Journal of Chemical Ecology, Vol. 21, No. 3, 1995

LEAF PROFILE OF MAIZE RESISTANCE FACTORS TO EUROPEAN CORN BORER, O s t r i n i a nubilalis I

DAVID

J. B E R G V I N S O N , 2"* R O B E R T JOHN

I. H A M I L T O N , 2 a n d

T. ARNASON 3

2plant Research Centre, Agricuftare Canada Central Experimental Farm. Bl~g. 121 Ottawa, Ontario, Canada KIA 0C6 3Department of Biology University of Ottawa, Ottawa Ontario, Canada KIN 6N5 (Received September 21, 1994; accepted November 29. 1994) Abstract--The feeding preference of European corn borer larvae for immature whorl tissue of maize was examined by conducting leaf bioassays and quantifying resistance factors along the length of mid-wborl leaves from the maize synthetic BS9(C4) developed by recurrent selection for resistance. Potential resistance factors that were quantified included percent foliar nitrogen, gravimetric determination of soluble metabolites and fiber, soluble phenolics and hydroxamic acids, cell-wall-bound phenolics, leaf toughness, and UV absorbance of the epidemaal cell wall determined by microspectrophotometry. Larvae consumed immature tissue at a higher rate than more mature tissue outside of the whorl, despite higher levels of DIMBOA in immature tissue. Consumption rate was highly negatively correlated with epidermal cell wall absorbance and leaf toughness. Fiber content and phenolic fortification of cell walls are proposed as the major resistance components that influence European corn borer feeding preference within the resistant synthetic BS9(C4). Key Words--Ostrinia nubilalis. Lepidoptera, Pyralidae, European corn borer, maize, resistance, phenolics, fiber, difemlic acid, truxillic acid, DIMBOA, toughness. INTRODUCTION M a i z e r e s i s t a n c e to t h e E u r o p e a n c o r n b o r e r ( E C B ) , Ostrinia nubilalis ( H b n ) , (Lepidoptera: Pyralidae) has been extensively studied, For over two decades, the hydroxamic acid 2,4-dihydroxy-7-methoxy-(2H)-t,4-benzoxazin-3-(4H)-one *To whom correspondence should be addressed. Present address: CIMMY'I~ Apdo Postal 6-64, 06600 Mexico DE Mexico. 1PRC contribution no. 1562. 343 (1098-0331195/0300-0343S07.50/0 ~.! 1995 Plenum Publishing Coq0oration

344

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(DIMBOA) has been recognized as the major component in leaf-feeding resistance (Klun and Robinson, 1969) and is a feeding deterrent in both field and laboratory studies (Robinson et al., 1978). DIMBOA acts on growth and development of corn borer larvae by noncompetitive inhibition of digestive proteases (Campos et al., 1989: Houseman et al., 1992). Other resistance factors that have been less extensively studied include silica, lignin, and fiber content, which may act by reducing the nutritional quality of the leaf or increasing tissue toughness and thereby rendering nutrients less accessible (Rojanaridpiched et al., 1984: Coors, 1987; Peterson et al., 1988; Buendgen et al., 1990), Breeding for resistance to ECB has resulted in the release of several resistant inbred lines, cultivars, and synthetics, BS9 is a synthetic developed by open pollination of 10 inbreds that had good agronomic traits as well as moderate resistance to ECB damage (Russell and Guthrie, 1982). Using an artificial infestation technique for ECB, four consecutive cycles of selection were conducted to select plants with reduced leaf feeding damage by ECB larvae, Since this germplasm was developed specifically for ECB resistance, changes in its phytochemical composition over cycles of selection have been investigated to better understand maize resistance mechanisms (Buendgen et al., 1990). Intraplant variation in secondary compounds and nutrients has been studied in maize and would be of value to further our understanding of insect feeding behavior and feeding site preference, The objective of this study was to monitor changes in various secondary compounds and nutritional parameters along the length of mid-whorl leaves of ECB-resistant maize and to relate these changes to ECB feeding performance in both laboratory bioassays and the field. Resistance factors investigated included foliar nitrogen content, leaf toughness, fiber content, soluble and cell-wall-bound hydroxycinnamic acids, hydroxamic acids, and cell wall absorbance using a microspectrophotometer to localize biochemical resistance factors along the entire length of the leaf.

