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Allelopathy and the Secret Life ofAilanthus altissima "

Rod M. Heisey -

Although the reputation of the tree-of-heaven as an ornamental has declinedover the past century, investigations now underway may discover a new rolefor the species as the source of a natural herbicide.

Ailanthus altissima (Simaroubaceae) has been

extremely successful in invading and dominat-ing certain habitats since its introduction to theUnited States in 1784. In parts of the northeast-ern United States, especially in southern Con-necticut, southern Pennsylvania, and the lowerHudson Valley of New York,A. altissimaformsnearly pure stands that are resistant to invasionby other tree species.A number of characteristics contribute tothe invasiveness and success ofAilanthus

altissima, often called tree-of-heaven. First, the

versatility of its reproduction methods providesa decided advantage. The trees regularly producelarge crops of winged seeds that are widely dis-persed by the wind.A. altissima can also spreadrapidly by sprouting from stumps or from itswide-ranging lateral roots, particularly in open-ings or at the edges of forested areas.Anotherof its advantageous characteristics is theextremely rapid growth rate that enables it tooutcompete many other species, especiallywhen reproducing from root or stump sprouts.Average heights reported for one-year-old trees

in south and central Pennsylvania were 1.3 feetfor seedlings, 2.7 feet for root sprouts, and6.0 feet for stump sprouts; two-year-old treesaveraged 3.9 feet, 5.6 feet, and 9.2 feet, respec-tively.2 These rates makeA. altissima one of thefastest-growing trees in the temperate zone.

Allelopathy: The Secret Weapon?Another contributor to the invasivenessand success ofAilanthus altissima may be a

secondary metabolite that provides competitivesuperiority through a process known as "allel-

opathy." Many plants produce chemical com-

pounds that haveno

apparent role in lifeprocesses or plant structure; hence, these com-pounds are called secondary metabolites.Astechniques for identifying naturally producedchemicals have improved in recent decades, ithas become apparent that plants manufacture agreat diversity of secondary metabolites, includ-ing terpenoids, alkaloids, glycosides, flavonoids,coumarins, quinones, saponins, and phenoliccompounds. Humans have found a variety ofuses for some of these compounds, includingmenthol, a terpenoid produced by mint; nico-tine, an alkaloid produced by tobacco; caffeme,an alkaloid produced by the coffee plant andother species; and salicin, a phenolic compoundhaving analgesic properties, from the willowtree. Why do plants produce secondary metabo-lites ?An early hypothesis suggesting that theywere simply waste products of normal metabo-lism has been largely discounted, since it doesnot explain the wide variety of secondarymetabolites. Much evidence now indicates thatsome of these allelochemicals, as they are

termed, play a defensive role for the producerorganism, protecting plants from herbivores bymaking the plant tissues toxic, perhaps, or byreducing their palatability.3 Other compoundshave antimicrobial effects and may protect

plants from invasion by pathogens.Another role that secondary metabolites may

play is that of allelopathy (Greek, allelo-, of oneanother; patheia, suffering), the inhibition ofone plants growth by another through the pro-duction and release of toxic chemicals into theenvironment. Many secondary metabolites have

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A typical stand ofAilanthus altissima showing a sparse understory with scatteredAilanthus root sprouts

been shown to inhibit seed germination or plant

growth in laboratory tests, and entire bookshave been written attributing a broad range ofeffects to allelopathy;4 however, some research-ers question how widespread or important itreally is in natural habitats.~ Two exampleswhere the argument for allelopathy seemsmost convincing are the purple sage (Salvialeucophylla) in the coastal sage scrub commu-

nity of California and the black walnut (Juglansnigra) in the eastern Umted States.~ But in fact,allelopathy has not yet been proven to exist in

any plantto

all researcherssatisfaction.

Testing forAllelopathic Effects

My research has focused on two areas: ( 1)deter-mining whetherAilanthus altissima actuallyis allelopathic under natural conditions, and

(2) evaluating the potential of its secondarymetabolite as a natural herbicide.

