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Evaluating maize for allelochemicals that affect European corn borer (lepidoptera: crambidae) larval development.

TRADITIONAL FIELD SCREENING of sweet corn germplasm effectively increased ear resistance to European corn borer (Davis et al., 1993; Warnock et al., 1998). Sweet corn germplasm with improved native resistance to ear feeding is available (Davis et al., 1993) and the use of hybrids with improved ear resistance can significantly limit ECB damage (Bolin et al., 1996). Sweet corn genotypes with native resistance factors can limit ECB feeding damage at levels comparable with Bacillus thuringiensis (Bt) transgenic sweet corn genotypes (Burkness et al., 2001). The resistance mechanism(s) of these hybrids, however, is unknown. Both biophysical components, such as tight, narrow, silk channels or tight husk cover, and biochemical components are thought to be involved (Grier and Davis, 1980; Joyce and Davis, 1995; Warnock et al., 1997), but the components are not easily separated in field studies. Laboratory bioassays, without the confounding effects of biophysical components, may facilitate the detection of allelochemicals in sweet corn tissues affecting ECB larval development (Warnock et al., 1997).

Three types of compounds occurring in corn tissues have been shown to affect insects detrimentally in laboratory bioassays: caffeic acid derivatives, flavonoid glycosides, and the hydroxamic acid family typified by chlorogenic acid, maysin, and DIMBOA (2,4-dihydroxy7-methoxy-1, 4-benzoxazine-3-one), respectively (Gueldner et al., 1991). Wiseman et al. (1996) identified maysin as the active component in several corn lines resistant to corn earworm and fall armyworm. However, maysin, initially extracted from corn silk (Waiss et al., 1979; Elliger et al., 1980), has little activity against ECB.

DIMBOA, a compound found in corn, is active against first generation ECB (Klun and Brindley, 1966; Russell et al., 1975), but is not a major resistance factor for second generation ECB populations as levels decrease with plant maturity (Klun and Robinson, 1969). Other researchers hypothesized that phenolic acid derivatives increase host plant resistance to ECB damage by strengthening plant cell walls (Bergvinson et al., 1994). Abou-Zaid et al. (1993) indicated that flavones and flavonols were active against ECB. Several compounds similar to flavones and flavonols are found in corn tissues at varying levels (Ceska and Styles, 1984). However, the relationship between flavone and flavonol levels in corn tissues and insect resistance is unknown.

The breeding strategy of combining multiple forms of resistance into a single variety is termed pyramiding. Attempts to pyramid native and transgenic forms of resistance may limit ECB population shifts toward increased tolerance. Isolation and identification of allelochemicals may allow breeders to select for novel forms of native resistance in maize to be combined with transgenic resistance. Bioassays incorporating the resistance component(s) are necessary for proper identification of allelochemicals affecting ECB larval development. The resistance components, however, must be isolated and identified before commercial availability or production techniques for the purified component can be developed. As an initial step in the isolation and identification of active allelochemicals, this research was designed to (i) identify maize genotypes and ear tissues with biological activity against ECB larval development, (ii) isolate silk tissue extracts detrimentally affecting ECB larval development, (iii) determine if absorption peaks detected through HPLC are associated with biological activity, (iv) determine if ferulic acid and p-coumaric acid affect larval development in bioassays, and (v) determine the relationship between resistance in the laboratory with resistance to ear damage in the field, as well as to the level of specific elements in ear tissues. To accomplish these objectives, a series of laboratory bioassays and field experiments were conducted.

MATERIALS AND METHODS

Tissue Preparation

One susceptible field (Su) corn inbred (W182E) and nine sweet (su) corn types (two inbred lines--MN 3152 and MN 3153 and 7 [F.sub.1] hybrids--Apache, Jubilee, MN 270, MN 272, MN 275, MN 276, and MG 15) with field resistance levels varying from commercially acceptable to unacceptably susceptible were field grown at St. Paul, MN. For each genotype, varying amounts of seed were hand planted into 33-m rows on 0.75-m centers on 27 and 28 May in 1994 and 1996, respectively. The number of rows for each genotype varied from one to six, depending on the availability of seed. Upon emergence, each row was thinned to approximately 130 equally spaced plants. Standard dry-land production methods for the Midwest were used except that no insecticides were applied. Overhead irrigation was applied as needed in 1996.

For each genotype, silk tissue from the primary ear of each plant, less the first 15 to 25 plants in the first row of each genotype, was bulk harvested 3 to 5 d after emergence from the ear tip, placed in paper bags, frozen, and stored at -20 [degrees] C. Ears of the remaining 15 to 25 plants of each genotype were grown to the commercial processing stage (~720 g [kg.sup.-1] kernel moisture) and bulk harvested within genotype. Kernel tissues were cut from the cob, frozen, and stored at -20 [degrees] C. The frozen silk and kernel tissues were kept separate and were lyophilized over a 3- to 4-mo period, as facilities permitted, and subsequently ground with a Wiley mill to pass through a 0.6-mm mesh screen. The ground lyophilized tissues were then stored at -20 [degrees] C and subsequently used in the bioassays described below.

Tissue Bioassay

Tissues harvested in 1994 and 1996 were used in assays conducted in the winters of 1995 and 1997, respectively. Silk and kernel tissues, maintained separately for each genotype, were divided into three replications and individually incorporated into a meridic diet (1 g tissue/20 mL diet) for rearing ECB larvae (Warnock et al., 1997). A cellulose control was maintained throughout the assay to obtain 21 diet-tissue combinations, including controls, per replication. Thirty individual 35-mL plastic cups containing 15 to 20 mL of a diet-tissue treatment combination were each infested with a single ECB neonate, capped with a plastic lid, and placed in a growth chamber at 25 [degrees] C, 24-h photoperiod, and 75% relative humidity as described by Warnock et al. (1997). Treatment combinations were arranged in a split-split-plot design with genotypes as main plots, tissues as sub-plots, and cups as sub-sub-plots. Replications were separated over time within year.

Larval weight was recorded at 10-d intervals on a cup basis, and individual larvae were transferred to fresh diet-tissue medium. Time to pupation, time to moth emergence, survival, and sex of moths were noted for each insect. Initial data analysis, indicated the cup (main) effect was not a source of variation. Thus, the cup variable was included in the error mean to simplify further analysis. Data were combined across years and analyzed as a split-split-plot design with year as main plot, genotype as sub-plot, and tissue as sub-sub-plot, by the general linear models (GLM) procedure of SAS (SAS Institute, 1990). Significantly different means were separated by Fischer's protected LSD ([alpha] = 0.05).