METHODS AND MATERIALS

Plant Samples'. The fourth cycle of selection from the maize population BS9 was used with the method of selection outlined by Russell and Guthrie (1982). BS9 is a synthetic that has the following inbreds in its genetic background: B49, B50, B52, B54, B55, B57, B68, CI31A, Mo17, and SD10. Planting occurred in mid-May of 1992 at the Central Experimental Farm, Agriculture Canada, Ottawa, Ontario, Canada. The rows (4.5 m) were spaced 0.9 m apart with 30 plants per row in a sandy loam soil. Three blocks of four rows were planted with one row from each block being infested at the mid-whorl stage with four egg masses (ca. 200 eggs) for field observations. Univoltine ECB normally oviposit at this time on the undersurface of the later-emerging leaves

MAIZE RESISTANCE TO CORN BORER

345

(leaves 10 through 13). At the mid-whorl stage (approx. 40 days from sowing), the 13th leaf was harvested by pulling the 1 lth leaf and above whorl out of the plant. Leaves were unwrapped from the whorl to expose the 13th leaf within the whorl. This leaf was green along one quarter, green-yellow along one half, and yellow along one quarter of its length and would emerge from the whorl within four days. Fifteen leaves from each block were harvested for pbytochemical analysis. The midrib was removed and the leaves were then cut into eight sections of equal length along the leaf length with four sections being predominantly green and four sections being predominantly yellow. Section 1 was located at the leaf tip and section 8 was located at the auricles. Tissue was frozen at - 2 0 ° C , thawed for 1 hr to release DIMBOA from its glucoside, refrozen, freeze-dried, and milled on a UD cyclone mill (UD Corp., Boulder, Colorado) with a l-mm screen. Milled samples were stored at - 2 0 ° C until analyzed. Bioassays. Thirty plants were dissected and sectioned as above. Tissue was stored in water for up to 6 hr prior to incorporation into bioassays. A modified Ascher et al. (1981) apparatus was used to determine consumption of a 1.25cm 2 disk of the leaf tissue exposed to two third-instars. The bioassay apparatus consisted of two top and one bottom halves of plastic Petri plates (5 cm diam.) with the bottom plate having a 1.25-cm-diam. hole. The top plate was inverted and wet cotton was placed inside and covered with filter paper. Leaf tissue was placed onto the filter paper with the top surface facing down. The bottom plate with the hole was taped on top to expose a defined feeding surface. Two early third-instars were placed into the apparatus, a small plastic covering was placed inside to shade the larvae, and the third plate was taped over the top to seal the larvae into the apparatus. The larvae were allowed to teed for 48 hr under controlled conditions of 25: 18°C light-dark, 85 % relative humidity, and light regime of 16:8 hr light-dark. Mean area consumed was determined from 30 leaf disks per section. Area consumed was measured by placing the consumed disk on top of 1-mm-grid graph paper. Leaf Toughness. Using the method reported in Bergvinson et al. (1994a), force measurements were taken from the abaxial leaf surface between veins using a 1-mm-diam. probe mounted to an instron (model TM-M, Instron Corp., Canton, Massachusetts). Leaf tissue was held in place using a stainless steel platform fitted with a Ptexiglas plate with a 2-cm-diam. hole for probe penetration. Ten samples for each of the eight sections were measured and the peak force recorded. Leaf samples were stored in distilled water at 4°C for up to 2 hr before force measurements were taken. Protein Determinations. Protein content was estimated with an automatic micro-Kjeldahl nitrogen analyzer (Tecator model 1030, H6gan~is, Sweden) using the conversion factor 6.25 to estimate percent protein from percent nitrogen. Cell Wall UV Absorbance. Five samples from leaf sections 2, 4, 6, and 8

346

BERGVINSON ET AL.

