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The allelopathic phenomenon, a dynamic process.

Francisco A. Macías, Alberto Oliveros-Bastidas, David Marín, Diego Castellano and José M.G. Molinillo

Grupo de Alelopatía, Departamento de Química Orgánica, Universidad de Cádiz. Facultad de Ciencias, C/ República Saharaui s/n, 11510 Puerto Real, Cádiz, Spain. Email:


The concentration of representative allelochemicals in wheat, rye, and maize in their first development stages was studied. The evolution of allelochemical concentrations in the whole plant shows the root exudation capacity to be directly dependent on the capacity to translocate chemicals from shoot to root, but not on the total concentration in the aerial parts of the plant. During plant growth, wheat accumulates more allelochemicals in roots than rye or maize, a fact which is related to wheat’s higher exudation capacity. HPLC-DAD analysis of target plants cultivated in non sterile and sterile conditions shows the product initially liberated by the donor plant, in addition to their degradation intermediates and final products, to be responsible for the phytotoxic activities observed. The complete cycle of these allelochemicals, from their synthesis and accumulation in donor plants to their absorption in target species is studied, thereby demonstrating accurately, under laboratory conditions, the effects of the chemicals liberated by the donor plants and their direct effect on the target organism.

Media summary

Allelochemical production, translocation and release of wheat, rye, and maize were studied. Plants exposed to benzoxazinones under sterile conditions were analysed.

Key Words

Plant-plant interaction, allelopathy, root exudates, DIBOA, DIMBOA, Triticum aestivum L., Secale cereale L.


Plants show their lack of movement capacity as a characteristic and evident feature, which eliminates escape as a defence mechanism. As a consequence, plants evolved different defence mechanisms which can be described as mechanical, phenological and chemical (Coley 1983; Aide 1993; Rosenthal and Berembaum 1991). All defences are used in synergistic action, and provide an excellent adaptation capacity for plants in many ecological situations. Chemical defence is based on secondary metabolites (SM), which, in addition to their defence role, are responsible for the interaction of plants with their surroundings, by means of plant-plant, plant-insect and plant-microorganism interactions. These metabolites can be synthesized by the plant from the beginning of its growth and development (constitutive defence), as in the case of intraspecific plant-plant interaction, or after exogenous stimulation (herbivore action). This last process is mediated by a metabolic alarm which starts or catalyses the secondary metabolites biosynthetic routes (induced defence) (Agrawal et al. 1999; Karban and Baldwin 1997; Tallamy and Raupp 1991). It is not surprising that a wide variety of bioactivities, measured in vitro or in vivo, are associated with these SM (Singh et al. 2003). Studies of the dynamics of these chemical defences, from an ecological point of view, include two main aspects: first, the observation of the natural phenomenon, and after this, (due to the great variety of conditions found in nature), imitation in the laboratory of the observed effect, by controlling various parameters; and second, to describe the phenomenon in an accurate manner. These studies are a part of allelopathy research, and the mediators of these interactions are allelochemicals (Rice 1984). Allelopathic science applies the fundamentals of ecological studies: observation of the natural phenomenon, and the simulation of them under controlled bioassay conditions. These phenomena can be described by an ecological component (the evidence of their existence in nature), a chemical component (to isolate, identify and characterise allelochemicals), and a physiological component (the mechanism of interference of chemicals with organisms, at both cell, and molecular level ). Following this direction, many different bioassay designs have been optimized for the study of allelochemicals , isolated or liberated by plants (donor plants), together with the growth and allelochemical distribution in target plants and micro organisms (Inderjit and Nilsen 2003; Inderjit and Del Moral 1997; Torquebiau 1994; Vandermeer 1989). This work describes the interference of these SM on the conventional metabolic pathways and physiology of the target plant. Most of the bioassays direct towards the identification of potential allelochemicals, and the evaluation of their phytotoxic effects on standard target species or weeds. The main objective of these works is to develop structure-activity relationships for secondary metabolites which include allelochemicals or synthetic analogues of them. Commercial herbicides or plant growth regulators are included in these assays as positive controls (Hoagland and Williams 2004). These successful bioassays are developed by fixing some parameters like target species and their development stages, or growth media, or other parameters to be measured. This permits discovery of the relationships between structure and activity of the compounds under study (Fay and Duke 1977; Fujii 2001; Fujii et al. 1990; Hashem and Adkins 1998; Navarez and Olofsdotter 1996; Putnam and Duke 1974; Rimando et al. 2001; Wu et al. 2000; Wu et al. 2001). Despite the wide spectrum of parameters analysed, their extrapolation to the natural environment is a current discussion topic (Inderjit and Weston 2000). In recent times, this fact has been taken into consideration, and has provoked several methodology optimizations towards the description of allelopathic interactions (Belz and Hurle 2004; Belz and Hurle 2005). The study of the allelopathic phenomenon has been directed to the detection and quantification of allelochemicals liberated from donor plants, and to their effects on target plants, but the natural phenomena associated with these interactions are much more complex. From an ecological point of view, these studies should imply the tracing of allelochemicals to target plants, their liberation mechanisms and the recording of some effects on the target plant (Kobayashi 2004; Inderjit and Nilsen 2003; Rice 1984). Some of these studies, dealing with allelochemical quantifications in donor plants, and their exudation through roots, have been reported (Belz and Hurle 2005; Copaja et al. 1999; Reberg-Horton et al. 2005; Wu et al. 1999; Ryan et al. 2001), but the distribution of these compounds inside donor plants, the correlation of this with the liberated concentrations, the dynamics of them under bioassay conditions, and the absorption of them by target plants has not been described yet. In this work, we present recent advances reached on the tracing of classical wheat, rye, and maize allelochemicals, their accumulation dynamics inside the plant, their liberation through root exudates, and their absorption by target plants (Lactuca sativa L., Sinapis alba L., Lolium rigidum Gaud. and Avena fatua L.) under different bioassay conditions, which include hydroponic, inert solid substrate and soil cultures.



