Maysin in Corn, Teosinte, and Centipede Grass - American Chemical

Chapter 16

Maysin in Corn, Teosinte, and Centipede Grass 1

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R. C . Gueldner , Maurice E . Snook B. R. Wiseman , N . W. D. S. Himmelsbach , and C . E . Costello 2

Downloaded by UNIV MASSACHUSETTS AMHERST on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch016

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Widstrom ,

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Tobacco Quality and Safety Research Unit, and Plant Structure and Composition Research Unit, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 5677, Athens, G A 30613 Plant Resistance—Germplasm Enhancement Research Unit, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 748, Tifton, G A 31793 Department of Chemistry, Massachusetts Institute of Technology, Cambridge, M A 02139

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Maysin, a flavonoid C-glycoside originally found in the silks of the Mexican corn, Zapalote Chico, is also found in the leaves of corn, teosinte and centipedegrass, which are resistant to the corn earworm and the fall armyworm, the major insect pests of corn in the southeastern United States. The two insect pests feed in the whorl which has a lower maysin content than earlier leaves. The maysin content of corn leaves is highest in the tips of the leaves connected just below the ear-bearing nodes. In some varieties of corn luteolin C-glycosides related to maysin are shown to be present by H P L C - U V analysis in silks and/or leaves. These varieties are potential sources for the variant luteolin C-glycosides that are likely resistant factors for the corn earworm and the fall armyworm. The two most important pests of corn in the southeastern United States are the corn earworm (Heliothis zea [Boddie]) and the fall armyworm (Spodoptera frugiperda [J. E . Smith]). The corn earworm damages the leaves in the early stages of corn growth, and later, as the corn develops, infests the ears. The fall armyworm damages all parts of the plant and in late season causes severe damage in the form of broken leaves as well as reduced leaf photosynthetic activity. Grasses are also targets of the fall armyworm which is a leaf feeder on relatively susceptible plants such as bermudagrass (Cynodon dactylon [L.] Pers.). Variations in resistance have been studied in different clones of bermudagrass (1). Centipedegrass (Eremochloa ophiuroides (Munro [Hack.]) (2,3) and teosinte (Zea mays L.ssp. mexicana [Schrad.]) are examples of Gramineae which are very resistant to the fall armyworm. Species of Gramineae that are resistant to the fall armyworm sustain less damage than susceptible plants and are readily identified by lack of feeding damage. Factors that cause resistance may be complex and often require prolonged scrutiny by teams of entomologists and chemists to define these factors. These factors may be physical or chemical. For instance, Walter's White, a sweet corn, is an example of mechanical resistance to the corn earworm which rarely reaches the kernels of the ear because of the large quantity and excellent quality of silks that provide the larvae with adequate nourishment. In the realm of chemical resistance just three types of compounds occurring This chapter not subject to U.S. copyright Published 1991 American Chemical Society

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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NATURALLY OCCURRING PEST BIOREGULATORS

in corn have been shown to be inhibitory to insects in laboratory bioassays: the hydroxamic acid family, Hx, typified by D I M B O A (2,4-dihydroxy-7-methoxy-2H-l,4-benzoxazin-3[4H]-one) (Fig. la) which occurs in corn in the form of a glucoside, caffeic acid derivatives typified by chlorogenic acid (Fig. lb) and flavonoid glycosides typified by maysin, (Fig. l c ) . The inhibition of growth of the larvae in laboratory bioassays is taken as evidence of a resistance factor in corn when it can be shown that the factor occurs at a significant level in the area of the plant that the larvae feed upon. The criterion of larval growth inhibition as the measure of the contribution of a chemical factor to resistance in a plant remains at least partially subjective because it is impossible to exactly match the nutritive value of the intact plant in the artificial medium used in the laboratory bioassay. By the above criteria D I M B O A is not very inhibitory to the corn earworm and the fall armyworm, but is effective against the first brood European corn borer (Ostrinia nubilalis [Hubner]) when D I M B O A is at its highest level at the seedling stage. Since the sizeable literature concerning the H x family has been recently reviewed (5) no further consideration of the Hx family will be presented in this discussion. Chlorogenic acid (Fig. lb), the main representative of the second class of inhibitor, is a derivative of caffeic acid, and shows growth-inhibiting activity against the corn earworm (6,7,8). The effect of growth inhibition seems to be a combination of inhibition of feeding and post-ingestive phenomena (9). Chlorogenic acid is a growth inhibitor of the fall armyworm also (3). Maysin (Fig. lc), the main representative of the third class of inhibitory compounds, the flavonoid glycosides, was shown to be active against the corn earworm and was identified as the antibiotic factor in the exotic strain of corn, Zapalote Chico (10). The mode of action of maysin in inhibiting the growth of the corn earworm is similar to that of chlorogenic acid (9). Maysin is a luteolin C-glycoside identified by Elliger, et al (11) as 2 -0-a-rhamnosyl-6-C-(6-deoxy-xylo-hexos-4-ulosyl)-luteolin (Fig. l c ) . Elliger, et al (12), also showed that the presence of [3\ 4'J-ortho dihydroxy groups on the B ring of the aglycone enhanced the inhibitory activity of flavonoids in contrast to the monohydroxy (apigenin) compounds. This structural feature contributes to activity against both the corn earworm and the fall armyworm and is also present in chlorogenic acid which has the ortho-dihydroxy grouping on the caffeic acid moiety. When the 3* hydroxy group of maysin is methylated, activity is cut in half; when the luteolin moiety is methylated at both the 3* and 4' positions the resulting compound is reduced in activity against the corn earworm (12). In searching for resistance factors in the resistant plants, teosinte and centipede grass, and, in examining the leaves of corn varieties, we conclude that the C-glycosyl flavonoids, typified by maysin, are a major biosynthetic pathway, one which we believe can possibly be genetically amplified to develop corn with resistance to both the corn earworm and the fall armyworm. We wish to report on the occurrence of maysin and related compounds in the leaves and silks of corn and discuss possible relationships to corn resistance for the corn earworm and the fall armyworm. M

