Valuation of Phytotoxic Activity of Naturally Occurring Phenolic Compounds*

J. Weed Sci. Tech. Vol. 43 (4) 341～348 (1998) 雑草研究 Original Report

Yuka Kobayashi** and Misako Ito**

Abstract: Relative phytotoxic activity of sensitivity of test plants did not relate to the 21 phenolic compounds previously reported family of species, but seemed to have some as allelochemicals was evaluated with aque- relationships with their seed size. The larger ous solution test and soil incorporation test. seed species tended to be less sensitive to At 1.0mM, aqueous solutions of juglone, both compounds, while sensitivity of the coumarin, t-cinnamic acid, 0-hydroxy- small seed species varied over a wide range phenylacetic acid and 3-phenylpropionic acid (Fig. 4). were most inhibitory to the radicle elongation of lettuce (Lactuca sativa) and Japanese barn- Key words: phenolic compounds, coumarin, yard millet (Echinochloa utilis), followed by juglone, soil incorporation, sensitivity of test salicylic acid, 3-resorcylic acid and benzoic plants acid (Fig. 1). The effect greatly decreased at Introduction 0.1mM for all compounds except juglone. Lettuce was more susceptible than E. utilis. Phenolic compounds are secondary com- The most effective 8 compounds were then pounds synthesized by vascular plants, incorporated in soil and bioassayed using composing lignins in cell walls and occurring germinating lettuce (Fig. 2). Inhibitory activ- as either a free or conjugated style of ity on lettuce radicle elongation was highest glycosides and esters in the various plant in juglone, followed by coumarin, as in the tissues23). Numerous naturally occurring solution test. The effect on 14 plant species of phenolic compounds, mostly phenolic acids, soil-incorporated coumarin and juglone was have been studied extensively from the al- also determined. Although juglone fatally lelopathic point of view23). They have been inhibited radicle elongation of all test species shown to be phytotoxic and are regarded as at 2.0tc mol/g soil, phytotoxic activity of responsible for allelopathic phenomena in coumarin at 2.0umol/g soil differed greatly weed-crop interference and population shifts among the species, and the activity of both of weeds, due to their activities in bioassay. compounds at 0.2 and 0.4umol/g soil also However, bioassay methods to determine differed among them (Fig. 3). In this case the their activity were so diverse in concentration of compounds, ways of application and * Part of this paper was presented at the 33rd test species, that growth inhibitory levels Annual Meeting of the Weed Science Society of reported often differed by 10 times or more Japan and at the 15th Asian Pacific Weed Science Society Conference, Tsukuba for the same compound, e. g. benzoic acid2,14), ** Laboratory of Weed Science, Graduate School of salicylic acid2,5,14), p-hydroxybenzoic acids, p Agriculture, Kyoto University, Kyoto 606-8502, -coumaric acids and vanillic acids. Conse- Japan (Accepted January 12, 1998) quently, the results were not always reliable 342 J. Weed Sci. Tech. Vol. 43 (1998) enough for the compounds to be categorized millet (Echinochloa utilis Ohwi et Yabuno, as allelopathic agents, although a few strong cv. Green millet), 20 seeds each, were placed inhibitors such as juglone have been con- in 6cm glass petri dishes, and 4ml of the test firmed as allelochemicals. solution was added to each dish with 3 re- Real allelochemicals necessarily have high plications. The dishes were then placed in an phytotoxic activity to certain plant popula- incubator controlled at 25C, light (34umol/ tions under certain natural conditions. For secm2). Radicle length of Japanese barnyard conclusive proof of allelopathy, lab bioassays millet and lettuce was measured on the 4th must demonstrate the effect of chemicals on and 6th days, respectively. associated plant species. Large numbers of controlled lab bioassay have adopted simple Soil incorporation test. The eight most conditions such as aqueous solution and a few effective compounds, 3-phenylpropionic acid, test species. But these bioassay methods t-cinnamic acid, o-hydroxyphenylacetic would lead to overestimation of the activity acid, salicylic acid, benzoic acid, Q-resorcylic of compounds, because bioassay in aqueous acid, coumarin and juglone, were selected solution would exclude the effect of adsorp- according to the result of the solution test tion and/or decomposition of compounds in and used in a further bioassay with soil. soil and the effect on a few test species might Plastic cups, 6cm in diameter and 3.5cm not apply to other species. deep, were filled 30g in each cup with non- Therefore, the objectives of this study were sterilized sandy loam soil filtered with 2.0 to evaluate: 1) the relative phytotoxic activ- mm mesh (pH:5.9, clay content: 12%, ity of 21 naturally occurring phenolic com- organic matter content: 2%, CEC: 10.2me/ pounds which had been implicated in al- 100g) collected from the Experimental Farm lelopathy, and 2) how the activity changes of Kyoto University. The compounds were when applied in soil and to various test added as aqueous solutions and mixed with species. soil carefully, final concentrations being 0.2, 0.4 and 2.0umol/g soil and soil moisture Materials and Methods being at 79.7% of the field capacity (pF= Aqueous solution test. Commercially avail- 2.0). Five germinating lettuce seeds per cup able phenolic compounds of analytical-grade were planted 5mm deep. The cups were (catechol, benzoic acid, salicylic acid, Q- covered with polyvinyl film and then incubat- resorcylic acid, gentisic acid, protocatechuic ed under 25C, light (12a mol/secm2). For acid, a-resorcylic acid, gallic acid, vanillic each treatment 4 cups were used. Radicle and acid, syringic acid, t-cinnamic acid, p- hypocotyl length was measured 5 days after coumaric acid, caffeic acid, ferulic acid, planting. sinapic acid, o-hydroxyphenylacetic acid, 3- phenylpropionic acid, coumarin, juglone and Comparison of test species. The phytotox- chlorogenic acid) were used. Each compound ic activity of juglone and coumarin, two of was initially dissolved in dimethylsulf oxide the most distinguished inhibitory compounds and diluted with distilled water to a final in the previous tests, was assayed on 12 weed concentration of 1.0mM or 0.1mM. Ger- species collected from agricultural land or minating seeds of lettuce (Lactuca sativa L., non-agricultural land in Kyoto prefecture cv. Great lakes 366) and Japanese barnyard (Poaceae: Sacciolepis indica (L.) Chase; Kobayashi et al.: Valuation of Phytotoxic Activity of Naturally Occurring Phenolic Compounds 343

