Journal of Chemical Ecology, Vol. 22, No. 9, 1996

H E M I C E L L U L O S E IS A N I M P O R T A N T L E A F - F E E D I N G R E S I S T A N C E F A C T O R IN C O R N TO THE F A L L ARMYWORM l

PAUL

A.

H E D I N , 2'* F R A N K RICKEY

P.

M.

DAVIS, 2 W.

HICKS, 3 and THOMAS

PAUL H.

WILLIAMS,

2

FISHER 3

"-Crop Science Research Laboratory, US Department of Agriculture Agricultural Research Service Mississippi State, Mississippi 39762 3Department of Chemistry, Mississippi State Universi~_ Mississippi State, Mississippi 39762 (Received December 18, 1995; accepted April 30, 1996)

Abstract--The fall armyworm, Spodopterafrugiperda (J. E. Smith), (FAW) is a major pest of corn, Zea mays L., in the southeastern United States. The damage to pretassel corn is caused by larvae feeding primarily on immature inner whorls. In this study, resistant lines were found to contain more crude fiber in whorls, mostly hemicellulose and cellulose. While hemicellulose, chiefly an arabinoxylan, was higher in resistant (R) lines than in susceptible (S) lines, the distribution of constituent neutral sugars was very similar in the lines. Both lines also contained p-coumaric and ferulic acids. These phenolic acids are known to occur both in the free state and in the cell wall as complexes bound by ester linkages to the arabinose moiety of the arabinoxylan, b~C NMR data showed that the intensity of the carbonyl carbon (184 ppm) in resistant hemicellulose was stronger, indicating a greater degree of cross-linking. Thus, resistant hemicellulose is both structurally different from susceptible hemicellulose and present in greater quantities. In two of three laboratory dietary tests, FAW larval weight gains were significantly higher on diets with (S) hemicellulose incorporated at the same level as (R) hemicellulose. Therefore, resistance to the FAW appears to be correlated with both a greater amount and a higher degree of cross-linking of the hemicellulose of (R) lines. Key Words--Corn, Zea mays (L.), fall armyworm, Spodoptera frugiperda (J. E. Smith), Lepidoptera, feeding resistance, hemicellulose. *To whom correspondence should be addressed. LMention of a trademark, proprietary product, or vendor does not constitute a guarantee of warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. 1655 (k')98-0331196f0900-1655509.50/()

~p 1996 Plenum Publishing Corl~Jr'atitm

1656

HEDIN ET AL. INTRODUCTION

The fall armyworm, Spodoptera frugiperda (J. E. Smith), (FAW) is a major pest of corn, Zea mays L., in the southeastern United States (Davis et al., 1988a,b). The damage to pretassel corn is caused by larvae feeding primarily on inner whorl leaf tissue. The damage caused by this insect can be extensive; in fact, late-planted corn is sometimes destroyed. A long-continuing corn breeding program at Mississippi State, Mississippi, has led to the release of eight germplasm lines with leaf-feeding resistance to the F A W and the southwestern corn borer (SWCB), Diatraea grandiosella Dyar (Davis et al., 1988a,b). These lines have also shown resistance to the sugarcane borer, Diatraea saccharalis (Fab.), the corn earworm, Heliothis zea (Boddie), and the European corn borer (ECB), Ostrinia nubilalis (Hubner) (Davis et al., 1988b). The resistance appears to be operating by the same mechanisms (antibiosis and nonpreference), suggesting that the same or similar chemical and physical factors may govern the resistance of corn to these insects (Wiseman et al., 1981, 1983; Williams et al., 1985, 1987; Davis et al., 1988a,b). Two major qualitative factors of resistance in corn to one or several corn insects are 6-MBOA-DIMBOA (Klun and Brindley, 1966) and maysin (Waiss et al., 1979). However, our investigations demonstrated that either these compounds possessed only limited toxicity to the F A W or SWCB or that they were present at minor levels in the corn lines found to have significant resistance to these two insects (Hedin et al., 1984, 1990, 1993). Resistance to a leaf disease in corn was also attributed to lignin by Lyons et al. (1993), but we could only find 1.5% lignin in susceptible corn whorls and 1.6% lignin in resistant whorls (Hedin et al., 1984). Waxes may also contribute to F A W resistance. The surfaces waxes of FAW-resistant lines were reported to be thicker (Davis et al., 1995). Earlier, Hedin et al., (1993) found the yield of waxes averaged 0.047% and 0.069% from fresh tissue for (S) and (R) lines, respectively. GLC and GLC-MS analyses revealed only minor differences in the composition of the waxes, but 6-MBOA, DIMBOA, and N-O-Me-DIMBOA were present in higher concentrations in (R) lines. However, these constituents are known not to be very toxic to the F A W and SWCB (Hedin et al., 1993). Thus, any contribution to resistance by the waxes is probably related to physical attributes that may modify the behavior of the larvae (Yang et al., 1991, 1992). Because qualitative defenses such as DIMBOA do not appear to contribute significantly to resistance of whorls to the F A W and the SWCB, quantitative defenses by cell wall constituents should be considered. The cell wall of plants has been characterized as consisting of cellulose microfibrils embedded in a lignin-hemicellulosic macromolecule to which acetyl and phenolic acid groups