Members of the Simaroubaceae, mcludingAilanthus, produce a class of bitter-tastingsecondary metabolites called quassinoids,which exhibit a wide range of biological activity

including negative effects on insects, fungi, pro-

tozoa, viruses, and cancer cells. In ChinaA.altissima has long been used as medicine and asinsect repellent.~ The first publications on allel-opathy byA. altissima were by Mergen (1959)and Voigt and Mergen ( 1962), who reported thatwater extracts of foliage and stems were injuri-ous to tree seedlings of other species. The majorphytotoxic compound produced byA. altissimawas recently identified as a quassinoid com-pound called ailanthone.9A major tool for research on allelopathy isthe

bioassay,a test that allows us to isolate

phy-totoxic compounds and quantify their effectsunder controlled laboratory conditions.A goodbioassay should possess high sensitivity, givereproducible results, and take a relatively shorttime to perform. I usually use seeds of gardencress (Lepidium sativum) for bioassays, becausethey germinate rapidly and are very sensitive tophytotoxins.A basic bioassay involves placinggarden cress seeds on filter paper in petri dishes,treating them with plant extracts, and thenincubating them under standard conditions.At

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Chemical structure of ailanthone (molecular weight376), the phytotoxic compound produced byAilanthus altissima.Allanthone is extremely bitterand belongs to the class of compounds calledquassmoids.

the end of the incubation period, the growth ofthe radicle (the initial root formation) of thetreated seedlings is compared to that of controlseedlings that received only deionized or dis-tilled water.

In order to learn whether ailan-thone could indeed serve as an allelo-

pathic agent forAilanthus altissima,I first needed to find out where in the

tree the phytotoxic compound isfound. This was important becausethe location helps determine thequantity of toxin released into thesoil as well as the release mecha-nism. I began by assaying waterextracts of differentA. altissima tis-

sues, using the method describedabove. Phytotoxic effects were high-est for the inner bark of the trunkand the bark of roots and branches,

intermediate for leaves, and lowestfor the thin outer bark of the trunkand for the wood of the trunkand roots.l These results suggestedtwo possible release mechanismsfor the phytotoxin: (1) extractionof toxin from bark and foliage byrain, followed by stemflow downbranches and trunks; or (2) exudationfrom roots.

Tests were designed to learn whichof these mechanisms, if either, could

deliver biologically effective amounts of thetoxin fromAilanthus altissima trees to nearbysoil. Stemflow collars were placed aroundA.altissima trunks, and rain was collected as it

flowed down the trees.At the same time, pre-cipitation was collected m open areas nearby toserve as a control. The water samples were thentested using the cress seed bioassay. Surpris-ingly, stemflow stimulated more cress radiclegrowth than either control precipitation ordeionized water.1 In retrospect this result wasnot unreasonable. The outer bark of A.

altissima (low in ailanthone) probably preventsthe toxin from being leached from the high-ailanthone inner bark in large enough quantities

to inhibit plant growth; the bark may also con-tribute inorganic nutrients or growth hormonesto the stemflow, thereby offsetting the effect ofany ailanthone that does reach the soil. In anycase, the results certainly did not supportstemflow as a mechanism responsible for allel-opathy under natural conditions.

Bioassay of water extracts ofAilanthus altissima root bark onseeds of barnyard grass (BYGR, Echinochloa crusgalli), corn(Zea mays), and garden cress (Lepidum satmum/. The seeds weremoistened with (left to mghtJ deiomzed water for control or awater extract of A. altissima root bark correspondmg to 1 gram ofbarkm 5000 and 500 milhhters, respectmely. Both concentrationsof extract caused considerable mhlbition of radicle growth of allthree species compared to the control.

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Effect of addmgAilanthus altissima leaflets and root bark to soil on growth of garden c,ress The pots rec.emed

(left to mght) no bark (=control); 2 grams of leaflets and 2 grams of root bark from which ailanthone had beenextracted with methanol, and 0.5, 1, and 2 grams of non-extracted root bark The non-extracted bark causedobmous inhibition of cress growth, but bark from which the allanthone was removed with methanolstimulated growth compared to the control contammg no bark. The leaflets reduced cress growth shghtly, buttmuch less than the bark.

To test whether significant amounts ofailanthone could be released byAilanthusaltissima roots, some roots were added to soil m

petri dishes. Control dishes contained identicalsoil, but no roots. The dishes containing rootswere stored m a refrigerator (to retard degrada-tion of the toxin by soil microorganisms) for 6 or13 days to provide time for the toxin from theroots to exude into the soil. The dishes were

then removed from refrigeration, seeds of gardencress were placed on the soil near the roots, andradicle growth was measured 3 days later. Theresults showed that significant amounts ofailanthone had been released from the roots.

Exposure of the soil for 6 days to fine roots (lessthan three millimeters in diameter/ reducedcress radicle growth to 50 percent of that in thecontrol soil, and exposure to larger roots (five to

ten millimeters in diameter) reduced cressradicle growth to 74 percent. In soil stored for 13

days, cress radicle growth was reduced by expo-sure to fine roots to 33 percent of growth in thecontrol soil and to 74 percent by exposure to

larger roots.In another testAilanthus altissima root bark

and leaves were added separately to soil in potsand the effect measured on garden cress seeds.Root bark strongly inhibited growth of theseeds, whereas leaves had a much weaker effect.

