Journal of Chemical Ecology, Vol. 15, No. 7, 1989

ISOLATION A N D IDENTIFICATION OF A COMPOUND FROM SOYBEAN CYST NEMATODE, Heterodera glycines, WITH SEX PHEROMONE ACTIVITY

H O W A R D J A F F E , l ROBIN N. H U E T T E L , 2 A L B E R T B. D E M I L O , 2 D O R A K. H A Y E S , l and R A Y M O N D V. REBOIS 2 USDA, ARS, Livestock and Poultry Sciences Institute 2plant Sciences Institute Beltsville, Maryland 20705

(Received June 21, 1988; accepted October 13, 1988) Abstract--A single compound with sex pheromone activity was isolated from the female soybean cyst nematode, Heterodera glycines, by a sequence of four high-performance liquid chromatographic steps and identified as vanillic acid by a combination of ultraviolet spectroscopy and chromatography. The structure was confirmed by gas chromatography-mass spectrometry. Both attractancy and coiling behavior in male soybean cyst nematode were elicited by authentic vanillic acid. Key Words--Soybean cyst nematode, Heterodera glycines, Nematoda, sex pheromone, vanillic acid, attractancy, coiling, bioregu]ation, behavior.

INTRODUCTION

The identification o f behavior-modifying compounds o f free-living, animal- and plant-parasitic nematodes has been the subject of intensive study since the late 1960s. H o w e v e r , to date, no complete structures o f any such compounds have been reported (for review see Bone, 1987; Huettel, 1986). Previous studies on nematodes generally have centered on sex pheromones, aggregation pheromones, or epideitic responses (Huettel, 1986). Bone et al. (1979), in extensive studies o f the animal parasitic nematode, N i p p o s t r o n g y l u s brasiliensis, tentatively identified a long-chain polypeptide as the sex attractant o f this nematode. In studies of the free-living nematode, Caenorhabditis elegans, compounds with physical and chromatographic properties 2031

2032

JAFFE ET AL.

similar to hydroxylated, short-chain fatty acids and bile acids were partially characterized as a primer or epideitic pheromone that causes the dauer larval formation in this species (Golden and Riddle, 1982, 1984). Bone (1986) fractionated the sex pheromone of a plant-parasitic nematode of soybeans, Heterodera glycines, into two components of different solubility. Our laboratory also has investigated pheromone production in H. glycines, the soybean cyst nematode (SCN). We have developed more sensitive bioassays than those used previously to detect responses by males to females of this species (Huettel and Rebois, 1986; Huettel and Jaffe, 1987). Unlike previous bioassays (Greet et al., 1968; Rende et al., 1982), which required at least 8 hr or more to complete, we developed a rapid bioassay based on a specific behavior that could be completed in several minutes. Furthermore, we demonstrated that this behavior could be induced in males only by the presence of females or their crude extracts (Huettel and Jaffe, 1987). This rapid method allowed us to screen large numbers of high-performance liquid chromatography (HPLC) fractions of female extracts. We report here the isolation and complete structural identification of a compound (SCNP) from female soybean cyst nematode with sex pheromone activity. This is, to our knowledge, the first structure of this type to be reported from a plant-parasitic nematode.

METHODS AND MATERIALS

Soybean Cyst Nematode Culture and SCNP Extraction. H. glycines Ichinohe (Nematoda), race 3, was maintained on monoxenic soybean root explant cultures, as previously described (Huettel and Rebois, 1985). Female nematodes (1000), ca. 10-12 days old, were removed manually from the roots and placed in 15 ml Milli-Q water (Millipore). The nematodes were gently agitated in the dark at room temperature for 24 hr. The aqueous extract was then filtered through a 0.22-tzm Acrodisc filter (Gelman) and frozen at - 8 0 ~ in a 20-ml polypropylene vial. Batches of extract from 20,000-40,000 females were lyophilized at 0-5 ~ SCNP Purification. The lyophilisate from 23,870 females was taken up in two 3-ml portions of the starting gradient mixture of HPLC step A (see below). The resulting solution was filtered through a 0.45-/~m Millex-HV filter (Millipore) prior to analysis by HPLC. HPLC Step A. The filtered sample (5.3 ml) was divided into five equal parts (4774 female equivalents) each of which was chromatographed on a 4.6 x 250 mm, 5/~m Supelcosil LC-18DB column with a Pelliguard guard column (Supelco) on a model 840 liquid chromatograph with autosampler (Waters). The sample was injected onto the column and eluted with a concave gradient (Waters