HEMICELLULOSE AS RESISTANCE FACTOR

1657

are bound (Morrison, 1979). Arabinoxylans are the characteristic hemicellulose of cereals (Fry, 1989). The phenolic constituents of the cell walls of graminaceous plants consist largely of p-coumaric and ferulic acids (Hartley and Jones, 1978; Sosulski et al., 1982). These acids are bound to cell wall polysaccharides through ester bonds (Fry, 1986). Based on polysaccharide hydrolase digests of sugar cane and corn cell walls, ferulic acid was shown to be linked to arabinose, which in turn is linked to xylose (Kato and Nevins, 1985). Classen et al. (1990) concluded that cell wall-bound ferulic acid was the only significant factor explaining the variation in kernel damage by larvae and in adult preference for oviposition by some grain storage insects. However, the constituent phenolic acids do not appear to be important because Bergvinson (1993) found that ferulic acid in meridic diets did not cause significant larval mortality of the ECB and that sugar conjugates of ferulic and p-coumaric acids extracted from resistant lines were phagostimulants. In recent years, a laboratory bioassay based on lyophilized corn whorl tissue has been developed and refined to evaluate the leaf-feeding resistance in lines of corn to the F A W , the SWCB, and other corn insects (Williams et al., 1990, 1995; Buckley et al., 1991; Williams and Buckley, 1992; Williams and Davis, 1995). These workers concluded that growth of larvae on diets based on lyophilized leaf tissue was correlated with the average performance of the different lines. Davis et al. (1995) studied the anatomical characteristics of corn resistant to leaf-feeding by SWCB and F A W . The most striking and consistent difference between resistant and susceptible inbreds was the thickness of the cuticle and the epidermal cell wall. The cell wall complex of resistant inbreds was 1.7 • thicker than that of susceptible inbreds. This finding is consistent with our chemical data that showed resistant lines contained higher levels of fiber, including hemicellulose (Hedin et al., 1984, 1990). This present work concentrated on the hemicelluloses that were found to be higher in resistant lines. They were extracted, purified, chemically defined, and reconstituted in diets.