Extraction Bioassay

Because silk tissue from 1994 was limited, a portion of the dried, ground silk tissue from 1996 was divided into three replications for the extraction bioassay. On the basis of results of the Tissue Bioassay and silk tissue availability, seven genotypes (three inbred lines--W182E, MN 3152, and MN 3153 and four [F.sub.1] hybrids--Apache, Jubilee, MN 276, and MG 15) that affected larval development were used in the extraction bioassay. The extraction procedure of Coseteng and Lee (1987) was modified such that for each replication, 4 g of prepared silk tissue from each genotype were combined with 80 mL 70% (v/v) methanol, boiled for 5 min, filtered through Whatman No. 1 filter paper under vacuum, resuspended in 80 mL 70% methanol, boiled for 10 min, filtered, and the supernatants combined within genotype. To create controls for solvent and residual effects, the extraction procedure was performed on 4 g of cellulose. Though a harsh extraction procedure, previous bioassays indicated (i) that the biologically active compounds remained in tissue residual when extracted with cold methanol, (ii) that some of the biologically active compounds remained in the tissue residual and some were captured in the supernate when extracted with room temperature methanol for 24 h, and (iii) that the biologically active compounds were pulled from the tissue residual and captured in the supernate when exposed to boiling methanol by the modified Coseteng and Lee method presented above (Warnock, 1998). For each replication, the eight supernatants were each condensed with a rotavapor-R (Buchl, Switzerland) from 160 mL to approximately 50 mL. The 50-mL aliquot was subdivided into a 5-mL sample for storage and a 45-mL sample that was coated onto 4 g of an inert cellulose carrier as described by Chan et al. (1978) and Reese et al. (1989) for analysis by means of a laboratory bioassay. The eight supernatants coated onto cellulose, the seven silk residuals, and the cellulose residual were dried at room temperature (-25 [degrees] C) for approximately 36 h before incorporation into a meridic diet (1 g tissue/20 mL diet) for rearing ECB larvae as described by Warnock et al. (1997). Cellulose and nonextracted, lyophilized silk tissues from Apache and Jubilee sweet corns were added to the meridic diet to create three bioassay controls. Thus, there were 19 treatment combinations, including the controls, per replication.

For each of the three replications, 32 individual 2-mL wells of a bioassay tray (Model #BIO-BA-128, C-D International, Pitman, NJ) were filled with 1.5 mL of a treatment combination before infestation with a single ECB neonate. The trays were sealed with vented covers (Model #BIO-CV-16, C-D International, Pitman, NJ) and placed in an incubation chamber at 25 [degrees] C, 24-h photoperiod, and 75% relative humidity. The 19 treatment combinations were arranged in a completely randomized block design with replications separated over time. In contrast to the procedure described by Warnock et al. (1997), the bioassay trays were not rotated within the incubation chamber on a daily basis. Because of limited amounts of maize tissues, the tissue extraction bioassay was limited to a 10-d period at which time larval weight and survival were recorded on a larva basis. Results from previous bioassays indicated that 10-d larval weights were correlated with final insect fitness on the basis of developmental times. Data were analyzed by the GLM procedure of SAS (SAS Institute, 1990), with mean separation by Fischer's protected LSD.

HPLC Analysis

For each replication, the 5-mL methanol sample from each genotype was placed in a microcentrifuge tube, labeled, wrapped in foil, and stored at -- 15 [degrees] C. After 4 wk, the samples were thawed, 1 mL was removed from each vial and the remainder returned to storage. After centrifugation for 15 min at 14 000 g, the supernate of each 1-mL sample was filtered through a 0.45-[micro]m syringe filter, placed into a microcentrifuge tube, and stored as above.

The HPLC system consisted of two pumps (Waters Models 590 and 510, Milford, MA), a Waters Model 680 gradient controller and a Hewlett Packard Co. (Avondale, PA) Model 1040A photodiode array detector. Separation was made with a Brownlee Aquapore [C.sub.8] RP-300 column (250- by 4.6-mm i.d.) manufactured by Applied Biosystems (Foster City, CA).

HPLC quality methanol and acetonitrile were obtained from Fisher Scientific (Pittsburgh, PA). Deionized, distilled water was withdrawn from a Millipore Milli-Q filtering system (Milford, MA). All other reagents were of analytical grade.

Elution was at 1.0 mL/min with a step gradient from 100% Buffer A (20 mM K[H.sub.2]P[O.sub.4] pH 5.0) to 100% Buffer B (20 mM K[H.sub.2]P[O.sub.4] pH 5.0: acetonitrile, 25:75, v/v) as described by Keukens et al. (1994). The column was maintained at room temperature, and the injection volume was 25 [micro]L. Detection of peaks was monitored at 220 nm. As an HPLC system control, an Apache silk tissue extract from the first replication was injected each day samples were analyzed.

Elemental Analysis

A 2-g portion of the remaining 1994 and 1996 silk and kernel tissues from each genotype was divided into two replications and analyzed for Kjeldahl nitrogen (Bremner, 1965), and for 16 additional elements by inductively coupled plasma-atomic emission spectrometry by the dry ash method (Munter and Grande, 1981). Cellulose was included in the elemental analyses as a control. For each element, ANOVAs of the main effects (year, genotype, and tissue) were determined by the GLM procedure of SAS (SAS Institute, 1990), followed by mean separation ([alpha] = 0.05) by Fischer's protected LSD. The relationship between elemental content and tissue bioassay activity, as well as between elemental content and resistance in the field was estimated by Spearman's rank correlation coefficients calculated on a plot mean basis for genotype and tissue.

Ferulic Acid/p-Coumaric Acid Bioassay

A sample of the 1996 Apache and Jubilee silk tissues was delivered to the USDA-ARS Forage Plant Cell Wall Chemistry Laboratory at the University of Minnesota and analyzed for phenolic acids as per Jung and Shalita-Jones (1990). HPLC analysis detected high levels of ferulic acid (FA) in the silk tissues of Apache and Jubilee, whereas the level was much lower for cellulose. A second phenolic acid, p-coumaric acid (PCA), possibly associated with ECB resistance in leaf tissue (Bergvinson et al., 1994), occurred at levels below 0.20 mg/g dried tissue for each genotype and cellulose.

Commercial ferulic acid and p-coumaric acid were obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI). Both chemicals were incorporated into the meridic diet at a rate comparable to those found in cellulose (<0.20 mg/g tissue for FA and PCA) and silk tissues from Apache (2.61 and <0.20 mg/g dried silk tissue for FA and PCA, respectively) or Jubilee (1.67 and <0.20 mg/g dried silk tissue for FA and PCA, respectively). Lyophilized silk tissues of each genotype and cellulose were maintained as controls during the bioassay. Two replications of 32 larvae per treatment combination were arranged in a randomized complete block, and the bioassay was conducted as described for the Extraction Bioassay except that the trays were rotated on a daily basis. Ten-day larval data were recorded and analyzed as previously described.

Field Evaluation

The three inbred lines and seven [F.sub.1] hybrids used in the bioassays also were planted in a completely randomized block design at St. Paul, MN, on 27 and 26 May in 1994 and 1996, respectively. To limit competitive effects, inbreds and hybrids were separated within the field. For both years, about 25 kernels of each genotype were hand-planted into three single-row plots (3.3 m), with rows spaced on 0.75-m centers. Plots were thinned to 13 equally spaced plants. Each 3.3-m plot represented a single replication for each genotype. Thus, three replications per genotype were planted and evaluated for each of 2 yr. The field layout was bordered by a commercial sweet corn hybrid. Cultural practices common to the production of dry-land commercial sweet corn were followed, except that no insecticides were applied and irrigation was applied in 1996 as needed.

