Charles J. Soderquisl Department of Environmental Toxicology University of California
Chemical ecology is the study of chemical interactions between living things. One type of interaction is termed allelochemic-that is, involving chemicals by which organisms of one species may affect the growth, health, or behavior of another species ( I ) . If one plant releases a chemical that inhibits the germination, growth, or occurrence of another plant, the interaction is allelopathic. Allelopathv has been observed in a wide variety of plant species. ~ c most e thoroughly studied case ( 2 ) is'the soft chipparal plant communities of southern California where mint and sagebrush dominate over grasses and herbs by releasing volatile terpenes such as camphor. Further examples include applL and peach trees (3),eucalyptus trees (4), and sunflowers (5). Additional examples plus discussions of the evolutionarv basis for and a d a ~ t a t i v eadvantages - of allelopathy are contained in excellent reviews (1, 6 ) . The classic example of allelopathv is the phytotoxic action of the black walnut tree (Juglam nigraj on the tomato plant. Pliny the Elder (23-79 A.D.) stated that "The shadow of the walnut tree is poison to all plants within its compass." It is now known that this allelopathic effect is comdue to the chemical 5-hydroxy-1,4-naphthoquinone, monly called juglone.
Juglone and Allelopathy He proposed that the observed toxicity was in fact due to leaf secretions carried to the soil by rain and not from the roots at all. In support of this, Bode noted that when tomato plants growing under walnut trees were covered to protect them from rain, no toxicity was observed. When the covers were removed, characteristic wilting and yellowing of the tomato leaves occurred. Furthermore, nutrient solutions previously used to grow walnut seedlings did not inhibit growth of tomato seedlings. Here we stand with two conflicting theories, both purporting to explain the allelopathic effect of juglone on tomato plants. There are a number of reasons for this discrenancv. First. some critical experiments vital to either proposition have only been conducted since both theories were ~uhlished.While the scientific literature did contain some necessary information, much of it was in rather obscure journals perhaps not available to workers a t the time. A close examination of the existing scientific literature will reveal data which may allow us to determine the validity of these two opposing hypotheses. These data include juglone history, synthesis, method of isolation, affected plants, producing plants, toxicity, distribution within the walnut tree, biosynthesis, and mode of action. In addition, this approach will supply a review of other interesting aspects of juglone chemistry.
. .
Literature JUGLONE
Juglone is a chemical of interest to scientists from a variety of disciplines. In the jargon of the botanist and ecoloeist. u , its effects are termed allelo~athic:to the pesticide chemist it is a naturally occurring herbicide; to the chemistrv instructor its facile synthesis and easily demonstrated effects on plants offers a unique bridge between the lab and the real world (7). Allelopathic chemicals may he transported from the producing plant to the target plant by a number of routes: from leaf surfaces via volatilization, rain wash, or fog drop; from decaying plant parts on or in the soil; or by secretion from root systems. Speculation as to the route of transport in the case of juglone has centered around two theories. The "root theory" was advocated by Cook in 1921 (8) and Massey in 1925 (9). Cook observed wilting and yellowing of the leaves, the characteristic symptoms of juglone toxicity, on tomatoes and potatoes. He noted that the range of toxicity corresponded to the spread of the walnut root system and concluded that the toxic factor resided in the roots. Massey observed that tomato plants within 50 feet of a hlack walnut tree were killed. He offered three possible explanations: (1) exhaustion of soil water by the walnut, (2) decay of leaves and shells releasing a toxin, or (3) secretion of a toxin from the roots. He "proved" that the first two cases were incorrect and therefore concluded that root secretion must he responsible. Massey further stated that affected plants must have their root systems in close contact with the roots of the walnut. The "root theory" was accepted for 30 years until Bode in 1958 (10) offered an alternate view: the "leaf theory." 782 / Journal of Chemical Education
