Crop Protection 23 (2004) 915–922

ARTICLE IN PRESS

*Correspondi

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doi:10.1016/j.cro

Assessment of phytotoxic action of Ageratum conyzoides L.(billy goat weed) on weeds

Tran Dang Xuana, Tawata Shinkichib, Nguyen Huu Hongc, Tran Dang Khanha,Chung Ill Mind,*

a Faculty of Agriculture, Miyazaki University, Miyazaki City 889-2192, Japanb Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0213, Japan

c Faculty of Agronomy, Thai Nguyen Agroforestry, Thai Nguyen City, Viet Namd Department of Crop Science, College of Life and Environment Science, Konkuk University, HwaYangDong, Seoul 143-701, Republic of Korea

Received 22 December 2003; received in revised form 20 January 2004; accepted 9 February 2004

Abstract

Ageratum conyzoides L. (billy goat weed), showed strong inhibition on Raphanus sativus L. (radish) germination and growth in a

bioassay. The leaves exhibited a greater suppression than the stem and root. The leaves of A. conyzoides applied at 2 t ha�1 reduced

about 70% of the growth of Echinochloa crus-galli var. formosensis Ohwi. and completely inhibited emergence of Monochoria

vaginalis (Burm.f. Persil var. plantaginea Solms.) and Aeschynomene indica L. in calcareous soil condition. Application of A.

conyzoides leaves at 2 t ha�1 in a paddy field 2 d after transplanting caused about 75% paddy weed reduction and increased yield by

14% compared with a herbicide treatment. Three phenolic compounds were identified in the leaves, stem and root including gallic

acid, coumalic acid, and protocatechuic acid, and catechin was found only in the stem. p-hydroxybenzoic acid was detected in both

A. conyzoides leaves and stem. Three additional putative allelochemicals were found in the leaves consisting of p-coumaric acid,

sinapic acid, and benzoic acid. The greater number of growth inhibitors found in the leaves might result in the stronger inhibitory

activity than the stem and root. A. conyzoides might be a natural herbicide for weed control in paddy fields to reduce the dependence

on synthetic herbicides.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: A. conyzoides; Allelochemicals; Inhibition; Phenolic acid; Rice; Weed control

1. Introduction

Ageratum conyzoides L. is an annual herbaceous plantnative to Central America and the Caribbean. It is nowfound in many places in tropical and sub-tropicalregions. This species has great morphological variation,and is highly adaptable to different ecological conditions(Sauerborn and Kock, 1988). It has a long history oftraditional medicinal uses in several countries. It hasinsecticidal and nematocidal activity and appears to be avaluable agricultural resource (Sauerborn and Kock,1988).

A. conyzoides is widely utilized in traditional medicineto treat pneumonia, and to cure wounds and burns(Durodola, 1977). Local communities in India use this

ng author. Tel.: +82-2450-3730; fax: +82-2446-7856.

ss: imcim@konkuk.ac.kr (C.I. Min).

front matter r 2004 Elsevier Ltd. All rights reserved.

pro.2004.02.005

species as a bacteriocide, antidysenteric, and antilithic(Borthakur and Baruah, 1987). Aqueous extracts ofleaves or whole plants have been used to treat colic,colds, and fevers, diarrhoea, rheumatism, spasms, or asa tonic (Oliveira et al., 1993). Several pharmacologicalinvestigations have been conducted to determine itsefficacy. Durodola (1977) verified inhibitory activities ofether and chloroform extracts against in vitro develop-ment of Staphylococus aureus. Almagboul et al. (1985)used methanolic extract of the whole plant and verifiedinhibitory action in the development of S. aureus,Bacillus subtilis, Escherichia coli, and Pseudomonas

aeruginosa.A. conyzoides has been reported to have potential use

in controlling pests. Shabana et al. (1991) used aqueousextracts of the whole plant to show reduction of larvaeemergence of Meloidogyne incognita. Pu et al. (1990) andLiang et al. (1994) indicated that this species sheltered

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predators of the spidermite Panonychus citri. Othercitrus spidermite populations, Phyllocoptruta oleivora

and Brevipalpus phoenicis were suppressed with main-tenance of A. conyzoides in the orchards and a reductionof leprosy virus was noted (Gravena et al., 1993).

