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G. Jelenkovic, S. Billings, Q. Chen, J. Lashomb, and G. Ghidiu

A chimeric construct, containing the synthetic cryIIIA (Btt) gene, the NPTII selectable marker and the uidA reporter gene, was incorporated via Agrobacterium tumefaciens into eggplant, variety Hibush. The synthetic cryIIIA gene, altered at the nucleotide level without changing the amino acids of the toxic protein by J. Kemp of New Mexico State Univ., Las Cruces, is adapted for high expression in plant cells. To verify the transgenic status, GUS assays were performed on over 300 plants, from which 185 were confirmed to be transgenic. Physical incorporation of the chimeric construct was further confirmed by Southern analysis of about 30 transgenic plants; both single and multiple site incorporation of the Btt gene were found. Resistance to Colorado potato beetle (CPB) was assessed by: a) placing egg masses of CPB on leaves of plants grown in the growth chamber; b) placing first-instar larvae on detached leaves; c) observing 173 transgenic plants under field conditions. About 60% of the transgenic plants displayed a very high level of resistance to CPB. No larvae survived on the resistant plants longer than 50–60 hours after hatching. Upon selfing, the transgenic plants with a single construct segregate in the S1 generation in a Mendelian fashion.

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Qi Chen, Gojko Jelenkovic, Chee-Kok Chin, Sharon Billings, Jodi Eherhardt, Joseph C. Goffreda, and Peter Day

Three constructs of a coleopteran toxic cryIIIB Bacillus thuringiensis gene were engineered and incorporated into eggplant (Solanum melongena L.). Southern blot analysis of the eight primary transformants and segregational analysis of their R, progenies indicated that the chimeric cryIIIB constructs in each of the transgenic plants were stably incorporated at a single locus or at multiple sites within the same linkage group and that they were regularly transmissible to the progeny. The results of Northern blot and RNase protection analyses demonstrated that transcription of the cryIIIB mRNA takes place in plant cells, but only a small amount of the expected entire length transcripts were produced. The amount of the 5' end mRNA fragment produced was at least 30 to 40 times more abundant than the amount of the 3' end mRNA fragment. This could be interpreted to mean that either the two ends of the mRNA are of different stability or that the transcription process is often interrupted and only a few mRNAs complete the entire process to the end. When the transgenic plant mRNA was reverse-transcribed, amplified by polymerase chain reaction, and hybridized to the cryIIIB probe, two smaller molecular weight mRNA species were identified. Thus, the preponderance of the cryIIIB mRNA in transgenic plants exists as a truncated species, a situation similar to that of cryI genes when expressed in transgenic plants. Seedlings from the eight independent transgenic plants were tested for Coleopteran insect resistance. However, they did not demonstrate any significant resistance to the first and second instar larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say).

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D.W.A. Hunt, A. Liptay, and C.F. Drury

Host plant selection by Colorado potato beetle [Leptinotarsa decemlineata (Say)] was examined on tomato [Lycopersicon esculentum Mill.] transplants fertilized with varying N, P, and K concentrations during greenhouse production. In choice tests conducted with beetles in the field and the greenhouse, the insect preference for plants increased with increasing leaf tissue N concentration, but P and K concentrations had no effect. Five-day, seedling acclimatization to outdoor spring temperatures before planting reduced the insect preference for plants.

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Kenneth L. Steffen, Michael S. Dann, Jayson K. Harper, Shelby J. Fleischer, Sizwe S. Mkhize, Doyle W. Grenoble', Alan A. MacNab, Ken Fager, and Joseph M. Russo