were collected and kept at - 2 0 ° C . Samples were embedded in water containing glycerol (5 drops/10 ml water). Sections 6 ~tm were prepared and loaded onto Zeiss quartz slides and cover slips using glycerol. A computer-controlled Zeiss UMSP 80 microspectrophotometer equipped with a high-pressure xenon lamp (XBO 75W/2) was used to determine the absorbance at 326 nm of the adaxial epidermal cell wall (see Bergvinson et al., 1994b for more details). Phytochemicat Analysis. Phenolic conjugates and hydroxamic acids were extracted from a 0.5-g sample of dry tissue. Samples were extracted for 20 sec at ambient temperature in 70% methanol (4 x 20 ml) and mixed with a polytron mixer (model PT-1200, Brinkmann, Westbury, New York). After centrifugation at 500g for 10 rain, the supernatants were pooled, methanol was removed by rotary evaporator (35°C), and the pH lowered to 2.0 using 1 M HCI. The aqueous fraction was extracted at ambient temperature by solvent partitioning with ethyl acetate (4 x 50 ml) (BDH, Omni-Solv grade). Ethyl acetate fractions were pooled, dried by a rotary evaporator, and stored at - 2 0 ° C until HPLC analysis. After extraction, the pellet that remained was washed in a Biichner funnel with 30 ml each of water, methanol, and ethyl acetate to remove chlorophyll and provide a crude cell wall preparation. Cell wall samples were dried in a desiccator for four days. This preparation was weighed, and the weight loss was used as the gravimetric measure of soluble metabolites. Cell wall preparations were shaken in 20 ml of 2 M NaOH for 4 hr under N2 and wrapped in foil to hydrolyze cell-wall-bound hydroxycinnamic acids. Samples were neutralized with 6 M HC1 and the pH lowered to 2.0. After centrifugation, the supernatant was extracted with ethyl acetate (3 x 50 ml). The pellet was resuspended in water and centrifuged twice, with both fractions pooled, and extracted with ethyl acetate (3 x 50 ml). Ethyl acetate fractions were pooled and dried by a rotary' evaporator under darkness and stored at - 2 0 ° C until HPLC analysis. The pellet that remained after extraction was dried and weighed to provide an estimate of fiber content. HPLC Analysis. All analyses were performed with a Perkin-Elmer (Beaconsfield, England) system consisting of an LC 480 diode scan array detector and a Perkin-Elmer LC250 binary pump fitted with a 10-/xl injection loop. Separations were achieved using a C18 ODS reverse-phase column (250 x 4.6 mm, 5-~m particle size, Beckman, Fullerton, California). Soluble extracts were suspended in 4 ml of 50% methanol and centrifuged at 500g for 5 min to remove excess chlorophyll. The supernatant was filtered and injected onto the column. The solvent system consisted of methanol (A) and 10 mM H2PO 4, pH 2.4 (B), at a flow rate of 1.5 ml/min using the following gradient: 25 to 55% A in 15 min, 55 to 80% A in 5 min, 80 to 100% A in 2 min, 100% A for 8 min, 100 to 25% A in 2 min, and 25% A for 3 min. DIMBOA (R, = 10.5 min) and MBOA (R, = 13.6 min) standards were syn-

MAIZE RESISTANCE TO CORN BORER

347

thetically prepared according to Atkinson et al. (1991). Peak identity was confirmed by on-line UV spectra and spiking of extracts with authentic standards. Cell-wall-bound phenolic acids were suspended in 1 ml of methanol, diluted 10-fold in methanol, filtered, and injected onto the column. The solvent system was the same as above except the starting mixture was 15 % methanol. Standards of (E)-p-coumaric (R, = 15.2 min) and (E)-ferulic acid (R, = 15.6 min) were purchased from Sigma (St. Louis, Missouri). Truxillic acid and 5,5'-diferulic acid were obtained from GHN Towers (Vancouver, Britich Columbia). Statistical Analysis. All statistical analyses were performed on SAS V.6.03 (SAS Institute, 1988). Data satisfied the assumptions of the general linear model and were not transformed. Statistical significance of data was assessed by analysis of variance (ANOVA). When ANOVA indicated there were significant effects, the Student-Neuman-Keuls (SNK) test was used to compare means. Regression analyses between larval consumption and phytochemical parameters were done using the mean values for each of the eight leaf sections.

