13 Mechanisms of Allelopathic Action in Bioassay 1
G. R. LEATHER and F. A. EINHELLIG
2
1
Agricultural Research Service, U.S. Department of Agriculture, Frederick, MD Department of Biology, University of South Dakota, Vermillion, SD 57069
21701
2
Bioassays to detect a wide range of concentrations of allelochemicals were developed to follow a l l e l o pathic a c t i v i t y during compound i d e n t i f i c a t i o n and to determine the biochemical mechanism(s) of plant growth i n h i b i t i o n by the allelochemicals. Compar ison of several bioassays f o r s e n s i t i v i t y to phenolic acids, flavanoids, and coumarins showed that the growth and reproduction of cultured Lemna species was i n h i b i t e d at concentrations as low as 50 μΜ. Other assays i n order of decreasing s e n s i t i v i t y were: sorghum seedling growth i n nutrient culture, seed germination, and radicle elongation. The Lemna assay was developed using 24-well culture dishes that provided s i x replications of each treatment. Beginning with three to f i v e fronds per w e l l , the rate of vegetative reproduction and growth rate were determined over a seven-day culture period. Lemna growing i n treatments containing high concentrations of phenolic acids (1000 µM) f a i l e d to produce new fronds and lacked chlorophyll. Lower concentrations (to 250 µM) reduced the growth rate 50% over a seven-day period. Determination of chlorophyll i n Lemna minor and anthocyanin i n Lemna obscura, increased the s e n s i t i v i t y of t h i s bioassay to 0.5 nM concentrations of allelochemicals. The Lemna assays were also useful for determining bioactive fractions extracted from a l l e l o p a t h i c plants. Bioassays are useful tools f o r detecting physiological a c t i v i t y of substances (allelochemicals) i n plant and soil extracts and f o r following a c t i v i t y as extracts are p u r i f i e d and the components separated into various f r a c t i o n s . Frequently, a bioassay detects physiological a c t i v i t y at concentrations much lower than the s e n s i t i v i t y of chemical t e s t s . The choice of a bioassay depends upon the amount of chemical a v a i l a b l e for testing, the suspected physiological a c t i v i t y of the allelochemical, and the s e n s i t i v i t y 0097-6156/ 85/0268-0197$06.00/ 0 © 1985 American Chemical Society
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THE CHEMISTRY OF ALLELOPATHY
needed for detection. A commonly used assay has entailed the use of leachates from sand-nutrient cultures of suspected allelopaths to i r r i g a t e target plants, using a s t a i r - s t e p array i n which pots containing donor plants are placed at a higher l e v e l than those holding the target plants (1). Other variations of sand culture include incorporation of plant residues into the rooting medium, i r r i g a t i o n with nutrient solutions containing plant extracts, and i r r i g a t i o n through surface-applied plant residues (2, 3). A great number of investigators of allelopathy have used e f f e c t s on seed germination and seedling morphology i n bioassays 03, 4^, 5^, 6). Although germination bioassays are simple and expedient, they often have been used without proper r e p l i c a t i o n and s t a t i s t i c a l analysis (7). Other kinds of bioassays have been used to detect the presence of s p e c i f i c allelochemical e f f e c t s (8), e f f e c t s on ^ - f i x a t i o n (j)), the presence of v o l a t i l e compounds (10) and of i n h i b i t o r y substances produced by marine microalgae (11). Putnam and Duke (12) have summarized the extraction techniques and bioassay methods used i n allelopathy research. Recent developments i n high performance l i q u i d chromatography (HPLC) separation of allelochemicals from plant extracts dictates the need for bioassays with s e n s i t i v i t y to low concentrations of compounds contained i n small volumes of eluent. E i n h e l l i g et a l . (13) described a bioassay using Lemna minor L. growing i n tissue culture c l u s t e r dish wells that maximizes s e n s i t i v i t y and minimizes sample requirements. The reported (14) mechanisms of action of allelochemicals include e f f e c t s on root ultrastructure and subsequent i n h i b i t i o n of ion absorption and water uptake, e f f e c t s on hormone-induced growth, a l t e r a t i o n of membrane permeability, changes i n lipid and organic acid metabolism, i n h i b i t i o n of protein synthesis and a l t e r a t i o n of enzyme a c t i v i t y , and e f f e c t s on stomatal opening and on photosynthesis. Reduced leaf water potential is one result of treatment with f e r u l i c and p-coumaric acids (15). Colton and E i n h e l l i g (16) found that aqueous extracts of velvetleaf (AbutiIon theophrasti Medic.) increased d i f f u s i v e resistance i n soybean [Glycine max. (L.) Merr.] leaves, probably as a result of stomatal closure. In addition, there was evidence of water stress and reduced quantities of chlorophyll i n i n h i b i t e d plants. E i n h e l l i g and Rasmussen (17) reported that i n addition to f e r u l i c and p-coumaric acids, v a n i l l i c acid reduced chlorophyll content of soybean leaves but did not a f f e c t chlorophyll i n grain sorghum [Sorghum b i c o l o r (L.) Moench.]