During the initial season of implementation, four tomato production systems differing in soil management, pest control practices, and level of inputs, such as labor, materials, and management intensity were evaluated. These systems were CON, a low input (no mulch, no trellising, overhead irrigation, preplant fertilization, scheduled pest control), conventional agrichemical system; BLD, a high input [straw mulch, trellising, trickle irrigation, compost fertility amendment, integrated pest management (IPM)], ecologically-oriented system that emphasized the building up of soil organic matter levels and used no agrichemicals to supply fertility or for pest control; BLD+, a system similar to BLD, except that agrichemical pesticides were used; and ICM, a high input system (black polyethylene mulch, trellising, trickle irrigation, fertigation, IPM pest control) that used agrichemicals to supply fertility and for pest control. Soil characteristics and fertility levels in the BLD and BLD+ systems were modified with extensive amendments of spent mushroom compost and well-rotted cattle manure. Levels of agrichemical NPK calculated to meet current crop needs were supplied to the CON and ICM systems, with 75% of fertility in the ICM system supplied through the trickle irrigation lines (fertigation). The BLD system had a greater soil water holding capacity and sharply reduced irrigation requirements. During a wet period, fruit cracking and evidence of water-mold root rot were significantly higher in the ICM system than the BLD and CON systems. Defoliation by Alternaria solani was greatest in the BLD system and least in the ICM system. The BLD and ICM systems resulted in a 1 week earlier peak yield compared to the CON system. The yield of No. 1 fruit was 55% to 60% greater in the BLD+ system than the other three systems, which were comparable in yield. Net return was highest in the BLD+ system, although the benefit/cost ratio was greatest in the CON system. This multidisciplinary study has identified important differences in the performance of diverse production systems during the unique transitional season.

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Simon Lachance and Conrad Cloutier

Predators and parasitoids used for biological control must possess good dispersal potential in order to ensure spatially uniform and cost-effective control. The rate of dispersal of Perillus bioculatus (F.) (Hemiptera: Pentatomidae), a predator of the Colorado potato beetle (Leptinotarsa decemlineata), was measured following central release in 0.025-ha potato plots. Factors influencing predator dispersal were also studied under controlled conditions in plant growth chambers. Temperature, predator size as affected by instar, and physiological age with respect to the completion of feeding during the intermolt stage were found to be significant factors. Predator density was also evaluated because of the strong tendency for this species to aggregate, thereby influencing dispersal. Results can be used to develop predictive models for inundative releases of P. bioculatus.

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Félix H. França and Ward M. Tingey

The influence of light level on the expression of resistance in Solanum berthaultii Hawkes (accessions PI 473331 and PI 473334) to the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), was studied by exposing plants to two levels of photosynthetically active radiation: 272 ± 37 μE/m2 per sec and 1025 ± 150 μE/m2 per sec. Over 26 days, shading generally reduced densities of type A and B glandular trichomes, volume of trichome exudate, and phenolic oxidation activity of type A trichomes. In both light regimes, larvae reared on S. tuberosum L. were heavier, developed more rapidly, and had greater survival than those reared on S. berthaultii. Similarly, females reared on S. tuberosum were heavier and produced 10- to 20-fold more egg masses and 68 to 472 times more eggs than those reared on S. berthaultii grown at the same light level. Light level did not affect larval weight, developmental time, survival, adult weight, or fecundity of CPB on either host plant species.

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Dominique Michaud, Serge Overney, Binh Nguyen-Quoc, and Serge Yelle

In the past few years, transformation of plant genomes with proteinase inhibitor (PI) genes has been proposed as an effective way to produce insect-tolerant plants. For such a control approach, however, biochemical studies are necessary to assess the effect of PIs on not only insect digestive proteinases (target enzymes) but also plant endogenous proteinases (nontarget enzymes). As an example, transformation of potato (Solanum tuberosum L.) with oryzacystatin (OC) genes, two cysteine PIs, was considered for controlling Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). The use of electrophoretic approaches and standard assays showed that CPB uses at least 14 cysteine proteinases for protein digestion throughout its development. Proteinases of the same class were also detected in sprouting potato tuber extracts, suggesting a potential interference of cPIs in transgenic plants. While OCs inhibit a significant fraction of CPB digestive proteinases, no inactivation of potato proteinases was detected. This apparent absence of direct interference suggests the real potential of OCs for producing CPB-tolerant transgenic potato plants.