Eleusine indica (L.) Gaertn.; Panicum bisul- Very active: 3-phenylpropionic acid, t-cin- catum Thunb.; Digitaria ciliaris (Retz.) Koel., namic acid, o-hydroxyphenylacetic acid, Asteraceae: Erigeron sumatrensis Retz.; coumarin and juglone at 1.0mM caused Artemisia indica Willd., Polygonaceae: Per- almost complete inhibition of radicle elonga- sicaria lapathifolia (L.) S. F. Gray, tion of both test plants. Juglone was recog- Lamiaceae: Leonurus japonicus Houtt., nized the most active compound from its Amaranthaceae: Amaranthus patulus Ber- activity at 0.1mM, followed by coumarin. toloni, Onagraceae: Oenothera biennis L., Relatively active: salicylic acid, benzoic acid Portulaceae: Portulaca oleracea L., and Cyper- and h-resorcylic acid were ranked in this aceae: Cyperus iria L.), lettuce and Japanese group from the responses of both species at barnyard millet. Seed germination of all the 1.0mM. species was over 80%, and only germinating Slightly active or non-active: catechol, p- seeds were used. All other procedures were hydroxybenzoic acid, a-resorcylic acid, the same as described above. gentisic acid, protocatechuic acid, gallic acid, vanillic acid, syringic acid, p-coumaric acid, Results caffeic acid, ferulic acid, sinapic acid, and Comparison among compounds. Phytotox- chlorogenic acid, caused little or no inhibition ic effects varied greatly among the 21 of radicle elongation in Japanese barnyard phenolic compounds (Fig. 1). Lettuce was millet, although the activity in lettuce varied. more sensitive than Japanese barnyard millet Phytotoxic symptoms in the test plants were on the whole. According to the response of different between the compounds: t-cinnamic both species at 1.0mM, these compounds acid, o-hydroxyphenylacetic acid, 3-phenyl- could be classified into the following three propionic acid and coumarin caused swelling categories in their phytotoxic activity: of lettuce radicles; salicylic acid and Q-resor-

1.0mM 0.1mM

Fig. 1. Effects of aqueous solutions of phenolic compounds on the radicle elongation of Japanese barnyard millet and lettuce. Abbreviations: o-HPAA=o-hydroxyphenylacetic acid, 3-PPA=3-phenylpropionic acid, p -HBA=p-hydroxybenzoic acid, Cat=catechol, Ben=benzoic acid, Sal=salicylic acid, 3-Res=3-resorcylic acid, Gen=gentisic acid, Pro=protocatechuic acid, a-Res=a-resorcylic acid, Gal=gallic acid, Van=vanilic acid, Sry=syringic acid, t-Cin=t-cinnamic acid, p-Cou=p-coumaric acid, Caf=caffeic acid, Fer=ferulic acid, Sin=sinapic acid, Cou=coumarin, Jug=juglone and Chl=chlorogenic acid 344 J. Weed Sci. Tech. Vol. 43 (1998)

cylic acid caused necrosis at the top and Effects of soil-incorporated application of around the base of lettuce radicles, respec- the 8 compounds most active in the solution tively; curving of cotyledons occurred in jug- test are shown in Fig. 2. Inhibitory activity on lone treated lettuce; lateral expansion of leaf lettuce radicle elongation was highest in jug- blades occurred in coumarin treated Japanese lone, followed by coumarin, as in the solution barnyard millet. test. Juglone both applied as aqueous solution

