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The differential analysis on allelochemicals extracted from root exudates in different allelopathic rice accessions

H.B. He1,2, W.X. Lin1,2, X.X. Chen2, H.Q. He1,2, J. Xiong2, X.L. Jia2 and Y.Y. Liang1

1Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fuzhou, 350002, China, Email: alexhhb@163.com
2
School of Life Sciences, Fujian Agriculture and Forest University, Fuzhou, 350002, China, Email: wenxiong181@163.com

Abstract

The root exudates of two rice accessions (allelopathic rice PI312777 and non-allelopathic rice Lemont) grown in paddy soil under natural environment conditions were collected by using a circulation system approach. The ether extracts of the root exudates were detected with GC-MS and the individual compounds were identified by comparison of their mass spectra with those from NIST and the Wiley Library of mass spectral database. The results showed that there were 36 compounds detected in PI312777 and 93 compounds in Lemont, the species of substances were terpenoids, phenols or quinones, aldehydes or ketones, heterocycles, alcohols, ethers, and hydrocarbons. There were differences in quantity, content and chemical composition between the two rice accessions. The result suggested that the allelopathic potential of rice resulted from the interaction among the compounds, performing the synergistic or antagonistic effect. Finally, the possible metabolism pathways of the compounds were further discussed.

Media summary

The root exudates of rice grown in paddy soil under natural environmental conditions were detected by GC-MS and the components of the root exudates were discussed.

Key Words

Rice (Oryza sativa L.), allelopathy, allelochemical, metabolism pathway

Introduction

Allelopathy is defined as biochemical interactions among plants through the release of chemical compounds into the environment, showing either negative or positive effects (Rice 1984). It is clear that allelopathy results in allelochemicals produced by one plant (donor), which escaped into the environment and influenced the growth and development of another plant (acceptor or receiver). The allelochemicals could be released by such processes as volatilization, root exudation, leaching and decomposition of plant residues (Rice 1984; Putnam et al. 1986).

Since Dilday and his workshop (1989a, 1991, 1994) have reported some rice accessions posses allelopathic activity in weed suppression, rice allelopathy has received a great deal of attention. The allelopathic effect of rice itself on weeds could be applied to reduce use of chemical herbicides which might result in improved water quality and less environmental contamination. There have been some research work on allelochemicals extracted from rice tissues, including leave, stem and root (Rice 1987; Chou et al. 1991; Inderjit 1996; Mattice et al. 1998; Blum 1998). Most of these studies were conducted by using organic solvent to extract the allelochemicals from the rice tissues (fresh or dried) such as leave, stem, or root. Few information is available on the root exudates released from the living rice into the environment under natural condition. Kim et al. (2000) have identified the allelopathic substances in root exudates of rice (Oryza sative L. cv. Kouketsumochi) which was exposed to distilled water under growth chamber condition, the results showed that the long chain fatty acid esters were a major species of the allelochemicals along with several benzene derivatives and aldehydes. Hisashi et al. (2002) have also isolated and identified the inhibitory momilactone B from the exudates of rice (Oryza sative L. cv. Koshihikari) grown in distilled water under growth chamber condition. However, the results obtained from laboratory experiments tended to be very different from those in field trials, which was attributed to the two different growing conditions. It has been reported that most of the rice accessions did not show allelopathic effect on weed in the field even though they significantly inhibited weed growth under laboratory conditions (Wang et al. 2000), and it was also found that the allelopathic effect of rice was higher on the root growth of weed than on the shoot growth (Wang et al. 2000; He 2000). Planting and growing rice in paddy soil are still commonly practised in many Asian countries and the root of rice is the primary route of transferring chemical substances among plant, soil and water, so the root exudates of living rice would be more important factors in studying rice allelopathy. In this paper, the root exudates of different allelopathic rice accessions growing in paddy soil under natural environment condition were studied.

Methods

The experiment was conducted at the Experimental Station of Fujian Agriculture and Forestry University, China under natural environmental conditions in July, 2003.

Collection and Extraction of rice root exudates

Both allelopathic rice PI312777 and non-allelopathic rice Lemont were used as test materials. Seeds of the two rice accessions were pre-germinated and seeded in seedling plates respectively. At 3 leaf- stage, 30 uniform seedlings of each entry were transplanted spacing at 5cm5cm in each pot (30cm30cm15cm) with 5 kg soil (five replications), in which the bottom was connected to a circulation system for the collection of leaching water from the pot soil in the period of rice growth. Distilled water with given amounts (100 ml, each time) was added as needed. Five days after transplanting, the collected liquid from five pots was gathered together and filtered through filter paper; the filtrates were extracted 3 times with an equal volume of ether. The extracts were combined, concentrated by a rotary evaporator in water bath at 40 C. The concentrated samples were detected by GC-MS.

GC-MS Analysis

One microliter of the concentrated samples was injected into GC-MS instrument (Varian Saturn 3900/2100, USA). The conditions were set as follows: DB-5 capillary column, 30m0.25mm, 0.25μm film thickness; temperature programmed from 50 C (5min) to 200 C (20min); flow rate 1 ml/min of helium. The MS method was EI-Auto scan mode with 70eV voltage, scan range 40-500 amu. The individual compounds were identified by comparison of their mass spectra with those from NIST and the Wiley Library of mass spectral database.