METHODS AND MATERIALS

Corn Lines and Harvesting. Corn lines used in these studies were developed by the Corn Host Plant Resistance Research Unit at Mississippi State, Mississippi. They were selected for desirable resistance properties to the F A W and grown using normal agronomic practices in the area. Whorls were excised on site from plants in the midwhorl Vs_~0 (Ritchie and Hanway, 1982) stage of

1658

HEDIN ET AL.

growth, transported to the laboratory where they were frozen at - 2 0 ~ and dissected with minimal warming to obtain the inner green-yellow tissue that constitutes the feeding site. This section was then freeze-dried, ground in a Wiley mill (40 mesh), and the powder stored in a freezer until used in a diet, fractionated, or analyzed. Dietary Evaluations. Larval growth on several experimental diets was compared to that of larvae fed a standard casein-wheat germ diet that had been established by Davis (1989) for the SWCB and F A W . The casein-wheat germ diet contains 17.0% casein and 14.3% wheat germ, with the remainder consisting of agar, alphacel, and other nutritional solids. Because wheat germ contains hemicellulose, it was excluded from the experimental diets and the casein was increased to 22.1% of total solids to provide an equal amount of protein. The hemicellulose and cellulose isolates were included in the diet at 15% of total solids, about half of their content in tissue. This variance was necessary because larger quantities caused diets to be too viscous for successful blending. Two diets, the first containing a mixture of 0.1% p-coumaric acid and 0.1% ferulic acid, and the second containing a mixture of 1.0% p-coumaric acid and 1.0% ferulic acid, were also included. The treatments (diets) were arranged in a randomized complete block design with 10 replications. Each treatment was represented by four larvae fed individually in 30-ml plastic cups, each containing approximately 10 ml of diet. The F A W and SWCB larvae were fed for 10 and 14 days, respectively, before weighing. Their rearing environment was 27.6~ 50-60% relative humidity, with a 16L : 8D photoperiod. The data were subjected to analysis of variance (ANOVA), and the least significant difference (LSD) tests was used to compare treatment means. Analytical Procedures. Association of Official Analytical Chemists (AOAC) methods (Horwitz, 1975) were used for the following analyses: total solids (moisture), 14.083; crude fat, 14.019; crude fiber, 14.118; acid detergent fiber, 7.056, 7.057, ash, 14.114; total protein, 2.049 (percent nitrogen x 6.25); nitrogen-free extract (NFE) by difference from 100%. AOAC methods were also used for analysis of acid detergent fiber, 973.18; and lignin (by loss on ignition), 973.18C (Helrich, 1990). Neutral detergent fiber was determined by the methods of Van Soest and Wine (1967). From these procedures, lignin, cellulose, and hemicellulose were determined directly, and soluble cell wall contents by difference from 100%. p-Coumaric and ferulic acids were determined by GLC-FID of their silyl derivatives, and their presence was confirmed by GLC-MS. Using the general procedures of Bergvinson (1993), freeze-dried corn whorl (5 g) was first extracted with 50% aqueous ethanol to remove chlorophyll. The residue was extracted in the dark for 4 hr with 2 N aq. NaOH (300 ml). Then the NaOH solution was extracted exhaustively with ethyl acetate. The ethyl acetate solution was dried