3-Phenyl- o-Hydoroxy- Benzoic Salicylic β-Resorcylic t-Cinnanuc Coumarin Juglone acid acid acid acid propionic phenylacedc acid acid

Fig. 2. Effects of soil-incorporated phenolic compounds on radicle elongation of lettuce. *and **indicate signifi- cant difference from control at the 5% and 1% levels by t-test, respectively.

Juglone Coumarin

Fig. 3. Effects of soil-incorporated juglone and coumarin on radicle elongation of 14 tested species.

-■-Sacciolepis indica ---◇--Lactuca sativa

--●-- Eleusine indica -★-Persicaria lapathifolia

-▲-Panicum bisulcatum --☆--Leonurus japonicus

---◆--Digitaria ciliaris -◎-Amaranthus patulus

-□-Echinochloa utilis --♂--Oenothera biennis

--○--Erigeron sumatrensis -♀-Portulaca oleracea

-△-Artemisia indica --×-Cyperus iria Kobayashi et al.: Valuation of Phytotoxic Activity of Naturally Occurring Phenolic Compounds 345

Juglone Coumarin

Fig. 4. Relationship between seed size and susceptibility of tested species to juglone and coumarin soil-incorporated at 0.4mol/g soil. The letters near each circle are the first two letters of the genus name of the test plants. Test conditions were the same as Fig. 3. at 1.0mM (Fig. 1) and soil-incorporated at species of Japanese barnyard millet, Persicar- 2.0umol/g soil (Fig. 2) exhibited almost ia lap athifolia, Digitaria ciliaris, Leonurus equally strong inhibition, while the inhibitory japonicus, lettuce and Panicum bisulcatum activity of the other 7 compounds decreased tended to be less sensitive to both compounds, when incorporated in soil. Q-resorcylic acid while sensitivity of the small seed species which showed a very strong effect in the varied widely (Fig. 4). Sensitivity of the solution test had little effect in the soil test. small seed species, except for Portulaca oleracea, also differed greatly between the two Comparison among test plants. Responses compounds. to juglone and coumarin of 14 species, 12 Discussion weed species belonging to different genera, lettuce and Japanese barnyard millet, were The phytotoxic activity markedly varied determined by soil-incorporated treatments among the compounds: juglone and coumarin (Fig. 3). At 2.01umol/g soil, juglone fatally were the most phytotoxic compounds in the inhibited radicle elongation of all test species, aqueous solution test (Fig. 1). The strong but the activity of coumarin varied among activities of juglone, one of the most the species. At 0.2 and 0.4umol/g soil, sensi- phytotoxic allelochemicals produced by tivity differed greatly among the species, black walnuts (Juglans nigra), as well as ranging from no inhibition to almost com- coumarin were also proved in many other plete inhibition in the two compounds. The studies1,5,11,23)Among the other compounds, 3 phytotoxic activity of juglone and coumarin -phenylpropionic, t-cinnamic and o-hydroxy- was generally reduced at 0.2umol/g soil, but phenylacetic acid also showed strong activ- these compounds were still inhibitory to most ity. Reynolds reported that methylation of the species tested. increased activity of aromatic compounds Sensitivity of the species to juglone and while hydroxylation decreased it, but excep- coumarin as evaluated from their responses tions were seen with ortho-hydroxylated at 0.4umol/g soil is presented in Fig. 4, in compounds. Our result supported this because relation to their seed weight. The larger seed 3-phenylpropionic and t-cinnamic acid do 346 J. Weed Sci. Tech. Vol. 43 (1998) not have a hydroxyl substituent and o- 3). This different sensitivity among species hydroxyphenylacetic acid has an ortho- did not relate to the botanical family of the hydroxyl substituent. Other studies compar- test species, but to the seed size (Fig. 4). As ing several phenolic compounds also showed shown in Fig. 4, relatively large seed species a similar phytotoxic tendency to our tended to be tolerant of juglone and coumar- results. in. This tendency was also obvious in the Furthermore, the result of the soil incorpor- result obtained by Williams and Hoagland23, ating test showed a decrease of phytotoxicity in which cultivated species (cotton, sorghum, of most of the compounds in soil (Fig. 2), corn and cantaloupe) and relatively large although they had similar activity to juglone seed weed species (sicklepod and hemp ses- in the aqueous solution test. When the com- bania) were tolerant to both these compounds were added to the soil at concentra- pounds at a level of which small seed species, tions of 0.2 and 0.4umol/g soil, most of them velvetleaf, redroot pigweed, prickly sida, except coumarin and juglone did not show were very susceptible. Redroot pigweed strong phytotoxicity. Levels of individual (Amaranthus retroflexus) was most suscep- phenolic acids were usually found far below tible to juglone in their experiment like 0.11mol/g soil2,3,13,2,21,22) even under a Amaranthus patulus in this study. Decreased condition such as stubble mulch or just after sensitivity of the larger seed species was also plant residues were incorporated. Results of found for the herbicides atrazine15), benth- the soil incorporating test, together with that iocarb9), alachlor8) and oxadiazone10). In con- of the aqueous solution test (Fig. 1), there- trast, as indicated in Fig. 4, responses of fore suggested that most of the phenolic acids small seed species all of them weeds to jug- have little effect. lone and coumarin varied over a wide range, All the above findings seem to support the and