The bark and husks of walnuts have a long history of use as a source of yellow dye. In this century in the American South i t was common practice to cut the husks from young nuts and throw them immediately into a pond, thereby stunning the fish which would rise to the surface and were easily collected (11). The Greeks and Romans knew of the walnut's fungicidal and bactericidal properties, and American doctors as late as 1917 prescribed juglone for treatment of various skin diseases, including ringworm. The first scientific report of juglone's allelopathic effects came from Stickney in 1881 (121, although this wax probably common knowledge to farmers long before. For example, in the July 1, 1871 issue of the Pacific Rural Press, a Mr. Snowberger wrote "I feel satisfied that I have seen three apple trees destroyed by black walnuts, and I believe they destroy grape vines. I judge it is the water dropping from the walnut leaves that does the work." Davis in 1928 (13) injected extracts of hlack walnut into tomato and alfalfa seedlings and observed toxic effects identical to those observed under field conditions. He also attempted to apply juglone's fungicidal properties as a seed coating with little practical success. Bernthsen and Semper in 1887 (14) first synthesized 5hydroxy-1,4-naphthoquinoneand characterized it as a yellow solid, melting point 155"C, slightly soluble in hot water, soluble in ether or alcohol, and steam distillable. Jesaitis and Krantz (7) recentlv described a simple svnthesis via oxidation of 1,5-dihydi~xyna~hthalene. k chemical termed "nucin" was isolated from walnuts in 1856 by Vogel and Reischauer (15), and characterized by Comhes in 1907 (16) as iugloue. Modem methods for the isolation of juglone are based on ether extraction followed by sublimation, or paper or thin-layer chromatography (17, 18). Juglone is reported to he toxic to tomatoes, potatoes, al-
Juglone Content in Walnut Parts (17)
V C , C H ?
II
0 HO JOH
falfa, azaleas, blueberries, and apples-all important economic crops. While many other plants are probably also susceptible, juglone is not a broad spectrum herbicide. For instance, grasses commonly grow under black walnut trees. Juglone occurs in the black walnut (Juglans nigra), butternut (J. cinerea), English or Persian walnut (J. reeia). walnut (J. sieboldiana). . . and hickor, .. JaDanese . (dhrya ouata). Juglone is highly phytotoxic. For example, a 10 part per million (ppm) aqueous solution gives 50% inhibition of growth of tomato seedlings, and a 100 ppm solution results in comulete mortalitv. .(17). . In addition. walnut extracts sedate goldfish, Daphnia magnu, rats, rabbits, perch, and frogs (19). Synthetic juglone is toxic to fungi, bacteria, and yeast (20), and an absolute lethal dose of 0.1 to 1.0 g juglone per kg body weight has been established in mice 1211. ~--,-
In 1969, Lee and Campbell (27) performed an experiment lone overdue. Thev auantitated the distribution of juglone in various parts of two-year-old walnut seedlings (see table. Dart A). I t is a~Darentthat most of the iuelone iesides in'ihe roots and h;ils. Data based on olderkalnut trees indicates only a small seasonal variation in content in leaves or hulls (see table, part B). Other workers have demonstrated that no juglone occurs in the edible walnut fruit (20). The walnut tree, like any other plant or animal containing a toxic substance, must in some way avoid autotoxicity. Plants commonly "tie up" potentially toxic chemicals as amino acid conjugates and/or glycosides. For example, the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is found in treated plants as the amide of aspartic acid and the ester of glucose. The first clue to the walnut's detoxication mechanism came from Gries ( l l ) , who in 1943 pointed out that the inner root bark and husks contain no juglone a t all, but are quite rich in a-hydrojuglone and that unon exnosure to air. a-hvdroiuelone is oxidized to . .juglone and then further t o dark, non-extractable products. In 1950. Daelish reexamined the situation (22). With careful exclusionbf moisture and air, he was able to isolate the 5-glucoside of 1,4,5-trihydroxynaphtbalene.Since the uv spectrum of a-hydrojuglone and the glucoside differ, Daglish was able to demonstrate the absence of free a-hydrojuglone in walnut extracts by the lack of its characteristic absorption. The glucoside is extremely labile, being readily hydrolyzed to glucose and a-hydrojuglone which can then be oxidized either by air or by oxidizing materials in soil or plant roots to the active toxic principle-juglone.
-
-.
