogen infection, and this finding could lead to improved microbial controls.

Research on this project addresses several components of National Program 304: Component II, Biology of Pests and Natural Enemies (Microbes); Component III, Plant, Pest and Natural Enemy Interactions and Ecology; Component V, Pest Control Technologies; and Component VI, Integrated Pest Management Systems and Areawide Suppression Programs.


4.Accomplishments
1. Measurement of predator diets using new molecular tools.

The diet of many beneficial insect predators needs to be investigated to determine which natural enemies are most valuable as controls for key pests. IIBBL and other ARS scientists used quantitative polymerase chain reaction (qPCR) to detect and measure pest DNA markers, to quantify predation by beneficial predators. In lab experiments with eggs of Colorado potato beetle, a key vegetable pest, and the generalist lady beetle predator Coleomegilla maculata, IIBBL scientists tested the following effects on pest DNA quantity detected: quantity consumed, time since consumption, "chaser" diet following pest consumption, and methods for sample fixation and preservation. Quantity of pest DNA detected was related to quantity consumed and to time elapsed since eating. Pest DNA disappearance was also dependent on chaser diet. How the predator sample is handled is of critical importance in proper use of the qPCR technique, and therefore also with conventional PCR, which is widely used to assess predator gut contents. Because the DNA marker is aggressively digested in the predator gut, it is more important in predator diet assessment than in most forensic or medical uses of qPCR, that this degradation be arrested promptly. Among seven methods tested, storing the predator immediately in 70% ethanol prechilled to -20ºC yielded the highest pest DNA quantity, about 23%. Other methods in widespread use such as freezing samples without solvent at either -80ºC or -20ºC performed significantly less well by comparison, and room temperature ethanol averaged below 1% recovery of target DNA. Results show both the value and complexity for applications of the newly-applied qPCR technique to studies of predation in the field.

304 National Program Component III, Plant, Pest and Natural Enemy Interactions and Ecology, Problem Area IIIA, Understanding the Complex Interactions.

2. Demonstration that bacteria in the gut of Colorado potato beetle inhibit an otherwise promising biological control agent.

Several insect pathogens are not very effective against Colorado potato beetle, a key potato pest. If the reasons can be discovered, improved controls may result. Gut bacteria of field-collected Colorado potato beetle, were cultured and identified by IIBBL scientists. The bacterial community of the gut appears to be composed of potato endophytes (bacteria living within tissues of the plant). Most of the gut bacteria inhibited the growth of the insect-pathogenic bacterium Photorhabdus temperata, and several also inhibited the insect-pathogenic fungus Beauveria bassiana. Because growth of the bacterium Photorhabdus inside the beetle host is necessary for the successful reproduction of the insect-pathogenic nematode Heterorhabditis marelatus, the results explain this nematode’s failure to reproduce in Colorado potato beetle. The gut bacteria discovered may serve an important role in defending the pest from pathogen infection; if so, this defense might be a target in development of novel microbial controls. 304 National Program Component II, Biology of Pests and Natural Enemies (Microbes), Problem Area IIA, Basic Biology.

3. Construction of an infestation responsive gene.

The production of plants that express an insecticidal gene at the site of attack by insect pests would lower the overall level of genetically engineered plant material released to the environment. Researchers at the Invasive Insects Biocontrol and Behavior Laboratory have cloned the regulatory region of an infestation-induced gene from the poplar tree, and fused it to a marker gene. Transformation of Arabidopsis (a model plant for testing gene action) with this transgene was performed to test whether the regulatory region was sufficient for induction by infestation. Indeed upon infestation the marker gene was expressed. This accomplishment makes possible the construction of an insecticidal gene that, when cloned into potato, creates a plant that expresses this transgene only when and where insect feeding occurs, thus diminishing the overall amount of genetically engineered plant material produced in the potato plant. 304 Crop Protection and Quarantine: Component V: Pest Control Technologies; Problem Area VD, Other Biologically-Based Control.


6.Technology Transfer

Number of Active CRADAs1
Number of Non-Peer Reviewed Presentations and Proceedings10
Number of Newspaper Articles and Other Presentations for Non-Science Audiences3

Review Publications
Rowley, D.L., Coddington, J, Norrbom, A., Ochoa, R., Vandenberg, N., Greenstone, M.H. 2007. Vouchering specimens for documenting arthropod barcodes: a non-destructive method for DNA extraction. Molecular Ecology Notes.(7):915-924.

Walsh, G.C., Weber, D.C., Mattioli, F., Heck, G. 2008. Qualitative and quantitative responses of Diabroticina (Coleoptera: Chrysomelidae) to cucurbit extracts linked to species, sex, weather and deployment method. Journal of Applied Entomology. Vol. 132(3):205-215.

Last Modified: 10/24/2014
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