A number of secondary metabolities of A. conzyoides

have been identified. Vyas and Mulchandani (1986)isolated flavones. Horie et al. (1993) reported hexame-toxyflavone and showed the presence of cumariniccompounds including 1-2 benzopirone in this plant. Inaddition, several alkaloids, including 1,2-desifropirroli-zidinic and licopsamine, which can have hepatotoxicactivity, were also identified.

Allelopathy is defined as the interaction betweenplants in the ecosystems by chemical exudation into theenvironment (Rice, 1984). In agricultural practice,allelopathy has been exploited as a tool of weedmanagement (Kohli et al., 1998). Numerous plants areobserved to have allelopathic activity. However, only afew among them: Medicago sativa L. (alfalfa), Hordeum

vulgare L. (barley), Fagopyrum esculentum Moench(buckwheat), Asparagus officinalis L. (asparagus), Oryza

sativa L. (rice), Zea mays L. (corn), Colocasia esculenta

Schott (taro), Vicia villosa (hairy vetch), and Mucuna

pruriens (velvet bean) own strong allelopathic properties(Fujii, 2001; Tsuzuki, 2001; Xuan et al., 2002). Xuanet al. (2002) found that alfalfa pellets at 1–2 t ha�1

completely inhibited the emergence of Dopatrium

junceum Hamilt. (abunome), Lindernia pyxidaria L.(false pimpernel) and Elatine triandra Schk.var. pedicel-

lata Krylov (long stemmed water wort). The numberand the dry weight of emerged Echinochloa oryzicola

plants were significantly reduced. V. villosa was recog-nized as the most promising cover crop for weedcontrols in fields, grassland, and orchard in the centraland southern part of Japan (Fujii, 2001). M. pruriens

was thought a desirable material for weed control inpaddy field (Fujii, 2001).

The possession of allelopathic properties of theseplants mentioned above has been known for somedecades. In the natural ecosystems, many higher plantsmay hold stronger allelopathic potential and mayexhibit a higher weed reduction. A screening onallelopathic potential of higher plants from SoutheastAsia was carried out during 1999–2001 (Hong et al.,2003). A. conyzoides was assessed to have a stronginvasion capacity in plant communities, significantlyreducing natural growth of weeds in its vicinity. Theleaves were reportedly used by local farmers, as atraditional manure in paddy field for soil improvement.

The objectives of this study were (i) to test theinhibitory effects of A. conyzoides on germination andgrowth of radish and some paddy weeds, (ii) to evaluatethe application of this species leaves for weed control inpaddy field, and (iii) to identify potential allelochemicalsin this plant.

2. Materials and methods

A. conyzoides was grown in the Botanical garden ofThai Nguyen University of Agroforestry, Thai Nguyencity, Vietnam in 2000–2001. At the full bloom stage, theplant was harvested and the leaves, stem and root wereseparated. They were well cleaned, chopped into 1 cmpieces, dried at 120�C for 2 h and at 80�C for 12 h andthen crushed into powder. Whole 1 cm piece of dried A.

conyzoides leaves were used in field experiment. Theplant powder was used in laboratory experiments.

2.1. Effects of A. conyzoides on germination and growth

of radish

This experiment was carried out in the Plant BiologyLaboratory, Crop Science faculty, Thai Nguyen Uni-versity of Agroforestry, Vietnam in March–June, 2001.Raphanus sativus L. (radish) was used as the test plantbecause it is strongly sensitive to allelochemicals at lowconcentration (Tsuzuki et al., 1995). Commercial seedsof radish were provided by seed center of Thai Nguyenprovince, Vietnam. The seeds were randomly selectedand germination tests were conducted.

