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Allelopathic effects of extracts from wheat and its secondary metabolite 2,4-dihydroxy- 7- methoxy -1, 4-benzoxazin-3-one on weeds

Zheng Yong-quan1, Zhao Yuan 2, Dong Feng-shou1, Yao Jian-ren1 and Karl Hurle3

1Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094 China; yqzheng@ippcaas.cn
2
College of Plant Protection, Northwest Sci-Tech Universit of Agriculture and Forestry, Yangling 712100 China;
3
University of Hohenhmei, Stuttgart 70599 Germany

Abstract

The allelopathic effects of wheat aqueous extracts and the wheat secondary metabolite 2,4-dihydroxy- 7- methoxy -1, 4-benzoxazin-3-one (DIMBOA) on several weeds were studies. The results showed that when intercropped with weeds, wheat had strong allelopathic effects on Digitaria sanguinalis, Amaranthus retroflexus L., Echinochloa crusgalli L., Poa annua L., and Avena fatua. There was a negative correlation between the germination ratio of weeds with the density of wheat seedlings. There was a significant allelopathic effect of wheat aqueous extracts on weed seedlings, D. sanguinalis, Poa annua L., A. retroflexus L., E. crus-galli L., A. fatua L., with IC50 (root)< 1.5 mg.ml-1 and IC50 (stem) <3.0 mg.ml-1. But the inhibitory effects to Lolium multiflorum Lam. and Ipomoea hederacea var. integriuscula L. were not detected. There was no significant correlation between aqueous extracts concentration and weeds germination inhibitory ratio, except for the weed of D. sanguinalis. The general trends of allelopathic effects of DIMBOA to different weeds species were: A. retroflexus L. >D. sanguinalis >A.a fatua L., with IC50 (root, stem) < 1.5 mg.ml-1 and IC50 (seed germination) <3.5 mg.ml-1. The impact sequence was as follows, DIMBOA > aqueous extracts (in terms of IC50).

Media summary

The allelopathic effects of wheat aqueous extracts and the wheat secondary metabolite DIMBOA on several weeds were studied by Chinese researchers.

Key Words

Allelopathy; wheat seedlings; DIMBOA; weeds; secondary metabolite; bioactivity.

Introduction

The term “allelopathy”, first coined by Molisch (1937), refers to both detrimental and beneficial biochemical interactions among all classes of plants, including those mediated by microorganisms. Allelopathy denotes biochemical interactions among all types of plants and microorganisms. The allelopathic effects are due to inhibitory substances that are released directly from living plants into the environment through root exudation, leaching, volatilization, and passively liberated through the decomposition of plant residues (Rice 1984). Whittaker and Feeny (1971) termed these phytotoxic substances as allelochemicals. The potential of allelopathy for weed control has been a particularly intense area of study during the past several decades (An et al.1998; Wu et al. 1999). A number of classes of allelochemicals causing inhibition of germination and growth have been identified (Wu et al. 1999). Allelopathy among crop plants, which has potential in integrated weed management, is identified as a category of active agents in the study on chemical ecology. Crop plants have the capability to produce and exude allelochemicals into their surroundings to suppress the growth of weeds in their vicinity. To date, progress has been made in understanding the bioactivity of crop allelopathy, and successful bioassay of this trait has also been demonstrated. The genetic enhancement of compounds of crop plants offers potential implications for weed management. Research is underway to identify genetic markers, associated with allelopathy (Wu et al. 1998; Lemerle et al. 1996.). Once the allelopathic genes have been located, a breeding programme could be initiated to transfer the genes into modern cultivars varieties for weed suppression, thereby reducing over-reliance on herbicides (Belz and Hurle 2002). Incorporation of allelopathic traits together with other plant interference potential into commercial cultivars could be a major step towards further development of sustainable crop production (Lemerle et al. 1996). In the future it is expected that the positive allelopathic effects against weeds will be fully explored, and the negative effects of residues minimized by an efficient chemical ecology management package, being another new promising area of IPM (Belz et al. 2002).