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Conrad Cloutier

Tests in experimental plots over two seasons have shown that it is possible to obtain excellent control of eggs and larvae of the Colorado potato beetle (CPB) (Leptinotarsa decemlineata) under Quebec growing conditions by augmentative releases of a generalist predator, the twospotted stinkbug P. bioculatus. The stinkbugs were mass-reared on CPB eggs and larvae in the laboratory, and were released as second or third instar nymphs at the time of peak beetle oviposition. They were introduced manually at ratios of 2-4 predators : beetle egg mass in plots comprising ≈1000 `Kennebec' potato plants. Short-interval sampling after introduction indicated good rates of establishment and survival of the released P. bioculatus nymphs. Analysis of CPB egg recruitment and mortality indicated high rates of destruction of CPB eggs by the stinkbug. Egg destruction was followed by significant predation of late-instar bugs on CPB larvae, resulting in significant reductions of CPB prepupal and adult densities, and excellent foliage protection in treated plots compared to untreated controls. The results will be discussed with reference to traits of P. bioculatus that make it a good candidate for biocontrol of the CPB, and to problems yet to be resolved before augmentative releases of the predator can be used as an integrated pest management tactic in larger scale potato production.

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Serge Overney, Dominique Michaud, Binh Nguyen-Quoc, and Serge Yelle

In recent years, several studies have demonstrated the potential of proteinase inhibitors (PIs) for controlling insect pests. Used as a component of an integrated pest management program, such an approach must, however, be considered with care, given the potential risks of interference on other control approaches. In particular, the effect of PIs on digestive proteinases of beneficial insects must be determined. As an example, this study analyzed the effect of oryzacystatins (OCs), two cysteine PIs isolated from rice, on digestive proteinases of Perillus bioculatus, a predator of the Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), a major pest. Electrophoretic analyses showed the existence of several cysteine proteinase forms in the digestive tract of P. bioculatus. For each developmental stage, OCs dramatically inhibited proteolytic activity, showing an affinity between these inhibitors and the digestive proteinases of the predator. Despite their potential for controlling CPB, the two rice cystatins thus represent possible growth-suppressing compounds for the beneficial insect P. bioculatus. Work is currently under way to assess the compatibility of the two control approaches.

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Joseph J. Coombs, David S. Douches, Wenbin Li, Edward J. Grafius, and Walter L. Pett

The Colorado potato beetle [Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)] is a destructive pest of the cultivated potato (Solanum tuberosum L.) in northern latitudes. Combining resistance mechanisms of leptine glycoalkaloids and glandular trichomes with the synthetic Bacillus thuringiensis Berliner (Bt) cry3A gene in potato may be an effective strategy for controlling the Colorado potato beetle. Bt-cry3A transgenic plants were developed for three potato lines with differing levels of resistance to Colorado potato beetle ['Yukon Gold' (susceptible control), USDA8380-1 (leptine glycoalkaloids), and NYL235-4 (glandular trichomes)]. Polymerase chain reaction, and Southern and northern blot analyses confirmed integration and transcription of the cry3A gene in the transgenic lines. Detached-leaf bioassays of the cry3A engineered transgenic lines demonstrated that resistance effectively controlled feeding by first instar Colorado potato beetles. The susceptible `Yukon Gold' control suffered 32.3% defoliation, the nontransformed high foliar leptine line (USDA8380-1) had 3.0% defoliation, and the nontransformed glandular trichome line (NYL235-4) had 32.9% defoliation. Mean percentage defoliation for all transgenic lines ranged between 0.1% and 1.9%. Mean mortality ranged from 0.0% to 98.9% among the Bt-cry3A transgenic lines, compared to 20% for the susceptible `Yukon Gold' control, 32.2% for USDA8380-1, and 16.4% for NYL235-4. Results indicate that genetic engineering and the availability of natural resistance mechanisms of potato provide the ability to readily combine host plant resistance factors with different mechanisms in potato.