About 1 g powder of the leaves, stem and root of A.

conyzoides was extracted with 10 ml of distilled waterand placed for 24 h at 25�C. These aqueous extractswere distilled several times by filter papers and were setas original condition (100 g l�1) and its dilutions of 50%,20%, 10%, and 5% were made (50, 20, 10, and 5 g l�1),respectively. A 10 ml aliquot of the extract was put in aPetri dish (9 cm in diameter) lined with filter paper, and30 seeds of radish were sown in each dish. Controlreceived only distilled water. Treatments were placedin a growth chamber (set at: 25�C, 4000 lx, light time:7:00–19:00). After 7 d, plant number, shoot height, rootlength and dry weight of radish were determined. Threereplicates were used.

2.2. Effects of A. conyzoides leaves on growth of some

paddy weeds in calcinated soil

This experiment was conducted in Laboratory ofCrop Science, Faculty of Agriculture, Miyazaki Uni-versity, Japan in 2002. Three paddy weeds (Echinochloa

crus-galli var. formosensis Ohwi., Monochoria vaginalis

(Burm.f.) Persil var. plantaginea Solms., and Aeschyno-

mene indica L.) were collected in paddy fields in 2000.These are among the world’s worst paddy weeds andcause serious rice yield reduction (Holm et al., 1997).Empty and underdeveloped seeds were discarded byfloating in tap water. The remaining seeds were then airdried and hermetically stored in a refrigerator set at�20�C to keep the seed fresh. In September 2002, seedsof the E. crus-galli were treated with a solution of H2SO4

(90%) for 2 min to loosen the seed coat, and rinsed

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many times with distilled water. Seeds of Monochoria

were cleaned and placed in a growth chamber (25�C,4000 lx) in a 200 ml glass pot filled with distilled water,and 20 seeds of rice were also added to the pot. After2 d, these seeds were used in all treatments. The seedcoat of Aeschynomene was removed and the seeds werecleaned several times with distilled water before use.Germination of the three paddy weeds was tested usingdistilled water at over 70%.

Commercial heated soil (Yamamune commercialAssociation, Miyazaki City, Japan, which did not haveany microorganisms and weed seeds) was used in thisexperiment [pH: 6.3; EC (ms cm�1): 0.45; NO3-N(mg 100 g dry soil�1): 19.1; exchangeable CaO, MgO,K2O, and available P2O3 (mg 100 g dry soil�1): 251.7,76.0, 111.4, and 5.21, respectively].

Soil (300 g) was put in a 500 ml plastic pot (diameter:9 cm) and saturated with tap water. Twenty seedseach of Echinochloa, Monochoria and Aeschynomene

were sown at a 2 cm depth in the soil. The powdersof A. conyzoides at 0.5, 1.0, 1.5, and 2.0 t ha�1 weresimultaneously applied in the pots, whilst controlreceived only tap water. Treatments were placedin a growth chamber (set at: 25�C, 4000 lx, light time:7:00–19:00). After 15 d, weed plants were counted, andplant height and radicle length of the three weeds weredetermined. This experiment was designed in a com-pletely randomized design with three replications andrepeated twice.

2.3. Application of A. conyzoides leaves in paddy field

This experiment was conducted in the ExperimentalFarm of Thai Nguyen University of Agroforestry,Vietnam during summer and spring seasons in 2002and 2003, respectively [pH: 6.6; total loam: 1.62%; totalN, P, and K (0.1, 0.07, and 0.15, respectively), cationexchange capacity: 8.6 me (milligram equivalent) per100 g soil, respectively]. The rice field was divided intoplots with an area 2� 3 m2 and the plots received noprevious herbicide treatment during 3 years or anyspecial treatment for the soil. A 30 d old seedling ofKhang Dan 18, a common rice (O. sativa L. var. indica)variety in Thai Nguyen province, Vietnam, was trans-planted into the experimental plots at a density20� 10 cm2, respectively. The field experiment had threetreatments. One treatment had 2 t ha�1 dried materialsof A. conyzoides leaves applied at 2 d after transplanting.Simultaneously, the herbicide treatment received3 kg ha�1 butachlor (formulated as Butataf 600 g l�1).The control treatment received neither plants norherbicide treatments. All plots were treated withconventional methods except for weed management.All treatments of greenhouse and field experiments wereapplied with fertilizer (N, P2O5, and K2O at the rate of7.5, 12.6, and 9.0 g m�2, respectively) at 1 d before

saturating the soil with water. A completely randomizedblock design with three replicates was used. Thirtydays after application, weed species, plant numberand dry weight of weeds, number of tillers and plantheight of rice were determined. Rice yield was alsomeasured.