. It is not known whether these reported mechanisms are primary or secondary events i n the i n h i b i t i o n of plant growth by allelochemicals. We investigated the value of d i f f e r e n t bioassays for elucidating the mechanisms of action of allelochemicals i n i n h i b i t i n g plant growth. Materials and Methods Seed germination. Tests for allelochemical i n h i b i t i o n of wild mustard [Brassica kaber (DC.) L.C. Wheeler var. p i n n a t i f i d a (Stokes) L.C. Wheeler] seed germination were made according to the methods of Leather (3). Germination of white clover (Trlfolium
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199
repens L.) and radish (Raphanus sat!vus L.) seeds was evaluated i n 5-cm p e t r i dishes with one ml of test solution which saturated a 4.9 cm disc of Whatman No. 3 f i l t e r paper. Each experiment contained 6 replications of 50 clover or 25 radish seeds. The germinator was maintained at 25° C with 8 hr of fluorescent l i g h t . Radicle elongation. Pre-germinated sorghum seeds were placed ten each at the top of a 4.5 by 5.0 cm piece of Whatman No. 3 f i l t e r paper saturated with 1 ml of test solution. The seeds were positioned with the emerging r a d i c l e d i s t a l to the paper and aligned with the long axis of the paper. The paper with seeds was placed between the glass plates of a sandwich chromatography chamber and the plates clamped. The plates were placed at a 45° angle i n a germinator f o r 4-6 hr. After the radicles made contact with the paper, the t i p position was indicated by an ink mark on the glass. Measurements of r a d i c l e elongation were made 24 hr l a t e r . This procedure was a modification of the bioassay described by Parker (18). Sorghum seedling growth. Seeds of sorghum were germinated i n vermiculite under greenhouse conditions (35/25° C day/night temperature, 14 hr supplemental l i g h t from metal hallde lamps). After 6 days, the seedlings were transplanted to 80-ml opaque v i a l s f i l l e d with f u l l strength Hoagland and Arnon's (19) solution containing 1.6 times the normal concentration of iron. After 3 days acclimatization, seedlings of uniform size were selected and treated with an allelochemical. Each treatment was replicated 15 times i n a completely randomized design. The plants were harvested 7 days a f t e r treatment and dry weights of the roots and shoots determined. Lemna bioassay. A bioassay f o r allelochemicals using Lemna minor L. (duckweed) growing i n 24-well tissue culture c l u s t e r dishes was previously described by E i n h e l l i g et a l . (13). In addition, tests were made with allelochemicals to determine their e f f e c t s on chlorophyll production by L^. minor. Chlorophyll content of duckweed was determined a f t e r 7 days i n culture using the method described by E i n h e l l i g and Rasmussen (17). In other experiments, t o t a l anthocyanin production by L^. obscura cultured as above was assayed. Harvested L. obscura were soaked i n 3 ml of O.1 M HC1 for 4 hr i n the dark. The solution was f i l t e r e d and the absorbance of the supernatant at 550 rim was determined. The plant residue was dried at 70° C f o r 24 hr and weighed. Anthocyanin content was expressed as ug of anthocyanin per mg dry weight of I J . obscura. In preliminary studies, we observed that ethanol reduced anthocyanin production i n t h i s bioassay. Since many crude fractions need to be s o l u b i l i z e d i n alcohol or other solvent (20), we examined the effects by adding 5 μΐ of ethanol solution to each well. A l l Lemna experiments contained 6 replications of each treatment and were repeated at least one time. Results and Discussion Seed germination. Germination of wild mustard seeds with a dormancy l e v e l of about 50 percent was stimulated a f t e r 10 days to
200
THE CHEMISTRY OF ALLELOPATHY
near 70 percent by 500 and 100 μΜ f e r u l i c acid (Table I ) . High concentrations (750 and 1500 μΜ) of benzoic acid i n h i b i t e d wild mustard germination throughout the 10 day test, but at 150 μΜ germination was delayed at 3 days and by 10 days stimulation was apparent· TABLE I.