RESULTS AND DISCUSSION

Field observations of recently hatched European corn borer larvae showed that approximately 80% of the larvae move towards the center of the whorl during daylight hours (unpublished data). Similar observations have been reported for other stem borers such as Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) in which 95-100% of live larvae are within the whorl (Ampofo and Kidiavai, 1987). A possible explanation for this behavior is avoidance of the hot, dry microenvironment on the exposed whorl leaves, which can result in desiccation of neonate larvae. This explanation is supported by the fact that egg mortality is higher at lower relative humidities (Lee, 1988) and larval mortality often exceeds 80% during the first 48 hr after hatching (Ross and Ostlie, 1990). A profile of leaf consumption by ECB on BS9(C4) is depicted in Figure 1. ECB larvae show the highest consumption rate on immature tissue (sections 6 to 8). The leaf section with the lowest consumption was at the point where the leaf subtends from the whorl (section 4). By conducting leaf feeding bioassays in growth chambers, the effect of relative humidity over the leaf length is controlled and the degree of feeding on mature tissue is obviously lower even when relative humidity is not a restricting factor. This suggests that factors other than microenvironment are influencing larval preference for feeding within the whorl of maize. Other parameters thought to be associated with feeding behavior are shown in Figure 1. Protein was lower for the sections around the green-yellow interface, reaching a level as low as 17%. Given the low protein levels at the greenyellow interface, one would expect consumption to be higher for this tissue so

348

BERGVINSON

ET AL.

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FIG. 1. Maize leaf profile for European corn borer consumption, protein, soluble metabolites, and fiber content lbr resistant synthetic BS9(C4). Leaf tip is section 1 and leaf attachment is section 8. Standard error of mean shown by vertical lines.

that it is able to fulfill nutritional requirements for development (Scriber and Slansky, 1981), but this was not observed during the 48-hr bioassay. Soluble metabolites washed from milled leaf tissue included sugars, soluble proteins, chlorophyll, phenolic conjugates such as flavonoids and hydroxycinnamic acids, and hydroxamics such as DIMBOA. The trend for soluble metabolites follows that of leaf consumption. Phenolic conjugates from maize have been shown to be phagostimulants (Bergvinson, 1993) and may account for higher consumption, as the level of sugar conjugates ofp-coumaric acid are higher for immature whorl tissue (Figure 2). Other soluble secondary metabolites such as ferulic acid conjugates or HMBOA fluctuate and show no consistent trend. DIMBOA was found to be at the highest levels within the yellow whorl tissue, which was also the most preferred by ECB larvae (Figure 2). Based on previous feeding preference studies, the converse would be expected (Robinson et al., 1978). Nutritional studies have shown that DIMBOA incorporated into meridic diet increases larval consumption while reducing the efficiency of nutrient assimilation and various fitness parameters (Houseman et al., 1992). This in part may explain the higher consumption rate of immature, yellow whorl tissue with elevated DIMBOA levels. Elevated levels of DIMBOA do not appear to be a significant deterrent to larval feeding during a 48-hr bioassay but may affect insect performance through reduced fecundity and prolonged development if feeding was restricted to this tissue throughout larval development. The high feeding preference for tissue with elevated levels of DIMBOA can be rationalized by observing the relative absence of physical defense mechanisms in immature whorl tissue. Fiber content in immature tissue is very low

349

MAIZE RESISTANCE TO CORN BORER

(Figure I), and the relative absence of phenolic fortification in epidermal cell walls, as demonstrated by the low microspectrophotometer readings (Table 1), renders nutrients within the leaf more accessible and hence makes the tissue more desirable (Scriber and Slansky, 1981; Bernays and Barbehenn, 1987). The tissue toughness profile found in Figure 3 best illustrates the absence of mechanical resistance factors vis-h-vis fiber and hydroxycinnamic acid fortification of cell walls (Figure 4). The toughness profile could account for field observations of neonate behavior. Immature whorl tissue would be easier to consume by neonates than tougher, mature leaf tissue. By migrating to the inner whorl, larvae would not

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FiG. 2. Maize leaf profile of soluble secondary metabolites for resistant synthetic B S 9 ( C 4 ) . L e a f tip is section I and leaf attachment is section 8. Standard error of mean

shown by vertical lines.