2.4. Identification of potential allelochemicals in

A. conyzoides

The experiment was carried out in the laboratory ofCrop Science, faculty of Agriculture, Miyazaki Uni-versity, Japan during February 2002–2003. Five g of A.

conyzoides powder of each of the leaves, stem and root,were extracted with 80% methanol and shaken with 80times/min by a bath shaker for 24 h at 25�C. Thesupernatant was separated and was fixed to pH 2.0 with2 M HCl. The solution was extracted 3 times with 50 mlof ethyl acetate. Later, dried by sodium sulfate(anhydrous), filtered and evaporated to dryness on arotary evaporator at 40�C. The residues were calculatedand added by MeOH to be 1 mg ml�1 (1000 ppm). Theacidic fraction was filtered through a 0.20 mm filter(Dismic-25cs, Toyo Roshi company, Japan) for HPLC(high pressure liquid chromatography).

Eighteen standard chemicals: benzoic acid, caffeicacid, chlorogenic acid, coumalic acid, ferulic acid,gallic acid, protocatechuic acid, p-coumaric acid,p-chlorobenzoic acid, p-hydroxybenzoic acid, salicylicacid, sinapic acid, syringic acid, trans-cinamic acid,trans-o-coumaric acid, vanillic acid, catechin andvanillin [Nacalai nesque (Japan), Sigma (Germany)and Wako (Japan) chemical companies] and thesamples were injected in 5 ml quantities and subjectedto HPLC analysis. Concentrations were calculated bycomparing peak areas of samples with those of thestandards. Conditions of HPLC were as follows:Column: TSK gel ODS-80Tm (4.6 mm F� 15 mm),temperature in column: 40�C, mobility phase: Asolution: acetic acid 2%, B solution: methanol (100%),wave-length of ultraviolet absorption of detector:240 nm. Retention times of the standard compoundsand the major peaks in the extracts were recorded. Thisexperiment was conducted with three replications andrepeated twice.

The inhibition percentage of the study was calculatedas follows:

Inhibition percentage ð%Þ

¼ ½1 � ðsample extracts=controlÞ� � 100:

Data of this study were analyzed using SAS6.12 version (SAS Institute, 1997) using ANOVA withthe least significant difference at the 0.01 probabilitylevel.

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3. Results and discussion

3.1. Effects of A. conyzoides on germination and growth

of radish

All plant parts of A. conyzoides inhibited radishgermination (Table 1). It was significantly suppressed byany concentration of the leaves, as compared with thecontrol. However, at low doses (5–20 g l�1) of stem androot material, there was negligible and non-significantreduction in germination. At higher dose (50–100 g l�1),radish germination was markedly decreased. Only thehighest dose of the leaves completely controlled radishgermination and growth. There was a slight inhibitionon shoot elongation of radish for the 50 g l�1 dose of theleaves, whereas the other applied doses of the leaves,stem and root promoted the shoot growth (30–80% ofstimulation) as compared with the control (Table 1).Radish root was more sensitive to the effects of A.

conyzoides than the shoot, and A. conyzoides leavesdisplayed stronger suppression than the stem and root.At 50 g l�1, radish root elongation was significantlyreduced by the leaves (59.8% of inhibition). Yet, theother doses of the leaves, stem and root eitherstimulated or inhibited root growth of radish butthose were not significantly different from the controls

Table 1

Effects from different parts of A. conyzoides on germination and growth of

Concentration (g l�1) Germination (%) Shoot height (m

Leaves

0.0 97.7a (0.0) 4.1b (0.0)