Dry root bark added in quantities of 0.2, 0.4, and

0.8 percent of dry soil weight reduced cress seed-ling emergence to 39, 21, and 5 percent respec-tively of that m pots containing no root bark;cress shoot biomass was reduced to 55, 25, and5 percent of biomass in the control pots. Dryleaflets added to soil m similar quantities onlyreduced cress emergence to 94, 88, and 93 per-cent of that in control pots and shoot biomass to

81, 81, and 64 percent. These results support the

hypothesis that exudation of ailanthone fromA.altissima roots is a mechanism whereby allel-opathy could occur, whereas exudation fromleaves probably is not.The experiments described so far had a seri-

ous weakness: theAilanthus altissima tissues

used had been removed from the trees and

injured by cutting or drying. It could thereforebe objected that the experiments did not mimic

natural situations.Another investigation wasperformed to assess more realistically thepotential for allelopathy caused by root exuda-tion of ailanthone. Soil within two centimetersofA. altissima roots was collected m a twenty-year-old stand of trees and assayed usmg gardencress seeds. Control soil was collected from a

nearby forested area containing fewA. altissimatrees. Cress radicle growth in soil from nearA.altissima roots was 85 percent of radicle growthin control soil. The bioassay was repeatedbecause the difference was so small; in the

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Effect of treatments of ailanthone pre-emergence, photographed 6 days afterspray (top row of flats), andpost-emergence, 5 days after spray (bottom rowof flats).Apphcation rates are (left to mghtJ 0 (control), 0.5, 1, 2, 4, and 8kilograms of ailanthone per hectare Plant species are (front to back of flats)redroot pigweed (Amaranthus retroflexus), garden cress, velvetleaf, foxtail~Setaria glaucal, barnyard grass, corn, and (m post-emergence flats only)seedlmgs ofAilanthus altissima.All plants were killed m the post-

emergence treatments,even at

05

kg/ha, except velvetleaf,which was

m~ured at the higher rates of apphcation, andA. altissima seedlmgs, whichremarkably showed no m~ury even at 8 kg/ha

second test, cress radicle growth in soil nearA. altissima roots was 77 percent of control

radicle growth.The investigations of root exudation provide

evidence that ailanthone may be released

into the rhizosphere ofAllanthus altissimain amounts sufficient to influence the growthof other plants. Before concludmg that A.altisslma is allelopathic, however, another fac-tor had to considered. Many organic compoundsare rapidly degraded by soil microorganisms;juglone, for example-the allelopathic com-pound from black walnut-can be degraded rap-idly by soil bacteria to concentrations belowwhich phytotoxicity would occur.2 If the sameis true of ailanthone, its biological effectivenesscould be greatly reduced.The persistence of ailanthone in soil was

therefore examined. In one investigation, asolution of ailanthone was mixed with soil in

petri dishes. In some dishes, soil that had beensterilized by autoclaving was used, whereas

nonsterile soil was used in other dishes. Thedishes were then incubated at 25 degrees Centi-grade for time periods ranging from 0 to 21 days,and the soil was subsequently tested for phyto-toxicity with a cress seed bioassay. Strongtoxicity persisted for 21 days in the dishes con-taining sterile soil; by contrast, it persisted foronly 2 or 3 days and rapidly disappeared thereaf-ter in the dishes containing nonsterile soil.Asimilar pattern was observed when powdered

Ailanthus altissima root barkwas

mixed withsoil and incubated. These results clearly demon-strate that the toxic effects of ailanthone in

soil are short-lived, probably because of micro-bial degradation, and raise questions aboutallelopathic potential ofA. altissima undernatural conditions.

Ailanthus altissima as a Herbicide

Regardless of its ecological role, ailanthone is avery powerful herbicidal compound: in thestandard garden cress bioassay, radicle growth

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is typically reduced to 50 percent by a solutioncontaining only 0.7 milligrams of ailanthoneper liter (0.7 parts per million~.3Ailanthoneis therefore being evaluated for commercial

use, since a natural herbicide could have severaladvantages over synthetic ones: (1) rapiddegradation of the herbicide in soil or water,resulting in less environmental pollution;(2) reduced dependence onfossil fuels since the herbicidecould be made biosyntheticallyrather than from petrochemi-cals ; and, perhaps (3), lowertoxicity of the herbicide to non-

target organisms.Ailanthone can be described

as a broad-spectrum herbicidethat is toxic to many plants,both weeds and crop species.It has its greatest effect onannual plants shortly after theyhave emerged, but it also hasa significant pre-emergenceeffect.Ailanthone is toxic toboth monocots and dicots, butdicots tend to be the more

sensitive. It has a very low

degree of selectivity; however,Ailanthus altissima seedlingsand certain species in theMalvaceae such as cotton

(Gossypium hirsutum) and vel-vetleaf (Abutilon theophrasti)are resistant.

