Joumal of Chemical Ecology. HII. 25. No. II. 1999

ALLELOCHEMICALS ISOLATED FROM TISSUES OF THE INVASIVE WEED GARLIC MUSTARD (Alliaria petiolata) I

STEVEN F. VAUGHN* and MARK A. BERHOW

Bio(lc/ive Agents Research USDA. ARS. Nll/ional Cemer jiJl' Agricultural Utilization Research 1815 N. University 51.. Peoria. Illinois 61604

Abstract-Garlic mustard (Alliaria {'etiolala) is a naturalized Eurasian species that has invaded woodlands and degraded habitats in the eastern United States and Canada. Several phytotoxic hydrolysis products of . principally allyl (AITC) and (BzITC). were isolated from dichloromethane extracts of garlic mustard tissues. AITC and BzITC were much more phytotoxic to wheat (Triticulll aestil'lllll) than their respective parent glucosinolates and glucotropaeolin. However. garden cress (Le{'idium sativlIIn) growth was inhibited to a greater degree by glucotropaeolin than BzITC. possibly due to conversion to BzITC by endogenous myrosinase. Sinigrin and glucotropaeolin were not detected in leaf/stem tissues harvested at the initiation of flowering. but were present in leaves and stems harvested in the autumn. Sinigrin levels in roots were similar for both sampling dates. but autumn-harvested roots contained glucotropaeolin at levels over three times higher than spring-harvested roots. The dominance of garlic mustard in forest ecosystems may be attributable in part to release of these phytotoxins. especially from root tissues.

Key Words-Garlic mustard. AIlia ria {'etiolala. . glucosinolates. allelopathy. phytotoxins. . benzyl isothiocyanate. sinigrin. glucotropaeolin.

"To whom correspondence should be addressed. I Names are necessary to report factually on available data; however. the USDA neither guarantees nor warrants the standard of the product. and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.

2495

0098-0331/99/1100-2495516.00/0 1999 Plenum Publishing Corporation 2496 VAUGHN AND BERHOW

INTRODUCTION

Garlic mustard [Alliaria petiolata (Bieb) Cavara & Grande (Brassicaceae)] is an herbaceous biennial that has invaded, and now dominates, much of the hardwood forest understory in the eastern and midwestern United States and southeastern Canada (Cavers et a!., 1979; Nuzzo, 1991, 1993, 1998). It is displacing native flora, and it is unlikely that its elimination from heavily infested areas is possible (Anderson et a!., 1996). Populations of native understory plants have been found to decline in areas with a heavy infestation of garlic mustard, which can be as high as 20,000 seedlings/m2 (Trimbur, 1973; Yost et a!., 1991). Groundcover by native ephemerals declined as cover by garlic mustard increased (Nuzzo, 1998). Recent studies have shown that garlic mustard may also pose a threat to organ­ isms other than higher plants, as Porter (1994) reported that adults of the endan­ gered West Virginia white butterfly [Pief·is virginiana (w. H. Edwards»), which normally feed on several Dentaria spp. (Brassicaceae), preferentially laid their eggs on garlic mustard plants. This occurs even though the plant appears to be moderately toxic to the developing larvae (Haribal and Renwick, 1998). Glucosinolates and/or their degradation products appear to be primarily responsible for the pesticidal activity of species in the Brassicaceae (syn. Cru­ ciferae) (Brown et a!., 1991; Grossman, 1993; Brown and Morra, 1995; Mayton et a!., 1996; Mancini et a!., 1997; Vaughn and Boydston, 1997). Glucosinolates are a class of glucose- and sulfur-containing organic anions whose biologically active degradation products are produced when plant cells are ruptured and the glucosino­ lates, which are present in vacuoles, are hydrolyzed by the enzyme myrosinase ((3­ thioglucosidase glucohydrolase; EC 3.2.3.1) (VanEtten and Tookey, 1983). These metabolites include substituted , nitriles, , and oxazo­ lidinethiones, which vary depending on the side-chain substitution, cell pH, and cell iron concentration (Cole, 1976; Daxenbichler and VanEtten, 1977; Fenwick et a!., 1983; Uda et aI., 1986; Chew, 1988). Some ofthese degradation products have been found to be potent phytotoxins (Wolf et a!., 1984; Oleszek, 1987; Bialy et a!., 1990; Yamane et a!., 1992a,b; Brinker and Spencer, 1993; Brown and Morra, 1995; Vaughn et a!., 1996; Vaughn and Boydston, 1997; Vaughn and Berhow, 1998). The leaves and seeds of garlic mustard have been previously shown to contain a high percentage ofglucosinolates (up to 3% offresh weight in seeds), with the predomi­ nant being allyl glucosinolate (sinigrin) (Nielsen et aI., 1979; Larsen et a!., 1983; Daxenbichler et a!., 1991). It is presently unclear whether the dominance of garlic mustard in forest groundlayers is due to competition, allelopathy, or both (Randall, 1996), although a recent report by McCarthy and Hanson (1998) discounted allelopathy as a pri­ mary mechanism. To further elucidate if allelopathy plays a role in garlic mustard dominance, we present results from a bioassay-guided isolation and identifica­ tion of phytotoxins from garlic mustard plants. GARLIC MUSTARD ALLELOCHEMICALS 2497