5.0 75.5b (22.7) 5.7a (�39.0)

10.0 47.7c (51.2) 6.1a (�48.8)

20.0 43.3c (55.7) 5.4ab (�31.7)

50.0 12.2d (87.5) 3.5b (14.6)

100.0 0.0d (100.0) 0.0c (100.0)

LSD 0.01 13.7 1.4

Stem

0.0 97.7a (0.0) 4.1a (0.0)

5.0 88.8ab (9.1) 5.8a (�41.5)

10.0 83.5ab (14.5) 6.8a (�65.9)

20.0 78.8ab (19.3) 7.1a (�73.2)

50.0 72.2b (26.1) 7.4a (�80.5)

100.0 37.7c (61.4) 5.3a (�29.2)

LSD 0.01 21.2 3.6

Root

0.0 97.7a (0.0) 4.1d (0.0)

5.0 84.4a (13.6) 5.6bc (�36.6)

10.0 90.0a (7.9) 7.0a (�70.7)

25.0 83.3a (14.7) 6.8ab (�65.9)

50.0 61.1b (37.5) 7.2a (�75.6)

100.0 18.8c (80.8) 5.4c (�31.7)

LSD 0.01 16.2 1.3

Values in the column with same letter are not significantly different at Po0

Values in the parentheses are inhibition percentage over control.

Values in the parentheses with (�) are promotion percentage over control.

(Table 1). Dry weight of radish was significantly reducedat the dose of 10 g l�1 (58.3% of inhibition) by theleaves. The highest dose (100 g l�1) entirely reduced (theleaves) and significantly inhibited radish dry weight (thestem and root). However, no major reduction of radishdry weight by other applied doses of the stem and rootwas observed (Table 1).

Results in Table 1 indicated that all plant parts of A.

conyzoides contain allelopathic potential and inhibitedgermination and growth of radish. The leaves exhibitedstronger suppression than the stem and root. Theinhibitory magnitude of the plant stem was a littlegreater than the root. The inhibitory degree of thisspecies was proportional to the increase of applieddoses.

3.2. Effects of A. conyzoides leaves on growth of some

paddy weeds in calcinated soil

Weed number of E. crus-galli was not significantlyaffected at 0.5–1 t ha�1 dose (Table 2). However, at thisdose weed number of A. indica, and that of M. vaginalis

at 1 t ha�1, were significantly reduced as compared withthe control (Table 2). At 2 t ha�1, weed number of E.

crus-galli was strongly decreased (78.4% of inhibition)while emergence of both M. vaginalis and A. indica was

radish

m) Root length (mm) Dry weight (g)

9.2b (0.0) 1.2a (0.0)

12.5a (�35.9) 0.9a (25.0)

9.7b (�5.4) 0.5b (58.3)

7.5b (18.5) 0.5b (58.3)

3.7c (59.8) 0.1c (91.7)

0.0d (100.0) 0.0c (100.0)

2.6 0.4

9.2ab (0.0) 1.2a (0.0)

11.0a (�19.6) 0.9ab (25.0)

10.2a (�10.9) 0.9ab (25.0)

10.3a (�12.0) 0.8ab (33.3)

9.1ab (1.1) 0.8ab (33.3)

5.2b (43.5) 0.5b (58.3)

4.2 0.5

9.2b (0.0) 1.2a (0.0)

12.9a (�40.2) 0.9ab (25.0)

13.4a (�45.7) 0.9ab (25.0)

13.3a (�44.6) 0.8ab (33.3)

11.2ab (�21.7) 0.8ab (33.3)

8.3b (9.8) 0.3b (75.0)

3.5 0.8

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completely controlled (Table 2). Plant height of E. crus-

galli was significantly inhibited at the highest concentra-tion. At the other applied doses, it was not differentfrom the control (Table 2). The A. conyzoides leavessignificantly suppressed plant height of both M.

vaginalis and A. indica at 1 t ha�1 dose. At 1.5 t ha�1,plant height of the two weeds was reduced toapproximately 90%. The lowest dose of A. conyzoides

leaves significantly stimulated E. crus-galli’s root elon-gation (26.3% of promotion) but significantly inhibitedthat of A. indica (Table 2). At the other applied doses,root length of the three weeds was markedly reduced ascompared with the control, except 1 t ha�1.