The University of Minnesota Department of Entomology supplied ECB larvae from a laboratory colony established and reared to maintain genetic diversity, vigor, aggressiveness, and freedom from pathogens, as described by Reed et al. (1972). Within 3 d after silk emergence, a single application of ~50 neonate ECB larvae mixed with dry, ground, maize cobs (grits) were applied to the silks on the top ear of each plant with a Davis inoculator (Wiseman et al., 1980). Infestation was conducted over 23 d in 1994 and 20 d in 1996, as a reflection of plant-to-plant and plot-to-plot variation in silking date. After several days of open pollination, infested ears were protected from bird damage with color-coded paper pollinating bags, which served also to identify each ear by infestation date. Each infested ear was evaluated for feeding damage after an accumulation of ~225 heat units (10 [degrees] C base) (Grier and Davis, 1980) by a 1-to-9 visual rating scale based on economic damage assessed as degree of failure to meet the requirements of the sweet corn processing industry, where 1 = no damage and 9 = damage to >10% of kernels on the ear (Davis et al., 1994; Warnock and Davis, 1998)

Data, collected on a plant basis, were combined across years before analysis by the GLM procedure of SAS (SAS Institute, 1990). Means were separated ([alpha] = 0.05) by Fischer's protected LSD. For each genotype, the relationship between tissue bioassay activity and field ear damage was estimated by the CORR procedure of SAS.

RESULTS

Tissue Bioassay

Larval survival at 10 d did not differ by year, genotype, or tissue, and there were no main effect interactions (Table 1). Seventy-three, 95, and 99% of the larvae reared on diet with silk, kernel, and cellulose, respectively, had pupated 20 d after inoculation, thus reflecting a developmental delay associated with the influence of silk tissue in the diet that carried through to pupation. The number of larvae surviving to pupation varied (F = 25.78; df = 1; P [is less than or equal to] 0.01) by tissue (Table 1), averaging about 88 [+ or -] 0.7 and 81 [+ or -] 0.9% on diets containing kernel and silk, respectively, but larvae on neither diet differed from larvae on the diet containing the cellulose control (84 [+ or -] 2.7%).

Mean 10-d larval weight differed by genotype (F = 5.04; df = 9; P [is less than or equal to] 0.01) and tissue (F = 341.74; df = 1; P [is less than or equal to] 0.01), with significant year x genotype, year x tissue, and genotype x tissue interaction (Table 1), while larval weights recorded on Day 20 were considered biased as more than 70% of the larvae had pupated. Larvae reared on diet containing silk tissue weighed 45 to 65% less after 10 d, averaging 25.9 mg, than larvae reared on diet containing kernel tissue (57.8 mg), and the latter did not differ from the cellulose control (62.8 mg). Thus, silk tissue may contain allelochemical(s), which detrimentally affect larval growth.

Silk tissue of all genotypes reduced 10-d larval weight, varying from 26.0% (Jubilee) to 80.4% (W182E), compared with the cellulose control (Table 2). Larvae reared on diet with W182E silk tissue had the lightest weight followed closely by larvae reared on diet modified with silk tissue from Apache (Table 2). Larvae reared on diet modified with kernel tissue had 10-d weights equally above and below the cellulose control mean (Table 2).

Across silk and kernel tissues, year x genotype and year x tissue interactions (Table 1) also affected mean 10-d larval weight. Larvae reared on diet containing Jubilee, MN 270, or MG 15 tissues were heavier in 1994 than larvae on all other diet tissue combinations except the cellulose control. While in 1996, Jubilee, MN 272, MN 275, and MN 276 tissues provided the heavier larval weight means. The range of means varied by year across genotypes and may be a reflection of different field environmental regimes between 1994 and 1996.

Mean 10-d larval weight was reduced when larvae were reared on diet containing the 1996 cellulose or kernel tissue versus the 1994 cellulose or kernel tissue. Mean 10-d weights of larvae reared on diet containing silk tissue did not differ between 1994 and 1996.

Mean pupal weight differed by year (F = 28.38; df = 1; P [is less than or equal to] 0.01), genotype (F = 2.24; df = 9; P [is less than or equal to] 0.05), and tissue (F = 53.51; df = 1; P -< 0.01) (Table 1). Mean pupal weight increased from 85.4 mg in 1994 to 102.1 mg in 1996, even though males outnumbered females in 1996 on the basis of Chi-square analysis (df = 1, P = 0.196; df = 1, P = 0.013, for 1994 and 1996, respectively). Female pupae normally are larger than males (Beck, 1989). Thus, an unequal ratio of females to males may bias larval weight.

Larvae reared on diet containing cellulose (control) attained the heaviest pupal weight (102.6 mg), followed by 96.0 mg with kernel tissue and 89.8 mg with silk tissue. The percentage of larvae reaching pupation on cellulose-, kernel-, and silk-modified diet was 84.4, 88.3, and 81.2, respectively. Chi-square analysis indicated that the expected 1:1 gender ratio was maintained on the cellulose control (df = 1, P = 0.417) but not on treatments containing silk (df = 1, P = 0.042) and kernel tissues (df = 1, P = 0.041). Males outnumbered females on the kernel and silk modified diets indicating that pupal weights for these treatments could be lower than expected.

Across tissues, all maize genotypes reduced pupal weight (F = 53.51; df = 1; P [is less than or equal to] 0.01) compared with the cellulose control (Tables 1 and 2). Pupal weight ranged from 83.3 to 102.6 mg for larvae reared on meridic diet with silk tissue from W182E and cellulose, respectively (Table 2). Some corn genotypes producing low 10-d larval weight, such as W182E and Apache, differed from one another and the cellulose control for pupal weight. Thus, a reduced 10-d larval weight did not guarantee a reduced pupal weight (Table 2).

The time to pupation differed for genotype (F = 7.42; df = 9; P [is less than or equal to] 0.01) and tissue (F = 249.03; df = 1; P [is less than or equal to] 0.01), and year x tissue, genotype x tissue, and year x genotype x tissue interactions occurred (Table 1). Across genotypes, larvae reared on diet with silk tissue had an increased mean time to pupation (19.1 d) compared with larvae reared on diet containing kernel tissue or cellulose (16.2 and 15.9 d, respectively). These results paralleled those for 10-d larval weights, reflecting the significant negative correlation (r = -0.67) found between 10-d larval weight and time to pupation. Larvae reared on diet containing silk tissue from W182E, Apache, MN 3153, or MN 276 had increased times to pupation compared with larvae reared on silk tissue of all other genotypes or the cellulose control (Table 2). Larvae reared on diets modified with kernel tissues had similar times to pupation (Table 2).

The year x tissue interaction reflected variable responses of larvae reared on the 1994 versus 1996 tissues. Larvae reared on diet with 1994 and 1996 silk tissues pupated 18.8 [+ or -] 0.14 and 19.3 [+ or -] 0.11 d after inoculation, respectively, while larvae reared on diet with kernel or cellulose tissues pupated 2 to 4 d earlier.

The year, genotype, and tissue main effects and their interactions significantly affected moth emergence (Table 1). Year x genotype and year x genotype x tissue interactions had small mean square values (61.65 and 87.18, respectively) when compared with the mean square values of year, tissue, genotype x year, and year x tissue effects (2,482.35, 7,344.85, 250.40 and 1,491.88, respectively).

Adults from larvae reared in 1996 emerged 26.5 [+ or -] 0.07 d after infestation, 2.8 d longer than larvae reared in 1994. Larvae reared on diet with kernel tissue or cellulose provided moths ~3 d earlier than larvae reared on diet with silk tissue (26.8 [+ or -] 0.10 d), suggesting that the delay in larval development noted at 10 d and pupation continued through to moth emergence. The relationship between time to pupation and time to moth emergence was relatively strong (r = 0.86).

