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B. Sosinski and D.S. Douches

DNA from 46 North American potato (Solanum tuberosum L.) cultivars was examined using the polymerase chain reaction (PCR) with 16 arbitrary primers of 10 nucleotide length (10 mers) to determine the efficiency of randomly amplified polymorphic DNA (RAPD) in delineating cultivars, both sexually derived and clonal variants. The 16 primers yielded 43 useful polymorphisms that were evaluated according to the presence or absence of fragments of equal size. All cultivars were discriminated with as few as 10 primers. The russet sport of Burbank was distinguished from a white-skinned clone by one band. More primers (29) were examined to identify a band polymorphism among six Russet Burbank clonal variants. When the cultivars were grouped by tuber type (excluding the russet clonal variants), three to four primers discriminated these commonly grown cultivars. Determination of cultivar integrity was accomplished with PCR amplification, regardless of tissue source (leaf vs. tuber) for DNA extraction. Cluster analysis based on RAPD markers was performed to examine pedigree relationships of the cultivars. Genetic relationships correlated with some pedigrees; however, many exceptions were noted.

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Maurice L. Vitosh, George H. Silva, Richard D. Leep, and David S. Douches

A procedure for rapid determination of nitrate in the fresh petiole sap using an ion specific electrode was developed. A highly significant correlation (R2-0.92) was obtained between the nitrate measured by the sap test and the conventional oven-dried tissue method.

The effects of five nitrogen(N) rates ranging from 0 to 268 kg ha-1, and five dates of sampling dates beginning at tuber initiation, on the sap nitrate concentration were investigated. The nitrate level increased in proportion to N fertilizer rate. The nitrate level was generally higher at tuber initiation and decreased as the season progressed. The rate of decrease was related to the N supply in the soil. At N rates of 0 and 67 Kg ha-1, the average weekly decrease in the nitrate level was greater than 100 ppm. Based on yield response, the nitrate levels were partitioned as deficient adequate and excessive, and a critical nutrient range was established. The sap test offers a tactical approach for corrective in-season fertilization and a means to increase the efficiency of both fertilizer and available soil N.

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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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D.S. Douches, A.L. Westedt, K. Zarka, B. Schroeter, and E.J. Grafius

Potato tuber moth (Phthorimaea operculella Zeller) is a highly destructive pest of the cultivated potato (Solanum tuberosum L.) in the tropics and subtropics and causes significant damage to both leaves and tubers. Development of host plant resistance is a central component of an integrated pest management (IPM) program for potato tuber moth. The purpose of this research was to augment natural resistance by transforming potato with a codon-modified CryV-Bt gene using Agrobacterium-mediated techniques. `Lemhi Russet' potato and two clones with different host plant resistance mechanisms, USDA8380-1 (leaf leptines) and L235-4 (glandular trichomes), were transformed with the CryV-Bt gene. Gene integration of regenerated plants was confirmed by polymerase chain reactions and Southern analyses; gene transcription was evaluated by northern analyses. Detached leaf bioassays showed that high levels of Bt expression occurred in the CryV-Bt transgenic lines (`Lemhi Russet' and L235-4), providing up to 96% control of potato tuber moth larvae, compared with 3% and 54% control in L235-4 and USDA8380-1, respectively. These transgenic lines can be used for breeding purposes to develop cultivars for (and eventual introduction into) IPM systems.

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Kimberly J. Felcher, D.S. Douches, W.W. Kirk, R. Hammerschmidt, and W. Li

Research was done to determine if enhanced resistance to potato (Solanum tuberosum L.) late blight could be obtained by combining host plant resistance and engineered resistance. Late blight susceptible cultivars, Atlantic, and Spunta and the partially resistant cultivar Libertas were transformed with a fungal glucose oxidase gene, resulting in lines which ranged in transgene copy number from 1 to 8. Glucose oxidase enzyme activity ranged from 0.00 to 96.74×10-5 units/mg plant tissue. There was no correlation between copy number and level of transgene mRNA, level of transgene mRNA and enzyme activity, or between level of enzyme activity and disease resistance. Field and growth chamber evaluation of late blight response demonstrated little to no effect of the glucose oxidase transgene in either late blight susceptible or partially late blight resistant cultivars. However, enzyme activity levels were much lower than levels reported in previous research, which may account for the lack of effect of glucose oxidase against Phytophthora infestans. Twenty-one percent of the transgenic lines were phenotypically off-type compared to nontransgenic controls. Most of the off-type transgenic lines (four out of seven) were derived from `Libertas'. Because several off-type lines did not express the glucose oxidase protein, this phenomenon could not be attributed solely to the glucose oxidase transgene. Based on these results, transgenic lines produced for this study do not increase resistance to P. infestans even in combination with moderate host plant resistance. However, production of greater numbers of transgenic lines with the current construct or, production of transgenic lines in which a different constitutive promoter drives the expression of the glucose oxidase gene might result in greater disease resistance. However, the usefulness of any small increase in resistance would need to be evaluated against the time and cost required for development of transgenic potato cultivars and the potential for off-type tubers and plants.

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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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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.