Allelopathy of Barnyardgrass (Echinochloa crux-galli L. Beauv. var. crux-galli)

Transcription

1 Weed Research, Japan Vol. 37 (2) (1992) Report Allelopathy of Barnyardgrass (Echinochloa crux-galli L. Beauv. var. crux-galli) Hai-Hang Li*, Mimako Urashima*, Midori Amano*, Labunmi Lajide*, Hiroyuki Nishimura**, Koji Hasegawa * and Junya Mizutani* Abstract: Barnyardgrass (Echinochloa crusgalli L. Beauv. var. crus-galli) is a very serious weed throughout the world. Its allelopathy through its root exudates was studied. The cultural of barnyardgrass showed strong inhibition on seedling of lettuce. and mung bean. The acidic-neutral fraction extracted from this cultural of barnyardgrass strongly inhibited seed germination and seedling of lettuce at 500ppm concentration. The basic fraction inhibited the of etiolated lettuce seedlings, but did not inhibit its seed germination. p-hydroxybenzaldehyde and p- hydroxybenzoic acid were identified by HPLC and 1H-NMR as the main allelochemicals in the cultural released by the root of barnyard grass. 3,4-Dihydroxybenzoic, vanillic acids and another unidentified inhibitor were also isolated from the. Key words: barnyardgrass, allelopathy, phenolic compounds, inhibition Introduction Barnyardgrass was reported to be one of the 10 worst weeds in the worlds'. It was a serious weed in 32 countries, a principal weed in 10 countries, and a common weed in 4 countries. It has * Mizutani Plant Ecochemicals Project, ERATO, Research Development Corporation of Japan, Eniwa Research Center, Meguminokita, Eniwa, Hokkaido , Japan. ** School of Engineering, Hokkaido Tokai University, Sapporo 005, Japan. (Received January 28, 1992) strong and propagative abilities and invades fields of crops very quickly. It caused yield reduction from 19-79% depending on the crop, environmental conditions, and control methods and their effectiveness12). Wellhausen15) reported that extensive amounts of virgin land was cleared in Venezuela in the late 1950s and early 1960s and planted with rice or corn. After a few years of continuous rice production, Barnyardgrass began to dominate the fields so that little or no grain was produced. In rice fields, barnyardgrass grows much quickly than rice. It has strong competitive ability to absorb light and nutrients7,9). This might be one of the reasons for the decreasing production of rice. It was reported that the water extracts of seeds4) and the residues1) of barnyardgrass inhibited seed germination and plant. Moreover, residues (1% w/w) which were incorporated into three different types of soil, reduced the of corn and soybean. These results indicated that barnyardgrass might have allelopathic effects on other plants, and inhibit the of crops. In many cases, allelochemicals were released not only by plant residues but also actively by the plant root3,11,14) Barnyardgrass grows well in both dry land and paddy land, and is easily cultured in nutrient. In this paper, Barnyardgrass was cultured with a nutrient in the greenhouse, and its allelopathic effect and allelochemicals in the root exudates were studied under laboratory conditions.