NEMATODE PHEROMONE

2033

curve 7) from 10 to 60% acetonitrile (0.1% v/v trifluoroacetic acid) against 0.1% aqueous trifluoroacetic acid (TFA) over 1 hr at ambient temperature and 1.0 ml/min. The eluent was monitored spectrophotometrically at 214 nm. Fractions were collected over l-rain intervals with both the autosampler and fraction collector cooled to 0-5~ Fractions with the same retention times from the five runs were pooled in the fraction collector, and those fractions (17-19 min) with biological activity were lyophilized at 0-5 ~ HPLC Step B. Each of the pooled lyophilized 1-min fractions was purified further by reverse-phase HPLC on a 4.6 x 250-mm, 7-/zm Zorbax C-8 150 SP column (Dupont) on a 1090 liquid chromatograph equipped with a photodiode array detector and Chemstation (Hewlett-Packard). The column was eluted with a linear gradient from 2 to 15 % acetonitrile in 0.25 N triethylammonium phosphate (TEAP), pH 2.20, over 1 hr at 28~ and 0.4 ml/min. Fractions were collected at 1-min intervals, and the fractions (49-50 min) containing SCNP were processed in vacuo in a model SVC200H Speed Vac concentrator (Savant) to remove acetonitrile. HPLC Step C. Fractions containing partially purified SCNP were purified further by reverse-phase HPLC using the same 1090 chromatograph and Zorbax column with a linear gradient from 2 to 20% acetonitrile (0.1% v/v TFA) against 0.1% aqueous TFA over 1 hr at 28~ and 0.4 ml/min. Fractions were collected at 1-min intervals and the fraction (46-47 min) containing SCNP was evaporated to dryness in the Speed Vac. HPLC Step D. Final purification of the SCNP was achieved by reversephase HPLC of the partially purified SCNP fractions resulting from HPLC step C using the same 1090 chromatograph and Zorbax column, but using a linear gradient from 2 to 20 % 2-propanol (0.1% v/v TFA) against 0.1% aqueous TFA over 1 hr at 28~ and 0.4 ml/min. The resulting fractions containing the SCNP peak were evaporated to dryness in the Speed Vac and stored at - 8 0 ~ Control HPLC Isolation Experiments. Three control samples were prepared and analyzed by HPLC as follows: (1) Milli-Q water (200 ml) was lyophilized, taken up in starting gradient mixture of HPLC step A, and analyzed by HPLC step A. Fractions eluting between 17 and 20 min were pooled, concentrated, and analyzed by HPLC step B. (2) Sterile Glycine max (L.) CV Kent root (t g) was stirred in 15 ml Milli-Q water at room temperature in the dark for 24 hr. The aqueous extract was filtered through a 0.22-#m Acrodisc filter and lyophilized. The residue was analyzed by HPLC step A. Fractions eluting between 16 and 20 min were pooled, concentrated, and analyzed by HPLC step B. (3) Gamborg's B-5 medium Agar (Gibco) was prepared according to the manufacturer's instructions. A 1-g sample was stirred in 15 ml Milli-Q water at room temperature in the dark for 24 hr. The aqueous phase was filtered and analyzed by HPLC step A. Fractions eluting between 16 and 20 min were concentrated and analyzed by HPLC step B.

2034

JAFFE ET AL.