MATERIALS AND METHODS Collection, Storage and Treatment of Plant Material Individual corn silks, approximately 5 g per ear, were analyzed by reversed


GUELDNER ET AL.

Mayan in Corn, Teosinte, and Centipede Grass


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c Figure 1.

Structures of three insect inhibitory compounds from corn.


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phase H P L C by the method of Snook et al (13). The silks, in methanol, were stored at 0 ° C , allowed to warm to room temperature, and ultrasonicated for 20 min just prior to sampling for H P L C analysis. Corn leaves were collected at various stages except for the large samples intended for bulk extraction which were extracted at the 10 to 14 leaf stage. Various drying methods were used for the leaf materials. For obtaining leaf phenolic profiles freeze drying was used. For small analytical samples (1-10 g) microwave drying (3-5 min at high power with a standard kitchen microwave oven) was found to be convenient and preserved the profile of fresh plant material very well. Large samples for bulk extractions were most conveniently air-dried, which, however, caused a 10-20 % reduction in chlorogenic acid and flavonoids as indicated by H P L C analysis. For H P L C analysis chrysin was added as an internal standard to the dried leaf samples (also used with silks) which were then sonicated in the extraction solvent, 50:50 methanolrwater for 30 min. The mixture was filtered and the solution used for H P L C analysis. Fresh leaf material was ground with a Polytron grinder and sonicated as for the dried plant material. The methanol used was from Burdick and Jackson (Muskegon, M I , U S A ) * "distilled-in-glass grade". Leaf extracts were analyzed by reversed phase H P L C similar to the method of Snook et al (9) using a Hewlett-Packard 1090M liquid chromatograph equipped with a 1040A diode array detector and Chem Station computerized data collection system. The column was a Nucleosil 5/x (4.6 X 25 cm) O D S . A solvent gradient was used starting at 20:80 methanol: water and ending at 100% methanol in 40 min. Both solvents contained 0.1% H P O . The flow rate was optimized at 0.8 mL/min for best resolution according to Meyer (14). 3

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Spectra U V spectra were taken on-the-fly with the 1040A diode array detector at a monitoring wavelength of 340 nm. Infrared spectra were recorded with an Analect Model FX-60 F T I R spectrophotometer. IR samples were prepared as K B r pellets. Carbon-13 N M R spectra were recorded with a Bruker A M - 2 5 0 spectrometer at 62.9 m H z using composite pulse decoupling. Samples were disolved in d6-dimethylsulfoxide (ca. 40 mg/ml) and referenced to the center peak of the solvent at 39.5 ppm. Positive ion fast atom bombardment ( F A B ) mass spectra were obtained with the J E O L HX110/HX110 tandem double-focusing mass spectrometer with J E O L gun and collisionally induced decomposition (CID) M S / M S . The J E O L instrument was operated at +10 k V with -20 k V postacceleration at the detector. The xenon neutral beam had 6 k V acceleration from the J E O L gun. C I D M S / M S was performed with 1:1000 resolution in both MS-1 and MS-2. Helium was used as the collision gas at a pressure sufficient to reduce precursor ion abundance by 75%. Samples were dissolved in methanol-glycerol (1:1, v/v) for all F A B M S analyses. Bioassays Insect bioassays were conducted according to the method of Wiseman, et al (15). Large-Scale Extraction and Purification Teosinte was grown to about the 12 leaf stage in Tift Co. Georgia. The leaves were stripped from the stalks and allowed to air-dry. T o extract, 4.3 kg of the dry teosinte leaves were ground in a Wiley mill and soaked with intermittent stirring in 8 L 50:50 methanol: water. After 24 hours most of the



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GUELDNER ET AL.