2 H. H. Li et al.: Allelopathy of Barnyardgrass 147 Materials and Methods Plant material Seeds of barnyardgrass, Echinochloa crus-galli L. Beauv. var. crus-galli, sterilized with 1% sodium hypochloride for five minutes and washed with distilled water, were germinated and grown on filter paper moistened with distilled water in a plastic tray covered with saran wrap in a chamber (25C, 3,000 Ergs/cm-2/sec-1 irradiation, 14 hours light and 10 hours dark) for one week. One-week-old plants were transplanted onto a stainless steel net fixed at the surface of the nutrient in a cultural tank with a total volume of 4.5 liters and 3.5 liters of Hoagland's nutrient in it, and cultured in the greenhouse. Except for the aerial part of the seedling, all the systems were wrapped with aluminium foil to prevent the of algae. The nutrient was replenished to the original level every day. Two-month-old plants with unripe seeds were used for the detection of allelopathy. Root exudate tests on seedling of lettuce and mung bean Surface sterilized seeds of mung bean (Phaseolus vulgaris L.) and lettuce (Lactuca scariola L. var. sativa Bisch) were sowed on filter paper moistened with distilled water in a plastic tray covered with saran wrap, germinated and grown for one week in the chamber (conditions were the same as described above). Uniform one-week-old seedlings were selected for evaluation of allelopathy. The allelopathy of barnyardgrass was tested using Stevens and Tang's recirculating device of cultural 13). The cultural was renewed before testing. Four one-week-old seedlings of mung bean or lettuce for each test were planted in the acceptor pods of both the control (no plants were cultured in its donor pods) and the treatment (barnyardgrass was cultured in its donor pods) devices. All other conditions were the same as described in the plant material above. As the ph value of the Fig. 1. Change in ph value of the cultural of barnyard grass during one week culture. culture decreased slightly during culture (Fig. 1), it was adjusted to ph 5.0 once every two days during testing. The, fresh and dry weights of the seedlings of mung bean and lettuce were measured after two weeks of treatment. Each test was repeated twice. Extraction, isolation and identification of active substances Four litres of the cultural of barnyardgrass was concentrated into 1 litre in vacuo at 40C. The concentrated was adjusted to ph 2.0 with 5M HCl and fractionated with ethyl acetate three times (acidic-neutral fraction). The aqueous fraction was adjusted to ph 11.0 with 5M NaOH and fractionated with ethyl acetate three times (basic fraction). These two fractions were evaporated into dryness in vacuo at 40t and dissolved in CH3OH (10,000ppm) for bioassay and for the isolation of active substances. The acidic-neutral fraction was analysed and separated by HPLC (YMC-packed ODS column 10mmX300mm). The sample was eluted (2ml/ min., UV detection at 254nm) with H2O: acetonitrile:acetic acid 80:20:0.1 (v/v, A) from 0 to 20min., linear increase to H2O:acetonitrile 9:1 (B) from 20 to 40min. and B from 40 to 70

3 148 Weed Research, min., Isolated compounds of HPLC were identified by the and 1H-NMR spectra retention times compared with those of the authentic Seed germination and seedling All seed germination were done on Toyo Effects tests No. 1 filter paper on tests was (10,000ppm) 500ppm) of the CH3OH of the root the exudates of barnyardgrass of mung bean and lettuce seedlings In the tests with Stevens in 33mm ting petri dishes with 1ml test. For each test, 50pl (the final concentration of the test Vol. 37 (1992) Results compounds. and seedling Japan device of cultural and Tang's 13), recircula- the seedling of mung bean and lettuce was significantly inhibited in the test sides in which the plants of barnyardgrass of the sample was placed on the filter were cultured pots paper in a 33mm petri dish. The dishes with samples were dried in a desiccator overnight. compared One ml of 100ppm bitions could be seen from the third day following Tween 80 aqueous to the controls in their donor cultured in their donor was added to the dishes for over four hours before the beginning they were All the controls bean and lettuce were tested 80 aqueous fresh used for bioassay. in 100ppm Tween solu- tion. tests, 50 seeds of lettuce for each test were added to the test petri after of treatment. weight dish. Germinated 24 hours incubation For seedling form etiolated the petri dish containing hours incubation of hypocotyls in a uni- After 48 in the dark at 25C, the lengths and roots test was repeated were measured. Each at least 2 times. decreased Effect of the cultural of barnyard the beginning slightly did not effect on the test results. of the seedling same whether of in Fig. 1, the ph value in of barnyardgrass mung bean by the root exudates The of lettuce and were almost the the ph value was adjusted to ph 5.0 once every two days or not. Effects of the extracts of barnyardgrass etiolated The Fig. 2. after of mung by 80 and 65% in during culture but this decrease inhibitions of lettuce were planted in test. As shown cultural have a significant seeds were measured in the dark at 25C. tests, five 36-hour-old seedlings the weeks These inhi- The were inhibited two treatment. For seed germination in which no plants were pots (Fig. 2). seedling acidic-neutral from cultural s on seed germination fraction and of lettuce of the extracts grass on the seedling of mung bean (left) and lettuce (right) in the Stevens and Tang recirculating device of the cultural One-week-old seedlings were treated for two weeks..

