Field Experiment

The experiments were conducted at the Sweet Potato Research Station, Louisiana State University Agricultural Center, Chase, Louisiana, in 1997 and 1998. “Beauregard” and “Excel” were used because Beauregard, a major cultivar in the region, is susceptible to SPW and Excel has shown a moderate level of resistance (Story et al. 1996). The treatments were 2 × 2 factorial combinations of water treatment (drought stressed and irrigated) by genotype arranged in a randomized complete block design with 4 replications. Each plot consisted of four 25-plant rows. Uniform transplants were mechanically transplanted on 30 June, 1997, and 27 June, 1998 in a Gilbert silt loam with a pH of 5.6 at 0.3-m spacing within rows on 1.0-m centered beds. The fields were fumigated with Telone™ C-17 (1,3-dicholropropene) 2 weeks before transplanting. Standard cultural practices were followed throughout the growing season (Boudreaux 1994).

The drought stress treatment was initiated 50 days after transplant (DAT) by constructing moveable rain shelters over the plots to exclude natural precipitation. The shelters were placed over the plots wherever there was more than 30% chance of precipitation in the local weather forecast. Otherwise, the plots were left open. The irrigated plots were watered starting 3 weeks after transplant with drip tubes (3.8 ml/min). Storage roots were harvested at 120 DAT, cured (30°C, 90% RH for 7 d), and stored at 15 ± 2°C for about 30 d before the bioassays and chemical analyses were started.

Insect Rearing

A SPW colony was established in January of 1997 from a field-collected population (about 500 adult insects) and maintained on storage roots of Beauregard in plastic containers (5.6 L) with screen covers at 28 ± 2°C and 85 ± 10% RH in the laboratory located at Louisiana State University Baton Rouge campus. In preparing experimental insects, 5 fresh storage roots (U.S. #1) were exposed to about 1000 unsexed adults for 5 d and then removed and kept under the conditions described above. Emerging adults were collected weekly and held with fresh storage roots. Female adults 3-4 wk old were used in the bioassays to ensure adequate egg-laying capability (Wilson et al. 1988).

Adult Feeding and Oviposition Bioassay

The bioassay technique was an adaptation of one previously described by Mullen et al. (1980) that has been used in several SPW feeding and oviposition studies (Wilson et al. 1988). The apparatus consisted of a 24-well tissue culture plate (12.5 × 8.5 × 2.0 cm, Falcon® Model 3047, Becton Dickenson & Co., Lincoln Park, NJ) placed in a rectangular clear plastic container (17 × 12 × 6 cm, Tri-State Plastic, Dixon, KY). Cores were cut from storage roots with a cork borer (1.6 cm diameter) and were inserted into the wells so that only the periderm was exposed. The diameters of the cores and the wells were the same, providing a close fit. Female adults were kept without food for 3 h before being introduced into the arena at a density of 2 weevils per root core. A moist cotton ball was placed in the container to prevent desiccation of the cores. After 24 h the number of feeding punctures on each root core was counted, and after 48 h the number of eggs was counted. No-choice tests were conducted by presenting a single root core in the arena. Free-choice tests were conducted by presenting 4 root cores in the arena which were cut from one root (U.S. #1) randomly selected from each treatment combination. Before testing, the roots were gently washed with tap water and allowed to dry. All tests were conducted at 28 ± 5°C, 85 ± 10% RH under total darkness to eliminate light as a variable. For each treatment, the tests were repeated 4 times with 4 roots (sampling units). Roots from four field blocks were tested in 4 consecutive weeks.

Larval Survival and Development Bioassay

SPW were reared individually in Petri dishes by transferring a single egg into a root section (about 1.5 × 1.5 × 1.5 cm) in a cavity (1-2 mm deep, 4.0 mm diameter) cut with a cork borer. Eggs were obtained by exposing Beauregard storage roots to a large number of females for 24 h. A pair of needle-nosed forceps was used to transfer eggs. At 12 d after the eggs were deposited, root sections were examined to determine if eggs had hatched. Nonviable eggs or rotten root sections were discarded. At about 25 d after oviposition, root sections were dissected for pupae. Larval survival and pupal weight were recorded. Two replications of each treatment combination were conducted with sample sizes ranging from 18 to 32 pupae each. The bioassays were conducted under conditions of 28 ± 5°C and 85 ± 10% RH in total darkness.