Wheat has been successfully used as a cover crop for weed control in various cropping systems (Putnam et al. 1983). It has been found that the aqueous extract of wheat residues is allelopathic to a number of weeds, and has consistently reduced weed emergence and growth. Both wehat resiude allelopathy and wheat seedling allelopathy can be exploited for managing weeds.Under laboratory conditions, aqueous extracts from wheat straw are allelopathic against a broad spectrum of weed species (Steinsiek et al. 1982). Cyclic hydorxamic acids (Hx), a class of alkaloids, were identified as a category of biologically active agents in weed suppression (Bohidar et al. 1986; Leszcynski et al. 1995). DIMBOA is an important secondary metabolite in wheat. Perez (1990) found DIMBOA inhibited root growth of wild oat (Avena fatua L.) by 50% at a concentration of 0.7 mM (Perez 1990). Bioassay is necessary to the study on allelopathic effects. In this study, wheat was examined for allelopathic potential to determine the differences exist between weed species in their IC50. We developed a fast and reliable laboratory screening bioassay for wheat living seedlings, aqueous extracts and DIMBOA on several kinds of weeds that includes dose–response considerations as an integral part of the experimental design. A set of experiments with Triticum aestivum L. as donor species was carried out to optimize the protocol.

Methods

Instruments and reagents

Centrifuge (Sigma 1-15, Germany), Non-tissue culture treated plate (MULTIWELL, flat bottom with low evaporation lid, USA), tissue ultra-turrax T 25 basic, Germany, rotary vacuum evaporator (RE 121, Switzerland), pipette (1ml,10ml), PVDF membrane Syringe Filter UnitsΦ 0.45 m, Scout Pro electronic balance (SPS2001F, Japan) and rotary shaker (HY-5) were employed for the experiment.

Self-prepared standard sample of DIMBOA (purity: 95%), methanol, pure water, anhydrous ether and anhydrous Na2SO4 (A.R.) were used for extraction of DIMBOA.

The germinated wheat and weed buds were selected and incubated in wet vermiculite beforehand, with the density ratio of wheat to weeds (plant/plant) was 0/50, 10/40, 20/30, 30/20, 40/10, respectively.

Experimental Design

(a) Identification of allelopathy effects by intercropping wheat seedlings with weeds

Exactly 50 seeds (wheat+ weed) were grown in one pot, and the treatments were replicated 3 times. The seedlings were kept wet and supplied with adequate sunlight, water and space so as to overcome the influence from competence among plants. The ratios of seeds germination were determined after 6 days cultivation.

(b) The allelopathic effects of wheat aqueous extracts on weeds

The preparation treatment of aqueous extracts from wheat seedlings: The shoot tissue of wheat was mixed with distilled water with the ratio of 1 to 9 (fw.w-1), and then homogenized with the tissue ultra-turrax at the speed of 9,500 rpm. The filtrate i.e. original aqueous extracts, was condensed after being surged in a rotary shaker for 24 h. The aqueous extracts were then diluted into 0.2, 0.4, 0.6, 0.8, 1.0 mg.ml-1 with distilled water, respectively. The dissolved residue was then centrifuged at 14,000 rpm at 15C for 20 min, and the final supernate, i. e. aqueous extracts with different kinds of concentration, and reserved in -20C refrigerator for bioassay.

The bioassay on allelopathic effects of aqueous extracts from wheat seedlings: The test weed species were listed in Table 1. The seeds of weed species were arrayed in good order in a non-tissue culture treated plate (24 well, flat bottom with low evaporation lid), with Φ2.1cm, and 2.8 cm high. In the experiment, 0.4 ml of aqueous extracts was added to every hole of the culture treated plate with Avena fatua L. and Ipomoea hederacea. var .integriuscula L., and 0.2 ml of aqueous extracts was added to the holes with other seeds. The plate was sealed with parafilm to keep seeds wet and breathing freely. Every treatment was replicated 3 times, with 5 seeds in each hole. The seeds were cultivated in a greenhouse for 5 days, and the length of root and stem were measured and the inhibitory ratio were calculated, respectively (Li Xiangju et al. 1998; Wu et al. 2000).

(c) The bioassay on allelopathic effects of DIMBOA

The seeds of weed species, i. e. Digitaria sanguinalis (L) Scop, Amaranthus retroflexus L. and Avena fatua L., were placed on a non-tissue culture treated plate. Every treatment was replicated 3 holes. Every 5 seeds were inserted into one hole. In the experiment, DIMBOA sample was dissolved in methanol and diluted into 0.2,0.4,0.6,0.8 mg.ml-1 with distilled water, respectively. The plate was sealed with parafilm after 30 min (methanol was fully volatile) and cultivated in the greenhouse at 22C.In the experiment, 0.4 ml of DIMBOA solution was added to every hole of the culture treated plate filled with Avena fatua L., and 0.2 ml was added to the holes filled with smaller seeds. The seeds were then cultivated in greenhouse for 5 days, and the length of root and stem were measured and the inhibitory ratio calculated.

