Combining Natural and Engineered Host Plant Resistance Mechanisms in Potato for Colorado Potato Beetle: Choice and No-choice Field Studies

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 fi ve different host plant resistance mechanisms were evaluated for 2 years in a fi eld situation under natural colorado potato beetle pressure in Michigan and New York, and in a no-choice fi eld cage study in Michigan. In the fi eld studies, the S. chacoensederived 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 Btcry3A 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 fi eld trials and no-choice fi eld 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. The colorado potato beetle is the most serious insect pest of potatoes throughout the eastern and north central United States and Canada. 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 the ability to adapt to every insecticide used for its control (Bishop and Grafi us, 1996). Currently, it has developed resistance to 41 insecticides, including organophosphates, carbamates, organochlorines, pyrethroids, hydrogen cyanide, and more recently the neonicotinoids imidacloprid and thiamethoxam (Byrne et al., 2004; Georgiou and Lagunes-Tejeda, 1991; Whalon et al., 2004). Host plant resistance is a central component of a practical longterm solution for controlling the colorado potato beetle in potato. No potato varieties resistant to colorado potato beetle are currently available. Glandular trichomes and leptine glycoalkaloids are two of the most thoroughly investigated natural insect host plant resistance mechanisms available in potato. The glandular trichomes of the wild Bolivian potato, Solanum berthaultii Hawkes, confer resistance to at least ten major insect pests, including the colorado potato beetle (Tingey, 1991). The presence of Type A and B trichomes in S. berthaultii leads to entrapment and death of Received for publication 4 Mar. 2005. Accepted for publication 7 May 2005. This publication was made possible through support provided by the Michigan Agriculture Experiment Station, USDA/ARS cooperative agreement 59-07902-060, and the Michigan Potato Industry Commission. small-bodied insects and reduces developmental time, survival, and oviposition of colorado potato beetle (Yencho and Tingey, 1994). However, in a no-choice situation, colorado potato beetle rapidly adapted to S. berthaultii (Groden and Casagrande, 1986). NYL235-4, the glandular trichome clone evaluated in this study, is the result of a series of backcrosses from S. berthaultii, with reported resistance to colorado potato beetle (Plaisted et al., 1992). Leptine glycoalkaloids have been defi ned as effective natural resistance mechanisms of potato against colorado potato beetle, but are found in only a few accessions of S. chacoense (Sinden et al., 1986a, 1986b). The North Dakota State University breeding line ND5873-15 (ND4382-19 x ʻChipetaʼ) evaluated in this study has shown resistance to colorado potato beetle in the fi eld, derived from S. chacoense (Lorenzen et al., 2001). The insecticidal proteins produced by Bt target specifi c orders of insects and have no known toxicity to mammals or birds (Lavrik et al., 1995). Bacillus thuringiensis subsp. tenebrionis produces the Cry3A δ-endotoxin, which is toxic to Coleoptera, including colorado potato beetle (Krieg et al., 1983). Bt-transgenic potatoes have been developed for resistance to colorado potato beetle and potato tuberworm (Phthorimaea operculella Zeller) (Douches et al., 1998; Perlak et al., 1993; Sutton et al., 1992). Coombs et al. (2002) transformed potato clones that have natural host plant resistance mechanisms (glandular trichomes and leptine glycoalkaloids) with a synthetic Bt-cry3A gene. 554 857 10/18/05 4:34:47 PM 858 J. AMER. SOC. HORT. SCI. 130(6):857–864. 2005. Use of combined resistance mechanisms in a single variety has been suggested as a strategy for delaying the development of insect adaptation to host plant resistance mechanisms, especially Bt (Cooper et al., 2004; Gould, 1998; McGaughey et al., 1998; Tabashnik, 1994). The colorado potato beetle has demonstrated the ability to develop resistance to Bt Cry3A in the lab (Whalon et al., 1993) and to glandular trichome-based resistance (Groden and Casagrande, 1986). Our strategy was to combine in potato genotypes, different sources of natural and engineered host plant resistance mechanisms. These natural host plant resistance mechanisms, combined with engineered Bt-transgenic resistance, should increase the durability and effi cacy of 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 to control colorado potato beetle and reduce defoliation in the fi eld and in 8-m3 screen cages. Materials and Methods Six different potato clones representing fi ve different host plant resistance mechanisms were evaluated in this study (Table 1). Nontransgenic potato clones included: NYL235-4 with glandular trichomes (Plaisted et al., 1992); ND5873-15 with fi eld resistance to colorado potato beetle (provided by Drs. R. Novy and J. Lorenzen at North Dakota State