Field Evaluation of Natural, Engineered, and Combined Resistance Mechanisms in Potato for Control of Colorado Potato Beetle

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 Btcry3A transgenic lines (‘Russet Burbank Newleaf,’ RBN15, and YGc3.1). The glandular trichome (NYL235-4) and Btcry5+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. needs compared to foliar Bt applications (Lambert and Peferoen, 1992). Currently, there are 40 major groups of Bt endotoxins, designated Cry1 through Cry40 with 108 holotypes (Crickmore et al., 2002), offering toxicity toward Lepidoptera, Coleoptera, and Diptera. The Bt-Cry3A protein is effective against Coleoptera (Adang et al., 1993; Perlak et al., 1993; Sutton et al., 1992) and BtCry5 (Cry1Ia1) is active against Lepidoptera and Coleoptera (Tailor et al., 1992). Glandular trichomes and leptine glycoalkaloids are two natural host plant resistance mechanisms of potato that are candidates for pyramiding with Bt-transgene(s). The presence of type A and B trichomes leads to entrapment and death of small-bodied insects (Gregory et al., 1986) and reduces developmental time, survival, and oviposition of colorado potato beetle (Casagrande, 1982; Wright et al., 1985; Yencho and Tingey, 1994). The trichomes of S. berthaultii Hawkes are associated with insect responses to host plant resistance including host avoidance, restlessness, reduced feeding, reduced development, reduced adult weight, reduced reproductive performance, decreased longevity, and increased mortality (Tingey, 1991). Glandular trichomes have been bred into cultivated potato from the wild species S. berthaultii and are available as a breeding line NYL235-4 (Plaisted et al., 1992). Glycoalkaloids are the most common form of antibiosis in potato (Sinden et al., 1986a). Steroidal glycoalkaloids (solanine and chaconine) are present in all potato plant parts, including the tubers, and may cause human health concerns if present at high levels (Sinden, 1987). Glycoalkaloid levels below 20 mg/100 g FW Received for publication 24 June 2002. Accepted for publication 13 Nov. 2002. This publication was made possible through support provided by the Michigan Agriculture Experiment Station and the National Potato Council. The research was also supported by the Office of USAID/CAIRO/AGR/A, under cooperative Agreement No. 263-0152 A-00-3036-00. Corresponding author; e-mail [email protected]. The colorado potato beetle, Leptinotarsa decemlineata (Say), is the most serious insect pest of potatoes (Solanum tuberosum L.) throughout the eastern and north central United States. Control of the colorado potato beetle has relied almost entirely on pesticides for over 125 years (Casagrande, 1987). Throughout its history, the colorado potato beetle has shown an ability to adapt to every insecticide used for its control (Forgash, 1985). Currently, it is reported worldwide to be resistant to 37 insecticides, including organophosphates, carbamates, organochlorines, pyrethroids, and hydrogen cyanide (Georgiou and Lagunes-Tejeda, 1991). Bacillus thuringiensis (Bt) is an aerobic, gram-positive, soil bacterium that accumulates high levels of insecticidal proteins during sporulation (Barton and Miller, 1993; McGaughey and Whalon, 1992). Different Bt toxins target specific orders of insects and have no known detrimental effects on mammals or birds (McGaughey and Whalon, 1992). The insecticidal crystal proteins produced by Bt are products of single genes, which have been isolated, analyzed, characterized, cloned, and codon-modified to increase expression in plants (Adang et al., 1993; Perlak et al., 1993; Sutton et al., 1992). Transgenic plants deliver the Bt toxin with increased efficacy, reduced application costs, and minimal scouting 9282-Genet 1/10/03, 2:24 AM 219 220 J. AMER. SOC. HORT. SCI. 128(2):219–224. 