METHODS AND MATERIALS

Spectroscopy. Gas chromatography-mass spectrometry (GC-MS) was per­ formed on a Hewlett-Packard (HP) 6890 GC system attached to a HP 5972A Mass Selective Detector. Columns used were fused silica HP-5MS capillaries (0.25-JLm film thickness, 30 m x 0.25 mm ID). The GC operating parame­ ters were as follows: splitless injection mode; temperature programmed from 40: to 315 D C at SOC/min with a 2-min initial and a lO-min final temperature hold; He carrier gas flow rate at 1.1 mljmin, with the injector temperature set at 250 D C. Spectra were compared with known standards or by computer with the Wiley/NBS Mass Spectral Registry (McLafferty and Stauffer, 1989). Extract Preparation. Garlic mustard tissues (100 g samples) were sequen­ tially extracted using a Soxhlet apparatus with hexane, CH2Cb, and MeOH, and concentrated by rotoevaporation at low (20:C for hexane and CH2Ch extracts, 50°C for MeOH extracts) water bath temperatures, preventing possible loss of volatile extraction products. A water extract was obtained by soaking the solvent­ extracted tissues in 250 ml of distilled water overnight in a refrigerator at 2°C, after which the marc was washed with two additional 250-ml aliquots, and the extracts lyophilized. Compounds in the crude CH2Ch extract, subsequently found to be active in the bioassays, were separated on a lipophilic Sephadex LH­ 20 (Supelco, Inc., Bellefonte, Pennsylvania) column into three separate fractions using 100% CHCl}; 50% CHCh/50% MeOH; and 100% MeOH as solvents. Seedling Radicle Elongation Bioassay. Wheat (Triticum aestivufll L., Car­ dinal) and cress (Lepidium sativum L. Curly Cress, Brassicaceae) seeds were used in routine bioassays of extracts. Wheat and cress seeds were surface steril­ ized with 0.5% (wIv) commercial chlorine bleach for 15 min, rinsed with sterile distilled water (SDW) twice and subsequently soaked with additional SDW for 2 hr. Seeds were wrapped in sterile paper towels saturated with water and incu­ bated overnight in darkness at 25'C. All crude extracts were assayed by adding extracts to autoclaved water agar in 9.0-cm plastic Petri dishes at the concen­ tration of 1 mg extract/ml agar after the agar had cooled to - 40°C. Column fractions from crude extracts were assayed at concentrations of 0.1 and 0.5 mg extract/ml agar. After the agar had solidified and all solvent had evaporated from the agar, six germinated seedlings of each bioassay species per plate were placed on the agar in the Petri dishes. Dishes were incubated in darkness at 25:C on 45: slants for 24-48 hr, then evaluated for inhibition of radicle growth. Allyl isothiocyanate (AITC), benzyl isothiocyanate (BzITC), and sinigrin standards were obtained from a commercial source (Sigma, St. Louis, Missouri). The glucotropaeolin standard used in bioassays was extracted and purified to greater than 98% from cress seeds by the method of Thies (1988). Solutions of the isothiocyanates (dissolved in acetone) and intact glucosinolates (dissolved in water) were added to cooling water agar to give final concentrations of 0, 2498 VAUGHN AND BERHOW