T A B L E 1. EPIDERMAL C E L L W A L L ABSORBANCE OF M A I Z E SYNTHETIC B S 9 ( C 4 ) FRO/Vl SECT[ONS ALONG L E A F L E N G T H

Leaf sections" 2 4 6 8

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_+ 0.11 a ± O. 1 0 a _+ 0.06 b + 0.01 c

" L e a f sections are from the leaf tip (section t) to the point of attachment to the stalk (section 8). J'Means followed by the same letter are not significantly different. Student-Newman-Keuls test, P < 0.05.

350

B E R G V I N S O N ET AL.

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FIG. 3. Maize leaf toughness profile of abaxial epidermis (dashed line) from resistant synthetic BS9(C4) in relation to leaf consumption by European corn borer larvae (solid line). Standard error of mean shown by vertical lines.

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FIG, 4. Maize leaf profile of cell-wall-bound hydroxycinnamic acids for resistant synthetic BS9(C4). Total diphenolic acids include dehydrodifemlic acid and related dimers. Total cyclobutane dimers include both tmxillic and truxinic acids. Leaf tip is section 1 and leaf attachment is section 8. Standard error of mean shown by vertical lines.

only be in a higher humidity microenvironment but would also be able to easily ingest water and nutrients to avoid desiccation and starvation during early stages of development. During the field study, another insect, the corn leaf aphid, Rhopalosiphum maidis, was observed to be most prominent within the whorl of several maize varieties where higher levels of D I M B O A are found (Bergvinson, 1993). D I M B O A in wheat has been identified as a feeding deterrent for aphids (Niemeyer et al., 1989) as well as accounting for varietal differences in maize resistance to the corn leaf aphid (Beck et al., 1983). However, like the ECB,

MAIZE RES[STANCE TO CORN BORER

351

there may be physical, environmental, or nutritional factors that are of greater importance to aphid performance that necessitate feeding within the whorl despite high DIMBOA levels. The major cell-wall-bound phenolic acids are (E)-ferulic and (E)-p-coumaric acids, which are attached to hemicellulose through pentose sugars (Kato and Nevins, 1985). Both phenolic acids reach their highest levels in sections 5 and 6, the yellow tissue that will emerge within two days (Figure 4). Cell-wallbound ferulic and p-coumaric acids can form dimers either enzymatically through peroxidase to form 5,5'-diferulic acid (Hartley and Jones, 1976) or through photochemical reactions to form truxillic and truxinic acids (Hartley et ak, 1988; Ford and Hartley, 1989). Formation of such dimers may increase the mechanical strength of the cell wall by cross-linking hemicellulose (Ford and Hartley, 1989). From the profiles in Figure 4 no individual biochemical component provides a suitable explanation for leaf consumption or toughness, However, when taken together, hydroxycinnamic acids provide a biochemical explanation for feeding performance. The leaf tip has been exposed to sunlight the longest and shows the highest levels of the photodimers tmxillic and truxinic acids, which could cross-link the hemicellulose of mature tissue to provide structural resistance. For sections 4-6, elevated levels of cell-wall-bound p-coumaric, femlic, and diphenolic acids sustain leaf toughness and thereby maintain a moderate leaf consumption rate. For sections 7 and 8, all cell wall phenolics are at their lowest levels, corresponding to increases in leaf consumption (Figure 1). It is of interest to note the dramatic reduction in cell-wall-bound p-coumaric acid, which has been shown to have a positive correlation with lignin in bromegrass, Brornus inermus Leyss. (Jung and Casler, 1990). It may be that cell-wall-bound p-coumaric acid is an indicator tbr lignin by facilitating its attachment to the cell wall, as has been proposed for ferulic acid in wheat (Iiyama et al,, 1990). Moreover, Akin et al. (1992) showed increased digestibility of bermudagrass leaf with alkaline release of phenolic monomers and their photoactivated dimers. This increase in digestibility was especially noticed in the epidermis. These observations support the hypothesis that phenolic acid monomers and dimers contribute to the mechanical resistance of maturing tissue. Simple linear regression analysis of leaf consumption against the 12 parameters studied identified three parameters that could account for over 90% of the variation in leaf consumption (Table 2). Epidermal cell wall absorbance, toughness, and fiber content are all components or indicators of mechanical resistance. Elevated fiber content would not only increase the bulk density of the insect's diet to make nutrient and water requirements less attainable (Bemays, 1986), but would also increase the area for phenolic cross-linking. Acting in concert, fiber and hydroxycinnamic acid fortification in epidermal cell wall tissue could increase leaf toughness of "apparent" (sensu Feeny, 1976) mature leaf tissue, making it inaccessible to neonate larvae, which are forced to feed on softer,

352

BERGVINSON ET AL.

