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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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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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Nazareno Acciarri, Gabriele Vitelli, Salvatore Arpaia, Giuseppe Mennella, Francesco Sunseri, and Giuseppe L. Rotino

Colorado potato beetle (CPB; Leptinotarsa decemlineata-Say) is a serious pest because it has developed resistance against insecticides. Three transgenic eggplant (Solanum melongena L.) lines bearing a mutagenized Bacillus thuringiensis Berl. gene coding for the Cry3B toxin, and the nontransformed control DR2-line were tested in field trials to assess their insect resistance. The transgenic lines 3-2, 6-1, and 9-8 were tested at two different locations in a randomized complete-block design. Samples were taken biweekly to assess the level of CPB and the presence of other insects. At harvest, total yield and fruit number per plot were recorded. Two transgenic lines showed high levels of resistance at both locations, as measured by CPB abundance and yield. Fruit production was almost twice as great in the highly resistant lines (3-2 and 9-8) as in the nontransformed control. The 6-1 transgenic line showed an intermediate level of resistance; it was similar to the control under heavy CPB pressure and was comparable to the other transgenic lines under milder infestations. Analysis by double antibody sandwich–enzyme linked immunosorbent assay (DAS–ELISA), performed on different tissues, revealed a lower amount of Cry3B protein in the 6-1 transgenic line than in lines 3-2 and 9-8. No detrimental effects on nontarget arthropods (including the chrysomelid Altica) were evident. Field observations confirmed that Bt may be able to control CPB infestation in eggplant, representing a potential effective and environmentally safe means of pest control.

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D.S. Douches, T.J. Kisha, J.J. Coombs, W. Li, W.L. Pett, and E.J. Grafius

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is the most serious insect pest of potatoes throughout the eastern and north central United States. Host plant resistance to the Colorado potato beetle has been identified in wild Solanum species and Bt-transgenic potato lines. Detached-leaf bioassays (72 h) were conducted on insecticide-resistant, first instar Colorado potato beetles to study the effectiveness of individual and combined host plant resistance traits in potato. Potato lines tested include non-transgenic cultivars (`Russet Burbank', `Lemhi Russet', and `Spunta'), a line with glandular trichomes (NYL235-4), a line with high foliar leptines (USDA8380-1), and transgenic lines expressing either codon-modified Bt-cry3A or Bt-cry5 (Bt-cry1Ia1). Bt-cry3A transgenic lines, foliar leptine line, and foliar leptine lines with Bt-cry5 had reduced feeding compared to non-transgenic cultivars. Glandular trichome lines and glandular trichome lines with Bt-cry5 did not reduce feeding in this no-choice feeding study. Some Bt-cry5 transgenic lines, using either the constitutive promoters CaMV35s or (ocs)3mas (Gelvin super promoter), were moderately effective in reducing larval feeding. Feeding on Bt-cry5 transgenic lines with the tuber-specific patatin promoter was not significantly different than or greater than feeding on the susceptible cultivars. Mortality of first instars was highest when fed on the Bt-cry3A lines (68% to 70%) and intermediate (38%) on the Bt-cry5 `Spunta' line SPG3 where the gus reporter gene was not included in the gene construct. Host plant resistance from foliar leptines is a candidate mechanism to pyramid with either Bt-cry3A or Bt-cry5 expression in potato foliage against Colorado potato beetle. Without multiple sources of host plant resistance, long-term sustainability is questionable for a highly adaptable insect like the Colorado potato beetle.

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Anusuya Rangarajan, A. Raymond Miller, and Richard E. Veilleux

Leptine glycoalkaloids in leaves of the weedy diploid potato, Solanum chacoense Bitt., have been shown to reduce feeding by Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). Development of cultivated potatoes with natural resistance to CPB has the potential to reduce costs and environmental impacts of production by reducing pesticide use. Through efforts to move the genes controlling leptine biosynthesis into cultivated potato, a series of hybrids was generated between the high leptine producing S. chacoense and a cultivated type, S. phureja Juz. and Buk. These hybrids were evaluated for solanine (+chaconine), leptinins, leptines, and total steroidal glycoalkaloid content. All hybrids contained leptines, but at different levels (ranging from 117 to 802 mg·g-1 dry weight of leptine aglycon). Some hybrids appeared to convert solanine (+chaconine) to leptinine and leptine efficiently and had no detectable solanine in sampled leaves. To verify the biological significance of these glycoalkaloids, leaf tissue was subjected to feeding assays with second instar CPB. CPB feeding rate ranged from 38 to 87 mm2·d-1 and was most closely correlated with leptine concentration. A minimum leptine level of 300 mg/100 g fresh leaves suppressed feeding by 50%, and levels below this had no effect on CPB feeding.