Effect of F e r u l i c (FA) and Benzoic (BA) Acids on the Germination of Wild Mustard Seeds Germination^/
Treatment
3 day
H2O Control 100 μΜ FA 500 μΜ FA 1000 μΜ FA
50.6 60.6 58.4 58.4
a a a a
"" 10 day 54.6 a 69.7 c 67.0 be 60.0 ab
H 0 Control 50.6 c 54.6 c 150 μΜ ΒΑ 41.0 b 68.6 de 750 μΜ ΒΑ 12.0 a 12.0 b 1500 μΜ ΒΑ 3.0 a 3.0 a l/ Means In each column followed by the same l e t t e r are not s i g n i f i c a n t l y different at the 5% l e v e l of probability according to Duncan*s Multiple Range t e s t . 2
The rate of germination of white clover seed was stimulated by 100 μΜ f e r u l i c acid through the i n i t i a l 24 hr of the test (Table I I ) . Germination of clover was delayed by 500 μΜ f e r u l i c acid, but a f t e r 36 hr no differences were observed at 100 to 500 μΜ concentrations. High concentrations (1000-2000 μΜ) were consis tently i n h i b i t o r y . Radish seed germination was not affected by f e r u l i c acid levels tested except at 24 hr when those i n 500 μΜ were less than controls. TABLE I I .
Effect of F e r u l i c Acid (FA) on the Germination of White Clover and Radish Seed
White clover Radish 24 hr 36 hr 18 hr 24 hr 36 hr X germination!' H 0 Control 30.0 be 51.4 b 62.0 b 58.0 a 78.0 be 86.7 a 100 μΜ FA 43.0 d 62.4 c 72.6 b 56.0 a 76.7 be 88.7 a 250 μΜ FA 37.0 cd 71.0 c 74.6 b 44.7 a 67.3 ab 82.7 a 500 μΜ FA 19.6 a 47.6 ab 62.0 b 51.3 a 58.0 a 86.7 a 1000 uM FA 19.4 a 41.2 a 45.6 a 49.3 a 74.7 be 85.3 a 2000 μΜ FA 21.4 ab 39.4 a 45.4 a 49.3 a 83.3 c 92.0 a 1/ Means i n each column followed by the same l e t t e r are not s i g n i f i c a n t l y different at the 5% l e v e l of probability according to Duncan's Multiple Range t e s t . Treatment
18 hr
2
Radicle elongation. The r a d i c l e elongation test was the least sensitive bioassay. Only very high (2000 μΜ) concentrations of f e r u l i c acid i n h i b i t e d the r a d i c l e elongation of pre-germinated grain sorghum (Table I I I ) . In e a r l i e r Investigations we observed i n h i b i t i o n of r a d i c l e elongation i n t h i s test using extracts of
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201
sunflower (Hellanthus annuus L.) at a l e v e l of 4 g/100 ml H2O whereas log d i l u t i o n s of the extract were not active (unpublished data). TABLE I I I . Effect of F e r u l i c Acid (FA) on Radicle Elongation of Pre-germinated Grain Sorghum Control
250 μΜ FA
25.8 + 1.0
500 μΜ FA 1000 μΜ FA mm + SEM a f t e r 24 hr 28.7 + 1.7 24.8 + 1.7 23.1 + 1.0
2000 μΜ FA 16.6 + O.9*