HPLC Analysis of Substituted Phenolic Acids. A group of commercially available substituted phenolic acids (Aldrich) was analyzed by the conditions of HPLC step D. The following absorbance maxima and retention times were observed: 2,3-dihydroxybenzoic acid, 206, 244, 247, 317 nm and 43.8 min; 2,4-dihydroxybenzoic acid (~-resorcylic acid), 208,256,295 nm and 43.0 min; 2,5-dihydroxybenzoic acid (gentisic acid), 212, 238, 330 nm and 34.5 min; 2,6-dihydroxybenzoic acid (3,-resorcylic acid), 2 0 8 , 2 1 5 , 2 4 7 , 3 0 8 nm and 40.5 min; 3,4-dihydroxybenzoic acid (protocatechuic acid), 206, 217,260, 294 nm and 23.4 rain; 3,5-dihydroxybenzoic acid (o~-resorcylic acid), 204, 220, 248, 307 nm and 23.4 min; 3,4-dimethoxybenzoic acid (veratric acid), 204, 219, 260,293 nm and 56.0 min; 3-hydroxy-4-methoxybenzoic acid (isovanillic acid), 2 0 5 , 2 1 8 , 2 6 1 , 2 9 4 nm and 40.4 min; 4-hydroxy-3-methoxybenzoic acid (vanillic acid), 2 0 5 , 2 1 8 , 2 6 1 , 2 9 3 nm and 37.0 min. Bioassay. Dried aliquots of HPLC fractions were taken up in Milli-Q water for bioassay by our previously described method (Huettel and Jaffe, 1987). The response of male SCN to various concentrations of vanillic acid (VA) was bioassayed as follows: male SCN were removed by hand from soybean root culture and aerated in 200 ml Milli-Q water to remove any effects of previous exposure to pheromone. For each concentration of VA in Milli-Q water, 10 males were removed from the water after aeration and placed in a 20-~1 drop of VA solution on a 1.5% Noble Agar plate (Difco). After 30 sec, the males were removed from the VA solution and observed for coiling behavior on the agar plate with a stereomicroscope (Nikon). Derivatization Procedure for GC-MS Analysis of SCNP. A dried aliquot of the purified SCNP peak eluting at 37.0 min in HPLC step D (see Figure 4) was transferred by means of MeOH washes (2 x 0.1 ml) to a 10-cm x 1.6cm-OD, Teflon-lined, screw-capped, glass culture tube for derivatization by a procedure adapted from the methods of Woolson and Harris (1967) and Metcalfe and Schmitz (1961). After the addition of 0.45 g BF3-MeOH reagent (Supelco), the tube was tightly capped and heated in a boiling-water bath for 3 min. After cooling, 6 ml of 10% Na2SO4 was added to decompose excess reagent. The aqueous phase was extracted with 3 ml isooctane-benzene (2 : 1). The organic extract was centrifuged for 5 min at 3500 rpm and the resulting supernatant transferred to a 15-ml conical flask for removal of solvents by rotary evaporation at 25~ The residue was transferred by means of two EtOAc washes (0.15 and 0.10 ml) to a 0.3-ml glass, tapered-tip, HPLC autoinjector vial (Waters). After removal of solvent on the Speed Vac, the residue was dissolved in 30 /xl EtOAc and 2 txl of the resulting solution was analyzed by GC-MS. The following control samples were derivatized and analyzed by GC-MS: (1) dried fractions eluting before (35-36 min) and after (39-40 min) the SCNP peak, (2) authentic VA (200 mg), and (3) a solvent/reagent blank.

NEMATODE PHEROMONE

2035

TABLE 1. G C - M S - S I M ANALYSIS OF METHYL ESTER DERIVATIVES OF S C N P ISOLATED FROM Heterodera glycines AND VANILLIC ACID

Relative Abundance(%) at IndicatedIon

m/z

m/z

m/z

m/z

Compound

Average retention time of GC peak (rain)a

108.1

123.2

151.3

182.2

SCNP Vanillic acid (VA) Methyl vanillate (MEVA)b

5.00 4.99 5.01

13.93 14.07 13.41

27.27 28.27 26.69

98.57 100.0 99.82

96.77 94.17 94.86

aFor SCNP, N = 3; VA, N = 3; MEVA, N = 11. hAuthentic sample (underivatized).