6 10-3, 10-4 , 10-5 , and 10- M (controls contained acetone only). Plates were sealed with Parafilm (American National Can, Neenah, Wisconsin) to prevent volatilization of the isothiocyanates. Radicle lengths (five plates of six seedlings each of wheat and cress) were measured after 48 hr of incubation, and Iso values (the amount of each compound required to reduce radicle elongation by 50%) were estimated from the intercept of 50% of the control with a best fit line of the data using nonlinear regression analysis (SlideWrite Plus, Advanced Graphics Software, Inc., Carlsbad, California). Glucosinolate Analysis. Glucosinolate concentrations were determined from leaf/stem and root tissues of garlic mustard plants that were harvested on May 7, 1998 (at initiation of flowering), and on October 30, 1998, from plants growing in a oak-hickory (Quercus-Carya) forest in Peoria, Illinois. The ana­ lytical method employed was a modification of a high-performance liquid chro­ matography (HPLC) method developed by Betz and Fox (1994). In brief, 5 g of freeze-dried plant material was added to 200 ml boiling 70% (vIv) MeOH with stirring for 15 min, and then cooled and filtered through Whatman No.2 filter paper. The marc was washed twice with 50 ml aliquots of 70% MeOH. The resulting extract was concentrated to 5-10 ml by rotoevaporation and was diluted to 25 ml to form a working solution. Glucosinolates were purified from the extracts through the use of disposable solid-phase extraction (SPE) columns (Sep-Pak tC 18 , Waters Corp., Milford, Massachusetts). Each column was pre­ conditioned with 5 ml 100% MeOH, followed by 5 ml 0.005 M tetrabutylam­ monium hydrogen sulfate (THS; Sigma). Five milliliters of the working solution was added to the column, and the column was subsequently washed with 5 ml of 0.005 M THS to remove unwanted compounds. Glucosinolates were eluted from the SPE column with 2 ml MeOH/THS (55: 45), and run on a Shimadzu 6A HPLC system using a CIS column (250 mm x 4.6 mm; RP-18, 511; Licrosorb, Alltech, Deerfield, Illinois). The glucosinolate peaks were detected with a Shi­ madzu SPD-M6A photodiode array detector set at 237 nm. The initial mobile phase conditions were 12% methanol-88% aqueous 0.005 M THS at a flow rate of 1 mljmin. The binary gradient was developed to 70% methanol-30% aqueous 0.005 M THS for 20 min. and held at these conditions for an additional 15 min. Concentrations of sinigrin and glucotropaeolin were calculated from standard curves developed for both compounds.

RESULTS AND DISCUSSION

Identification of Phytotoxins from Extracted Tissues. The crude CHzClz extract strongly inhibited cress and wheat radicle elongation. However, the methanol extract was only slightly inhibitory, while the hexane and water extracts had no effect. Fractionation of the crude CHzClz extract on the GARLIC MUSTARD ALLELOCHEMICALS 2499

CH2=CH-CH2-N=C=S Allyl isothiocyanate Benzyl isothiocyanate (20.4%) I @-CH2-N:=C:=S I (35.6%)

Ul C ::J o ~ (J) o c co S "0 /, C H2C-CH-CH2-C=N ::J .Cl« 2,3-epithiopropylnitrile /(8.1%) )~~

5 10 15 20 25 30 Time (min)

FIG. 1. Gas chromatograph and identification of major compounds In active 100% CH2Cl2 fraction from garlic mustard plants.