4 H. H. Li et at.: Allelopathy of Barnyardgrass 149 Table 1. Effect of the acidic-neutral fraction and the basic fraction of the extracts from the cultural of barnyardgrass on the seed germination of lettuce at 500ppm concentration. * For each test 50 seed were used. Each value is the mean of three repeats+se. from the cultural of barnyardgrass showed strong inhibition on the seed germination of lettuce; at 500ppm, it completely inhibited germination for up to 72 hours. The basic fraction, however, did not significantly inhibit the seed germination of lettuce (Table 1). Both the acidic-neutral fraction and the basic fraction showed strong inhibition on the of etiolated seedlings of lettuce. The acidic-neutral fraction inhibited the elongation of hypocotyls and roots of etiolated lettuce seedlings by 54 and 62%, respectively, and the basic fraction by 45 and 53%, respectively, at the concentration of 500ppm (Fig. 3), It seems that the inhibitions on roots were stronger than those on hypocotyls for both the two fractions. Isolation and identification of the main active substances in the acidic-neutral fraction The acidic-neutral fraction (24.2mg) of the extracts from the cultural was analysed by HPLC. Four main peaks were recognized by UV detector at 254nm. These four peaks and other parts of the eludate were separated into 8 fractions (Fig. 4). Fractions 2, 7 and 8 did not show any significant inhibition on the seed germination of lettuce at 200 and 500ppm concentrations (data not shown). Fractions 1(1.5mg), 3 (5.0mg), 4 (1.2mg) and 6 (6.0mg) were purified again by HPLC and identified as 3,4-dihydroxybenzoic acid, p-hydroxybenzoic acid, vanillic acid and p-hydroxybenzaldehyde, respectively, by Fig. 3. Effects of the extracts from the cultural of barnyardgrass on the of etiolated seedlings of lettuce. Each value was the mean of three repeats. Bar indicates SE. HPLC and 1H-NMR analysis compared with the authentic compounds. These substances have been demonstrated to be allelochemicals from many plant species3,11,14) Fraction 5 (1.5mg) inhibited seed germination of lettuce by 100% for up to 48 hours at 200ppm. At least five compounds were included in this fraction, and one of them was active (as indicated in Fig. 4), following

5 150 Weed Research, Japan Vol. 37 (1992) Fig. 4. HPLC spectrum of the acidic-neutral fraction of the extracts from the cultural of barnyardgrass. Column: YMC-pack ODS column (10mmX300mm); eluates: A (H2O:acetonitrile:acetic acid 80:20:0.1, v/v) from 0-20min., A linear gradient to B (H2O: acetonitrile, 10: 90) from 20-40min. and B from 40-70min. at 2ml/min.; detector: UV detector at 254nm.; peak 1:3, 4-dihydroxybenzoic acid, peak 3: p-hydroxybenzoic acid, peak 4: vanillic acid, peak 6: p-hydroxybenzaldehyde, the peak with an asterisk in fraction 5: the unidentified inhibitor. Retention time (min.) detailed analysis by HPLC. This unidentified inhibitor had high inhibitory activity on lettuce seed germination, however, the amount was too small to be identified. The basic fraction was a total amount of 5.1mg and was used up by bioassay. Its lesser amount and lower activity than the acidic-neutral fraction indicated that it was not as important in the allelopathy of barnyardgrass. In the four identified phenolic compounds, the amounts of p-hydroxybenzaldehyde and p- hydroxybenzoic acid were much greater than the other two compounds. They were considered as the main allelochemicals in the root exudates from barnyardgrass. Discussion Barnyardgrass is a typical weed in paddy fields. It can grow vigorously in both paddy and dry land. This property makes it easy to collect its root exudates using culture without water stress. In our experiment, the plants of barnyardgrass grew well in Hoagland's cultural in a greenhouse throughout their life cycle simply by replenishing the nutrient, without any adjustment of ph and nutrients. The cultural showed strong inhibition and a toxic effect on the of lettuce and mung bean seedlings (Fig. 2). However, the barnyardgrass itself did not show any poisonous symptoms when it was cultured in the same nutrient for over two months. These results indicated that barnyardgrass released allelochemicals to inhibit the of other plants, but did not inhibit its own. The acidic-neutral fraction of the extracts from two-month-old plants of barnyardgrass cultured in the greenhouse showed strong inhibition on the germination and seedling of lettuce, Chinese cabbage (Brassica rapa L. var. amplexicanlis), green amaranth (Amaranthus viridis L.), Digitaria adscendens Henr. and timothy (Phleum pratense L.). p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-coumaric acid, transaconitic acid and two unidentified inhibitors were isolated from this fraction. Among them, the amount of p-hydroxybenzaldehyde was much greater (30mg/kg fresh plants) than the other