different sensitivity of Denothera biennis, opinion that most of the phenolic acids, by Cyperus iria, Amaranthus patulus and themselves, should not be categorized as Eleusine indica was exhibited to both com- allelochemicals. It nevertheless cannot be pounds. Therefore, it is assumed that differ- denied that these compounds could be im- ent responses of species to the allelo- plicated in allelopathic phenomena in certain chemicals may depend on both seed size and situations. Synergistic interaction between intrinsic sensitivity of the species. some phenolic acids2,23) could be suggested, The study also suggested that bioassay of although Dukes) pointed out that these may phytotoxic compounds should be done under have been the result of improper statistical a certain standard by which the result could analysis. Another possible interaction of be evaluated impartially. Using a well- which Rice19 pointed out the importance is known strong inhibitor such as juglone or that of the environment: hig temperature coumarin for the check is useful device from increased the effect of ferulic acid on the this point of view. In addition, not one but seedlings of soybean and sorghums) and al- several test plants should be selected both lelopathy of Eupatorium odoratum was en- from crop and weed species covering a wide hanced under a shaded conditions range of seed size. Adoption of weed species In the case of coumarin and juglone, the for test plants is particularly important to most phytotoxic compounds, their phytotox- determine the allelopathic potential of the icity among the 14 test species differed (Fig. compounds which are assumed to implicate Kobayashi et al.: Valuation of Phytotoxic Activity of Naturally Occurring Phenolic Compounds 347 weed population shifts or weed-crop interfer- namic acid, its related phenolic allelochemicals, ence. and abscisic acid in seedling growth and seed germination of lettuce. J. Chem. Ecol. 19 (8), 1775-1787. References 13) Li H. H., L. Lajide, H. Nishimura, K. Hasegawa, 1) Audus, L. J. and J. H. Quastel 1947. Coumarin as and J. Mizutani 1993. Allelochemicals in the soil a selective phytocidal agent. Nature 159, 937- beneath Quercus mongolica Fisch var. grosseser- 944. rata Rehd. Wils. Weed Research, Japan 38 (4), 2) Chou, C. H. and Z. A. Patrick 1976. Identifica- 282-293. tion and phytotoxic activity of compounds 14) Lynch, J. M. 1980. Effects of organic acids of the produced during decomposition of corn and rye residues in soil. J. Chem. Ecol. 2, 369-387. germination of seeds and growth of seedlings. Plant, Cell and Environment. 3, 255-259. 3) Chou, C. H. and C. C. Young 1975. Phytotoxic 15) Mennega, R. and P. C. Nel. 1991. Tolerance of substances in twelve subtropical grasses. J. dry bean cultivars (Phaseolus spp.) to atrazine. Chem. Ecol. 1 (2), 369-387. Appl. Plant Sci. 4, 78-81. 4) Demos, I. K., M. Woolwine, R. H. Wilson and C. 16) Nakamura, N. and M. Nemoto. 1994. Combined McMillan 1975. The effects of ten phenolic effects of allelopathy and shading in Eupator- compounds on hypocotyl growth and mitochon- ium odoratum on the growth of seedlings of drial metabolism of mung bean. Am. J. Bot. 62, several weed species. Weed Research, Japan 39, 97-102. 27-33. (In Japanese with English summary) 5) Duke, S. O. 1986. Naturally occurring chemical 17) Putnam, A. R. and C. S. Tang. 1986. Allelopath- compounds as herbicides. Rev. Weed Sci. 2, pp. y: state of science. In "The Science of Al- 15-44. lelopathy" ed. by A. R. Putnam and C. S. Tang. 6) Einhellig, F. A. and P. C. Eckrich 1984. Interac- Jone Wiley, New York. pp. 1-19. tion of temperature and ferulic acid stress on 18) Reynolds, T. 1978. Comparative effects of aro- grain sorghum and soybeans. J. Chem. Ecol. 10, matic compounds of inhibition of lettuce fruit 161-170. germination. Ann. Bot. 42, 419-427. 7) Einhellig, F. A. and J. A. P. Rasmussen 1978. 19) Rice, E. L. 1984. Allelopathy, 2nd edition, Acad. Synergistic inhibitory effects of vanillic and p- Press, Orlando, 422 pp. hydroxybenzoic acids in radish and grain sor- 20) Shindo, H., S. Ohta and S. Kuwatsuka 1978. ghum. J. Chem. Ecol. 4, 425-436. Behavior of phenolic substances in the decaying 8) Harvey, R. G. 1974. Susceptibility of seven process of plants IX. Distribution of phenolic annual grasses to herbicides. Weed Res., 14, 51- acids in soils of paddy fields and forests. Soil 55. Sci. Plant Nutr. 22 (1), 23-33. 9) Ichizen N. 1979. Study on the difference in 21) Wang, T. S. C., T. K. Yang and T. T. Chuang susceptibility and growth response of rice and 1967. Soil phenolic acids as plant growth in- barnyardgrass against Benthiocarb. Ph D disser- hibitors. Soil Sci. 103, 239-246. tation, Kyoto University, Kyoto, Japan. (in 22) Whitehead, D. C. 1964. Identification of p- Japanese) hydroxybenzoic, vanillic, p-coumaric and fer- 10) Kawamura, Y. 1975. Studies on a new herbicide, ulic acids in soils. Nature. 202, 417-418. oxadiazone 3. Weed Res., Japan 20, 55-60. (In 23) Williams, R. D. and R. E. Hoagland 1982. The Japanese with English summary) effects of naturally occurring phenolic com- 11) Kitou, M. 1997. Effects of 16 kinds of phenolic pounds on seed germination. Weed Sci. 30, 206- compounds on the germination and root elonga- 212. tion of several plants. Jour. Weed Sci. Tech. 42 (Suppl.) 114-115. (In Japanese) 12) Li H. H., M. Inoue, H. Nishimura, J. Mizutani, and E. Tsuzuki 1993. Interactions of trans-cin- 348 J. Weed Sci. Tech. Vol. 43 (1998)