HEMICELLULOSE AS RESISTANCE FACTOR

1659

with Na2SO4 and concentrated to ca. 20 ml from which a 1-ml aliquot was silylated with N,N-bis(trimethyl-silyl)acetamide (Pierce Chemical Co., Rockford, Illinois). The analyses were quantitated by comparison with authentic, weighed samples of p-coumaric and ferulic acids (Fluka, Ronkonkoma, New York). Extraction and Analysis of Hemicellulose and Cellulose. The procedures were based on those of Selvendran and Ryden (1990). Typically, 100 g of ground freeze-dried whorl was extracted at boiling by Soxhlet with 50% aqueous ethanol, yielding about 20% solubles including the pigments, lipids, sugars, and salts. The residue from the aqueous ethanol extraction was extracted with l N KOH (1:25 w/v) for 24 hr, centrifuged, and the resulting residue was resuspended in water and centrifuged a second time. The supernatants were combined and adjusted to pH 5.0 with glacial acetic acid so that the hemicellulose precipitated. The residues from the centrifugation (chiefly cellulose) and the HOAC precipitate (hemicellulose) were transferred to dialysis tubing and dialyzed with daily changes of deionized water for seven days, after which they were freezedried and weighted to determine yield. Glycosyl compositions (sugars) were determined via hydrolysis with 2 mol/ liter trifluoroacetic acid and subsequent preparation and analysis of their alditol acetates according to the methods of Sturgeon (1990). Solid-and Liquid-State NMR Experiments. The 13C solid-state NMR spectra of freeze-dehydrated whorl powders and their isolated hemicelluloses were recorded at 75.49 MHz on a Bruker AMX-300 spectometer with a Level I Solids Accessory using a 4-mm rotor. The Bruker pulse program cp21ev was used with a prepulse delay of 3 sec and a contact time of 3.0 msec. Each spectrum was obtained using a MAS spinning rate of 7.5 kHz and 6000 scans. Samples of resistant (Mp 708) and susceptible (SC 229) hemicelluloses were prepared by dissolving 80 mg of each in 2.5 ml of D20 containing 20% KOH (by volume). All spectra were referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). Isotopically enriched D20 was purchased from Isotec Inc. (Miamisburg, Ohio), and 2,2-dimethyl-2-silapentane-5-sulfonate was purchased from Cambridge Isotope Company (Woburn, Massachusetts). Both compounds were used without further purification. All NMR experiments were conducted with a Bruker AMX-300 spectrometer. The one-dimensional proton spectra were acquired at a frequency of 300.1355342 MHz using a spectral width of 6024 Hz, with 16K data points yielding a digital resolution of 0.37 Hz/point with a total acquisition time of 1.35 sec/scan. A total of 32 scans (1 sec delay between scans) were acquired and then processed with an exponential multiplication defined by a 1-Hz line broadening. The one-dimensional carbon13 spectra were acquired at a frequency of 75.4685870 MHz using a spectral width of 19,230 Hz, with 32K data points yielding a digital resolution of 0.6 Hz/point with a total acquisition time of 3.4078 sec/scan. A total of 35,000

1660

HEDIN ET AL.

scans (1 sec delay b e t w e e n scans) was acquired and then processed with an exponential multiplication defined by a 3-Hz line b r o a d e n i n g . Statistical Methods. Data were c o m b i n e d for analysis o f variance or comparison o f standard errors o f the mean.

RESULTS AND DISCUSSION The results from attempts to c h e m i c a l l y characterize the (S) and (R) whorl powders are s u m m a r i z e d in T a b l e 1. Crude fiber was 2 7 % higher on average in the (R) whorls than in the (S) whorls. P r o x i m a t e analyses o f A B 24E (S), SC 229 (S), M p 704 (R), and M p 708 (R) gave results similar to those previously reported for several o t h e r (S) and (R) lines (Hedin et al., 1984, 1990). T a b l e 1 also shows results for the contents o f lignin, cellulose, hemicellulose, and cell wall solubles from d e t e r m i n a t i o n s based on the use o f A O A C neutral and acid detergent fiber procedures. Tissue from (R) plants was 2 0 % h i g h e r on average in cellulose and 31% h i g h e r in hemicellulose, again similar to o u r earlier findings (Hedin et al., 1984). Lignin was very low ( 1 . 8 - 2 . 0 % ) , and not different in (S)

TABLE 1. CONSTITUENTS OF FAW SUSCEPTIBLE AND RESISTANT CORN INNER WHORL LEAF TISSUE a

Percent

Ash Protein Fat Fiber Nitrogen-free extract Cellulose Hemicellulose Lignin Cell wall contents Isolated HemicelluloseI' Free p-coumafic acid' Free ferulic acid'

AB 24E (S)

SC 229 (S)

Mp 704 (R)

Mp 708 (R)

8.8 25.6 1.6 24. I 40.0 28.0 25.5 1.9 44.6

6.9 26.9 1.7 22.9 41.5 24.6 23.8 1.8 47.6 22.3 J: 4.0 0.97 1.21

8.0 17.8 1.4 28.6 44.1 31.8 32.9 2.0 33.2

8.5 18.2 1.4 31.3 40.6 31.1 31.7 1.9 35.3 26.7 + 3.6 0.95 1.34

"Association of Official Analytical Chemists (AOAC) methods (Horwitz, 1975; Helrich, 1990). Analyses in duplicate. t'Hemicellulose isolated on five occasions by procedures based on those of Selvedran and Ryden (1990). "GLC-FID and GLC-MS analysis of silylated isolates.