6 H. H. Li et al.: Allelopathy of Barnyardgrass 151 substances, and displayed the main inhibiting activity of this fraction (Li et al. unpublished data). trans-aconitic acid, an antifeedant identified from barnyardgrass8), and p-coumaric acid were not found in the cultural of barnyardgrass. Inoue et al.6) identified salicylic acid, coumarin and other plant inhibitors in dormant seeds of barnyardgrass (Echinochloa crus-galli var. oryzicola). These substances were not found in the cultural. p-hydroxybenzaldehyde and p-hydroxybenzoic acid were identified as the main allelochemicals secreted by the root of barnyardgrass. But we do not know whether p-hydroxybenzoic acid was secreted directly from the root or was transformed from p-hydroxybenzaldehyde in the cultural. The mechanisms and dynamics of the secretion of these allelochemicals by the root of barnyardgrass are under investigation. Phenolic compounds have been demonstrated to be general allelochemicals from root exudates, fallen leaves, and plant residues of many plant species2,3,10,11,14) These allelochemicals might have important roles in production in agriculture and forestry and in natural ecosystems. However, the mechanisms of actions of these compounds on plant and the resistance of plants to these chemicals are not quite clear. Arabidopsis, a model plant for studies on molecular and genetic mechanisms of plant and development, and lettuce are being used to study the modes of actions of these phenolic compounds. References 1) Bhowmik, P. C. and J. D. Doll Evaluation of allelopathic effects of weed and crop residues. Agron. J. 74, ) Chou, C. H. and Y. F. Lee Allelopathic dominance of Miscanthus transmorrisonensis in an alpine grassland community in Taiwan. J. Chem. Ecol. 17, ) Chou, C. H. and G. R. Waller Phytochemical Ecology: Allelochemicals, Mycotoxins, and Insect Pheromones and Allomones. Institute of Botany, Academia Sinica Monograph Series No. 9. Taipei, Taiwan. 4) Gressel, J. B. and L. G. Holm Chemical inhibition of crop germination by weed seeds and the nature of inhibition by Abutilon theophrasti. Weed Res. 4, ) Holm, L Weed problems in developing countries. Weed Sci. 17, ) Inoue, K., Y. Sarutatara and K. Yamasaki Growth inhibitor in dormant seeds of barnyard grass (Echinochloa crus-galli var. oryzicola). (in Japanese). Nippon Dojo-Hiryogaku Zasshi 46, ) Kawasaki, T., K. Jikihara, M. Moritsugu and T. Fujimoto Nutrient-absorbing capacities in crops and weeds. I. Rice and barnyard grass (in Japanese). Nippon Dojo-Hiryogaku Zasshi 49, ) Kim, M, H. S. Koh, T. Obata, H. Fukami and S. Ishii Isolation and identification of trans-aconitic acid as the antifeedant in barnyardgrass against the brown planthopper, Nilaparvata lugens (Stal) (Homoptera: Delphacidar). Appl. Entomol. Zool 11, ) Kleining, C. R. and J. C. Noble Competition between rice and barnyardgrass (Echinochloa). II. Response to nitrogen, phosphorus, and sulfur in pots. Husb. 9, Aust. J. Exp. Agr. Anim. 10) Kuiters, AT Effects of phenolic acids on germination and early of herbaceous woodland plants. J. Chem. Ecol. 15, ) Putnam, A. R. and C. S. Tang The Science of Allelopathy. New York. John Wiley & Sons, Inc. 12) Rice, E. L Allelopathy, 2nd ed., Academic Press, London. 13) Stevens, G. Jr. and CS. Tang Inhibition of seedling of crop species by circulating root exudates of Gidens pilosa L., J. Chem. Ecol. 11, ) Waller, G. R Allelochemicals: Role in Agriculture and Forestry. ACS Symposium Series, ) Wellhausen, E. J Weeds and man in Latin America. Weeds 10,

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