Extraction Bioassay

When larvae were reared on meridic diet containing lyophilized silk tissue, methanol extracts of silk tissue, or residual silk tissue, 10-d larval survival ranged from 94.8 to 100% (data not shown), but did not vary by replication or treatment (Table 3). However, mean 10-d larval weight varied by replication (F = 68.15; df = 2; P [is less than or equal to] 0.01) and by treatment (F = 11.42; df = 18; P [is less than or equal to] 0.01) (Table 3) on meridic diet containing either silk tissue extracts or silk tissue residuals of the 7 corn silks extracted with boiling 70% methanol.

For any given treatment combination, larvae in Replication 3 consistently were heavier than larvae in Replication 2 or Replication 1. Mean larval weight decreased from Replication 3 (38.6 mg) to Replication 2 (32.2 mg) to Replication 1 (26.2 mg), reflecting replication location differences within the incubation chamber.

Mean 10-d larval weight was reduced by all methanol extracts added to the meridic diet, except those from Jubilee, compared with the nonextracted cellulose control and the methanol extract from cellulose (Table 4). Furthermore, larvae reared for 10 d on any silk tissue residual, except the Jubilee residual, did not significantly differ in mean weight from larvae grown on the nonextracted cellulose control (Table 4), suggesting that allelochemicals present in silk tissues effectively were captured by methanol extraction. As presented earlier, the Tissue Bioassay results indicate that silk tissue from all of the genotypes, except Jubilee, compared with kernel and cellulose tissues slowed ECB development (Table 2). Only the methanol extract from MG 15 silk tissue reduced 10-d larval weight as much as the nonextracted Apache silk tissue control (Table 4). Also, larvae reared on diet containing the methanol extract from Apache silk reached greater mean weights than larvae reared on diet containing nonextracted silk tissue of Apache. These results suggest that some allelochemical activity may have been lost during the extraction procedure.

HPLC Analysis

Preliminary fractionation HPLC of the most consistently bioassay-active methanol extracts showed the presence of three to four large peaks appearing 20 to 23 min after injection with high absorbance at 220 nm (Fig. 1a and 1b). These peaks were absent in the less active methanol fraction from Jubilee (Fig. 1c) and in the inactive methanol fraction from cellulose (Fig. 1d). Although HPLC results do not prove that the peaks identify allelochemicals that reduce 10-d larval weights, the circumstantial evidence is supportive. Additional partitioning with baseline separation of the methanol fraction of silk tissues and absorbency spectrums at varying wavelengths is necessary before association between the detected peaks and bioassay activity can be defined.

[FIGURE 1 OMITTED]

Elemental Analysis

Results of the Tissue and Extraction Bioassay indicate that larvae reared on diet containing kernel tissue or cellulose were heavier than larvae reared on diet containing silk tissue. The question arose as to whether silk tissue may be limiting in its nutritional value and thus may slow larval development and weight gain.

Elemental analysis indicated that nine of the 17 elements tested were below detectable levels in some samples of the 10 corn genotypes (Table 5). Because elemental amounts below detectable levels may be greater than zero, the lowest detectable level is used when calculating average elemental means. This standard procedure causes the calculated mean for an individual genotype to be larger than the actual mean (Table 5). Consequently, statistical procedures were biased and mean separation unreliable for the nine elements with at least one sample below detectable levels. The remaining eight elements, however, were above the minimum detectable levels in all samples, allowing proper mean separation.

Of the eight elements, mean elemental level across ear tissue varied by genotype for potassium (F = 693.95; df = 1; P [is less than or equal to] 0.01), calcium (F = 639.86; df = 1; P [is less than or equal to] 0.01), manganese (F = 53.35; df = 1; P [is less than or equal to] 0.01), silica (F = 949.76; df = 1; P [is less than or equal to] 0.01), and zinc (F = 729.78; df = 1; P [is less than or equal to] 0.01), suggesting nutritional differences could have affected larval growth and development (Table 6). Of the eight elements with unbiased means, potassium, calcium, magnesium, manganese, silica, and zinc were not limiting in silk tissue, because levels of these 6 elements were greater in silk tissue than in kernel tissue or cellulose (Table 5). However, possible toxic effects of one or more of these elements, while unlikely, cannot entirely be dismissed in this bioassay. Although increased silica levels have been shown to improve leaf feeding resistance to ECB in some corn varieties (Rojanaridpiched et al., 1984), high silica levels in the current bioassay were not associated with detrimental effects on larval development. Calcium levels were not associated with reduced larval growth. Generally, as potassium and zinc levels increased in maize tissues, mean 10-d larval weight increased. Manganese was the only element correlated negatively (r = -0.70) with 10-d larval weight, suggesting that manganese levels may have reached toxic levels within some genotypes (Table 6). Thus, most of the elements did not reduce larval growth and development even if they were present at higher levels in silk tissue than in kernel tissue or in cellulose.

FA/PCA Bioassay

The addition of FA or PCA at levels found in silk tissues of Apache or Jubilee did not affect 10-d larval survival (Table 7). Survival ranged from 95.3 % on diet containing PCA at the level found in cellulose to 100% on diet containing either FA or PCA at the levels found in Apache silk, diet containing lyophilized silks of Apache or Jubilee, and diet containing cellulose.

Although 10-d larval weight varied (F = 15.42; df = 8; P [is less than or equal to] 0.01) by meridic diet additives (Table 7), neither the addition of FA nor PCA reduced larval weight compared with the cellulose control (Table 8), suggesting that free FA and PCA were not the active allelochemicals in Apache silk tissue. The addition of lyophilized silk tissues from Apache and Jubilee to the meridic diet reduced mean 10-d larval weight by 60.8 and 14.7%, respectively, compared with the cellulose control (Table 8), as in the Extraction Bioassay.

Field Resistance

Several of the Minnesota hybrids had less damage (F = 17.27; df = 9; P [is less than or equal to] 0.01) than Jubilee, the susceptible control (Table 2). Field damage and bioassay activity were not correlated (P > 0.05). Though tissue changes in storage or during the extraction procedure cannot be ruled out as a cause of poor correlation between field damage and bioassay activity, morphological features, such as lengthened silk channels and tight husks, are factors more likely contributing to this lack of correlation. Two lines, MN 3152 and W182E, which had little ear resistance in the field, had high levels of bioassay activity based on the 10-d weights of larvae reared on meridic diet with silk tissue or silk tissue extracts (Tables 2 and 4). Some field susceptible lines, therefore, may contain allelochemicals that reduce larval growth and development. Though not measured quantitatively, husk tightness was similar for all lines except W182E and Jubilee, which had loose husks. Tight husks may limit insect penetration to the kernel tissues, while loose husks allow insects to bypass silk tissues containing allelochemicals that negatively affect insect development. MN 270 had high resistance in the field but less bioassay activity than the most active genotypes, Apache and W182E, suggesting that the field resistance of this line mainly was of a biophysical nature. MN 270 had a slightly longer silk channel (6-7 cm) than Apache (5-6 cm) or Jubilee (2-4 cm), which may confer ear resistance. Apache and MN 272, which had high levels of ear resistance as well as high bioassay activity (Table 2), may combine biophysical and biochemical resistance, thus contributing to the observed field resistance against ECB.

