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Dilson A. Bisognin and David S. Douches

An understanding of the genetic relationship within potato germplasm is important to establish a broad genetic base for breeding purposes. The objective of this study was to assess the genetic diversity of potato (Solanum tuberosum subsp. tuberosum Hawkes) germplasm that can be used in the development of cultivars with resistance to late blight caused by Phytophthora infestans (Mont.) de Bary. Thirty-three diploid and 27 tetraploid late blight resistant potato clones were evaluated for their genetic diversity based on 11 isozyme loci and nine microsatellites. A total of 35 allozymes and 42 polymorphic microsatellite fragments was scored for presence or absence. The germplasm was clustered based on the matrix of genetic similarities and the unweighted pair group means analysis of the isozyme and microsatellite data, which were used to construct a dendrogram using NTSYS-pc version 1.7. Twenty-three allozymes and DNA fragments were unique to the wild species. The diploid Solanum species S. berthaultii Hawkes and S. microdontum Bitter formed two distinct phenetic groups. Within S. microdontum, three subgroups were observed. The tetraploid germplasm formed another group, with S. sucrense Hawkes in one subgroup and the cultivated potato and Russian hybrids in another subgroup. Based upon the genetic diversity and the level of late blight resistance, S. microdontum and S. sucrense offer the best choice for strong late blight resistance from genetically diverse sources. This potato germplasm with reported late blight resistance should be introgressed into the potato gene pool to broaden the genetic base to achieve stronger and more durable resistance.

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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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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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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, 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 C. Kuhl, Kelly Zarka, Joseph Coombs, William W. Kirk, and David S. Douches

Late blight of potato (Solanum tuberosum L.), incited by Phytophthora infestans (Mont.) de Bary, is a devastating disease affecting tuber yield and storage. Recent work has isolated a resistance gene, RB, from the wild species Solanum bulbocastanum Dun. Earlier work in Toluca, Mexico, observed significant levels of field resistance under intense disease pressure in a somatic hybrid containing RB. In this study, five transgenic RB lines were recovered from the late blight susceptible line MSE149-5Y, from the Michigan State University (MSU) potato breeding program. Transgenic lines were molecularly characterized for the RB transgene, RB transcript, and insertion number of the kanamycin resistance gene NPTII. Transgenic lines and the parent line were evaluated for resistance in field and laboratory tests. Molecular characterization alone did not predict which lines were resistant. Three of the RB transformed MSE149-5Y lines showed increased resistance under field conditions at MSU and increased resistance in detached leaf evaluations using multiple isolates individually (US-1, US-1.7, US-8, US-10, and US-14). Transfer of RB into late blight susceptible and resistant lines could provide increased protection to potato late blight. The use of the RB gene for transformation in this way creates a partially cisgenic event in potato because the gene's native promoter and terminator are used. This type of transformation provides a chance to generate greater public acceptance of engineered approaches to trait introgression in food crops.

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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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Kelly A. Zarka, Ria Greyling, Inge Gazendam, Dean Olefse, Kimberly Felcher, Gurling Bothma, Johan Brink, Hector Quemada, and David S. Douches

Potato tuber moth (Phthorimaea operculella) is a serious pest of potatoes in tropical and subtropical regions of the world, including South Africa. The cry1Ia1 gene (from Bacillus thuringiensis) under the control of the 35S cauliflower mosaic virus promoter was transformed into the potato (Solanum tuberosum) cultivar Spunta to develop a cultivar with resistance to potato tuber moth for release in South Africa. Two transformation events, ‘SpuntaG2’ and ‘SpuntaG3’, were selected and subjected to extensive molecular analyses as required by the regulatory agencies of South Africa. Southern hybridization experiments indicated that ‘SpuntaG2’ and ‘SpuntaG3’ had one and three copies of the cry1Ia1 gene, respectively, and that the gene insertion was stable through multiple clonal generations. Furthermore, the sequence of the cry1Ia1 gene in ‘SpuntaG2’ was compared with the known sequence of the cry1Ia1 gene and found to be identical. Polymerase chain reaction (PCR) amplification using primers for plasmid “backbone” genes demonstrated that ‘SpuntaG2’ contained no backbone plasmid genes, whereas ‘SpuntaG3’ contained several backbone plasmid genes. Therefore, further analyses were limited to ‘SpuntaG2’, and event-specific primers were developed for this cultivar. Analysis of the left and right border regions in ‘SpuntaG2’ demonstrated that the insertion of the cry1Ia1 gene did not disrupt any functional genes nor did it create new open reading frames that encoded proteins with a significant match to the non-redundant sequence database queried by the BLASTP program. Enzyme-linked immunoabsorbent assays (ELISA) tests indicate that the cry1Ia1 gene was expressed at a mean concentration of 2.24 μg·g−1 fresh weight in leaf tissue and 0.12 μg·g−1 fresh weight in tubers. This study demonstrates the extensive molecular characterization that is necessary to apply for deregulation of a genetically modified crop and these data have been used in a regulatory package for the general release of ‘SpuntaG2’.