HEMICELLULOSE AS RESISTANCE FACTOR

1661

and (R) whorls. The higher content of cell wall solubles in (S) whorls (35% on average) suggested higher digestibility and availability of nutrients. To evaluate completeness of the extractions, the isolated hemicellulose and cellulose fractions were analyzed by the crude fiber procedure, which is defined as the loss on ignition of dried residue remaining after digestion of sample with boiling 1.25% H2SO4 and 1.25% NaOH (AOAC 14.118). As expected, the hemicellulose was solubilized (0.0-0.7%), whereas the cellulose was retained (58.8-60.4%), thus confirming that the two isolates were different and essentially free of each other; this was important information for the interpretation of subsequent laboratory bioassays. Free p-coumaric and ferulic acids (as extracted with 2 N NaOH) were found to be about equal, respectively, in (S) whorls (0.95-0.97%) and in (R) whorls (1.21-1.34%) (Table 1). They were present in somewhat higher concentrations than reported by Bergvinson (1993). However, alkaline extractable p-coumaric and ferulic acids could not be isolated from purified hemicellulose. This is not surprising, since extraction of the hemicellulose from whorls initially had utilized aqueous NaOH, which was subsequently discarded. To further characterize the hemicellulose, a series of isolates were prepared for determination of their substituent sugars and in sufficient quantities for dietary tests. Initially, sugar analysis via the alditol acetate procedure was performed on the untreated whorls. The recovery of neutral sugars (Table 2) (by summation) was about 21% for two (S) lines and nearly 29% for two (R) lines. Xylose and arabinose, from the hemicellulose, and glucose, chiefly from carbohydrates other than cellulose, were the major sugars. When several replications each of hemicelluloses from an (S) line (SC 229) and an (R) line (Mp 708) were analyzed for neutral sugar content, they were found to be significantly higher in the (R) line (52%) than in the (S) line (37%). However, the proportion of hemicellulosic xylose was not higher in the resistant lines. The approximate ratio of xylose to arabinose in the purified hemicellulose was 9 : 1 , defining whorl hemicellulose as an arabinoxylan. For comparison, a corn cob arabinoxylan (isolated and analyzed by R. B. Hespell, Northern Regional Research Laboratory, USDA-ARS, Peoria, Illinois) gave a somewhat higher recovery of sugars, but the ratio of neutral sugars was fairly similar to corn whorl. A commercial xylan sample (Sigma Chemical Co., St. Louis, Missouri) yielded 64.2 % neutral sugars and contained only a small amount of arabinose (Table 2). The solid-state taC NMR analyses of the (R) and (S) whorls (Figure 1, Table 3) showed significant differences in the samples, and indicated that the (R) hemicellulose had a greater degree of cross-linking than (S) hemicellulose. The following solid-state ~3C NMR peaks were present in both the (R) and (S) samples: 25 (R), 55 (CH30), 65 (CH20), 75 (CH--O), 103 (anomeric C), 173 (broad carbonyl 1), and 181 (carbonyl 2) ppm. In the (R) sample, the peaks at 181 and 25 ppm were more intense than the corresponding peaks in the (S)

1662

TABLE 2.