DISCUSSION

The U.S. Environmental Protection Agency recently approved the commercial release of both dent and sweet corn hybrids with resistance to Lepidopteran larvae through the expression of a Bt protein gene (Burkness et al., 2001; Ostlie et al., 1997; Lynch et al., 1999). Widespread planting of transgenic hybrids, however, may shift insect populations toward greater resistance (Alstad and Andow, 1995; Ostlie et al., 1997). Laboratory colonies of ECB have been selected for resistance to Bt toxins (Bolin et al., 1999; Huang et al., 1997), and at least two isolated larvae have been found feeding on transgenic plants in fields of Bt corn (Pierce et al., 1998). As transgenic corn acreage increases, methods to limit the development of ECB resistance may become increasingly important.

The improvement of maize resistance to ECB is a goal for many host plant resistance breeding programs. Field evaluations, while beneficial, are limited in determining if specific resistance factors are responsible for reduced feeding damage. Elimination of maize morphological features allows researchers the opportunity to screen for chemical resistance factors. Laboratory bioassays facilitate the detection of allelochemicals in sweet corn tissues affecting ECB larval development (Warnock et al., 1997). When combined, field and laboratory results provide a more complete picture of host plant resistance to ECB than either one alone.

The laboratory bioassays presented above give a better understanding of maize tissue impact on larval development when every stage of larval development is evaluated. The developmental delays noted for 10-d larval weight were apparent for time to pupation and time to moth emergence with the detrimental effects of silk tissues from W182E, Apache, MN 31.53 and MN 276. W182E and Apache, which produced the lightest larvae after 10 d of feeding, and the longest times to pupation may exceed the other genotypes in levels of allelochemical(s) affecting larval development. The allelochemicals may differ between genotypes, however, because larvae reared on diet containing tissues from Apache attained a high pupal weight (~cellulose control) but at a slower rate than larvae reared on diet containing W182E tissues. This form of resistance therefore may be classified as antibiosis, as larval growth and development are affected (Painter, 1951). In field environments, a slowing of larval development tends to decrease larval survival through increased exposure to predation and other detrimental environmental features (LaBatte et al., 1997). Silk tissue from Jubilee had little effect on larval development at any stage, suggesting that this genotype is undesirable from a host plant resistance standpoint.

Though tissue and genotype effects were greatest, significant differences existed between years for larval development. The variability between 1994 and 1996 might be attributable to the environmental conditions under which tissues were produced or the laboratory colony may have been inadvertently selected for various developmental delays. With the exception of 10-d pupal weight and moth emergence, larvae development was similar for both years and thus does not support the notion that the colony was altered. The environmental conditions under which the ear tissues were grown varied from normal in 1994 to excessively dry in 1996. Climatic conditions between years easily may have influenced tissue composition and hence larval development. Differences in levels of feeding damage in the field evaluations are common. For traits with complex inheritance patterns, such as ear feeding resistance (Warnock et al., 1998), breeders rely on multiple evaluation sites and years to ensure that the most desirable genotypes are utilized. Because tissues grown under field conditions can vary, laboratory bioassays conducted with tissue from a single season should be viewed as limited.

Just as chemical composition may vary annually, one may assume that tissue elemental content may be influenced by climate. The addition of tissues to a meridic diet may cause essential elements to become limiting or toxic. Adkisson et al. (1960), Chippendale and Beck (1964), and Vanderzant et al. (1962) examined the nutritional consequences of individual elements during the development of a meridic diet for ECB. However, discussion of the levels at which individual elements might become toxic was not included. Johnson (1980) found maize silk tissue reduced larval weight compared with kernel tissue, but could not separate allelochemical effects from nutritional effects because a nutritionally incomplete diet was used. Warnock et al. (1997) found that the larger volume of silk compared with the smaller volume of kernel tissue when tissues were incorporated on a weight basis into a complete meridic diet had no effect on ECB development. In the present study, the use of a complete diet decreased the possibility of reducing larval development through nutritional limitations. The eight elements with unbiased means were not limited in silk tissue and only manganese might be at toxic levels.

Bergvinson et al. (1994) concluded that free FA and PCA indirectly confer ECB leaf feeding resistance by strengthening cell walls. Cell wall strength in silk tissue may affect feeding resistance in some sweet corn genotypes. Grinding tissues before incorporating them into the larval diet should eliminate most of the biophysical components of resistance. However, if high cell wall integrity improves resistance against ECB, grinding may not completely eliminate this resistance factor. Tissue extraction or digestion with various solvents could alleviate possible cell wall effects. In the Extraction Bioassay, the removal of active allelochemicals with methanol eliminated potential cell wall physical effects on host plant resistance to ECB.

In the Extraction Bioassay, failure to exercise daily rotation of assay trays in the incubation chamber, a departure from the protocols presented by Warnock et al. (1997), created a temperature gradient. Differences in larval weight between replications can be explained by this temperature gradient. Larval development is temperature driven and larvae in Replication 3, located nearest the light/heat source, matured more quickly. Ellsworth et al. (1989) showed that an increase in temperature from 20 [degrees] C to 25 [degrees] C could result in a 55% increase in development rate. Larvae in the tissue and subsequent bioassays in which the trays were rotated had no larval weight differences between replications.

The Extraction Bioassay indicated that chemical resistance components were present in several maize genotypes and could be removed. The identity of these allelochemicals was unknown but FA and PCA affect feeding resistance (Bergvinson et al., 1994). Because Apache and Jubilee silk tissues contained high levels of FA and little PCA, the effects of FA and PCA were tested separately. On the basis of the current research, one may conclude that the active allelochemical(s) extracted from silk tissues of the various corn genotypes by methanol are neither FA nor PCA, but the identity of specific compounds and whether synergistic effects occur between FA and PCA is unknown.

The identification of maize genotypes, such as W182E, Apache, MN 3153, and MN 276, which possess silk tissue detrimentally affecting larval growth and development when incorporated into a nutritionally complete diet indicates that chemical resistance components may be involved in ear resistance. Future research with silk tissue extracts may lead to the identification of the active allelochemicals affecting ECB larval growth and development. Isolation, identification, and exploitation of these allelochemicals by breeding programs may lead to improved ear resistance in field environments. Host plant resistance breeding programs, however, should not neglect morphological features, such as an extended silk channel length and tight husks, which confer resistance to ECB. Maize genotypes, such as Apache and MN 272, with chemical resistance components and morphological resistance features have multiple defenses against ECB. The addition of previously unexploited native resistance to the already effective transgenic forms of resistance should enhance the durability of sweet corn resistance to ECB.

Abbreviations: Bt, Bacillus thuringiensis, ECB, European corn borer, FA, ferulic acid, PCA, p-coumaric acid.
Table 1. ANOVA summary for seven developmental parameters of ECB when
reared on diet containing silk or kernel tissue of 10 corn genotypes
evaluated for two growing seasons.

                        Larval survival   Larval weight

Source ([dagger])   df  10 day  Pupation  10 day  Pupal

Year (Y)             1    NS       NS       NS     **
Rep within
 Y = Error a         4    --       --       --     --
Genotype (G)         9    NS       NS       **      *
Y x G                9    NS       NS       **     NS
Rep x G within
 Y = Error b        40    --       --       --     --
Tissue (T)           1    NS       **       **     **
Y x T                1    NS       NS       **     NS
G x T                9    NS       NS       **     NS
Y x G x T            9    NS       NS       NS     NS
Rep x G x T within
 Y = Error c        40    --       --       --     --

                    Time to    Time to
Source ([dagger])   pupation  emergence

Year (Y)               NS         *
Rep within
 Y = Error a           --        --
Genotype (G)           **        **
Y x G                  NS         *
Rep x G within
 Y = Error b           --        --
Tissue (T)             **        **
Y x T                  **        **
G x T                  **        **
Y x G x T              **        **
Rep x G x T within
 Y = Error c           --        --

* Indicates significance at P = 0.05.