Maysin in Corn, Teosinte, and Centipede Grass

solvent was siphoned off and a fresh batch of solvent was used for further extraction. After the third soaking for a total of 72 hrs the liquid extract was concentrated on a rotary evaporator to 8 L of aqueous extract. The aqueous extract was applied to a column of bonded-phase octadecylsilane on silica gel ( O D S or C-18) from Waters Associates as a Prep-Pak 500 cartridge. After the adsorption of the sample the column was eluted with 1.8 L water, 2 L of 20% methanol in water, and 1 L each of 30,35,40,45,50,55,60,65 and 70% methanol. The compounds of interest eluted mainly in the 40-50 % methanol-water fractions. About 9 g of residue from the 40-50% fractions was deposited on 15 g of silica gel (J. T. Baker) and this sample was placed at the top of 100 g silica gel in a chromatographic column. Stepwise gradient elution with hexane, ethyl acetate, and ethyl acetate-acetone yielded two compounds which were further separated by droplet counter-current chromatography ( D C C C ) using C H C l : M e O H : n - P r O H : H 2 0 (5:6:1:4). The upper aqueous layer was the stationary phase and the lower, mainly C H C 1 layer was the mobile phase. Fractions (10 mL) were collected every hour. Fractions 71-79 contained 35.2 mg of a maysin isomer and fractions 89-% contained 38.3 mg of maysin. Walter's White corn plants were grown until the silks had dried. The leaves were processed and extracted in a fashion similar to teosinte. The crude extract, however, was deposited directly onto silica gel, eluted from a silica gel column as with the teosinte extract and then further purified on an open reversed phase O D S column. Luteolin glycosides were obtained in 30-50% methanokwater fractions. Further purification of small amounts for F A B - M S was achieved by thin-layer chromatography using aluminum backed silica gel plates (Whatman cat. # 4420 222) with EtOAc:MEK:formic acid: water, 5:3:1:1 as developing solvent. The compounds in the scraped bands were eluted with methanol. 3

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R E S U L T S A N D DISCUSSION Maysin was found in the leaves of teosinte, corn, and centipede grass as shown by the H P L C chromatograms (Fig. 2) with U V spectra (see Fig. 3) acquired on-the-fly. In these H P L C separations obtained with a watenmethanol gradient with 0.01% H P O ^ in each solvent, the early peaks were identified as caffeic acid derivatives, mainly chlorogenic acid and its isomers, and the later peaks were identified as mainly flavonoids with the detecting wavelength at 340 nm. In centipede grass the major phenolic compound was chlorogenic acid but maysin and other luteolin derivatives (Fig. 2c) were present as indicated by peak retention times and U V spectra. Teosinte (Fig. 2a), on the other hand, contained a high level of maysin in the leaves and silks, and two other luteolin derivatives in the leaves which eluted just before and just after the maysin peak in the H P L C chromatograms. Very little chlorogenic acid or its isomers was found. Maysin was isolated from teosinte leaves and its C N M R spectrum (Fig. 4a) compared and found to be identical to the original C N M R spectrum (unpublished) obtained by Elliger (11). In addition another isomer of maysin was separated from maysin by D C C C and its C N M R spectrum recorded (Fig. 4b). Maysin has been detected at various levels by H P L C in the lower, early (pre-whorl) leaves of all but 24 out of more than 300 varieties, inbreds and populations. However, in the silks, maysin was usually much more concentrated in the 297 samples of inbreds and populations analyzed. The fall armyworm and corn earworm both prefer to feed on whorl leaf tissue. However, late in the season when population densities are high, the fall armyworm will also feed more towards the tip of the leaf which contains higher levels of maysin. 3

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Teosinte Leaves

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Centipede Grass

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Walter's White At Dry Silk Stage

U4W —r~ 10

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Time (min) Figure 2.

H P L C profiles of leaf extracts (340 nm).



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GUELDNER ET AL.

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Maysin in Corn, Teosinte, and Centipede Grass

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Wavelength (nm) Figure 3.

U V spectra of luteolin (maysin) and apigenin compounds.


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Maysin 6"

H

sjX^OH

" ° ^ J ^ O ^


Me

a-Rha

Maysin

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Maysin in Corn, Teosinte, and Centipede Grass - American Chemical

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