Field and Storage Evaluations of ‘SpuntaG2’ for Resistance to Potato Tuber Moth and Agronomic Performance

‘SpuntaG2’ is a transgenic potato (Solanum tuberosum) cultivar that contains the cry1Ia1 gene for resistance to potato tuber moth (Phthorimaea operculella), which is a serious pest of potato in many parts of the world. Previous studies have characterized ‘SpuntaG2’ at the molecular level and evaluated it for safety as a human food source. The objective here was to determine the efficacy of ‘SpuntaG2’ against the potato tuber moth in the field and in storage and to evaluate its agronomic performance. Efficacy trials at seven South African locations over 5 years indicated that ‘SpuntaG2’ gave complete control of potato tuber moth in the field and storage. The agronomic performance (tuber size and yield) of ‘SpuntaG2’ was not statistically different from ‘Spunta’ or was better than ‘Spunta’ at all locations/years with two exceptions. Three years of trials in Michigan further support these conclusions. Evaluations done during the 2006–07 South African season showed that ‘SpuntaG2’ did not differ from non-transgenic ‘Spunta’ for the following traits: maturity, growth habit, eye morphology, tuber size distribution, tuber shape, skin and flesh color, growth cracks, internal defects, specific gravity, chip color, cooking quality, disease resistance, and pollen fertility. Based on the results of these trials, it was concluded that ‘SpuntaG2’ provides the agronomic performance of ‘Spunta’ with the added benefit of resistance to potato tuber moth. Potato tuber moth is a serious pest of potato in South Africa. This insect causes damage during the growing season and in potato storage. The life cycle of potato tuber moth can be completed within 20 to 30 d and there may be as many as 12 generations in a single year (Raman, 1980). Adult tuber moths live for 10 to 15 d and during this time the females lay clusters of eggs on potato plants (leaves, stems, and tubers), storage containers (sacks, boxes, etc.), and dirt or debris surrounding tubers (Raman, 1980). Larvae typically emerge from the eggs after 5 d of incubation (Goldson and Emberson, 1985) and begin feeding on leaves, stems, and tubers. Larval mining on the plant results in the loss of leaf tissue, death of growing points, and weakening or breakage of stems (Raman, 1980), all Received for publication 19 Jan. 2010. Accepted for publication 2 Apr. 2010. Corresponding author. E-mail: [email protected]. J. AMER. SOC. HORT. SCI. 135(4):333–340. 2010. 333 of which can reduce yield. Tuber mining results in tubers that are not marketable or consumable and renders tubers susceptible to infection by potato pathogens (Alvarez et al., 2005). After 8 d of feeding, the larvae pupate and emerge as adults after 6 to 9 d (Alvarez et al., 2005). Host plant resistance to potato tuber moth is highly desirable, and components of resistance such as glandular trichomes and acetylated glycoalkaloids (leptines) have been identified in wild potato species such as Solanum berthaultii, Solanum polyadenium, Solanum tarijense, and Solanum chacoense. However, attempts to introduce these resistance components into cultivated potato have been difficult due to linkage with undesirable traits (Kalazich and Plaisted, 1991). Currently, traditional breeding has not produced a commercial potato cultivar that is resistant to potato tuber moth (Lagnaoui et al., 2000). Therefore, commercial producers rely on insecticide application for tuber moth control in the field, and subsistence level farmers that cannot afford insecticides have no recourse. Aldicarb (a carbamate) is applied at planting and organophosphates or pyrethroids are generally applied at weekly intervals starting when the first potato tuber moths appear or when specific tuber moth thresholds are reached. Insecticides are applied from eight to twelve times per season, with the number of applications depending on the season of the year as well as on the level and progression of the infestation. Tuber moth infestation is typically lower and slower to progress during the winter season thus fewer applications are necessary. However, control is not always satisfactory and damage levels vary between seasons and years, depending largely on the survival of overwintering moths and their reinfestation of newly planted fields (Visser, 2004). Adult tuber moths can migrate from infested fields to cull piles and storages to lay eggs. Tuber moth eggs are destroyed by low temperature (1.7–4.4 C) exposure for 4 months, while all other life stages are destroyed by temperatures in excess of 36 C for 15 d (Trivedi and Rajagopal, 1992). Therefore, in cull piles and storage, where temperatures are often ideal and no insecticides are applied, all life-stages of tuber moths are likely to survive the winter and to infest potato fields the following season (Alvarez et al., 2005). Potato tuber moth not only attacks tubers in the soil, but also in storage. Unfortunately, there are no insecticides registered for use in South Africa to control potato tuber moth under storage conditions, including Bt sprays (Nel et al., 2002). The only control strategy that gives consistently good control against the potato tuber moth is the use of genetically modified, insectresistant potatoes containing the cry1Ia1 gene (Visser, 2004). ‘SpuntaG2’ is a transgenic potato cultivar containing the cry1Ia1 gene from Bacillus thuringiensis for resistance to potato tuber moth. This cultivar has been characterized at the molecular level (Zarka et al., 2010) and has been thoroughly evaluated for safety as a human food source (Quemada et al., 2010). The current study focuses on the agronomic performance of ‘SpuntaG2’ and its efficacy against potato tuber moth in the field and in storage. Materials and Methods FIELD TRIAL DESIGN. Five years of field trials were conducted at seven South African sites to evaluate ‘SpuntaG2’ for resistance to potato tuber moth and for agronomic performance. The sites were chosen to represent the various growing regions in South Africa and included Ceres, Dendron, Roodeplaat, Kokstad (irrigated), Kokstad (dry land), Patensie, and Petrus Steyn. All field trials were randomized block designs with four replicates and are summarized in Table 1. ‘Spunta’, ‘Mnandi’, and ‘BP1’ were used as control