Results and Discussion

The compounds identified in the extracts by ether solvent from the root exudates of PI312777 and Lemont rice accessions are shown in Table 1. There were 36 compounds found in the rice accession PI312777, including 9 terpenoids (peak area 10.97%), 8 phenols or quinones (5.87%), 6 esters (10.68%), 3 aldehydes or ketones (1.44%), 4 heterocycles (68.04%), 2 alcohols (1.23%), 2 ethers (0.57%) and 2 others (1.20%). There were 93 compounds detected in the rice rice accession Lemont, including 19 terpenoids (peak area 20.99%), 6 phenols or quinones (4.25%), 18 esters (17.30%), 11 aldehydes or ketones (4.20%), 7 heterocycles (27.14%), 13 alcohols (16.48%), 2 ethers (0.27%), 14 hydrocarbons (6.86%) and 3 others (2.51%). There are 18 same compounds found in PI312777 and Lemont rice accessions took up 60.04% and 46.97% respectively (table 1), included terpenoids, phenols or quinines, esters, hetercycles and others.

The results show that terpenoids, phenols or quinones, esters and aldehydes or ketones exist in the two exudates of the rice roots. Generally speaking, terpenes showing allelopathic effect are volatiles and have low water solubility. However the present results show that most terpenoids in our rice root exudates are terpene derivatives in hydroxided or oxided formes so their water solubility was high enough to express allelopathic activity (Weidenhamer et al. 1993). Maicias (1995) also reported that the activity of promising allelochemicals varied greatly depending on the class of allelochemicals, and the most potential natural allelochemicals in terms of bioactivity were terpernoids and fatty acids with an activity range of 0.25-105 ppb, rather than the traditionally considered phenolics, quinones or alkaloids.

Table 1. Compounds identified in the extracts by ether from the root exudates of PI312777 and Lemont rice accessions

Species of Substance

Compound

Peak area %

PI312777

Lemont

Terpenoid

Carvone oxide,cis-

1.32

0.65

Cedrol

1.18

0.30

5-Isopropenyl-2-methyl-7-oxabicyclo[4,1,0] heptan –2-ol

1.23

1.00

Limonene dioxide

1.23

9.48

Phenol or Quinone

Phenol,2-(1,1-dimethylethyl)-4-methyl-

1.13

0.29

2,6-di-t-Butyl-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one

1.67

1.07

7,9-di-t-Butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione

0.82

0.88

Butylated hydroxytoluene

0.20

0.41

Methyl 3-(3,5-di-t-butyl-4-hydroxyphenyl)propionate

0.68

0.29

Phenol,2,4-bis(1,1-dimethylethyl)-

0.21

1.31

Ester

Fumaric acid,ethyl 2-(2-methyl enecyclopropyl)propyl ester

2.56

1.51

1,2-Benzenedicarboxylic acid bis(2-methylpropyl) ester

3.79

1.24

Dibutyl phthalate

3.08

2.99

Heterocycle

Benzothiazole

0.04

3.64

2(3H)-Benzothiazolone

37.11

19.35

1,3,5-Triazine-2,4,6(1H,3H,5H)-trione,1,3,5-tri-2- propenyl-

2.59

1.37

Other

Benzenamine,4,4'-methylenebis[2-methyl-]

0.44

0.03

Methoxy phenyl,oxime

0.76

1.16

Total

 

60.04

46.97

The heterocycles, which have not been reported as allelopathic substances previously, were detected as major components in the root exudates of rice accessions, showing 68.04% in allelopathic rice PI312777 and 27.14% in non-allelopathic rice Lemont. These heterocycles were considered to have important influence on the expression of rice allelopathy. The inhibitory activity of allelochemicals such as terpenoids, phenols and esters in the suppression on target weeds, might be regulated by those less allelopathic substances such as heterocycles, alcohols, ethers etc. These compound even though less allelopathic are produced and may interact with the other allelopathic compounds, and act as molecular companions to increase or decrease allelopathic effect on weeds, resulting in synergistic or antagonistic effect . Increasingly studies show that most allelopathic effects are a joint action of several compounds in a mixture (Einhellig 1987; Indrejit 1996; Rice 1987; Chou et al. 1991; Lin et al. 2000). The joint action of all substances in the mixture would be more significant for use of allelopathy in paddy field of integrated weed management and in manipulated ecosystems.

The present results show that the peak area of the same compounds in PI312777 and Lemont were 60.04% and 46.97% respectively, this findings suggested that the rice accession, whether it refers to allelopathic rice PI312777 or non-allelopathic rice Lemont, had the same or similar metabolic pathway. It has been reported that most of the plant chemicals associated with allelopathic activity are secondary metabolites from shikimic acid, acetate or terpenoids pathways (Rice 1984, 1985; Rizvi et al. 1992; Macias et al. 1995). Similarly the present results show that there were terpenoids, phenols and esters in the rice root exudates which are produced from the three metabolic pathways as mentioned above. Some scientists have documented that allelopathic activity in rice is the result of the joint action of several secondary metabolites that might act synergistically (Olsfsdotter et al. 1995; Geally et al. 2000). It hints that the allelopathic rice (such as PI312777) has a self- adjust mechanism through several secondary metabolic pathways under environment stresses that could modulate the quantity, content and structure of chemical substances in best state so as to have advantage in competition with other plants or environment. We need to further understand how to regulate and control the allelopathic ability in rice, with aid of genomic, proteomic and metabolomic approaches, which would promote the application of allelopathy into practical use in the near future.

Acknowledgements

This work was co-supported by the grants of National Natural Science Foundation (30471028), Fujian Provincial Scientific and Technological Program (K04038)of China

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