H E D [ N ET AL,

N E U T R A L SUGARS IN F A W

SUSCEPTIBLE AND RESI STANT C O R N P L A N T INNER W H O R L

TISSUES AND THEIR HEMICELLULOSE FRACTIONS

Sample"

Neutral sugars (%)1,

Distribution of neutral sugars (%)' A

X

M

Ga

GI

Othe

Ab 24E whorl (S) SC 229 whorl (S) Mp 704 whorl (R) Mp 708 whorl (R) SC 229 whorl hemicellulose (S) Mp 708 whorl hemicellulose (R) NRRC corn cob xylan Xylan, Sigma Chemical Co.

20.6 22.3 29.4 28.2 37.3 + 4.3 52.4 + 5.4 75.3 64.2

9.5 9.0 9.8 10.3 9.5 8.9 15.0 0.8

52.4 51.4 61.3 61.7 80.8 82.6 76.0 91.9

2.9 2.5 1.6 2. I 0.3 0. I 0 0.3

4.6 3.9 3.5 4. I 1.4 0.9 5.0 0.8

29.5 33.2 23.8 21.8 6.7 6.4 5.0 5.1

1.1 0 0 0 1.4 1.(I 0 0. I

"Analyses performed in duplicate except for hemicellulose and cellulose. Two xylans were isolated and am lyzed. 1'Percent neutral sugars in sample. Differences in total from 100% largely attributable to cellulose, which resistant to acid hydrolysis, and to xylan, which on acid hydrolysis yields only about 65% of theoretic~ value. ' Sugars determined by GLC of the alditol acetates (Sturgeon, 1990). A, ar'abinose; X, xylose; M, mannos~ Ga, galactose; GI, glucose.

156R

I

Solid State NMR Spectra

Liquid State NMR Spectra

Carbonyl Region

IS6R

1

ppm

180

] 150

180

150

9, . . . . . . . . . . . . . r. . . . . . . . . . . . . . . . . . . ppm

180

160

156S

| ppm

!

,,,,,,,,'H'I''''''H'H'''H''' I ppm

180

160

FIG. 1. Solid- and liquid-state ~3C NMR spectra of the carbonyl region (150-220 ppm) of SC 229 (156S) and Mp 708 (156 R) purified hemicelluloses.

HEMICELLULOSE AS RESISTANCEFACTOR

1663

TABLE 3. LIQUID- AND SOLID-STATE CARBON-13 NMR ASSIGNMENTS FOR RESISTANT (Mp 708) AND SUSCEPTtBLECORN (SC 229) WHORL HEMICELLULOSES Peak No.

R (ppm)~

S (pprn)"

Possible assignment

1 2 3 4 5 6 7 8 9 10 11 12

184.00 175.8 br 111.79 w 104.70 104.28 88.80 w 82.85 w 80.95 w 78.72 76.81 75.65 75.38 65.80 65.53 25.92 w 25.75 w

184.02 175.95 br 111.77w 104.67 104.27 88.68 w 82.86 w 80.79 w 78.69 76.75 75.64 75.34 65.76 65.61 25.92 w 25.75 w

Carbonyl, possibly water Carbonyl, possibly carboxylate Possible aromatic or alkene carbon or/3 isomer of C-I carbon (ketal linkage) c~ or/3 isomer of C-I carbon (ketal linkage) Peaks 6, 7, 8 most likely correspond to carbons contained in the furanose (five membered ring) sugars Most of the contributions to peak intensity in this region (9- I I ) correspond to carbons contained in the pyranose (six membered ring) Sugars with some overlap with the carbons contained in the furanose sugars

13

14 15 16

Peaks 15 and 16 correspond to alkyl compounds

"br = broad singlet; w = resonance which is less than 20% of most intense resonance.