cultivars. ‘Mnandi’ is favored by small-scale farmers and has potato late blight (Phytophthora infestans) resistance. The cultivar BP1 is the top commercial cultivar in South Africa and ‘Spunta’ is the non-transgenic cultivar from which ‘SpuntaG2’ was derived. Each year greenhouse-grown, virusand disease-free mini-tubers were planted. The only exception was the 2005–06 season during which tubers harvested from the previous year’s trials were used for planting. Plots consisted of three rows of test material bordered on each side by non-transgenic controls. Rows were 3 m in length with 1 m spacing between rows and 10 seed pieces were planted per row. However, during the 2005–06 and 2006–07 seasons, plot sizes were altered at Petrus Steyn, Dendron, and Roodeplaat to accommodate the practices and equipment of the farm operation. At the Dendron site, the plot rows were 5 m in length with 0.8 m spacing between rows and 16 seed pieces planted per row. The Roodeplaat 2005–06 site was planted in rows that were 11 m long with 1.34 m row spacing and 36 plants per row. At the Petrus Steyn site, rows were 9 m long with 1.65 m spacing between rows and 36 tubers were planted per row. Supplemental irrigation was applied as needed with the exception of the Kokstad dry land site and the Petrus Steyn site. At all sites, natural infestations of potato tuber moths were used with the exception of the 2002–03 Roodeplaat site where 30,000 tuber moths were released over the course of the growing season. Fields were inspected regularly for tuber moth infestations by scouting the non-transgenic control plots to look for the presence of leaf mines. When more than 15% of the plants in the control plots were attacked, the rest of the plots were scouted as well. During scouting, the following data were recorded from 25 randomly selected haulms per plot: the number of haulms attacked, the number of leaf mines present, and the number of live larvae. Two weeks before harvest, all haulms were destroyed by herbicide application (paraquat) or by slashing or pulling the haulms. At harvest, all tubers from each plot were kept separate in clearly marked containers. Weighing and sorting were done on sorting tables in the potato stores at the South African Agricultural Research Council’s (ARC) facilities in Roodeplaat, and by hand in the field at all other locations. Tubers from all plots were weighed separately after sorting into standard categories: large (>200 g), medium (>80 and <200 g), small (<80 g), and unmarketable (small enough to fall through the sorter or rotten/damaged). While sorting, all tubers with any sign of tuber moth damage were sorted out into the nonmarketable group. These nonmarketable tubers were inspected later and all tubers with potato tuber moth damage were weighed. All tubers were stored in temperature-controlled cooling facilities. All rotten or discarded tubers were killed by freezing or burning and were then buried in 2to 3-m-deep holes. During the 2005–06 and 2006–07 seasons in Roodeplaat, ‘Spunta’ and ‘SpuntaG2’ plots were evaluated for several agronomic properties by A. Visser. These properties included maturity, growth habit, foliage cover, mature plant height, tuber size distribution, tuber dimensions/shape, skin color, eye morphology, growth cracks, skin cracking/mechanical damage, flesh color, internal qualities, specific gravity, chip scores, cooking quality, and response to Fusarium dry rot (Fusarium spp.), stem-end rot (Fusarium spp.), common scab (Streptomyces scabies), potato late blight, potato early blight (Alternaria solani), and root knot nematode (Meloidogyne spp.). All traits 334 J. AMER. SOC. HORT. SCI. 135(4):333–340. 2010. were evaluated in accordance with the protocols established by the International Union for the Protection of New Varieties of Plants (UPOV, 1986). In addition, 64 pollen samples from both cultivars were tested for fertility with the lacto phenol-acid fuchsin pollen fertility staining technique (Alexander, 1980) and with pollen germination on artificial media (Mortenson et al., 1964). Statistics were conducted using analysis of variance (ANOVA) and Fisher’s protected least significant difference (LSD) for means separation (a = 0.05). All ANOVA were performed using the SAS (release 8.20; SAS Institute, Cary, NC) general linear model procedure at a = 0.05. Agronomic evaluations of ‘SpuntaG2’ were also conducted in Michigan during the 2000, 2001, and 2002 seasons. The field experiments were planted in a randomized complete block design with four replications at the Montcalm Research Farm, Entrican, MI. The plots were 7 m long with 0.3 m spacing between plants and an interrow spacing of 0.86 m. Supplemental irrigation was applied as needed. In each of the trials, the yield was graded into four size classes: A = 113 to 285 g, B = <113 g, oversize = >285 g, and pickouts = unmarketable. Tubers >8.3 cm in diameter or >285 g in weight were scored for incidence of external and internal defects, and samples of A-sized tubers were taken to determine specific gravity. Statistics for the replicated field trials were conducted using ANOVA and LSD for means separation (a = 0.05). All ANOVA were performed using the SAS (release 8.20) general linear model procedure at a = 0.05. STORAGE TRIALS. After the completion of the field trials in 2002–03, 2003–04, and 2004–05, diffused light storage trials were started, using undamaged tubers harvested from the field. Three different storage facilities were used to simulate the types of storage set-ups used by small-scale and commercial growers in South Africa: a rustic out-building (Roodeplaat), an out-building in good condition with one window (Kokstad), and a commercial storage facility (Ceres). Tubers were stored for 6 months under diffused light storage conditions to evaluate the efficacy of the cry1Ia1 gene under long-term storage. Ten tubers of each treatment were stored in mini-crates stacked in a randomized block design. Four replicates were used (one replicate per stack or rack). In addition to natural infestations, 20 to 40 tuber moths were released into all the crates at 2to 3-week intervals during the first 2 months of the storage experiment. Tuber moths for these experiments were collected from the farms and were reared on potato tubers until released into the storage crates.