Table 1 The test weed species of bioassay 1)

Serial number

Scientific name

English name

Seed size

Amount of seeds

1

Digitaria sanguinalis

Large crabgrass

middle

90

2

Poa annua L.

Annual bluegrass

middle

90

3

Amaranthus retroflexus

Redroot amaranth

small

90

4

Echinochloa crusgalli

Barnyardness

middle

90

5

Avena fatua L.

Wild oat

large

90

6

Ipomoea hederacea

Entireleaf morningglory

large

90

7

Lolium multiflorum

Annual ryegrass

middle

90

1) “*”represents monocotyledon, and “**” represents dicotyledon.

(d) Statistical analysis

Data on bioassay experiment was analyzed with EXCEL software. Every test weed species had a responsive logarithmic model, i.e. ya1nx+b, the values of IC50 and relative coefficient on every kind of test weeds were calculated according to the regression model.

Results

The allelopathic effects of pot culture experiment

The result of pot culture experiment was listed in Table 2. The result showed that intercropped with weeds, wheat had strong allelopathic effects on Digitaria sanguinalis, Amaranthus retroflexus L., Echinochloa crusgalli L., Poa annua L., Lolium multiflorum Lam.and Avena fatua. There was a negative correlation between the germination ratio of weeds and the density of wheat seedlings. The more wheat plants, the lower the germination ratio of the seeds of weed species. Poa annua L. was the most sensitive among the test six weed species. Its seed germination ratios were all the lowest at different wheat seed densities.

Table 2. The experimental results of allelopathic effects by pot culture1)

Weed species

Average weeds germination ratio in different densities(wheat /weed) (%)

0/50

10/50

20/50

30/50

40/50

Digitaria sanguinalis Amaranthus retroflexus
Lolium multiflorum
Lam.
Avena fatua
L.
Poa annua
L.
Echinochloa crus-galli
L.

95.4%Aa
90.3%Aa
91.7%Aa
90.9%Aa
86.2%Aa
89.3%Aa

94.3%Aa
88.3%Aa
90.1%Aa
89.3%Aa
82.3%Aa
84.2%Aa

94.4%Aa
85.1%Ab
92.2%Aa
84.6%Ba
75.4%Bb
79.0%Bb

87.7%Ab
78.1%Bb
90.3%Aa
76.8%Bb
71.3%Cb
74.3%Cb

83.9%Bb
75.1%Bb
87.4%Aa
71.9%Bb
66.5%Cc
69.5%Cc

1) Capital letters and small letters mean the significance of difference at the level of P0.05 and P 0.01 by LSD. Different letters means significant difference and the same letters means insignificant.

The allelopathic effects of aqueous extracts on 7 weed species

The results of the influence of test 7 weed species on the seed germination and the growth of root and stem were listed in Table 3 (The values of IC50 were listed on Fig 1). From Table 3, a significant allelopathic effect of wheat aqueous extracts was observed on weeds seedlings, Digitaria sanguinalis, Poa annua L., Amaranthus retroflexus L., Echinochloa crus-galli L., Avena fatua L., with IC50 (root)< 1.5 mg.ml-1, IC50 (stem) <3.0 mg.ml-1 and R≥0.977. But weak inhibitory effects to Ipomoea hederacea and no inhibitory effects to Lolium multiflorum Lam. were detected. There was no significant correlation between aqueous extracts concentration and weeds germination inhibitory ratio, except for the weed of Digitaria sanguinalis. For Digitaria sanguinalis (L) Scop. and Echinochloa crus-galli L, stronger response was detected on root other than on stem.