** Indicates significance at P = 0.01.

*** Indicates significance at P = 0.001.

NS = nonsignificant.

([dagger]) Error terms for the variables were (i) replication within
year for year, (ii) replication x genotype within year for genotype,
year x genotype, and (iii) replication x genotype x tissue within
year for tissue, year x tissue, genotype x tissue, and year x
genotype x tissue.
Table 2. Mean 10 d larval weight, pupal weight, time to pupation, and
time to moth emergence of ECB larvae reared on meridic diet containing
silk or kernel tissue from 10 corn genotypes, and mean visual estima-
tion (1-9) of ear damage at ~225 heat units (base 10 [degrees] C)
after manual infestation in the field.

                           10 Day
                       larval weight          Pupal weight

                       Mean [+ or -]
Genotype  Tissue       SEM ([dagger])       Mean [+ or -] SEM

                                        mg
W182E     Silk       12.3 [+ or -] 0.63a    83.3 [+ or -] 2.00a
Apache    Silk       14.8 [+ or -] 0.92b    92.7 [+ or -] 1.74cd
MN 3153   Silk       18.0 [+ or -] 1.03c    91.0 [+ or -] 1.84bcd
MN 276    Silk       18.0 [+ or -] 1.00c    86.6 [+ or -] 1.91b
MG 15     Silk       20.3 [+ or -] 0.77d    90.2 [+ or -] 1.67bc
MN 3152   Silk       20.9 [+ or -] 0.92d    88.0 [+ or -] 1.88b
MN 272    Silk       24.3 [+ or -] 0.93e    90.1 [+ or -] 1.64bc
MN 275    Silk       25.2 [+ or -] 0.97ef   87.9 [+ or -] 1.69b
MN 270    Silk       28.5 [+ or -] 0.95g    89.6 [+ or -] 1.76bc
Jubilee   Silk       46.5 [+ or -] 1.00h    93.1 [+ or -] 1.06de
W182E     Kernel     51.0 [+ or -] 2.07i    92.1 [+ or -] 1.73cd
MN 272    Kernel     52.7 [+ or -] 1.56ij   96.4 [+ or -] 1.95el
Apache    Kernel     53.5 [+ or -] 1.91j    98.4 [+ or -] 1.83fg
MN 3152   Kernel     54.8 [+ or -] 1.86jk   92.9 [+ or -] 1.71d
MN 275    Kernel     57.0 [+ or -] 1.99l    98.9 [+ or -] 1.73g
Jubilee   Kernel     57.5 [+ or -] 1.17l    95.0 [+ or -] 1.10ef
MN 270    Kernel     58.5 [+ or -] 2.26l    95.9 [+ or -] 1.87ef
MN 3153   Kernel     62.2 [+ or -] 2.21m    98.7 [+ or -] 2.09g
Control   Cellulose  62.8 [+ or -] 1.47m   102.6 [+ or -] 1.83h
MN 276    Kernel     62.9 [+ or -] 2.23m    94.8 [+ or -] 1.79ef
MG 15     Kernel     68.6 [+ or -] 2.23n    98.7 [+ or -] 1.77g

              Time to                 Time to
              pupation               emergence

Genotype  Mean [+ or -] SEM      Mean [+ or -] SEM

                              d
W182E     21.8 [+ or -] 0.33j    29.7 [+ or -] 0.38i
Apache    21.2 [+ or -] 0.32i    29.2 [+ or -] 0.32h
MN 3153   21.1 [+ or -] 0.45i    28.8 [+ or -] 0.51h
MN 276    21.3 [+ or -] 0.44i    28.4 [+ or -] 0.40h
MG 15     19.3 [+ or -] 0.24h    27.4 [+ or -] 0.31g
MN 3152   18.8 [+ or -] 0.24g    26.5 [+ or -] 0.23f
MN 272    18.8 [+ or -] 0.21g    26.8 [+ or -] 0.26f
MN 275    18.8 [+ or -] 0.23g    26.7 [+ or -] 0.28f
MN 270    18.5 [+ or -] 0.20f    26.9 [+ or -] 0.27f
Jubilee   16.4 [+ or -] 0.11cde  24.2 [+ or -] 0.12e
W182E     16.7 [+ or -] 0.23e    24.2 [+ or -] 0.25e
MN 272    16.0 [+ or -] 0.14bc   24.0 [+ or -] 0.19de
Apache    16.3 [+ or -] 0.19bcd  24.2 [+ or -] 0.21e
MN 3152   16.1 [+ or -] 0.24bc   23.6 [+ or -] 0.22bc
MN 275    16.6 [+ or -] 0.28de   24.0 [+ or -] 0.25de
Jubilee   16.1 [+ or -] 0.11bc   23.7 [+ or -] 0.15cd
MN 270    16.4 [+ or -] 0.22cde  23.8 [+ or -] 0.26cd
MN 3153   16.0 [+ or -] 0.25bc   23.2 [+ or -] 0.21a
Control   15.9 [+ or -] 0.14b    23.7 [+ or -] 0.20cd
MN 276    16.2 [+ or -] 0.27bcd  23.7 [+ or -] 0.24cd
MG 15     15.6 [+ or -] 0.18a    23.3 [+ or -] 0.23ab

             Field ear
          damage ([double
             dagger])

Genotype  Mean [+ or -] SEM

               1 to 9
W182E
Apache
MN 3153
MN 276
MG 15
MN 3152
MN 272
MN 275
MN 270
Jubilee
W182E     6.91 [+ or -] 0.26e
MN 272    3.96 [+ or -] 0.40d
Apache    4.25 [+ or -] 0.20d
MN 3152   7.56 [+ or -] 0.21f
MN 275    3.58 [+ or -] 0.23bcd
Jubilee   6.75 [+ or -] 0.18e
MN 270    3.23 [+ or -] 0.28b
MN 3153   3.47 [+ or -] 0.23bc
Control           --
MN 276    3.90 [+ or -] 0.31d
MG 15     2.91 [+ or -] 0.22a

([dagger]) Means within column followed by the same letter do not
differ at P [less than or equal to] 0.05 on the basis of Fischer's
protected LSD. Means based on 180 observations, except for ear
damage in the field, which was based on 90 observations, across 3
replications and 2 years.

([double dagger]) 1 = no damage to husks, silks, or kernels; 2 = silk
and/or husk damage only; 3, 4, and 5 = less than 1%, 1-5%, and 6-10%
of tip kernels damaged, respectively; 6 and 7 = up to 5% and 6-10% of
tip and side or side only kernels damaged, respectively; 8 = cob
tunneling [greater than or equal to] 1 cm from the tip or shank; and
9 = more than 10% of the kernels damaged.
Table 3. ANOVA summary for mean 10 d survival and weight
of ECB larvae reared on meridic diet containing either supernatants
or extraction residues of seven corn genotype silk tissues extracted
with boiling 70% (v/v) methanol for 15 min (Treatment).