Data in Table 2 show that the inhibitory magnitude ofA. conyzoides leaves on the three paddy weeds wasspecies dependent. Its inhibition on the growth of M.

Table 2

Growth percentage of some paddy weeds treated with A. conyzoides

leaves in calcareous soil as compared with the respective control

Concentration

(ton ha�1)

Weed number Plant height Root length

E. crus-galli

0.0 100.0a (0.0) 100.0ab (0.0) 100.0b (0.0)

0.5 103.4a (�3.4) 114.7a (�124.7) 126.3a (�26.3)

1.0 91.5ab (8.5) 98.2ab (1.8) 102.4b (2.4)

1.5 71.2cd (28.8) 83.6bcd (16.4) 68.3c (31.7)

2.0 21.6f (78.4) 55.4e (44.6) 17.5e (82.5)

M. vaginalis

0.5 91.6ab (8.4) 89.3bc (10.7) 97.6b (2.4)

1.0 67.3d (32.7) 71.7cde (28.3) 46.7d (53.3)

1.5 12.2fg (87.8) 9.1f (90.9) 2.1ef (97.9)

2.0 0.0g (100.0) 0.0f (100.0) 0.0f (100.0)

A. indica

0.5 82.5bc (17.5) 87.4bcd (12.6) 67.5c (32.5)

1.0 51.3e (48.7) 68.7de (31.3) 36.7d (63.3)

1.5 7.3fg (92.7) 11.1f (89.9) 3.4ef (96.6)

2.0 0.0g (100.0) 0.0f (100.0) 0.0f (100.0)

LSD 0.01 14.69 19.36 15.47

Values in the column with same letter are not significantly different at

Po0:01:Values in the parentheses are inhibition percentage over control.

Values in the parentheses with (�) are promotion percentage over

control.

Table 3

Effects of A. conyzoides leaves on weed emergence in paddy field

Treated material Weed species Weed number (plant

Control 7 131.0a (0.0)

A. conyzoides 5 19.0b (85.5)

Herbicide 2 17.0b (87.0)

LSD0.01 35.7

Values in the column with same letter are not significantly different at Po0

Values in the parentheses are inhibition percentage over control.

vaginalis and A. indica was almost identical but greaterthan that of E. crus-galli. At the dose 1.5–2 t ha�1, thesuppressive degree of A. conyzoides leaves on emergenceof M. vaginalis and A. indica was much stronger thanthat of E. crus-galli and was significantly different. Itsinhibitory magnitude on the three weeds was propor-tional to the increase of applied doses (Table 2).

3.3. Application of A. conyzoides leaves in paddy field

3.3.1. Paddy weed growth

Paddy weed number was drastically reduced by A.

conyzoides (86% of inhibition) (Table 3). However,butachlor application gave a higher weed control (87%),as compared with the control. Both fresh and dry weightof paddy weeds was significantly inhibited by A.

conyzoides (about 75% of inhibition); however, theherbicide showed a significant increase of weed suppres-sion than this species did (about 20%, Table 3).Observations denoted that at the applied dose of2 t ha�1, A. conyzoides was a promising material forpaddy weed control and with more than 75% paddyweed reduction, although butachlor gave greater levelsof control (around 95%).