Table 3. The results of wheat aqueous extracts on the seed germination, growth of root and stem of weeds1)

Weed species

Root length of weed seedlings

Stem of weed seedlings

Seed germination

y=alnx+b

IC50

R

y=alnx+b

IC50

R

y=alnx+b

IC50

R

1

y=28.56lnx +53.84

0.87Aa

0.983

y=29.49lnx+52.01

0.93Aa

0.985

y =31.95lnx+54.57

0.87Aa

0.970

2

y =6.65lnx+52.87

0.90Aa

0.984

y=25.18lnx+52.87

0.89Aa

0.994

y =22.13lnx+38.70

1.67Bb

0.947

3

y =19.53lnx+51.40

0.93Aa

0.988

y=24.67lnx+52.20

0.92Aa

0.977

y= 8.72lnx+19.42

33.34Dd

0.900

4

y =22.89lnx+49.13

1.04Bb

0.995

y=16.87lnx+31.43

3.01Bb

0.946

y=11.49lnx+25.23

8.63Cc

0.960

5

y =20 .47lnx+44.23

1.33Bb

0.997

y=23.90lnx+47.54

1.11Aa

0.983

y=4.24lnx+13.82

-

0.878

6

y =13.83lnx+25.34

5.95Cc

0.956

y=13.46lnx+28.27

5.03Cc

0.997

y=7.70

-

0

7

y =7.70lnx+9.47

193.18Dd

0.971

y=7.70lnx+9.47

-

0.776

y=3.97lnx+7.95

-

0.842

1) y = calibration inhibitory rate (%); x = the concentration of chemicals(mg.ml-1); IC50 = the concentration to reach 50% inhibitory effects (mg.ml-1); ND = not detected; “-”= invalid concentration.

The allelopathic effects of DIMBOA on 3 test weed species

The allelopathic effects of DIMBOA on the 3 weed species tested were listed in Table 4 (The value of IC50 was listed on Figure 2). The results suggested that DIMBOA presented inhibitory effects on root elongation, stem elongation and seed germination on all test weed species. The general trends of allelopathic effects of DIMBOA to different weeds species were: Amaranthus retroflexus L. >Digitaria sanguinalis >Avena fatua L., with IC50 (root, stem) < 1.5 mg.ml-1; IC50 (seed germination) <3.5 mg.ml-1. The impact sequence was as follows, DIMBOA > aqueous extracts (in terms of IC50). The results showed that DIMBOA, contributing the allelopathic effects on weed growth, was the main secondary metabolite in wheat root exudates.

Table 4. The inhibitory results of DIMBOA on the seed germination, growth of root and stem of 3 weed species

Weed species

Root length

Stem length

Seed germination

y=alnx+b

IC50

R

y=a lnx+b

IC50

R

y=alnx+b

IC50

R

1

Y=42.31lnx+76.54

0.53Aa

0.985

y=34.78lnx+70.04

0.56Aa

0.986

y=12.77lnx+36.51

2.88Bb

0.937

3

Y=27.95lnx+57.13

0.78Aa

0.974

y=25.44lnx+61.58

0.63Aa

0.979

y=36.14lnx+59.76

0.76Aa

0.957

5

Y=23.10lnx+43.07

1.35Bb

0.978

y=22.69lnx+40.44

1.52Bb

0.970

y=15.80lnx+30.70

3.39Cc

0.967

Figure 1. IC50 of wheat aqueous extracts on 6 weed species

Figure 2. IC50 of DIMBOA on 3 weed species

Conclusion

There was a high correlation between the density of wheat and the seed germination ratio. The germination ratio decreased with the increase in the number of wheat plants. This showed that the inhibitory ability may be improved by increasing the density of wheat in production, which really provided scientific bases for the ecological management of weed-suppression.

There was a significant allelopathic effect of wheat aqueous extracts on weed seedlings, Digitaria sanguinalis, Poa annua L., Amaranthus retroflexus L., Echinochloa crus-galli L., Avena fatua L., with IC50 (root)< 1.5 mg.ml-1 and IC50 (stem) <3.0 mg.ml-1. But the inhibitory effect to Ipomoea hederacea was week,and to Lolium multiflorum Lam. was not detected.

The general trends of allelopathic effects of DIMBOA to different weeds species were: Amaranthus retroflexus L. >Digitaria sanguinalis >Avena fatua L., with IC50 (root, stem) < 1.5 mg.ml-1, IC50 (seed germination) <3.5 mg.ml-1.

Acknowledgements

This work was financially supported by the National Key Project of Fundamental Research (973 Project: 2002CB410806).

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