Sephadex LH-20 column yielded only one fraction (100% CHCI 3) that was highly inhibitory to radicle elongation at 0.5 mg extract/ml agar and that was also very active at the 0.1 mg extract/ml concentration. This fraction contained three major peaks as determined by GC-MS (Figure 1), which were identified by comparison with published mass spectra (Kjrer, 1963; Spencer and Daxen­ bichler, 1980) and comparison with spectra obtained from commercial standards as AITC, BzITC and 2,3-epithiopropylnitrile. After further fractionation on a Sephadex LH-20 column using CH2CI2, separate fractions containing AITC and BzITC were bioassayed as active at 0.1 mg extract/ml agar. A fraction contain­ ing primarily 2,3-epithiopropylnitrile was not active at 0.5 mg extract/ml agar. Prominent diagnostic mass spectral ions and their relative intensities for the iso­ lated AITC and BzITC are as follows: AITC: EI-MS [m/z (%)]: 99 (M+, 88), 72 (34), 45 (12),41 (100). BzITC: EI-MS [m/z (%)]: 149 (M+, 18),91 (100), 65 (15), 51 (6). Toxicity ofIsothiocyanates and Parent Glucosinolates. The calculated I50s for wheat and cress, respectively, were as follows: for sinigrin, >1 x 10-3 and 4.4 x 10-4 M; for glucotropaeolin, >1 x 10-3 and 6.6 x 10-4 M; for AITC, 4.1 x 10-5 and 8.5 x 10-4 M; and for BzITC, 5.2 x 10-5 and 1.9 x 10-4 M (Fig­ ure 2). The higher toxicities of sinigrin and glucotropaeolin to cress may be due 2500 VAUGHN AND BERHOW

125 -e- ....1;.,.,: -0- c,= i 125 g .1 g -0- '\'l-,,,o:! -0- e:".....: c: ._0T-V c: 0 100 ... 1 .i 0 100 f--l i T '0" ~ " ..... , ,J '0 r---!>

125 -0- ...~,,::: -0- ere:.:. 125 e -0- Wb"..: -0- c'~ c ec 0 100 0 100 i ~ '0" l----~- 0 ~'''''' T __b I ',1 C 75 ~ c: c: 75 I I 1\\ ~ \ \ :§ Cl \ " 50 ·-~T \. "Cl c: 0 c: 50 1\ 0 1 . • 0 Ji\:l iii iii 25 U" AITC U'" 25 :c i5 BzlTC. - \ 0::" 0:: 1\ "\ " 10' 10' 104 10~ 10' 10· 104 10~ Concentration (M) Concentration (M)

FIG. 2. Inhibition of wheat and cress radicle elongation by sinigrin, glucotropaeolin, AITC and BzITC. Bars represent ± I SE.

to hydrolysis of glucosinolates in the cress seedlings by endogenous myrosi­ nase (which is absent in wheat), producing AITC and BzITC. Although both AITC and BzITC were quite phytotoxic in these bioassays, AITC in particu­ lar appears to be even more potent as a volatile, because Vaughn and Boydston (1997) reported the complete inhibition of seed germination and growth of seven plant species by this compound at a level of 1 ppm. Perhaps the bioassay that we employed in this study is less sensitive in determining toxicities of compounds with little or no water solubility such as AITC and BzITC. Glucosinolate Content of Garlic Mustard Tissues. There were significant differences in glucosinolate levels in spring-harvested garlic mustard tissues compared to those in autumn-harvested tissues (Table 1). Both sinigrin and glucotropaeolin were below detection limits «1 Itg/mg freeze-dried tissue) for GARLIC MUSTARD ALLELOCHEMICALS 2501