3.3.2. Weed species

Six weed species: E. oryzicola Vasing., Eleocharis

acicularis Roem. Et Schult. var. longiseta Sven.,Graticola japonica Miq., L. pyxidaria L., M. vaginalis

Presl. var. plantaginea Solms-Laub., Rotala indica

Koehne. var. uliginosa Koehne, were found in thecontrol (Table 4). A. conyzoides treatment completelycontrolled emergence of G. japonica, and L. pyxidaria.Five weed species were observed in the A. conyzoides

treatment (Table 4), but their plant numbers weredrastically reduced (5–10 times) as compared with thecontrol. In the herbicide treatment, E. oryzicola and R.

indica were found but E. oryzicola was almost controlled(only one weed plant), yet R. indica (16 weed plants)showed strong resistance to the herbicide (18 weedplants). The herbicide exhibited a greater inhibition(completely controlled emergence of E. acicularis,L. pyxidaria, and M. vaginalis) than A. conyzoides;

m�2) Fresh weight (g m�2) Dry weight (g m�2)

317.0a (0.0) 44.9a (0.0)

82.7b (73.9) 11.1b (75.3)

15.5c (95.1) 2.9c (93.5)

31.3 7.4

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however, its inhibitory magnitude on E. oryzicola wasalmost equal to that of A. conyzoides. Surprisingly, A.

conyzoides appeared to have stronger suppression on R.

indica (three weed plants) than the herbicide (16 weedplants). E. oryzicola and R. indica are among the mostdifficult to control species in the paddy field (Xuan et al.,2002, 2003).

3.3.3. Rice growth

Both A. conyzoides and herbicide stimulated tillernumber but A. conyzoides showed greater promotionthan the herbicide did (Table 5). Both panicle and grainnumber were significantly greater than those of thecontrol by A. conyzoides. Herbicide was noted to slightlystimulate these criteria of rice growth and they were notmarkedly different from the control. A. conyzoides

recorded a 22% significance of rice yield growth (Table 5).In Table 3, herbicide treatments gave around 95%

paddy weed control and it was about 20% greateramount of weed reduction than the A. conyzoides did.Nevertheless, A. conyzoides treatment gave 14% greaterrice yield than the herbicide. The nitrogen content ofallelopathic plants plays an important role in improvingrice yield than from the allelopathic factors. Xuan et al.(2003) demonstrated that nitrogen in M. sativa L. cv.Rasen when applied in paddy field caused 10% increaseof rice yield. Furthermore, when weeds are reduced byapplication of allelopathic plants, there would be morenutrients for rice growth left in the soil than the control

Table 4

Weed species emergence in field experiment

Applied

materials

Species

Control Echinochloa oryzicola (22), Eleocharis acicularis

(27), Graticola japonica (14), Lindernia pyxidaria

(16), Monochoria vaginalis (31), Rotala indica (18),

unknown (3)

A. conyzoides Echinochloa oryzicola (2), Eleocharis acicularis (5),

Lindernia pyxidaria (5), Monochoria vaginalis (4),

Rotala indica (3)

Herbicide Echinochloa oryzicola (1), Rotala indica (16)

Number in the parentheses indicates average number of weed plants

per plot.

Table 5

Effects of A. conyzoides leaves on growth and development of rice plant

Treated material Plant height (mm) Tiller number (plant hill�1) Panicle

Control 85.6a (100.0) 7.1b (100.0) 275.0b

A. conyzoides 90.0a (�4.9) 7.8a (�9.9) 304.0a

Herbicide 83.3a (2.7) 7.3b (�2.8) 297.0a

LSD 0.01 12.4 0.6 21.2

Values in the column with same letter are not significantly different at Po0

Values in the parentheses are inhibition percentage over control.

Values in the parentheses with (�) are promotion percentage over control.

(without weed management). It is possible to infer thatnutrient compounds in A. conyzoides might give greaterrice yield in A. conyzoides treatment than the herbicide.However, the question of how much allelochemicalscontained in allelopathic plants affect growth of riceplant, with the exception of nutrient factors, needsfurther research.

3.4. Identification of potential allelochemicals in A.

conyzoides

Table 6 showed that three phenolic acids wereidentified in A. conyzoides leaves, stem and rootincluding gallic acid, coumalic acid, and protocatechuicacid. The amount of gallic acid and protocatechuic acidwere the greatest (4.5 and 2.4 mg g�1, respectively).However, the amount of coumalic acid in the stem wasthe least (0.68 mg g�1). Only catechin (3.4 mg g�1) wasfound in A. conyzoides stem and p-hydroxybenzoic acidwas found in both the leaves and stem of this species(0.07 and 1.8 mg g�1, respectively). Three additionalphenolic compounds consisting of p-coumaric acid,sinapic acid, and benzoic acid were observed in theleaves of A. conyzoides (0.81, 1.28, and 19.9 mg g�1,respectively) (Table 6).