Donor and target plants.

Donor plants (Triticum aestivum, Secale cereale L. and Zea mays) and target plants (Lactuca sativa, Sinapis alba, Avena fatua and Lolium rigidum) seed surfaces were sterilized with 12 % calcium hypochlorite and pre-germinated in Petri dishes at 24°C, by using agar-agar as substrate, and under photoperiod conditions (16 h light, 8 h darkness).

Bioassay-hydroponic culture

The obtained plantula were aseptically transferred to sterilized glass containers (2 L). Glass perlite, rock wool or commercial soil were used as substrates (Figure 1). Bioassays were carried out in both sterile and non-sterile conditions. Different donor plant densities were co-cultured with target seedlings using Hoagland culture media (Sigma-Aldrich Co., 1.6 g/L, pH=5.6).

Figure 1. Containers used for the sterile co-cultivation, sampling and analysis of allelochemicals from root exudates.

Allelochemicals analysis

Donor plants (roots and shoots), culture solutions or substrates, and target plants, were extracted with methanol (1%AcOH x 4) and analysed by HPLC-DAD in previously validated conditions (Eljarrat et al. 2004). Under these instrumental parameters, representative allelochemicals, responsible for the allelopathic potential 2,4-dihydroxy-2H-1,4-benzoxazin-3-(4H)-one (DIBOA) 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) (Copaja et al. 1999; Reberg-Horton et al., 2005; Wu et al. 1999), in addition to their degradation products 2-aminophenol (APH), benzoxazolin-2(3H)-one (BOA), 6-methoxy-benzoxazolin-2(3H)-one (MBOA), 2-amino-3H-phenoxazin-3-one (APO) and 2-amino-7-methoxy-3H-phenoxazin-3-one (AMPO) (Macías et al. 2004, Macías et al. 2004), are separated, identified and quantified simultaneously (Figure 2). The identification was made by comparison of retention times and UV-VIS spectra at two wavelengths (95 % confidence) with pure standards, and standards addition. Quantification was made by the external standard method.

Fig.2.- HPLC-DAD (260 nm) chromatogram obtained alfer allelochemicals and degradation products isolation (0.25 μg of each chemical injected).


Allelochemicals in target plants and their liberation through root exudates

The representative allelochemicals DIMBOA and DIBOA were identified in roots, shoots and culture media for the three donor plant species (Figure 3). DIMBOA was the main allelochemical found in wheat and maize, while DIBOA was the main one in rye.

Other related chemicals, like HBOA and HMBOA were also detected under these analysis conditions, although as minor products. The same compounds are detected in plant culture media, and were consequently chosen for the dynamics study, from their accumulation in the donor to their absorption by the target plants. The evolution of the allelochemical concentrations inside the plants (roots and shoots) is in accordance with the model proposed by An (An et al. 2003). In this model, the allelochemical concentration inside the plant (Ap) decreases as the plant growth advances, as described by the following equation:

Where Apo is the starting allelochemical concentration, t represents the plant growth time and –k is the allelochemical accumulation constant. For the study of roots and shoots, this equation is transformed into the following one:

Where [Ar/As]0, which is obtained by regression, indicates the concentration of the allelochemical at the germination moment, and kr/s is a constant which measures the translocation potential for an allelochemical from shoot to root.


Zea Mays

Secale Cereale

Triticum aestivum L.








Figure 3. HPLC-DAD analysis of root, shoot and exudate in the culture media for each donor plant.

Table 1 shows the results obtained by applying this model for allelochemical accumulation in donor plants. This model predicts the allelochemical concentration at the moment of germination will be maize > rye > wheat. This fact has been confirmed after several determinations (Copaja et al. 1999; Reberg-Horton 2005; Wu et al. 1999). The model also indicates that at their mature stage, the plants will accumulate less allelochemical amounts due to their decay speed. Following the pathway of these allelochemicals in the bioassay system, and assuming their main release process to be root exudation, the translocation of the allelochemicals from aerial parts to roots before exudation is evident (Gransee and Wittenmayer 2000; Macias et al 2005a). Taking this into consideration, allelochemical levels in roots were correlated with their concentration in shoots (Figure 4). This analysis displayed a polynomial (second order) growth rate for wheat and rye, whereas this rate is constant in maize, with higher amounts than the former plants. This fact is reflected in the allelochemical translocation capacity to the environment from the donor plant. All the donors assayed produce significant amounts of these allelochemicals, but wheat, which has less metabolization and accumulation capacity for these compounds, shows a higher translocation from shoots to roots, and consequently higher exudation capacity. This fact is in good accord with the higher allelopathic potential of wheat in comparison to rye and maize. Correlation analysis (Figure 5) shows the allelochemical amount released to be directly connected with the amount translocated to roots. The amounts produced in shoots define neither the liberated amount nor the allelopathic potential of the plant.