TABLE 2. REGRESSIONSBETWEEN Ostrinia nubilalis CONSUMPTION OF LEAF TISSUE AND BIOCHEMICAL REStSTANCE FACTORS FOR MAIZE SYNTHETIC BS9(C4)

Parameter Absorbance Toughness Fiber Solubles HMBOA DIMBOA

Regression equation" LF LF LF LF LF LF

= = = = = =

126(_+5) - 58(_+4) x Abs 142(_+7) - 181(_+17) × T 540(:I:46) - 2688(-+312) x Wt -62(_+18) + 403~'_+52) x Ratio 29(-+10) + 58(_+I21 x HMBOA -13(_+25) + 198(_+58) × DIMBOA

r2

F value

P

0.98 0.94 0.93 0.89 0.76 0.61

181.1 111,9 74.2 59.1 23,1 11.7

0.005 0.001 0.001 0.001 0,03 0.01

"LF = leaf feeding (mm~/48 hr): T = leaf toughness in newtons (N); Wt = weight in rag/g, Ratio = grams of soluble/total weight; DIMBOA = mg/g dry weight. Regressions based on means from eight sections of leaf tissue from along the length of the leaf (N = 8).

i m m a t u r e w h o r l tissue that is d e f e n d e d by high levels o f D I M B O A .

A s the

insect m a t u r e s , its m a n d i b l e s m a y be better able to c o p e with t o u g h e r , m a t u r e tissue ( B e r n a y s , 1986) that h a s l o w e r levels o f D I M B O A . Based o n w i t h i n - l e a f variation o f f e e d i n g p r e f e r e n c e and b i o c h e m i c a l factors, leaf t o u g h n e s s and the b i o c h e m i c a l factors r e s p o n s i b l e for l e a f t o u g h n e s s a p p e a r to be the p r e d o m i n a n t factors that influence E C B f e e d i n g b e h a v i o r w i t h i n m a i z e d u r i n g the m i d - w h o r l s t a g e o f plant d e v e l o p m e n t .

Acknowledgments--We thank J. Gale for maintenance of the ECB cuhure and G. H. N. Towers for providing synthetic standards of truxillic acid and 5,5'-diferolic acid. This work was supported by an NSERC strategic grant (Arnason) and the Ontario Ministry of Agriculture and Food and by an NSERC Graduate Scholarship to D.J.B.

REFERENCES AKIN, D.E., HARTLEY, R.D., RIGSBY, L.L., and MORRISON, W.H., llI. 1992. Phenolic acids released from bermudagrass (Cynodon daco'lon} by sequential sodium hydroxide treatment in relation to biodegradation of cell types. J. Sci. Food Agric. 58:207-214. AMPOFO, J.K.O., and KIDIAVA~,E.L. 1987, Chilo partellus (Swinhoe) (Lepid., Pyralidae) larval movement and growth on maize plants in relation to plant age and resistance or susceptibility, J. Appl. EntomoL 103:483-488. ASCHER, K.R.S,, SCHMUTTERER, H., GLOTTER, E., and KIRSON, |. 1981. Withanotides and related ergostane-type steroids as antifeedants for larvae of Epilachna varivestis (Coleoptera: Chrysomelidae). Phytoparasitica 9:197-205. ATKINSON,J., MORAND, P., ARNASON,J.T,, NIEMEYER,H.M., and BRAVO, H.R. 1991. Analogues of the cyclic hydroxamic acid 2,4-dihydroxy-7-methoxy-2H-l,4-benzoxazin-3-one: Decomposition to benzoxazofinones and reaction with (3-mercaptoethanol. J. Org. Chem. 56:17881800. BECK, D.L., DUNN, G.M., ROUTLEY, D.G., and BOWMAN,J.S. 1983. Biochemical basis of resistance in corn to the corn leaf aphid. Crop Sci. 23:995-998.

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