                     10 d larval  10 d larval
Source           df   survival    weight (mg)

Replication (R)   2      NS           **
Treatment (T)    18      NS           **
R x T (Error)    56      --           --

* Indicates significance at P = 0.05

** Indicates significance at P = 0.01.

*** Indicates significance at P = 0.001.

NS = nonsignificant.
Table 4. Mean 10 d weight of ECB larvae reared on meridic diet
containing either methanol extracts from corn silk tissue or
the silk tissue residual. Bioassay controls were meridic diet
containing (i) extracted cellulose (methanol extract or residual),
(ii) non-extracted cellulose, (iii) non-extracted silks of
Jubilee, or (iv) non-extracted silks of Apache.

                                   Mean 10-d larval
                                    weight [+ or -]
Genotype   Fraction ([dagger])   SEM ([double dagger])

                                          mg
Jubilee    Residual              42.09 [+ or -] 1.45a
W182E      Residual              39.08 [+ or -] 1.35ab
Cellulose  Residual              38.53 [+ or -] 1.37ab
MN 3153    Residual              38.47 [+ or -] 1.01ab
Apache     Residual              38.23 [+ or -] 1.29abc
MN 3152    Residual              37.46 [+ or -] 0.91abc
MN 276     Residual              37.02 [+ or -] 0.78abc
Cellulose  Methanol              35.49 [+ or -] 1.13bc
MG 15      Residual              35.01 [+ or -] 0.93bc
Cellulose  Non-extracted         34.22 [+ or -] 1.05bc
Jubilee    Methanol              32.87 [+ or -] 0.93cd
Apache     Methanol              28.09 [+ or -] 1.03de
MN 276     Methanol              27.89 [+ or -] 1.02def
MN 3152    Methanol              27.61 [+ or -] 1.11def
Jubilee    Non-extracted         27.43 [+ or -] 0.91ef
MN 3153    Methanol              26.87 [+ or -] 1.05ef
W182E      Methanol              26.72 [+ or -] 1.17ef
MG 15      Methanol              22.48 [+ or -] 1.05fg
Apache     Non-extracted         19.20 [+ or -] 0.92g

([dagger]) Residual, methanol, and non-extracted = silk tissue residue
after extracting with 70% (v/v) boiling methanol for 15 min, the
methanol supernate from the silk tissue extraction, and dried silk
tissue not given the extraction treatment, respectively.

([double dagger]) Means within column followed by the same letter do
not differ at P [less than or equal to] 0.05 on the basis of Fischer's
protected LSD. Each mean was based on 64 observations across two
replications.
Table 5. Mean level ([+ or -] SEM) of 16 elements in corn silk or
kernel tissue and cellulose as determined by inductively coupled
plasma-atomic emission spectrometry using the dry ash method;
nitrogen levels were determined by the Kjeldahi digestion method.

                            Level in tissue ([dagger])

Element                    Silk                     Kernel

                                       mg/kg
Aluminum (Al)      26.75 [+ or -] 1.93       <3.74 [+ or -] 0.06
Boron (B)          13.67 [+ or -] 0.38        2.98 [+ or -] 0.17
Cadmium (Cd)       <0.13 [+ or -] 0.00       <0.12 [+ or -] 0.00
Calcium (Ca)      847.48 [+ or -] 38.60a     63.98 [+ or -] 4.68b
Copper (Cu)         5.23 [+ or -] 0.34        2.22 [+ or -] 0.33
Chromium (Cr)      <0.44 [+ or -] 0.02       <0.29 [+ or -] 0.00
Iron (Fe)         183.00 [+ or -] 70.33a     85.10 [+ or -] 30.89a
Lead (Pb)          <1.79 [+ or -] 0.07       <2.68 [+ or -] 0.18
Magnesium (Mg)  1 309.61 [+ or -] 24.83a  1 063.91 [+ or -] 26.87b
Manganese (Mn)     11.49 [+ or -] 0.60a       4.13 [+ or -] 0.26b
Nickel (Ni)        <0.56 [+ or -] 0.03       <0.49 [+ or -] 0.03
Nitrogen
 (N) ([double
 dagger])            2.22 [+ or -] 0.06         2.11 [+ or -] 0.06
Phosphorus (P)   3 721.10 [+ or -] 59.76a   3 602.50 [+ or -] 82.97a
Potassium (K)   18 694.50 [+ or -] 299.57a  9 144.30 [+ or -] 313.10b
Silica (Si)        606.92 [+ or -] 22.09a      23.88 [+ or -] 2.01b
Sodium (Na)        <56.09 [+ or -] 8.61        10.51 [+ or -] 1.76
Zinc (Zn)           49.36 [+ or -] 1.13a       23.28 [+ or -] 0.88b

Element               Cellulose

Aluminum (Al)   <3.58 [+ or -] 0.00
Boron (B)       <0.46 [+ or -] 0.00
Cadmium (Cd)    <0.12 [+ or -] 0.00
Calcium (Ca)   116.75 [+ or -] 0.63b
Copper (Cu)     <0.52 [+ or -] 0.00
Chromium (Cr)   <0.29 [+ or -] 0.01
Iron (Fe)       15.90 [+ or -] 0.31a
Lead (Pb)       <1.68 [+ or -] 0.00
Magnesium (Mg)  39.92 [+ or -] 1.05c
Manganese (Mn)   0.58 [+ or -] 0.01c
Nickel (Ni)     <0.44 [+ or -] 0.00
Nitrogen
 (N) ([double
 dagger])       <0.01 [+ or -] 0.00
Phosphorus (P)   2.40 [+ or -] 0.31b
Potassium (K)   30.40 [+ or -] 2.65c
Silica (Si)      6.60 [+ or -] 0.42b
Sodium (Na)    402.32 [+ or -] 9.92
Zinc (Zn)        0.29 [+ or -] 0.00c

([dagger]) Means within a row, based on four sample analyses across
two replications and 2 yr, followed by different letters are
significantly different, Least Squares Means (P [less than or equal
to] 0.05, SAS Institute, 1990). < Indicates that at least one of the
four samples analyzed was below detectable levels and thus biased
statistical procedures.

([double dagger]) Nitrogen levels are presented as percent total
Kjeldahl nitrogen per 150-mg sample.
Table 6. Mean ([+ or -] SEM) 10 d weight of ECB larvae reared on
meridic diet containing ear tissue of 10 maize genotypes. Mean level
([+ or -] SEM) of 5 elements that differed significantly across tissue
type for 10 maize genotypes as determined by inductively coupled
plasma-atomic emission spectrometry using the dry ash method.