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Abbas M. Lafta and James H. Lorenzen

Growth chamber and greenhouse experiments were conducted to investigate the effect of temperature and irradiance on foliar glycoalkaloids of three potato genotypes (Solanum tuberosum L.) that differ in glycoalkaloid content. Two genotypes (ND4382-17 and ND4382-19) produced the acetylated glycoalkaloids, leptine I and II, that contribute resistance to the Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). The glycoalkaloids were separated and quantified by high performance liquid chromatography. Exposure of plants to high temperature (32/27 °C, 14-hour day/10-hour night) for 3 weeks under a 14-hour photoperiod with an irradiance of 475 μmol·m-2·s-1 significantly increased the levels of leptines I and II, solanine, and chaconine compared to that at low temperature (22/17 °C). Increases in foliar leptines and total glycoalkaloids at high temperature were 90% and 169%, respectively. Growing potato plants at low irradiance (75% reduction) for 2 or 4 weeks resulted in a significant reduction in the levels of leptine I and II (46%), solanine (43%), and chaconine (38%) compared to nonshaded plants. Transferring plants from high to low irradiance or from low to high irradiance for 2 weeks caused a decrease and an increase in glycoalkaloid concentration, respectively. Therefore, both temperature and irradiance influenced foliar levels of glycoalkaloids in potato plants without changing the leptines and solanine to chaconine ratios. Thus, irradiance and temperature influenced glycoalkaloid compounds that can effect resistance to CPB, especially leptine I and II.

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D.S. Douches, W. Li, K. Zarka, J. Coombs, W. Pett, E. Grafius, and T. El-Nasr

The potato tuber moth (Phthorimaea operculella Zeller) is the primary insect pest of cultivated potato (Solanum tuberosum L.) in tropical and subtropical regions, causing both foliar and tuber damage. In contrast, the Colorado potato beetle (Leptinotarsa decemlineata Say) is the most important insect pest in the northern potato production latitudes. The codon-modified Bacillus thuringiensis Bt-cry5 gene (revised nomenclature cry1IaI), specifically toxic to Lepidoptera and Coleoptera, was transformed into cultivar Spunta using an Agrobacterium vector to provide resistance to both potato tuber moth and Colorado potato beetle. The Bt-cry5 gene was placed downstream from the constitutive CaMV35S promoter. Two transgenic 'Spunta' clones, G2 and G3, produced high levels of mortality in first instars of potato tuber moth in detached-leaf bioassays (80% to 83% mortality), laboratory tuber tests (100% mortality), and field trials in Egypt (99% to 100% undamaged tubers). Reduced feeding by Colorado potato beetle first instars was also observed in detached-leaf bioassays (80% to 90% reduction). Field trials in the United States demonstrated that the horticultural performance of the two transgenic lines was comparable to 'Spunta'. These Bt-cry5 transgenic potato plants with high potato tuber moth resistance have value in integrated pest management programs.

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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.

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Joseph J. Coombs, David S. Douches, Susannah G. Cooper, Edward J. Grafius, Walter L. Pett, and Dale D. Moyer

Colorado potato beetle (Leptinotarsa decemlineata Say) is the leading insect pest of potato (Solanum tuberosum L.) in northern latitudes. Host plant resistance is an important tool in an integrated pest management program for controlling insect pests. Field studies were conducted to compare natural host plant resistance mechanisms (glandular trichomes and Solanum chacoense Bitter-derived resistance), engineered [Bacillus thuringiensis (Bt) Berliner Bt-cry3A], and combined (glandular trichomes + Bt-cry3A and S. chacoense-derived resistance + Bt-cry3A transgenic potato lines) sources of resistance for control of colorado potato beetle. Six different potato clones representing five different host plant resistance mechanisms were evaluated for 2 years in a field situation under natural colorado potato beetle pressure in Michigan and New York, and in a no-choice field cage study in Michigan. In the field studies, the S. chacoense-derived resistance line, Bt-cry3A transgenic, and combined resistance lines were effective in controlling defoliation by colorado potato beetle adults and larvae. Effectively no feeding was observed in the Bt-cry3A transgenic lines. The glandular trichome line suffered less defoliation than the susceptible control, but had greater defoliation than the Bt-cry3A transgenic lines and the S. chacoense-derived resistance line. In the no-choice cage study, the Bt-cry3A transgenic lines and the combined resistance lines were effective in controlling feeding by colorado potato beetle adults and larvae with no defoliation observed. The S. chacoense-derived resistance line and the glandular trichome line suffered less defoliation than the susceptible control. Based on the results of the field trials and no-choice field cage studies, these host plant resistance mechanisms could be used to develop potato varieties for use in a resistance management program for control of colorado potato beetle.

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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.