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a Univ. of Illinois, Dep. of Natural Resources and Environmental Sciences, 1201 S. Dorner Dr., Urbana, IL 61802
b Univ. of Minnesota, Dep. of Entomology, 1980 Folwell Ave., St. Paul, MN 55108
c Univ. of Minnesota, Dep. of Horticultural Science, 1970 Folwell Ave., St. Paul, MN 55108
* Corresponding author (dwarnock{at}uiuc.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: Bt, Bacillus thuringiensis • ECB, European corn borer • FA, ferulic acid • PCA, p-coumaric acid
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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-dihydroxy-7-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.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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°C. Ears of the remaining 15 to 25 plants of each genotype were grown to the commercial processing stage (
720 g kg-1 kernel moisture) and bulk harvested within genotype. Kernel tissues were cut from the cob, frozen, and stored at -20°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°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°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 (
= 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 F1 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 (Büchl, 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°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°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°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-µm 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 C8 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 KH2PO4 pH 5.0) to 100% Buffer B (20 mM KH2PO4 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 µL. 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 ( = 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 F1 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°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 ( = 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.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.01) by tissue (Table 1), averaging about 88 ± 0.7 and 81 ± 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 ± 2.7%).
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0.01) and tissue (F = 341.74; df = 1; P 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).
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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 0.01), genotype (F = 2.24; df = 9; P 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 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 0.01) and tissue (F = 249.03; df = 1; P 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 ± 0.14 and 19.3 ± 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 ± 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 ± 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 0.01) and by treatment (F = 11.42; df = 18; P 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.
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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.
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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.
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0.01), calcium (F = 639.86; df = 1; P 0.01), manganese (F = 53.35; df = 1; P 0.01), silica (F = 949.76; df = 1; P 0.01), and zinc (F = 729.78; df = 1; P 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.
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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.
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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 |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 3153 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°C to 25°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.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication December 11, 2000.
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REFERENCES |
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-tomatine in tomato. J. Agric. Food Chem. 42:2475–2477.
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