2008 Annual Report
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.
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.
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.