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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 the leading insect pest of potato (Solanum tuberosum L.) in northern latitudes. Host plant resistance has the potential use in an integrated pest management program for control of colorado potato beetle. During the 1998 and 1999 seasons, field studies were conducted to compare natural (leptine glycoalkaloids and glandular trichomes), engineered (Bt-cry3A and Bt-cry5 transgenic potato lines), and combined (Bt-cry5+glandular trichomes) plant resistance mechanisms of potato for control of colorado potato beetle. Nine different potato clones representing five different host plant resistance mechanisms were evaluated under natural colorado potato beetle infestation at the Montcalm Research Farm in Entrican, Michigan. The Bt-cry3A transgenic lines, the high leptine line (USDA8380-1), and the high foliar glycoalkaloid line (ND5873-15) were most effective for controlling defoliation by colorado potato beetle adults and larvae. The Bt-cry5 line (SPc5-G2) was not as effective as the Bt-cry3A transgenic lines ('Russet Burbank Newleaf,' RBN15, and YGc3.1). The glandular trichome (NYL235-4) and Bt-cry5+glandular trichome lines proved to be ineffective. Significant rank correlations for the potato lines between the two years were observed for egg masses, second and third instar, and fourth instar seasonal cumulative mean number of individuals per plant, and defoliation. Egg mass and first instar seasonal cumulative mean number of individuals per plant were not strong indicators of host plant resistance in contrast to second and third instars or adults. Based on these results, the Bt-cry3A transgenic lines, the high leptine line, and the high total glycoalkaloid line are effective host plant resistance mechanisms for control of colorado potato beetle.

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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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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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Maria A. Estrada, Kelly Zarka, Susannah Cooper, Joseph Coombs, David S. Douches, and Edward J. Grafius

The potato tuberworm [Phthorimaea operculella (Zeller)] is one of the most destructive insect pests to potato (Solanum tuberosum L.) in tropical and subtropical regions, and it has recently become established in the Pacific Northwest of the United States. Combining natural resistance mechanisms with Bacillus thuringiensis (Bt) cry genes could be a potential solution to improve potato resistance to tuberworm. We have expressed Bt cry1Ac in two potato lines: Spunta, a susceptible potato line, and ND5873-15, a moderately resistant line with high foliar glycoalkaloids derived from Solanum chacoense. Putative transgenic lines of Spunta and ND5873-15 were developed using a vector construct pSPUD15 with the codon-modified Bt cry1Ac driven by the 35S CaMV promoter. Integration of Bt cry1Ac in Spunta and ND5873-15 transgenic lines was determined by PCR and Southern analysis. Protein expression in the transgenic lines (0–580 ng·g−1) was determined by ELISA. Plants expressing Bt cry1Ac were effective in controlling potato tuberworm first-instar larvae in the detached-leaf bioassays (up to 97% mortality) and in tuber bioassays (up to 99% mortality). Based on the assays conducted, the Bt cry1Ac Spunta lines were similar to the Bt cry1Ac ND5873-15 lines for potato tuberworm mortality. Constitutively expressed Bt cry1Ac would be a useful gene to use for host plant resistance to potato tuberworm.

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

The codon-modified cryV-Bt gene (cryV-Bt) from Bacillus thuringiensis subsp. kurstaki Berliner, which is specifically toxic to Lepidoptera and Coleoptera insects, and a potato virus Yo coat protein gene (PVYocp), in which the aphid transmission site was inactivated, were cotransformed into potato (Solanum tuberosum L.) `Spunta' via Agrobacterium tumefaciens Conn. We demonstrated the integration and expression of both genes by molecular analysis and bioassays. All cryV-Bt/PVYocp-transgenic lines were more resistant to potato tuber moth (Phthorimaea operculella Zeller) and PVYo infection than nontransgenic `Spunta'. Four cryV-Bt/PVYocp transgenic lines were equal in potato tuber moth mortality to a cryV-Bt transgenic line, but of these four only two lines were equivalent in PVYo titer levels to a PVYocp-transgenic line. We identified two transgenic lines, 6a-3 and 6a-5, which showed greater resistance to potato tuber moth and PVYo than the other cryV-Bt/PVYocp transgenic lines. This study indicated that multiple genes, conferring insect pest resistance and virus resistance, could be engineered into and expressed simultaneously in a potato cultivar.

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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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Anna L. Hale, J. Creighton Miller Jr., K. Renganayaki, Alan K. Fritz, J.J. Coombs, L.M. Frank, and D.S. Douches

The objective of this study was to differentiate six intraclonal variants of the potato (Solanum tuberosum L.) cultivar Russet Norkotah. One-hundred-twelve AFLP primer combinations producing 3755 bands and 79 microsatellite primers producing over 400 bands failed to identify any reproducible polymorphisms among the intraclonal variants and `Russet Norkotah'. The inability to detect differences between clones underscores the degree of genetic similarity between them, despite differences in phenotypic expression. This inability could be due to the tetraploid nature of the clones and/or to epigenetic differences not detected by the utilized procedures.