* D i f f e r s s i g n i f i c a n t l y from the c o n t r o l , Ρ • O.05 Grain sorghum seedlings. The growth of grain sorghum i n nutrient culture was inhibited by 250 μΜ concentrations of s a l i c y l i c a c i d (Table IV). Threshold concentrations were variable under greenhouse conditions but were consistently lower than i n seed germination or r a d i c l e elongation experiments. E i n h e l l i g and Rasmussen (17) reported the threshold concentration f o r i n h i b i t i o n of grain sorghum by v a n i l l i c , p-coumaric, and f e r u l i c acids, to be 500 μΜ. A recent study shows that 200 μΜ f e r u l i c acid i n h i b i t s grain sorghum growth when day temperature is 37° C (21), TABLE IV. Effect of S a l i c y l i c Acid (SA) on Grai. Sorghum Seedlings Growing i n Nutrient Solution Concentration SA (μΜ) 0 50 100 250 500 1000 ± J Values are the treatment. Means not s i g n i f i c a n t l y Duncan's Multiple
Experiment 1
Experiment 2
mg dry wt.l/ 209.8 cd 227.9 cd 213.7 d 220.1 cd 260.9 d 184.6 be 165.8 b 206.8 be 76.0 a 172.4 b 57.9 a 66.5 a means of 15 plants harvested 7 days a f t e r i n each column followed by the same l e t t e r are different at the 5% l e v e l of probability using Range t e s t .
Lemna bioassay. The growth of minor i n tissue culture dishes has been reported to have been i n h i b i t e d by 50 μΜ concentrations of allelochemical (13). Results of experiments designed to compare the a c t i v i t y of different allelochemicals on the growth of L. minor are shown i n Tables V and VI. A l l the compounds except chlorogenic acid decreased the f i n a l frond number and dry weight when supplied at 1000 μΜ concentration. However, 250 μΜ chlorogenic acid inhibited both of the measured growth parameters. The 250 μΜ concentration of e s c u l e t i n was also i n h i b i t o r y . These results compare with those reported previously for f e r u l i c acid (13). Another important feature of t h i s bioassay is the a b i l i t y to detect stimulatory levels of compounds. This was shown i n tests with catechin where stimulation of growth and frond production was measured at levels as low as 50 μΜ (Tables V and VI).
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THE CHEMISTRY OF ALLELOPATHY
TABLE V. Comparison of Allelochemicals on the F i n a l Frond Number of L. minor Growing f o r 7 Days i n 24-well Tissue Culture Cluster Dishes Concentration μΜ
Catechin
Esculetin
Chlorogenic Vanillic acid S copoletin acid #
0 35.7 c 39.5 a 52.5 a 54.5 ab 43.5 a 50 59.3 a 39.3 a 45.7 ab 43.5 b NSi/ 100 49.0 b 37.0 a 41.8 abc 55.0 ab 42.8 a 250 42.3 be 23.0 b 36.0 bed 58.0 a 36.2 ab 500 35.8 c 11.2 c 39.3 bed 28.3 c 24.5 b 1000 10.3 d 4.0 c 40.8 abc 4.2 d 4.0 c U Values are the means of 6 replications harvested 7 days a f t e r treatment. Means i n a column followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l using Duncan's Multiple Range t e s t . U No sample. TABLE VI. Comparisons of Allelochemicals on the Dry Weight of L^. minor Growing f o r 7 Days i n 24-well Tissue Culture Cluster Dishes Concentration yM
Chlorogenic Vanillic Esculetin acid Scopoletin acid Dry wt. 0 2.9 c 3.3 a 4.5 ab 3.9 ab 3.7 ab 50 5.4 a 3.6 a 4.8 a 3.4 b NSi/ 100 4.5 b 3.1 a 3.6 ab 4.5 a 3.8 a 250 4.3 b 1.4 b 2.8 c 4.5 a 3.7 ab 500 3.2 c O.6 b 4.2 ab 2.9 b 2.4 b 1000 1.9 d O.3 c 4.6 a O.6 c O.2 c L Values are the means of 6 replications harvested 7 days a f t e r treatment. Means i n each column followed by the same l e t t e r are not s i g n i f i c a n t l y different at the 5% l e v e l using Duncan's Multiple Range t e s t . U No sample. Catechin