sample, indicating that the (R) sample c o n t a i n e d more carbonyl 2 functionalities and more aliphatic c a r b o n s than did the (S) sample. C o m m o n carbonyl compounds that a p p e a r in this region include carboxylic acids, esters, and carboxylate salts (about 5 p p m more deshielded than their c o r r e s p o n d i n g acid). Since the liquid samples were a n a l y z e d in N a O H , these two carbonyl peaks can be attributed to an acid carboxyl a n d / o r carboxylate salt because the acid would have been c o n v e r t e d into the salt u n d e r the basic liquid N M R conditions. T h e liquid and solid N M R s h o w e d the s a m e relative a m o u n t s o f c a r b o n y l 1 and carbonyl 2 c o m p o u n d s . In liquid-state ~3C N M R studies (Figure 1, T a b l e 3), the c a r b o n - 1 3 spectra of M p 708 (R) and SC 229 (S) hemicelluloses were c o m p l i c a t e d by the relatively large m o l e c u l a r weights o f the h e m i c e l l u l o s e s and by the high pH o f samples. The use o f 2 0 % K O H , which was necessary to dissolve the c o m p o u n d s , caused line b r o a d e n i n g and baseline problems. T h e o n e - d i m e n s i o n a l carbon-13 spectra are qualitatively very similar, as seen in T a b l e 3, but the quantitative intensities of the c a r b o n resonances for (R) and (S) are m u c h different. As can be seen in Figure 1, the carbonyl resonance at approximately 184 p p m was m u c h stronger in the (R) hemicellulose than it was in the (S) hemicellulose. H o w e v e r , the relative intensity o f the broad carbonyl resonances centered at approximately 175.9 ppm was h i g h e r in (S) than (R). T h e intensity o f the resonances occurring

1664

HEDIN ET AL.

between 90 and 80 p p m also s e e m e d to be relatively greater in (S) than in (R). These features clearly differentiated b e t w e e n the two hemicelluloses. The proton spectra (not presented) o f both (R) and (S) gave only broad singlets with no o b s e r v a b l e couplings, and there were no clear differences between the proton spectra o f the two c o m p o u n d s . ~3C N M R spectra were also taken on the original whorl powders. H o w e v e r , no differences in carbonyl c a r b o n intensity between the (S) and (R) powders were evident, presumably because the hemicelluloses c o m p r i s e d only a relatively small fraction o f the whorl powders. Table 4 shows the results o f five laboratory feeding bioassays, three with the F A W and two with the S W C B for comparison. In two o f three bioassay tests, growth o f F A W larvae on diets c o n t a i n i n g (R) hemicellulose was significantly lower than on diets containing equal a m o u n t s o f the (S) hemicellulose. In the third test, larval weights on diets containing the (S) and (R) hemicelluloses were not statistically different. If it had been dietarily feasible to test the (S) and (R) hemicelluloses at h i g h e r levels, reflecting their contents in the whorl, the effect o f the (R) hemicellulose may have been greater. As with the F A W , weight gains of S W C B larvae fed the (R) hemicellulose diet tended to be lower, but not significantly so, than those fed the (S) h e m i cellulose diet. On the o t h e r hand, larval weights were increased significantly on diets containing the (R) cellulose.

TABLE 4. WEIGHTS OF F A W AND SWCB LARVAE FED CASEIN-BASED LABORATORY DIETS CONTAINtNG 15% CORN WHORL HEMtCELLULOSE AND CELLULOSE (milligrams)"

FAW Diet~'

Test 1

Casein-wheat germ Casein-wheat germ plus cellulose Casein Casein plus cellulose Casein plus S whorl hemicellulose Casein plus R whorl hemicellulose Casein plus S whorl cellulose Casein plus R whorl cellulose LSD 0.05

483.8 a 484.7 a 199.9 d 238.2 cd

SWCB

Test 2

Test 3

Test I

Test 2

353.6 a

226.6 bc

238.7 a 94.0 e

175.6 c

287.3 a 298.8 a 126.2 de 112.4 e

105.2 d

338.2 b

207.8 b

272.8 b

144.3 cd

145.5cd

189.1 d 254.0 c 363.9 b 54

161.5 c 180.6 bc 176.1 c 56

254.6 b 403.7 a 281.3 b 21

127.1 de 147.7 c 201.8 b 19

126.6 d 147.4 c 183. I b 18

"Means followed by different letters are significantly (P = 0.05) different according to Duncan's multiple range test. ~'Based on the laboratory diet of Davis (1989). Hemicellulose and cellulose isolated by procedures based on those of Selvendran and Ryden (1990).