Table 1. Obtained allelochemicals accumulation parameters in wheat (DIMBOA), maize (DIMBOA) and rye (DIBOA) during their growth, according to An model. (An et al. 2003).


Ap= Apo e –kt


Donor plant

Apo (t=0 days)

(mMol/Kg FW)



Secale cereale L.

26 ±10

0.38 ± 0.04

1.8 ± 0.2

Triticum aestivum L

24 ± 3

0.16 ± 0.02

3.9 ± 0.2

Zea mays L.

32 ± 4

0.09 ± 0.01

8.2 ± 0.9


[AR/AS]t = [AR/AS]0 e –Kr/st





Secale cereale

0.013 ± 0.006

0.16 ± 0.04

3.00 ± 0.50

Triticum aestivum

0.379 ± 0.194

0.24 ± 0.05

0.16 ± 0.04

*Time from germination, necessary for a 50 % decrease of allelochemicals inside the plant.

** Time from germination necessary to reach the same allelochemicals concentration in roots and shoots.

Figure 4. Relative accumulation of allelochemicals in roots and shoots (evolution with donor plants growth).

Figure 5. Relationships between accumulated allelochemical concentrations (root and shoot) and the amount exuded for Zea mays and Triticum aestivum (sterile conditions).

Exuded allelochemicals, their biotransformation and absorption by target plants.

The phytotoxic activity of the allelochemicals, once outside the plant, is modified by biological and abiotic factors. Most of these transformations occur in soil (Kobayashi 2004). DIMBOA and DIBOA evolution in soil has been already described (Macías et al. 2004; Macías et al. 2005a). These allelochemicals were found to degrade to benzoxazolinones MBOA and BOA, respectively. After that transformation, aminophenoxazines AMPO and APO, respectively, were found as final products. The phytotoxic activities of these degradation products have been found to be significantly different from the starting allelochemicals (Macías et al. 2005b). The relative concentrations of allelochemicals and their degradation products will vary depending on the media to which they are released (soil, hydroponic, inert substrate), and the biological features of them (microbial and fungal population). A mixture of compounds would finally act, and this mixture would include the starting allelochemical released by the donor plant, in addition to the final degradation products, and their chemical intermediates (Figure 6).

Bioassay Conditions






Figure 6. HPCL-DAD (λ=253nm) chromatogram of hydroponic solution and extract of Sinapis alba L. in sterile and non-sterile assays after 6 days of growth.


These results show that the concentration of the allelochemical inside the plant does not define exclusively its allelopathic potential, measured as the amounts released to the environment. These chemicals have been quantified in shoots, but the amounts released correlate better with the concentrations recorded in roots. Thus, roots are transit organs for the allelochemicals before their exudation. Starting allelochemicals, in addition to partial and complete degradation products are present in the root exudates, and all of them contribute to the measured bioactivities of the donor plants. The allelochemical DIBOA is transformed into benzoxazolinone BOA and aminophenoxazine APO also under sterile conditions. Thus, the measurement of the biological (allelopathic) activity is masked by the biotransformation products. This fact can be assumed in the case of any allelochemical. In the bioassays carried out with plantula, the target plant is never chemically analysed in the search for the allelochemicals released by the donor, but only the macroscopic effects (germination and growth), together with some enzymological properties are recorded. This kind of analysis can help to determine which chemicals truly act in an allelopathic phenomenon. The released allelochemical, in addition to its degradation products, can be absorbed by target plants, and this fact must be considered in biological activity studies. This is especially important when dealing with mode of action determination, since each chemical in these compound mixtures could have a different mode . It is also important to note that the activities recorded in a bioassay depend on the different biologically and non-biologically induced transformations of the studied allelochemical. All these factors complicate the study of the allelopathic phenomenon, but their control can offer a more accurate description of what really occurs in the environment. None of the different parameters is static, but allelopathic phenomena are instead dynamic and time-dependent. The bioassay conditions themselves induce variations, though some of them can be controlled. On the other hand, the chemical nature of the allelopathic agent can not be controlled, this determining its persistence and integrity in the environment.


This work was financially supported by the program ‘Quality of life and management of living resources (1998 to 2002)’ of the European Union, FATEALLCHEM Contract No. QLRT-2000-01967, and the Ministry of Education and Science (MEC, Spain), Project No. CTQ2004-08314C02-01. Fellowships from Universidad de Los Andes-Venezuela (A. O. B.), European Commission (European Union), and the Regional Gobernment of Andalusia-Spain (D. M.) are also gratefully acknowledged.


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