1
Additional research with Lemna species has indicated that parameters other than direct growth measurements may be more sensitive i n t h i s bioassay. Table VII compares growth and chlorophyll content of L^. minor as affected by catechin. F i n a l frond number was i n h i b i t e d by 1000 μΜ catechin and stimulated at lower concentrations of 50 and 100 μΜ. Chlorophyll content on a per-frond basis, however, was consistently i n h i b i t e d by catechin and was concentration dependent to 100 μΜ. Lemna obscura fronds contain high quantities of anthocyanin that are extractable by soaking the fronds i n O.1 M HC1. L. obscura growth i n the culture dish bioassay was similar to that of L^. minor and appeared to be more s e n s i t i v e to low l e v e l s of allelochemicals. Anthocyanin concentration was affected by low concentrations of s a l i c y l i c acid (Table V I I I ) . F i n a l frond number and dry weight were consistently reduced by 100 μΜ and 500 μΜ concentrations of s a l i c y l i c acid whereas anthocyanin formation was i n h i b i t e d by 50 yM concentrations of s a l i c y l i c acid and stimulated by O.5 μΜ.
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TABLE VII. E f f e c t of Catechin on the Growth and Chlorophyll Content of Lemna minor L. Growing i n 24-well Tissue Culture Cluster Dishes Concentration catechin, μΜ
Final frond no.
Chlorophyll ^g/frond)
% of control 50 125.1* 100 127.9* 250 110.0 500 98.7 1000 26.5* * D i f f e r s s i g n i f i c a n t l y from the control, Ρ - O.05.
100.6 80.8 61.8* 49.8* 21.3*
TABLE VIII. Effect of S a l i c y l i c Acid (SA) on Growth and Anthocyanin Content i n Lemna obscura L. Cultured i n 24-well Tissue Culture Cluster Dishes Concentration SA, (μΜ)
Final frond no.
Final Anthocyanin dry wt. (mg) (\i&/mg dry wt.) % of control O.5 96.3 93.1 134.4* 1.0 93.1 104.5 88.4* 5.0 84.4* 93.7 94.0 10.0 100.5 127.2* 102.7 50.0 88.7* 123.5* 48.5* 100.0 62.4* 84.6* 30.6* 500.0 13.3* 56.2* O.0* * D i f f e r s s i g n i f i c a n t l y from the controls, Ρ » O.05 by t test. The use of ethanol or other organic solvents f o r dissolving plant allelochemicals to permit bioassay may influence the results of sensitive bioassays. We examined the effects of several d i f f e r e n t concentrations of ethanol on frond production and anthocyanin content i n L^. obscura. The results show that both f i n a l frond number and anthocyanin content were decreased when the culture medium contained O.133 or greater percent ethanol (v/v) (Table IX). TABLE IX. Influence of Ethanol Amendments on F i n a l Frond Number and Anthocyanin Content of _L. obscura Growing i n 24-well Tissue Culture Cluster Dishes
O.0 Final frond no. Anthocyanin (Ug/mg dry wt.)