GC-MS Analysis. Samples were analyzed by GC-MS on a model 5985A GC-MS equipped with a model 18835 capillary inlet system, model 1000 computer, model 7920 and 7906 disk drives (Hewlett-Packard), and a DB5 30-m x 0.257-mm-ID fused-silica capillary (0.25/~m film) column (J&W), operated in the splitless mode and temperature programmed from 70 to 250~ at 18~ rain. For selected ion monitoring (SIM) experiments, the column was temperature programmed from 70 to 250~ at 30~ The column was held at 250~ for 20 min between runs for column burn-off. GC-MS instrument parameters were: injection port, 250~ carrier gas (He) inlet pressure, 8 psi; detector source temperature, 200~ electron multiplier voltage 2800 V. Major ions (relative intensities > 4.0) for methyl vallinate (MEVA) standard were observed at m/z 108 (12.6), 111 (5.3), 123 (23.5), 136 (4.8), 151 (base, 100.0), 152 (10.8), 167 (4.5), 182 ( M + , 55.9), and 183 (4.4). For SIM analyses, the instrument was calibrated to monitor exact masses of the following ions: m/z 108.1, 123.2, 151.3, and 182.2. A total of 200-msec dwell time (50 msec/ion) was used for each SIM scan. The GC retention times and relative abundances of the selected ions (Table 1) were averaged for the SCNP (N = 3), VA (N = 3), and MEVA (N = 11). RESULTS AND DISCUSSION

A single compound (SCNP) with sex pheromone activity in male SCN was isolated from aqueous extracts of females and purified by a four-step reversephase HPLC procedure. Biologically active 1-min fractions eluting between 17 and 19 min by HPLC step A (Figure 1) were each further purified by HPLC step B. As shown (Figure 2) for the fraction eluting between 18 and 19 min, the change of column and buffer from TFA to TEAP at this stage of purification

2036

JAFFE ET AL.

588,088

250.000

0.880 8

5

I

1

I

I

!

I

I

I

I

I

I

I

IO

15

29

25

30~

35

40

45

50

55

60

65

Mir~utes

500..888

250.088

0.000 I

10

11

ie

13

I

'14

Minutes

'16

I

I

17

18

19

FIG. 1. Elution profile of extract of 4774 female soybean cyst nematodes by HPLC step A (0.500A214 full scale) between 0 and 70 min (upper chromatogram) and 10-20 min (lower chromatogram). Bars indicate fractions with biological activity. caused the fraction to resolve into several major peaks. The peak eluting between 49 and 50 min (later shown to be biologically active) was purified further by HPLC step C (Figure 3), where it eluted between 46 and 47 min. Although the change of buffer from TEAP to TFA at this stage of purification caused a coeluting small peak in HPLC step B to shift to a lower retention time, satisfactory purification of the main peak was not achieved, as indicated by photodiode array UV spectroscopy. Final peak purification, however, was achieved in HPLC step D using the volatile TFA-2-propanol buffer (Figure 4). Superimposition

NEMATODE PHEROMONE

2037

90 80 70 60

50 D

EE

40

E

30 2010oi

0

1B

3(8

20 Time

40

513

6(8

70

(min,)

Fro. 2. Elution profile using HPLC step B (0.100A22 0 full scale) of pooled fractions from HPLC step A eluting between 18 and 19 rain from 23,870 females. Asterisk indicates peak with biological activity.

of the normalized upslope, apex, and downslope UV spectra (Figure 5) indicated peak homogeniety. Biological activity corresponded exactly with this peak. The structure of SCNP was determined by a combination of UV spectroscopy and HPLC and confirmed by GC-MS. The UV spectrum of SCNP between

18~ ]

:

12~7

L 402

i

!

Time

(min.)

FIG. 3. Elution profile using HPLC step C (0.200A220 full scale) of peak from HPLC step 13 eluting between 49 and 50 min. Asterisk indicates peak with biological activity.

JAFFE ET AL.

2038

J

se~

5~=

i/'

4G]

I

J

'1

34

FIG. 4.

36 Time

38

4.

42

(m4n.)

Elution profile using HPLC step D (0.100A260 full scale) between 32 and 42 min of purified SCNP from 23,870 females.

200 and 500 nm displayed four distinct maxima, characteristic of substituted phenolic acids (Doub and Vandenbelt, 1955; Scott, 1961). Analysis of a representative group of these compounds under HPLC conditions of step D (see Methods and Materials) revealed that VA and SCNP had identical HPLC retention times and UV spectra. In addition, in higher pH buffers, such as 0.01 M NH4OAc, at pH 6.55, both SCNP and VA exhibited identical changes in UV

807083-~

1

5G-

30

2.?