Initial investigations of theherbicidal effects of Allan-thus altissima were made

in the greenhouse using acrude extract of root bark.

Later, after the herbicidal com-

pound had been identified,purified ailanthone was used.Both the crude extract and

the purified ailanthone weresprayed onto the surface of soilsown with weeds and crop spe-cies to test for pre-emergenceherbicidal effects. The soil wasthen watered so that the herbi-cidal material would be carried

down into the seed zone. To test post-

emergence herbicidal effects, the crude extractor purified ailanthone was sprayed directly ontoemerged seedlings of weeds and crop species.

Strong herbicidal effects resulted from bothpre- and post-emergence applications, but thepost-emergence effects were especially striking:even the lowest application rate of ailanthone

Field trial ofpost-emergence spray with extract ofA. altissima bark mnedays after apphcation. The plot at top (control) received no extract, andthe plot belowrecemed the eqmvalent of 1 1 kilogram of ailanthoneperhectare. The predommant weed is Galinsoga ciliata. The crop plants are(front row to back row) corn, cauhflower (Brassica oleracea var. rtalica/,tomato (Lycopersicon esculentuml, and green bean (Phaseolus vulgans/.

A sigmficant reduction m weed populatlon of the treated plot isapparent, but m~ury to the crops is also evident.

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A matureA~lanthus altissima mAugust Showmg the charac,tenstrc compound leaves, gmmg a somewhat

palm-like appearance, and abundant clusters of mpening samaras.

(equivalent to 0.5 kilogram per hectare) causedcomplete mortality of most plant species tested.The most recent tests of ailanthone were

conducted in outdoor field plots. Because largeamounts of the herbicidal material were

required and isolation of pure ailanthone isexpensive and time-consuming, a crude extractofAilanthus altissima trunk bark was used.

Weeds and crops were planted in the field andsprayed after emergence with an extract con-taining a known amount of ailanthone. Symp-toms of damage were evident on many weedsand crop species within a few days of spraying.

As demonstrated previously in the laboratory,ailanthone does not persist long in the soil, sonew weeds germinated and some injured weeds

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recovered within a few weeks of spraying.Asingle application of ailanthone would thereforebe insufficient to control weeds over an entire

growing season. Future research will investigate

ways to extend the herbicidal effects over alonger time and to minimize toxicity to crops.

Conclusion

Despite the positive results of many laboratoryinvestigations, we do not yet have enough infor-mation to state unequivocally thatAilanthusaltissima is allelopathic: too little is known ofthe complex interactions and potentially miti-gating circumstances that occur in the naturalenvironment. However, from an evolutionarystandpoint it makes little sense that A.altissima would expend the energy to produce acompound unless it somehow conferred a selec-tive advantage. It is certain that ailanthone haspowerful herbicidal effects and may haveevolved to inhibit competing plants, but it mayalso have other functions.Anecdotal evidence

suggests that it is toxic to some fungi and maytherefore function to protect A. altissima

against fungal pathogens. It might also act as afeedmg deterrent to herbivores because of itsextremely bitter taste, a possibility suggested by

the fact that few animals feed onA. altissimaplants. Clearly, there is much we have yet tolearn about ailanthone and the secret role it

plays m the life ofA. altissima.

Endnotes

1 Illick and Brouse 1926, Hu 1979, Pelgler 1993.2 Illick and Brouse 1926.

3 Swam 1977, Bell 1981.4 Rice 1984, Putnam and Tang 1986.

5 Harper 19776

Muller and Muller 1964, Muller and del Moral 1966,Massey 1925, Fisher 1978.

7 Klocke et al. 1985, Polonsky et al. 1989; Hoffmann etal. 1992; Trager and Polonsky 1981, Pierre et al. 1980,Ogura et al 1977.

8Yang and Tang 1988.

9 Heisey 1996.10 Heisey 1990a.11 Heisey 1990b.12 Schmidt 1988.

i3 Heisey 1996.

References

Bell, E.A. 1981 The physiological role(s) of secondary(natural) products. The Biochemistry of Plants,ed. P. K Stumpf and E. E. Conn. NY:AcademicPress, vol. 7, 1-19.