Percent ethanol (v/v)!/ O.017 O.033 O.067 O.133 O.200 O.267 O.333
39.5
35.6
34.5
33.3
31.4
27.8
27.6
25.6
3.2
3.4
3.4
3.2
2.0
1.8
1.8
1.5
i/Results are the means of two experiments, 6 times.
each value replicated
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T H E C H E M I S T R Y OF A L L E L O P A T H Y
The results of our experiments with several bioassays f o r the determination of allelochemical a c t i v i t y show differences i n threshold levels for i n h i b i t i o n or stimulation. We have demonstrated that anthocyanin production by L^. obscura growing i n 24-well tissue culture c l u s t e r dishes is the most sensitive to allelochemicals. Other assays i n order of decreasing s e n s i t i v i t y were: 1) chlorophyll production and growth of L^. minor i n tissue culture dishes, 2) sorghum seedling growth i n nutrient culture, 3) seed germination, and 4) r a d i c l e elongation. Selection of a bioassay depends on the growth parameters to be measured and the quantity of allelochemical available. For example, the sorghum seedling bioassay used i n our studies may be the best for determining the uptake, d i s t r i b u t i o n , and metabolism of known allelochemicals where quantity of compound is not a factor and the use of radiolabeled materials would be expedient. Additional investigation is required to determine i f effects on these pathways are primary or secondary mechanisms of action and i f the same mechanisms are applicable to more anatomically complex plants and under a range of environmental conditions. The Lemna bioassay, as previously described (13), is p a r t i c u l a r l y useful for the determination of bioactive fractions collected during HPLC of extracts from a l l e l o p a t h i c plants. A c t i v i t y has been detected i n the Lemna bioassay with HPLC fractions of unknown allelochemicals from sunflower using quantities as low as 5 μΐ of a 28 ppm w/v solution (see Saggese et a l . this publication). Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13. 14. 15. 16.
B e l l , D. T., Koeppe, D. F. Agron. J. 1972, 64, 321. Rice, E. L. "Allelopathy"; Academic: New York, 1974; pp. 353. Leather, G. R. Weed S c i . 1983, 31, 37. Lawrence, T., K i l c h e r , M. R. Can. J. Plant S c i . 1962, 42, 308. Guenzi, W. T., McCalla, T. M. Proc. Soil S c i . Am. 1962, 26, 456. Macfarlane, M. J., Scott, D., J a r v i s , P. N. Zealand J. Agric. Res., 1982, 25, 503. Lehle, F. R., Putnam, A. R. Plant Physiol. 1982, 69, 1212. Fay, P. Κ., Duke, W. B. Weed S c i . 1977, 25, 224. Kapustka, L. Α., Rice, E. L. Soil B i o l . Biochem. 1976, 8, 497. Muller, C. H., Chou, C. H. In "Phytochemical Ecology"; Harborne, J. B., Ed.; Academic: London, 1972; pp. 201-16. Chan, A. T., Andersen, R. J., LeBlanc, M. J., Harrison, P. J. Marine Biology. 1980, 59, 7. Putnam, A. R., Duke, W. B. "Allelopathy i n Agroecosystems"; Ann. Rev. Phytopathol 16; Annual Reviews Inc. 1978; pp. 431-51. E i n h e l l i g , F. Α., Leather, G. R., Hobbs, L. L. J. Chem. E c o l . 1984, 10, ( i n press). Rice, E. L. Bot. Rev. 1979, 45, 15. E i n h e l l i g , F. Α., Stille, M. L. Bot. Soc. Amer. Misc. Publ. 1979, 157, 40. Colton, C. E., E i n h e l l i g , F. A. Amer. J. Bot. 1980, 67, 1407.
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17. 18. 19.
20. 21.
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E i n h e l l i g , F. Α., Rasmussen, J. A. J. Chem. E c o l . 1979, 5, 815. Parker, C. Weeds. 1966, 14, 117. Hoagland, D. R., Arnon, D. I. "The water-culture method f o r growing plants without soil," C a l i f . Agric. Exp. Stn. Manual 347, 1950. Davis, D. G., Wergin, W. P., Dusbabek, Κ. E. Pestic. Biochem. and Physiol. 1978, 8, 84. E i n h e l l i g , F. Α., Eckrich, P. C. J. Chem. E c o l . 1984, 10, 161.
RECEIVED June 12, 1984