               10-d larval
Genotype     weight ([dagger])                Potassium

                     mg                       mg/kg
Cellulose  65.06 [+ or -] 5.17a        30.4 [+ or -] 2.65e
Jubilee    49.22 [+ or -] 4.99b    15 259.5 [+ or -] 1 614.47a
MG 15      46.46 [+ or -] 12.13bc  15 141.1 [+ or -] 1 571.16ab
MN 270     43.61 [+ or -] 8.41c    12 196.8 [+ or -] 2 227.38d
MN 276     43.61 [+ or -] 11.92cd  13 740.5 [+ or -] 1 807.31abcd
MN 272     40.04 [+ or -] 6.71cd   13 493.0 [+ or -] 2 171.85bcd
MN 3153    38.98 [+ or -] 10.73cd  15 028.5 [+ or -] 2 425.43ab
MN 275     37.79 [+ or -] 6.49cd   13 219.6 [+ or -] 1 552.11cd
MN 3152    35.70 [+ or -] 8.29cde  13 711.4 [+ or -] 1 390.08abcd
Apache     34.01 [+ or -] 9.72de   13 890.3 [+ or -] 2 139.42abc
W182E      33.06 [+ or -] 11.47e   13 513.3 [+ or -] 1 945.21bcd

Genotype           Calcium                Manganese

                                mg/kg
Cellulose  116.8 [+ or -] 0.63d      0.6 [+ or -] 0.01e
Jubilee    631.5 [+ or -] 213.52a    7.5 [+ or -] 1.39bcd
MG 15      399.9 [+ or -] 133.80bc   6.6 [+ or -] 1.29d
MN 270     682.0 [+ or -] 242.53a    9.5 [+ or -] 2.45ab
MN 276     445.2 [+ or -] 150.50b    6.7 [+ or -] 1.03cd
MN 272     299.8 [+ or -] 91.94c     5.6 [+ or -] 0.61d
MN 3153    374.9 [+ or -] 121.61bc   7.1 [+ or -] 1.26bcd
MN 275     443.6 [+ or -] 138.54bc   7.9 [+ or -] 1.39abcd
MN 3152    456.0 [+ or -] 134.25b    9.2 [+ or -] 2.31abc
Apache     432.6 [+ or -] 143.28bc   7.9 [+ or -] 1.45abcd
W182E      391.8 [+ or -] 134.63bc  10.2 [+ or -] 2.55a

Genotype            Silica                   Zinc

                                 mg/kg
Cellulose    6.6 [+ or -] 0.42e       0.3 [+ or -] 0.02e
Jubilee    345.9 [+ or -] 118.85ab   42.3 [+ or -] 6.57a
MG 15      363.8 [+ or -] 127.00ab   36.3 [+ or -] 4.74b
MN 270     359.1 [+ or -] 130.88ab   38.1 [+ or -] 5.64ab
MN 276     254.4 [+ or -] 87.32cd    36.1 [+ or -] 5.33b
MN 272     298.8 [+ or -] 104.94bc   30.5 [+ or -] 4.11d
MN 3153    275.9 [+ or -] 107.86bcd  35.3 [+ or -] 4.78bc
MN 275     301.3 [+ or -] 106.07bc   38.3 [+ or -] 5.27ab
MN 3152    363.2 [+ or -] 129.96ab   37.7 [+ or -] 5.23b
Apache     391.4 [+ or -] 142.04a    37.8 [+ or -] 5.60ab
W182E      200.3 [+ or -] 67.31d     31.0 [+ or -] 5.68cd

([dagger]) Means within column followed by different letters differ
(P [less than or equal to] 0.05) based on Least Squares Means (SAS
Institute, 1990). Means of individual elements were based on four
observations across two replications and 2 yr, while 10 d larval
weight means were based on 360 observations across two tissues,
three replications, and 2 yr.
Table 7. ANOVA summary of mean 10-d weight of ECB larvae
reared on meridic diet containing either ferulic acid or p-coumaric
acid at levels found in cellulose and in silk tissues of Apache
and Jubilee sweet corn hybrids.

                     10-d larval  10-d larval
Source           df   survival      weight

                                      mg
Replication (R)   1      NS           NS
Treatment (T)     8      NS           **
R x T (Error)     8      --           --

* Indicates significance at P = 0.05.

** Indicates significance at P = 0.01.

*** Indicates significance at P = 0.001.

NS = nonsignificant.
Table 8. Mean 10-d weight of ECB larvae reared on meridic diet
containing ferulic acid (FA) or p-coumaric acid (PCA) at levels
found in cellulose and in the silk tissues of Apache and Jubilee
sweet corn hybrids. Bioassay controls were diet containing silk
tissue of Apache or Jubilee and diet containing cellulose.

                         Mean 10-d larval
Treatment      weight [+ or -] SEM ([double dagger])
([dagger])
                                mg
Cellulose              35.77 [+ or -] 0.82a
PCA-Apache             35.61 [+ or -] 0.71a
PCA-Jubilee            35.26 [+ or -] 0.95ab
FA-Apache              34.23 [+ or -] 1.00ab
FA-Cellulose           34.05 [+ or -] 0.99ab
PCA-Cellulose          33.60 [+ or -] 1.01ab
FA-Jubilee             31.38 [+ or -] 1.09ab
Jubilee-silk           30.52 [+ or -] 1.03b
Apache-silk            14.03 [+ or -] 0.79c

([dagger]) PCA-Apache, PCA-Jubilee, PCA-Cellulose = meridic diet with
PCA incorporated at a level equal to that found in the silk tissue of
an individual genotype (<0.20 mg/g dried tissue for Apache, Jubilee,
and cellulose, respectively). FA-Apache, FA-Jubilee, FA-Cellulose =
meridic diet with FA incorporated at 2.6, 1.67, and <0.20 mg/g dried
tissue for Apache silk, Jubilee silk, and cellulose, respectively.
Apache, Jubilee, and Cellulose = meridic diet with silk tissue of
Apache, Jubilee, or cellulose incorporated at 1 g/20 mL diet.

([double dagger]) Means within column followed by the same letter do
not differ at P [less than or equal to] 0.05 on the basis of Fischer's
protected LSD. Each mean is based on 60 observations across two
replications.


ACKNOWLEDGMENTS

We thank D. Andow and Y. Pang (Dep. of Entomology, Univ. of Minnesota) for supplying larvae and equipment. We thank P. Bolin (Dep. of Entomology, Univ. of Minnesota) for technical and logistical support and the Univ. of Minnesota Dep. of Soil, Water, and Climate Research Analytical Laboratory for the elemental analysis. We thank T. Joe of the USDA-ARS Forage Plant Cell Wall Chemistry Laboratory at the Univ. of Minnesota for help with the FA and PCA analysis and L. Bonilla of the Univ. of Minnesota Cancer Center for help with the HPLC analysis. We also thank J. Luby (Dep. of Horticultural Science, Univ. of Minnesota), T. Kurtti, and P. Bolin (Dep. of Entomology, Univ. of Minnesota) for critical review of an early draft of this manuscript.

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Daniel F. Warnock, * William D. Hutchinson, Cindy B. S. Tong, and David W. Davis

D.F. Warnock, Univ. of Illinois, Dep. of Natural Resources and Environmental Sciences, 1201 S. Dorner Dr., Urbana, IL 61802; W.D. Hutchison, Univ. of Minnesota, Dep. of Entomology, 1980 Folwell Ave., St. Paul, MN 55108; C.B.S. Tong and D.W. Davis, Univ. of Minnesota, Dep. of Horticultural Science, 1970 Folwell Ave., St. Paul, MN 55108. Funding for this research was provided, in part, by grants from the Midwest Food Processors Association, the Agricultural Utilization Research Institute (AURI), and the Univ. of Minnesota Agric. Exp. Stn. This research represents a portion of the senior author's dissertation. Received 11 Dec. 2000. Daniel F. Warnock, Corresponding author (dwarnock@ uiuc.edu).
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Author:Warnock, Daniel F.; Hutchison, William D.; Tong, Cindy B. S.; Davis, David W.
Publication:Crop Science
Article Type:Abstract
Geographic Code:1USA
Date:Nov 1, 2001
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