This process is vividly demonstrated when a green walnut husk is cut open. The opened, white husk will rapidly turn yellow as the glucoside is hydrolyzed and oxidized to juglone. Some work has been done recently on elucidating the biosynthesis of juglone. Various precursors such as ocarboxyphenyl pyruvic acid (231, o-succinylbenzoic acid (a), and shikimic acid plus 1,4-naphthoquinone (25) have
OH
-
o-SUCCINnBENZOIC AClD
-
SHIKIMIC AClD oCARWXYPHENYL PYRUVIC ACID
0
been implicated. By comparison, essentially nothing is known of the mode of action of juglone, although Bode (10) suggests one similar to indoleacetic acid (IAA). Interference with the respiratory process is not the mode of action as demonstrated by Perry (26) who grew tomato (juglone sensitide) and bean (juglone insensitive) plants in a M juglod? solution and observed a 50% inhibition of respiration (oxygen uptake) in both cases. Along with leaf wash and root secretion, it should be noted that the decay of dead walnut parts represents a possible transport mode. Winter and Brusewitz (27) have demonstrated that plants readily abstract naturally occurring constituents of other plants. In fact, they found that when walnut debris was placed on top of soil in which wheat seedlings were growing, juglone and two other unidentified chemicals were found in both the soil and wheat plants. Conclusion
It is well established that the black walnut tree and the tomato plant are involved allelopathically and that juglone is the active principle. The details are less clear. Discussion of the merits of the "leaf' and "root" theories may provide the student with some perspective of the way in which scientific theories change. In both the lab and the class room, juglone is a good example of chemical ecology because of the familiarity and availability of tomato and walnut plants, the straightforward isolation of juglone from walnuts, and the easily demonstrated phytotoxicity. Literature Cited Whittaker,R. H.. andFeny. P. P..Scienee. 171,757 li9711. Muller. C. H.. and daiMoral. R..Bull. Torre Bofan. Ciub. 93.130l19661. Borner,H.,Bofon. R a u . 26.393(19601. Florence. R. G., and Cracker. R. L..Ecology. 43. 670 (19621. Curtis, J.T.,andCottam, G.,Bull. TarrwBofon. Club. 77.167(19501. Sondheimer, E., and Simeonc, J. B.. (Editoral, "Chemical Emlow," Academic Press. New York. 1970. (71 Jesaitia, R.0..and Krsntr, A,. J . CHEM. EDUC., 49, 436 (19721. (61 Cook. M. T.. Phvfaoathoiow. 11.346 119211.
(11 (21 (31 (41 (51 I61
(141 (151 (161 (171 1181 (191 I201 (211 1221 I231 (24) (251 (261 I271
~ m t h a & , A . ,andsemper, A.,B~TDIYL.Chem. Oes., 30,934l18871. Vogel, A,. and Reiachausr. C.. B"ehnerNeuaa Rep. furPharm.. 5.106 116561 Comhes,R..Bull. S o c Chim., l.M0(19Wl.
h, K. C., and Campbell, R.W.. Hortscienca, 4.297 (19691. Thieiemann. H..Sci. Pharm.. 39. 146119711. Westfall. B . A . . S C ~ 134.1617(19611. ~~~~, lkekawa, T.. Wana. E. L.. Hsrnada. M., Takeuchi. T., and Umezawa. H., Chem. P h m . B d i . ( ~ i k y a ) .i s . 242(19671. Menheralova. A. C.. Trudy Xishine". Sa&hokhm. Inst., 11.37 (19561. Daglish, C..Biorhsm. J.. 47.152(19501. Iadue, M. M., Danaeffe, P. M., and Azerad, R. G., Eur J. Biochsm., 15, 428 (19701. Dsnsette, P. M.. and Azerad, R. G.. Bioehem. Biophys. Re8 Commun.. 40. 1090 (19701. hisfner, E..andzenk, M. H . . Z Ndurfomeh.. B. 23.259119681. Perry, S. F.,Bvll. Torrw Bof. Club. 94,2611967). Winter. A. G., and Brusewifz. G.. Noruruusewchafren, 47,139 (19601.
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November 1973
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