自然界にみられるフェノール化合物の性が最も高かった8種類のフェノール化合物を土壌

生育抑制作用の評価に混和して, レタス幼根の伸長に対する影響を調査した。juglone は最も高い阻害活性を示し, coumarin 小林由佳＊伊藤操子＊がそれに準じた (Fig. 2)。また, 14種の供試植物に

対して土壌に混和した juglone と coumarin が与え摘要る影響を調査した。juglone は2.0μmol/g土壌におアレロケミカルとして過去に報告されたことのあいて全ての供試植物の幼根伸長を強く阻害したが, る21種類のフェノール化合物の阻害活性を水溶液試同じ濃度における coumarin の阻害活性は供試植物験と土壌混和試験によって相対的に評価した。水溶間で異なった。また, 0.2および0.4μmol/g土壌に液試験では, juglone, coumarin, t-cinnamic acid, おいては両化合物の阻害活性が供試植物間で異なっ o-hydroxyphenylacetic acid および 3-phenyl- た (Fig. 3)。この場合, 供試植物の感受性は供試植 proponic acidは1.0mMにおいてレタスと栽培ヒエ物の科とは関係なかったが, 種子の大きさとは関係の幼根の伸長を顕著に阻害し, salicylic acid, β- があるようであった。両化合物の阻害活性は大きな resorcylic acid および benzoic acid の阻害活性がそ種子の供試植物では小さいが, 小さな種子の供試植れに準じた。0.1mMでは juglone を除く全ての化合物については種によってその感受性が大きく異なっ物の阻害活性が減少した。また, レタスは栽培ヒエた (Fig. 4)。より強く阻害された (Fig. 1)。次に水溶液の阻害活キーワード: フェノール化合物, coumarin, juglone, ＊京都大学大学院農学研究科雑草学研究室土壌混和, 供試植物の感受性