Univ.); and ʻNorwis ̓ (susceptible control). The Bt-cry3A (coleopteran specifi c) gene used for transformation of the transgenic potato lines in this study was supplied by Dr. J. Kemp at New Mexico State Univ. (Sutton et al., 1992). The constitutive ocs3mas promoter (Ni et al., 1995) was used to express Bt-cry3A and the Bt-cry3A transgenic potato lines were generated using Agrobacterium tumefaciens Smith and Townsend-mediated transformation (Coombs et al., 2002): The Bt-cry3A transgenic clones containing single or combined resistance factors (ND5c3.1, NYLc3.3, and NORc3.8) used in this study were developed in our lab (Table 1). Bt-Cry3A concentration in the foliage of the three Bt-cry3Atransgenic lines was determined using a Bt-Cry3A ELISA kit (Agdia, Elkhart, Ind.). Leaf samples (three to four leaves down from the top of the plant) were collected from 60-d-old fi eldgrown plants in the cage study. Samples (75 mg) were placed in Agdia tissue homogenizer envelopes and placed at –80 °C. Samples were homogenized in 20 μL of the extraction buffer and then diluted 1:500. ELISA was done using 100 μL of this 1:500 dilution according to the manufacturerʼs instruction. Absorbance of the ELISA plates were measured at 405 nm after a 30 min incubation period. The fi eld trials were conducted in 2000 and in 2001 at the Michigan State Univ. Montcalm Research Farm in Entrican, Michigan, and the Cornell Univ. Long Island Horticultural Research and Extension Center in Riverhead, N.Y. The Michigan fi eld location was chosen for its history of high levels of natural colorado potato beetle infestation. The colorado potato beetle population at the Montcalm Research Farm in Entrican, Mich., is known to be resistant to most insecticides, including organochlorines, organophosphates, carbamates and pyrethroids, similar to most populations present in commercial potatoes in Michigan (Bishop et al., 2001; Ioannidis et al., 1991). The experiment was replicated in Long Island, N.Y., to evaluate the performance of the host plant resistance clones against colorado potato beetle populations with high levels of resistance to most insecticides including imidacloprid (Zhao et al., 2000). The Michigan and New York research sites have been maintained and used for various colorado potato beetle fi eld research since the 1970s and 1950s, respectively (e.g., Grafi us and Blakeslee, 1978; Noling et al., 1984; Roberts et al., 1981). Potato seed tubers were obtained from fi eld-grown tissue culture transplants. The experimental design of the fi eld study was a randomized complete-block design with four replications of 10 plants each. Treatment plots (2.5 m long, 25 cm within-row spacing between plants) were planted between rows of a susceptible guard (ʻSnowden ̓in Michigan and ʻNorwis ̓in New York) (86 cm spacing between rows) to maintain an even distribution of colorado potato beetles across fi eld plots. Observations were recorded on a weekly basis for a visual estimation of percent defoliation by colorado potato beetles and the numbers of egg masses, larvae, and adults per plant. Data were collected until vine senescence or until defoliation levels reached 100% on the susceptible clone. A no-choice fi eld cage study was conducted in 2002 at Michigan State Univ., East Lansing. A no-choice study was important because it more closely models a commercial fi eld situation where only one variety would likely be grown and adults would not have a choice for feeding or egg laying. For example, in our fi eld trials, each treatment row was adjacent to a row of susceptible potatoes (ʻSnowden ̓in Michigan, ʻNorwis ̓in New York). Females may avoid ovipositing on a less-preferred line in a varietal trial; the potato line may have few larvae and appear resistant. However, in a no-choice situation (e.g., a commercial potato fi eld) the lesspreferred variety may be perfectly acceptable for oviposition, larval survival and development. Plots in cages were two rows by fi ve plants (86 cm between rows, 25 cm between plants in a row), with three replications Table 1. Host plant resistant potato clones (natural, engineered and combined) evaluated against colorado potato beetles (CPB) in choice field studies (Michigan and New York) and a cage no-choice field study (Michigan). Potato clone Host plant resistance mechanism Construct z Source y Parent clone ND5873-15 CPB-resistant (S. chacoense-derived) None-NT NDSU NA x NYL235-4 Glandular trichomes None-NT Cornell NA ‘Norwis’ Susceptible control None-NT NA NA ND5c3.1 CPB resistant (S. chacoense-derived) + Bt-cry3A ocs3mas/cry3A MSU ND5873-15 NYLc3.3 Glandular trichomes + Bt-cry3A ocs3mas/cry3A MSU NYL235-4 NORc3.8 Bt-cry3A ocs3mas/cry3A MSU ‘Norwis’ z Transgene construct information: ocs3mas: trimer octopine synthase and mannopine activator and promoter; cry3A: Bt-cry3A is a coleopteran-specific insecticidal crystal; None-NT = nontransgenic potato line. y Source of plant material: NDSU = North Dakota State University, Fargo; Cornell = Cornell Univ., Ithaca, N.Y.; MSU = Michigan State Univ., East Lansing.