\ aee

INave l e n g t h

(nm)

4;e

5oe

FIG. 5. Overlay of normalized UV spectra of the upslope, apex, and downslope of purified SCNP peak shown in Figure 4.

2039

NEMATODE PHEROMONE

spectrum and shifts to lower HPLC retention times characteristic of substituted phenolic acids (data not shown). Based on these data, SCNP was identified as VA (4-hydroxy-3-methoxybenzoic acid) (Scheme 1).

OH SCHEME 1.

The total isolated yield of VA from 23,870 female SCN was 315 ng or 3.2 pg/female. In further support of the structural identification, the elution profile of a sample containing equal amounts of SCNP and VA (analyzed by the HPLC conditions of step C or D) displayed only one peak (Figure 6). In each case, superimposition of the normalized upslope, apex, and downslope UV spectra was observed, indicating peak homogeneity. The identity of SCNP was confirmed by GC-MS. Because VA exhibited poor chromatographic behavior (broad, distorted peaks) on a variety of fusedsilica capillary columns adaptable to GC-MS, it was converted by treatment with BF3-MeOH to its methyl ester (MEVA). As expected, MEVA eluted as a sharp symmetrical peak and was easily detectable by GC-MS in the low nanogram range (1-5 ng) when analyzed in the selected ion monitoring (SIM) mode. Data in Table 1 show that BF3-MeOH reaction with the SCNP or an

4

30D E E 2~

4b Time

5b

6b

(mira.)

FIG. 6. Elution profiles between 30 and 60 min of equal amounts of purified SCNP and vanillic acid by conditions of HPLC step C (0.050A22ofull scale, lower chromatogram) and HPLC step D (0.050A260full scale, upper chromatogram).

2040

JAFFE ET AL.

authentic sample of VA produced a GC peak with essentially identical retention time and SIM profile as that observed for MEVA. In addition, dried fractions eluting before and after the SCNP peak in HPLC step D were also derivatized and analyzed by GC-MS-SIM. MEVA was not detected in either of these control fractions or the reagent/solvent blank. Since SIM experiments consumed a small portion of the derivatized SCNP sample, the remainder was concentrated and analyzed by GC-MS in the full spectrum mode. Peak I (Figure 7) in the total ion chromatograph was identified as MEVA based on a comparison of its retention time and mass spectrum to those of an authentic sample of MEVA. All major ions present in MEVA (mass spectrum B) were observed in the mass spectrum of peak I (mass spectrum A) in approximately the same relative intensity. Because the yield in the BF3-MeOH derivatization reaction was 55 %, we expected the presence of a significant amount of underivatized SCNP in the reaction mixture. Examination of spectra in the region (peak II, 5.85 min) in the total ion chromatogram where VA elutes as a broad, unsymmetrical peak (5.18-5.92 min) provided a spectrum (mass spectrum C) that contained all major ions observed for an authentic sample of VA (mass spectrum D), including the molecular ion m/z 168 and the base peak at m/z 153 corresponding to M CH 3. Clearly, the GC-MS and GC-MS-SIM data confirm VA as the SCNP. Peaks other than I and II in the total ion chromatogram (Figure 7) resulted from HPLC column bleed and/or impurities in the reagents/solvents. Male SCN displayed identical sex pheromone behavior to VA, i.e., the previously observed attractancy and coiling (Huettel and Rebois, 1986; Huettel and Jaffe, 1987) towards females or their extracts. Males responded to a concentration range of 10-5-10 -7 M VA. At concentrations > 10 -5 M VA, a paralytic-like effect was observed, resulting possibly from an "overload" of the pheromone receptor(s). It should be noted that VA is extremely active in our bioassay, with 60% of the males tested responding to 10 -7 M VA or a total of 336 pg. The phenolic acids including VA are ubiquitous compounds in nature, occurring in soil (Flaig et al., 1975) as products of plant lignin decomposition by fungi (Ishihara and Miyazaki, 1972; Ander and Eriksson, 1978). Although previous work (Harden and Stutte, 1980; Porter, 1983) had failed to detect VA in extracts of unhydrolyzed soybean leaf and root, it was considered essential nevertheless to conduct control experiments. These included the elimination of soybean root, Milli-Q water, and agar as a source of VA by our purification procedure. Both the weight of root and agar used in the control experiments were estimated to be substantially greater than the total weight of the females used in the SCNP isolation. The volume of Milli-Q water was about half that used in the SCNP isolation. Results of these control experiments were negative, confirming only the female SCN as the source of VA. Although a recent report (Bone, 1986) suggested a two-component sex

/ I

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minutes RET. TIME:

8(~]

4.97

A, Peak I

X~l

I% 28

184 i III

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123 ~ ,

:

188

139

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128

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.