2003. (mg%) are considered safe for human consumption. Solanum tuberosum generally contains only 2 to 10 mg% glycoalkaloids. Leptines are the most active form of glycoalkaloids present in potato for insect control (Lawson et al., 1993) and demonstrate antifeedant properties against colorado potato beetle at concentrations as low as 100 mg% (Sanford et al., 1996). Leptines and other acetylated glycoalkaloids are reported to be synthesized by only a few accessions of S. chacoense Bitter (Sanford et al., 1996). Human health concerns about high glycoalkaloids are not an issue as leptines are synthesized and present only in the leaves and not the tubers (Sanford et al., 1996). The high leptine-producing accession of S. chacoense, USDA8380-1 (PI 458310), is a diploid selection containing ≈300 mg% leptines (constituting 90% of the total glycoalkaloids) with strong antifeedant properties against colorado potato beetle (Deahl et al., 1991; Sinden et al., 1986a). These natural host plant resistance mechanisms, combined with engineered Bt-transgenic resistance, could increase the durability and efficacy of potato host plant resistance to the colorado potato beetle. The objective of this study was to evaluate various natural, engineered, and combined host plant resistance mechanisms in potato for control of colorado potato beetle under field conditions. Materials and Methods Nine different potato clones representing five different host plant resistance mechanisms were evaluated in a choice situation under natural colorado potato beetle pressure at the Montcalm Research Farm in Entrican, Mich. Table 1 describes the vector construct, source, and parent clones of the plant resistance mechanisms. Nontransgenic potato clones included: USDA8380-1 (S. chacoense Bitter P.I. 458310) with high foliar leptines (provided by L. Sanford, USDA–ARS, Beltsville, Md.); ND5873-15 with high foliar total glycoalkaloids (TGA) (data not shown) (provided by R. Novy and J. Lorenzen at North Dakota State University); NYL235-4 with glandular trichomes (Plaisted et al., 1992); and ‘Russet Burbank’. The two Bt genes used were Bt-cry3A (coleopteran specific) and Bt-cry5 (Bt-cry1Ia1) (leptidopteran and coleopteran specific). The Bt-cry3A transgenic potato clones used in this study included Btcry3A-Russet Burbank (RBN15) provided by J. Kemp at New Mexico State University; ‘Russet Burbank Newleaf’ (NatureMark); and the Bt-cry3A-Yukon Gold (YGc3.1) developed in our laboratory (Coombs et al., 2002). The Bt-cry5 transgenic clones used were SPc5-G2 (Douches et al., 2002) and NYL235-4c5.13, developed in our laboratory via Agrobacterium tumefaciens transformation as described by Douches et al. (1998). The synthetic Bt-cry3A gene used was supplied by J. Kemp at New Mexico State University (Sutton et al., 1992). The codon-modified Bt-cry5 gene was provided by Syngenta (formerly Zeneca) (Tailor et al., 1992). All potato lines were maintained in tissue culture by nodal propagation in 25 x 150 mm culture tubes or GA-7 Magenta vessels (Magenta Corp., Chicago, Ill.) in modified Murashige and Skoog (1962) medium [4.3 g·L MS salts, 3% sucrose, 1.4 mM sodium phosphate, 1.1 μM thiamine, 0.55 mM myo-inositol, pH 6.0, 8 g·L Bactoagar (Difco, Detroit, Mich.)], and sealed with Micropore surgical tape (3M Company, St. Paul, Minn.). The tissue culture growth environment was 16/8 L/D photoperiod under 30 μmol·m·s fluorescent bulbs at 25 ± 2 °C. Tissue culture plants were transplanted to greenhouse trays (50 per tray, 3 cm diameter), grown for four weeks in the greenhouse, and then transplanted to the field in 1998. Tubers were saved from the 1998 trial and used as seed for the 1999 trial, with the exception of USDA8380-1 (a nontuberizing line under long-day conditions), which were grown as tissue culture-derived transplants. Field trials were conducted in 1998 and in 1999 at the Michigan State University Montcalm Research Farm in Entrican, Mich. A randomized complete block design consisting of four replications of 10 plants each was used. Treatment plots (25.4 cm plant spacing) were planted between alternating rows (86.4 cm row spacing) of a susceptible guard, ‘Snowden’. In 1998, greenhouse-grown tissue culture plantlets were transplanted to the field. In 1999, tuber seed pieces (≈57 g each) were planted, except USDA8380-1, which was handled as previously mentioned. Observations were recorded about weekly for a visual estimation of percent defoliation by colorado potato beetles, number of egg masses, and number of first, second, third, fourth instars, and adults per plant. Data were collected until vine senescence; from 1 July to 8 Aug. (42 d) in 1998 and from 17 June to 26 July (39 d) in 1999. The colorado potato beetle population at the Montcalm Research Farm is known to be resistant to most insecticides including organochlorines, organophosphates, carbamates, and pyrethroids; and is similar to most colorado potato beetle populations found in commercial potato plantings in Michigan (Ioannidis et al., 1991). This Table 1. Potato lines indicating plant resistance mechanism.