Fisher, R. F. 1978. Juglone mhibits pme growth undercertam moisture conditions. Soil Science

Society of Amenca Journal 42: 801-803.

Harper, J. L. 1977. Population Biology of Plants NY:Academic Press, 369-381.

Heisey, R. M. 1990a.Allelopathic and herbicidal effectsof extracts from tree-of-heaven (Ailanthusaltissima)Amencan Journal of Botany 77:662-670

. 1990b. Evidence for allelopathy by tree-of-heaven(Ailanthus altissima) Journal of ChemicalEcology 16: 2039-2055.

. 1996. Identification of an allelopathic compoundfromAilanthus altissima (Simaroubaceae) andcharacterization of its herbicidal activity.

Amemcan Journal of Botany 83: 192-200.

Hoffmann,J J., S. D. Jolad, L. K Hutter, and S. P.McLaughhn. 1992. Glaucarubolone glucoside,a potent fungicidal agent for the control ofgrape downy mildew Journal ofAgriculturaland Food Chemistry 40 1056-1057.

Hu, S. Y. 1979.Allanthus.Arnoldia 39: 25-50. ~ -

Ilhck, J. S , and E F. Brouse. 1926 The Ailanthus Tree mPennsylvama. Bulletm 38. Harmsburg, PA.Pennsylvania Department of Forests and

Waters.

Klocke, J.A, M.Amsawa, S. S. Handa,A. D Kmghorn,G.A. Cordell, and N. R. Farnsworth. 1985.Growth mhibitory, msecticidal, andantifeedant effects of some antileukemic and

cytotoxic quassmoids on two species ofagricultural pests. Expementia 41: 379-382.

Massey,A. B. 1925.Antagomsm of the walnuts (Juglansmgra L. and J. cmerea L.) m certain plantassociations. Phytopathology 15: 773-784.

Mergen, F. 1959.A toxic principle m the leaves ofAilanthus Botamcal Gazette 121: 32-36.

Muller, C. H, and R. del Moral. 1966. Soil toxicityinduced by terpenes from Salma leucophyllaBulletm of the Torrey Botanical Club 93: 130-137

Muller, W. H., and C H. Muller 1964. Volatile growthinhibitors produced by Salma species. Bulletmof the Torrey Botanical Club 91: 327-330.

Ogura, M., G.A. Cordell,A. D. Kinghorn, and N R.Farnsworth. 1977. Potential anticancer agents.VI. Constituents of Ailanthus excelsa

(Simaroubaceae). Lloydia 40: 579-584.

Peigler, R. 1993.A defense of ailanthus.AmericanHorticultumst 72/2/: 38-43.

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Pierre,A., M. Robert-Gero, C. Tempete, and J. Polonsky1980. Structural requirements of quassinoldsfor the inhibition of cell transformation

Biochemical and Biophysical ResearchCommumcations 93: 675-686.

Polonsky, J., S. C. Bhatnagar, D. C Griffiths, J.A.Pickett, and C. M. Woodcock. 1989.Activity of

quassinolds as antifeedants against aphidsJournal of Chemical Ecology 15: 993-998

Putnam,A.R., and C. S. Tang, eds. 1986. The Science ofAllelopathy. NY: Wiley

Rice, E. L. 1984.Allelopathy NY:Academic Press.

Schmidt, S. K. 1988. Degradation of ~uglone by soilbacteria. Journal of Chemical Ecology 14:1561-1571

Swain, T. 1977. Secondary compounds as protectiveagents.Annual Remew of Plant Physiology 28:

479-501.

Trager, W., and J. Polonsky. 1981.Antimalarial activityof quassinolds against chloroquine-resistant

Plasmodmm falciparum m vitro.AmencanJournal of Tropical Medicme and Hygiene 30.531-537.

Voigt, G. K., and F. Mergen. 1962 Seasonal variation intoxicity of ailanthus leaves to pme seedlingsBotanical Gazette 123: 262-265.

Yang, R. Z., and C. S. Tang. 1988 Plants used for pestcontrol m Chma: hterature review. Economic

Botany 42: 376-406.

Rod Heisey, professor of biology at Penn State Umver-sity, grew up on a farm in Lancaster County, Pennsyl-vania. While a graduate student at the University ofCalifornia, Davis, and a postdoctoral researcher at thePesticide Research Center of Michigan State University,he became mterested in allelopathy and the use ofnatural products for controlling agricultural pests.His research focuses on plant ecology and microbialecology, with emphasis on allelopathy and natural-product pesticides.

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