,

148

. It,,.

,192 199 .

168

,.h.

,

:

188

2e8

m/z RET. TIME:

88]

4.95

B, methyl vanillate

151

68

182

I% 48

I~8

28

123

93

136

98

188

118

128

138

167

148

158

168

178

188

m/z RET. TIME:

88]

S.8S

C. Peak I T

68

153

l~e

184

I% 48

9?

28

12S

8 ,

.......... .

,

,I . . . . . . . . , 9

188

~1+.

13S l. . . .

12~

183 [I.t

I.

..

I.

191 ,,

168

14~

188

.

199

2~8

m/z RZT. TIME: 88

S.82

1~3

D, vanillic acid

B8 68

I%

125

97

4e

168

182

28 8

...... ! ......... ':: ............. ,! ......... ,,? ...... 9" '

98

188

118

128

138

148

,...!,...

IS8

,....,

.....

168

I,_..,H..,.

I78

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m/z FIG. 7. Total ion chromatogram of derivatized SCNP (upper trace) and associated mass spectra: (A) peak I; (B) methyl vanillate; (C) peak II; (D) vanillic acid.

2042

JAFFE ET AL.

pheromone for SCN, our isolation procedure yielded only a single compound that elicited both aspects of SCN sex pheromone behavior, i.e., attractancy and coiling. We do not, however, rule out the possibility of other active compounds undetected by us. To our knowledge, vanillic acid has not been previously identified as having sex pheromone activity in any species, although vanillin and vanillyl alcohol, the corresponding aldehyde and alcohol, have been identified (Aldrich et al., 1979) in gland secretions of male leaf-footed bugs (Hemiptera: Heteroptera) and vanillin has been identified (Ubik et al., 1975; Vrkoc et al., 1977) as a component of the sex pheromone of the bug Eurygaster integriceps (Heteroptera, Scutelleridae). With the present limited capabilities for chemical control of plant-parasitic nematodes (Feldmesser et al., 1985), we are hopeful the report here of the determination of the first structure of a compound with sex pheromone activity in nematodes might lead to development of future novel and environmentally safe control strategies for these pests. Acknowledgments--We gratefully acknowledge the assistance in mass spectral aspects of this work by Mr. R. Thomas of the Analytical Chemistry Laboratory, Office of Pesticide Programs, Environmental Protection Agency. We also are grateful for technical assistance provided by Ms. S.B. Majeed and for the helpful discussions with Dr. J. Lau of Hewlett-Packard Co., Dr. J. Sprouse of Sprouse Scientific Systems, Inc., and Dr. A. Gennaro of The Philadelphia College of Pharmacy and Science. This paper reports the results of research only. Mention of a proprietary product or a pesticide does not constitute a recommendation by the USDA, nor does it imply registration under FIFRA as amended.