Chemical Analysis

The chemical analysis was conducted in the USDA-ARS Vegetable Laboratory, Charleston, South Carolina. Storage roots were carefully washed under flowing water and allowed to dry. Periderm tissue was gently scraped off with a scalpel, dried at 50°C, and ground to a fine powder in liquid nitrogen with a mortar and pestle. Subsequently the powder was re-dried at 40°C and stored in vials under nitrogen at -20°C until analysis. Powder samples were weighed (200 mg) into Teflon-lined, screw-capped test tubes, and 2.0 ml of methanol were added containing 0.08 mg of chrysin (recrystallized from amyl alcohol) as an internal standard. Test tubes were ultrasonicated for 20 min while the surrounding water was ice-cooled. The tubes were centrifuged and the supernatant was filtered through Nylon-66 membrane filters (0.20 μm, Pierce Chemical Company, Rockville, IL) into auto injector vials. Resin glycosides and caffeic acid concentrations were analyzed by reverse-phase HPLC with 20 μl of the solution. For resin glycosides, a H₂O/MeOH linear gradient from 60% to 100% MeOH in 15 min was used and held at 100% MeOH for 25 min; flow rate was 1 ml min-1 and detection was at 230 nm. For caffeic acid, a second injection of 20 μl was made, with the same sample as was used for the resin glycosides analysis. A H₂O/MeOH linear gradient from 10% to 100% MeOH in 35 min was used and held at 100% MeOH for 25 min; flow rate was 1 ml min^-1 and detection was at 340 nm. Each solvent contained 0.1% H₃PO₄. The column used was a Beckman Ultrasphere C₁₈, 5 μm (4.6 × 250 mm, Beckman and Coulter, Fullerton, CA). Purified reference substances were used as external standards to determine response factor versus chrysin for quantification. Reference glycoside material was purified by Sephadex column chromatography followed by semi-preparative HPLC as described previously (Peterson et al. 1998). Reference caffeic acid was purchased from Aldrich Chemical Company (Milwaukee, WI).

Data Analysis

The data were analyzed by year using two-way analysis of variance (PROC GLM, SAS 1990). A square-root transformation was used for larval survival data. Year effect was evaluated by analyzing the data by one-way analysis of variance. The significance level was α = 0.05.

Adult Feeding and Oviposition

Drought stress significantly increased adult SPW feeding and oviposition in free-choice tests and oviposition in no-choice tests in 1997 (Table 1). In 1998, drought stress had no significant effect on oviposition or on feeding (Table 1). Year effect was significant on feeding (no-choice test: F = 23.08, df = 1,24, P < 0.0001; free-choice test: F = 10.09, df = 1,24, P = 0.0041) and on oviposition under both testing conditions (no-choice test: F = 50.51, df = 1,24, P <0.0001; free-choice test: F = 17.50, df = 1,24, P = 0.0003). Beauregard received more feeding punctures than Excel in free-choice tests, but not in no-choice tests in 1997. No significant cultivar effect was found on oviposition in 1997. In 1998, cultivar had a significant effect on both feeding and oviposition in free-choice tests where Beauregard was the preferred cultivar, but there was no cultivar effect in no-choice tests (Table 1). The interaction of drought stress and cultivar was significant for feeding in free-choice tests in 1997, but not in 1998 (Table 1).

Larval Survival and Development

Drought stress significantly reduced larval survival rate in 1997, but not in 1998 (Table 2). No significant drought effect was found on pupal weight in both years. Higher larval survival rate was found on Beauregard than on Excel in 1997 and in 1998 (Table 2). Weevils reared on Beauregard had lower pupal weight than that of Excel in 1997, but not in 1998. Drought and cultivar interaction effect was significant for larval survival in 1997, but not in 1998. No significant interaction effect was found with pupal weight (Table 2). The test for year effect was not significant for larval survival (F = 1.18; df = 1,16; P = 0.2936) and pupal weight (F = 1.34; df = 1,16; P = 0.2642).

Resin Glycosides and Caffeic Acid Contents

Drought stress did not have a significant effect on the level of resin glycosides in either year (Table 3). Drought stress significantly reduced the level of caffeic acid in 1997, but not in 1998. Excel tended to have a higher level of resin glycosides than Beauregard, but this difference was statistically significant only in 1998. Both genotypes contained similar levels of caffeic acid (Table 3). No significant interaction effect was found. Year effect was significant for caffeic acid (F = 88.45; df = 1,21; P < 0.0001), but not for resin glycosides (F = 0.01; df = 1,21; P = 0.9283).