These phenolic acids detected in the leaves, stem, androot of A. conyzoides are putative allelochemicals whichshow effects (promotion or inhibition) on paddy weeds.Chung et al. (2002) evaluated the effects of 23 phenolicacids on the growth of E. crus-galli. Ferulic, p-hydroxybenzoic acid, p-coumaric acid, and m-coumaricacid were among the most inhibitive compounds (Chunget al., 2002). Findings in Table 1 showed that the leavesof A. conyzoides had a great inhibition on radish growththan that of its stem and root. Greater amounts ofphenolic compounds identified in the leaves might resultin the stronger inhibitory exhibition than the stem andthe root. Moreover, benzoic acid with high concentra-tion (19.9 mg g�1) present in the leaves of A. conyzoides

might contribute an active role to greater inhibition ofthe leaves than the other parts of this species.

To date, there are few reports on the allelopathy of A.

conyzoides. Jha and Dhakal (1990) reported that anaqueous extract of aerial parts or root of this species

number (plant m�2) Grain number (seed panicle�1) Yield (t ha�1)

(100.0) 108.0b (100.0) 4.3b (100.0)

(�10.5) 125.0a (�15.7) 5.3a (�23.3)

b (�8.0) 110.0ab (�1.9) 4.7b (�9.3)

16.3 0.5

:01:

ARTICLE IN PRESS

Table 6

Retention time and concentration of phenolic compounds of A. conyzoides identified by HPLC (n ¼ 6)

Chemicals Retention time (min) Concentration (mg g�1)

Standard Leaves Stem Root Leaves Stem Root

Gallic acid 1.99 2.01 1.95 1.97 0.2970.01 4.570.1 0.1370.06

Coumalic acid 2.85 2.86 2.88 2.81 10.6470.5 0.6870.02 11.570.06

Protocatechuic acid 3.71 3.69 3.75 3.68 0.1670.01 2.470.1 0.0270.001

Catechin 4.35 — 4.31 — — 3.470.2 —

p-Hydroxybenzoic acid 5.75 5.72 5.74 — 0.0770.04 1.870.3 —

p-Coumaric acid 11.66 11.69 — — 0.8170.01 — —

Sinapic acid 14.54 14.49 — — 1.2870.02 — —

Benzoic acid 16.35 16.29 — — 19.970.5 — —

Data are means7standard errors.

T.D. Xuan et al. / Crop Protection 23 (2004) 915–922 921

inhibited germination of Triticum aestivum L. (wheat)and rice seeds. Parasad and Srivastava (1991) indicatedthat extracts of A. conyzoides caused lower reduction ofArachis hypogaea L. (peanut) seeds. However, this is thefirst study on the application of A. conyzoides for weedcontrol in paddy field detecting phenolic acids in thisspecies.

4. Conclusions

Findings in this research suggest that application ofA. conyzoides at 2 t ha�1 might be used as a naturalherbicide for paddy weed control and give increases ofrice yield above a herbicide treatment. This species wasreported to have potential of a natural fungicide (Puet al., 1990; Shabana et al., 1991; Gravena et al., 1993;Liang et al., 1994). Therefore, A. conyzoides might bringother beneficial effects to rice plant growth, yield, andquality improvement beside efficacy attained from weedcontrol when applied in paddy field. However, a broaderrange of doses and application times of A conyzoides inpaddy soil on rice plant should be examined to improvethe efficacy of this species for weed control. Itsenvironmental impact needs evaluation to see if it helpsto reduce usage of synthetic herbicides.

Acknowledgements

The authors wish to acknowledge in the financialsupport of the Rural Development Administrationmade in the Biogreen 21 project year of 2003.

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