TABLE 1. SINIGRIN AND GLUCOTROPAEOLIN CONTENT OF GARLIC MUSTARD PLANTS FROM SPRING AND AUTUMN HARVEST

Freeze-dried tissue (/lg/mg)(/

Tissue Sinigrin Glucotropaeolin

Spring leaf/stem O.Oc O.Oc Spring root 3.5a l5.3b Autumn leaf/stem 2.8ab O.Oc Autumn root 2.8ab 50.2a

"Means within a column followed by the same letter are not different at P = 0.05 according to Fisher"s protected LSD test. leaves and stems from spring-harvested plants, although autumn-harvested plants had detectable levels of both compounds. Root tissues from both spring and autumn had similar levels of sinigrin, but glucotropaeolin levels were more than three times greater in autumn-harvested than spring-harvested roots. McCarthy and Hanson (1998) suggested that allelopathy was not the pri­ mary method of interference with garlic mustard and that future research should focus on its competitiveness. As these authors point out, garlic mustard is a highly competitive plant that is self-compatible, sets large amounts of seed, germinates under a wide range of environmental conditions, and grows vigorously at near­ freezing temperatures in the late autumn and early spring when leaves are absent from deciduous trees. These researchers reported that a dilution series of aqueous extracts from garlic mustard roots and shoots did not reduce germination of the seeds of several plants, including rye (Secale cereale L.), hairy vetch (Vicia vi!­ losa Roth), and lettuce (Lactuca sativa L.), with only the strongest dilution tested, 1 : 10, being inhibitory to radish (Raphanus sativus L.). In their research, McCarthy and Hanson (1998) prepared extracts by macerating garlic mustard root and shoot tissue in a vegetable juicer and diluting the mixture with distilled water to form a dilution series. Presumably, most of the glucosinolates present in these tissue would be quickly hydrolyzed by endogenous myrosinase to form breakdown prod­ ucts during the extraction process. It is unlikely that AITC and BzITC, which are practically insoluble in water, would remain solubilized, or even be present, in these aqueous extracts. The slight activity seen by these researchers against radish seeds may be similar to the results found in this study against cress seeds, as resid­ ual intact glucosinolates could be converted to phytotoxic hydrolysis products by endogenous myrosinase present in both species. On several occasions when walking in areas heavily infested with garlic mustard plants, the senior author could detect the distinctive odor of AITC ema­ nating from the plants. We were able to collect measurable amounts of both AITC and BzITC once via solid-phase microextraction (SPME) collection from 2502 VAUGHN AND BERHOW plants in vivo, but unfortunately, we have not been able to repeat this observation (data not presented). AITC, which presumably has a very low odor threshold for humans, has been detected only on relatively warm days in the autumn, but curi­ ously not in either the spring or the summer. Perhaps the higher glucosinolate levels we found in the autumn-harvested plants were the cause of the detectable levels of breakdown products we observed. As both AITC and BzITC have been found to be inhibitory to fungi (Patil et aI., 1973; Holley and Jones, 1985; Gamliel and Stapleton, 1993), another poten­ tial role of these compounds, in addition to direct allelopathic effects on neigh­ boring plants, is the inhibition of mycorrhizal fungi. 4-Hydroxybenzyl isothio­ cyanate, the major hydrolysis product of the glucosinolate glucosinalbin, was found to be the predominant compound in roots of wild mustard [Brassica kaber (DC.) Wheeler], which inhibited germination of the vesicular-arbuscular mycor­ rhizal fungus Glo1llus etullicatu1Il (Shreiner and Koide, 1993a,b). Garlic mustard, like other members of the Brassicaceae, is nonmycorrhizal, while the majority of competing plant species present in the oak-hickory understory are mycorrhizal (Harley, 1969). Further study of volatiles released by garlic mustard plants in vivo, and their effect on mycorrhizal associations, is warranted.

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