HEMICELLULOSE AS RESISTANCE FACTOR

1665

Two diets, one containing 0.1% each ofp-coumaric and ferulic acids, and the other containing 1.0% each of these two phenolic acids added to the control diet were bioassayed in feeding tests with the FAW. The average larval weight at 10 days on diets containing 0.1% each of the phenolic acids was 184.9 mg. At the 1.0% level, the average weight was 127.6 mg. The lower level of this phenolic acid mixture was approximately that which was found by Bergvinson (1993), and the higher level reflected that found by us (Table 1). Freeze-dried whorl powders have been dispersed in laboratory diets to evaluate F A W resistance (Williams and Buckley, 1992; Williams et al., 1995). The aqueous residue from resistant whorls was associated with poor growth (Williams and Davis, 1995). However, to identify the actual basis of resistance, it is necessary to devise extractive procedures that conserve biological activity upon recombination of fractions. Unfortunately, no entirely satisfactory procedures could be established because extraction at reflux with presumably mild solvents, including water, ethanol, and 50% aqueous ethanol, failed to give extracts that elicited growth upon recombination with the residue. An all-purpose vitamin mixture partially restored activity, but this approach could not be optimized. Aqueous extractions at intermediate temperatures (65-70~ gave full activity upon recombination with the residue. However, further treatments of the extracts or residues resulted in lost activity, so efforts to use a bioassay based on conserving the activity of whorl powder were abandoned. An alternative strategy was then adopted to isolate fractions or components that differed in concentration in (S) and (R) lines, and then to evaluate their biological activities in laboratory diets. Hartley et al. (1988) proposed that a portion of the phenolic acids may become bound to hemicellulose through a mechanism in which a four-membered ring is formed from the double bonds of the side chains in either a head-to-tail fashion to form truxillic acids, or in a head-to-head fashion to form truxinic acids. Both phenolics may be attached to arabinose and xylose prior to the phytochemical reaction. The cyclobutane dimers consist of both ferulic and p-coumaric acid, and it has been hypothesized that they may constitute a defense against insect attack (Hartley and Ford, 1989; liyama et al., 1990; Bergvinson et al., 1994). The levels of these cyclobutane dimers are highest in tissues that intercept sunlight, with leaf blades having the highest levels, followed by sheath and stem tissue (Eraso and Hartley, 1990). Head-to-tail dimers are generally in the greatest abundance (Morrison et al., 1991). The formation of these dimers in full sunlight may explain why maize plants grown in greenhouses are more susceptible to the ECB feeding on leaves, as reported by Guthrie et al. (1986) and Bergvinson et al. (1994). In summary, more hemicellulose was found in (R) whorls than in (S) whorls. Using standard analytical procedures, (R) lines were also found to contain more crude fiber and cellulose, but less protein and cell wall solubles. NMR

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e v i d e n c e w a s o b t a i n e d f o r a h i g h e r level o f c a r b o n y l c a r b o n in (R) h e m i c e l l u l o s e . In dietary tests, larval w e i g h t g a i n s w e r e h i g h e r o n (S) h e m i c e l l u l o s e i n c o r p o rated in diets at the s a m e level as (R) h e m i c e l l u l o s e . T h e s e data s u g g e s t that the l o w e r w e i g h t g a i n s can be attributed to the relative a m o u n t s a n d d i f f e r e n c e s in structure o f h e m i c e l l u l o s e s . Acknowledgments--The authors thank Mr. Paul Buckley for performing preliminary insect bioassays, Mrs. Debbie B. Saebo for performing NMR analyses, and Mr. Douglas Dollar for performing GLC sugar analyses.

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