REFERENCES ALDRICH, J.R., BLUM, M.S., and FALES, H.M. 1979. Species-specific natural products of adult male leaf-footed bugs (Hemiptera: Heteroptera). J. Chem. Ecol. 5:53-62. ANDER, P., and ERIKSSON,K.E. 1978. Lignin degradation and utilization by microorganisms. Prog. Ind. Microbiol. 14:1-58. BONE, L.W. 1986. Fractionation of the female's pheromone of the soybean cyst nematode, Heterodera glycines. Proc. Helminthol. Soc. Wash. 53:132-134. BONE, L. 1987. Pheromone communication in nematodes, pp. 147-152, in J.A. Veech and D.W. Dickson (eds.). Vistas on Nematology. E.D. Painter, DeLeon Springs, Florida. BONE, L.W., HAMMOCK, B.D., GASTON, L.K., REED, S.K., and SHOREY, H.H. 1979. Partial purification of the aggregation pheromone nippolure from female Nippostrongylus brasiliensis (Nematoda). J. Chem. Ecol. 5:297-308. DOUB, L., and VANDENBELT,J.M. 1955. The continuity of the ultraviolet bands of benzene with those of its derivatives. Application to certain trisubstituted derivatives. J. Am. Chem. Soc. 77:4535-4540. FELDMESSER,J., KOCHANSKY,J., JAFFE,H., and CHITWOOD,D. 1985. Future chemicals for control of nematodes, pp. 327-344, in J.L. Hilton (ed.). Agricultural Chemicals of the Future (BARC Symposium 8). Rowman & Allanheld, Ottowa. FLAIG, W., BEUTELOPACHER,H., and RIETZ, E. 1975. Chemical composition and physical properties of humic substances, pp. 1-211, in J.E. Gieseking (ed.). Soil Components, Vol. I. Springer-Verlag, New York.

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2043

GOLDEN, J.W., and RIDDLE, D.L. 1982. A pheromone influences larval development in the nematode, Caenorhabiditis elegans. Science 218:578-580. GOLDEN, J.W., and R~DDLE, D.L. 1984. A Caenorhabiditis elegans dauer-inducing pheromone and an antagonistic component of the food supply. J. Chem. Ecol. 10:1265-1280. GREET, D.N., GREEN, C.D., and POULTON, M.E. 1968. Extraction, standardization, and assessment of the volatility of the sex attractants of Heterodera rostochiensis Woll., and H. schachtii Schm. Ann. Appl. Biol. 61:511-519. HARDEN, J.M., and STUTTE, C.A. 1980. Analysis of phenolic and flavonoid compounds by highpressure liquid chromatography. Anal. Biochem. 102:171-175. HUETTEL, R.N. 1986. Chemical communicators in nematodes. J. Nematol. 18:3-8. HUETTEL, R.N., and JAFFE, H. 1987. Attraction and behavior of Heterodera glycines, the soybean cyst nematode, to some biological and inorganic compounds. Proc. Helminthol. Soc. Wash. 54:122-125. HUETTEL, R.N., and REBOIS,R.V. 1985. Culturing nematodes--culturing plant parasitic nematodes using root explants, pp. 155-158, in B.M. Zuckerman, W.F. Mai, and M.B. Harrison (eds.). Plant Nematology Laboratory Manual. University of Massachusetts Agricultural Experiment Station, Amherst, Massachusetts. HUETTEL, R.N., and REBOIS, R.V. 1986. Bioassay comparisons for pheromone detection in Heterodera glycines, the soybean cyst nematode. Proc. Helminthol. Soc. Wash. 53:63-68. ISHIHARA,T., and MIYAZAKI,M. 1972. Oxidation of milled wood lignin by fnngal lactase. J. Jpn. Wood Res. Soc. 18:415-419. METCALFE, L.D., and SCHMITZ, A.A. 1961. The rapid preparation of fatty acid esters for gas chromatography analysis. Anal. Chem. 33:363-364. PORTER, P.M. 1983. Identification of phenolic acids and flavonoids in the roots of the soybean (Glycine max). MS thesis. University of Illinois at Urbana-Champaign, Illinois. RENDE, J.F., TEFFT, P.M., and BONE, L.W. 1982. Pheromone attraction in the soybean cyst nematode, Heterodera glycines. Race 3. J. Chem. Ecol. 8:981-991. SCOTT, A.I. 1961. The electron transfer absorption of substituted benzenes. Experientia 17(2):6869. UBIK, K., VRKOC,J., ZDAREK,J., and KONTEV,C. 1975. Vanillin: A possible sex pheromone of an insect. Naturwissenschaften 62:348. VRKOC,J., UBIK, K., ZDAREK,J., and KONTEV,C. 1977. Ethyl acrylate and vanillin as components of the male sex pheromone in Eurygaster intergriceps (Heteroptera, Scutelleridae). Acta Entotool. Behemoslov. 74:205-206. WOOLSON, E.A., and HARRIS, C.I. 1967. Methylation of herbicides for gas chromatography determination. Weeds 15:168-170.