Agronomy Journal 93:43-48 (2001)
© 2001 American Society of Agronomy
ALLELOPATHY SYMPOSIUM
Laboratory Bioassay for Phytotoxicity
An Example from Wheat Straw
Bader Al Hamdia,
Inderjitb,
Maria Olofsdottera and
Jens C. Streibiga
a Dep. of Agric. Sci. (Weed Sci.), The Royal Veterinary and Agric. Univ., Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark
b Dep. of Botany, Panjab Univ., Chandigarh 160014, India
Corresponding author (allelopathy{at}satyam.net.in)
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ABSTRACT
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Allelopathy involves complex plant x plant chemical interactions. Although a large number of laboratory bioassays have proposed to demonstrate allelopathy, most of them have little or no relevance in terms of explaining behavior in the field. In this paper, we discuss the phytotoxicity of wheat (Triticum aestivum L.) straw leachate to the seedling growth of perennial ryegrass (Lolium perenne L.). The objective of this study was to discuss the significance of (i) soil, (ii) leachate concentrations in bioassays of plant debris and soil, (iii) the role of N fertilizer in overcoming plant growth inhibition, (iv) organic molecules in plant inhibition, and (v) actual assay species. The results show the phytotoxic nature of wheat straw leachate (WSL) and the possible involvement of organic molecules in the growth inhibition of perennial ryegrass. However, laboratory studies can not demonstrate allelopathy as the sole factor responsible for the observed growth inhibition.
Abbreviations: FS, full strength • WSL, wheat straw leachate
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INTRODUCTION
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CONCERNS ARE OFTEN RAISED about the relevance of laboratory bioassays for allelopathy (Connell, 1990; Inderjit and Olofsdotter, 1998; Inderjit and Dakshini, 1995, 1999). Willis (1985)(p. 72) listed a six-point protocol necessary to demonstrate allelopathy in natural systems: "(i) a pattern of inhibition of one species or plant by another must be shown, (ii) the putative aggressive plant must produce a toxin, (iii) there must be a mode of toxin release from the plant into the environment, (iv) there must be toxin transport and/or accumulation in the environment, (v) the afflicted plant must have some means of toxin uptake, and (vi) the observed pattern of inhibition cannot be explained solely by physical factors or other biotic factors, especially competition and herbivory." Blum et al. (1999) recently concluded that no study has ever demonstrated all of these criteria. Nature is too dynamic to be solely explained by a mechanism of plant interference. The observed growth pattern is better explained by a synergistic action of several mechanisms of interference (Inderjit and Del Moral, 1997). It is almost impossible to demonstrate allelopathy by following the above six criteria. We, therefore, will restrict our discussion to phytotoxicity. We argue that laboratory bioassays can generate some meaningful data, provided that attention is paid to following points: (i) soil, (ii) several concentrations of phytotoxic material, (iii) elimination of possible inhibition by N deficiency due to added organic material, (iv) involvement of organic molecules in plant inhibition, and (v) assay species. A study with a wheat straw–perennial ryegrass system is designed to address the above criteria. Activated charcoal was added to the system, as suggested, to isolate the interference by organic molecules (Mahall and Callaway, 1992; Inderjit and Foy, 1999).
Many ecologists often argue that the addition of plant debris, leachate, or both into the soil results in enhanced microbial activity, which causes N depletion. Any growth suppression, they argue, is due to N depletion, rather than organic molecules (Harper, 1977). To address this concern and invoke the probable involvement of organic molecules in growth suppression, a series of experiments was conducted. These experiments investigated the effect of soil amended with WSL on the seedling growth of perennial ryegrass and whether the interference due to wheat straw is modified after the addition of activated charcoal and different amounts of N fertilizers. The objective of this paper is to demonstrate that laboratory bioassays for phytotoxicity can generate some meaningful data, provided that experiments are conducted under realistic conditions.
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Why Wheat Straw?
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Wheat straw has been reported to possess allelopathic activities (Guenzi and McCalla, 1962; Guenzi et al., 1967). Guenzi and McCalla (1966) found phytotoxicity of phenolic acids, particularly p-coumaric acid, from residues of wheat and other cereals. However, the bioassays conducted by these authors to demonstrate phytotoxicity had several shortcomings: (i) organic solvent was used to prepare the phenolic acid solution, (ii) authentic phenolic acids were taken, (iii) soil was not involved in the bioassays, and (iv) no consideration was paid to the significance of mixtures of chemicals (Einhellig, 1999). Another major problem with studies on phenolic acids is the lack of relevance of the tested concentration in field settings. The three common concentrations used by Guenzi and McCalla (1966) in their bioassays were 1250, 2500, and 5000 ppm. With these concentrations, each petri plate received 7.5, 15, and 30 mg of phenolic acid for the respective concentration, with 10 wheat seeds sown. This is an unrealistic amount for wheat seeds to experience in a natural setting. Alam (1990) studied the effect of wheat straw extracts on the germination and seedling growth of wheat. However, he ground the wheat straw to make the extract. Grinding may lead to the release of certain enzymes, amino acids, and other organic compounds that would have never been released from wheat straw in nature (Chou and Muller, 1972; Inderjit and Dakshini, 1995). Another problem with this study was the absence of soils. Soil is important because abiotic and biotic soil factors significantly influence the quantitative and qualitative levels of allelochemicals (Cheng, 1995; Inderjit et al., 1999). Steinsiek et al. (1982) reported that allelopathic interference of wheat to selected weed species was dependent on the extract, species, and temperature. They reported that ivyleaf morning-glory (Ipomoea hederacea Jacq.) was most affected and barnyardgrass [Echinochloa crus-galli (L.) Beauv.] was least affected. However, conclusive evidence for allelopathy is still lacking. Growth response due to soaked, agitated, or leached extracts at different temperatures cannot be attributed to the occurrence of allelopathic compounds. No soil was included in the bioassay of Steinsiek et al., and it is difficult to argue for allelopathy in the absence of soil. Using an extract for bioassay, an investigator can only demonstrate the potential phytotoxicity of the extract. More realistic experimentation is needed to determine whether the observed phytotoxicity of the extract is expressed in a natural environment. Hicks et al. (1989) reported allelopathic effects of wheat straw on the germination, emergence, and yield of cotton (Gossypium hirsutum L.). They found that the maximum inhibition in cotton germination and emergence occurred when wheat straw was mixed throughout the soil. Later, Opoku et al. (1997) implicated phenolics in the allelopathic interference of wheat straw to corn (Zea mays L.). They reported that the total phenolic levels of soil in surface-placed straw were higher compared with soil alone. However, the phenolic content of soil mixed with straw was not different from that of soil alone. Therefore, their results were not conclusive.
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Materials and methods
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General Procedures
Soil (sandy loam) was collected from a wheat-free field situated near the Royal Veterinary and Agricultural University Experimental Station in Høbakkegård, Denmark (55°40' N, 12°30' E). The soil was allowed to dry at room temperature and was sieved (1.8 mm sieve) and stored in paper bags.
Wheat straw (50 g) was obtained from Højbakkegård and soaked in 900 mL of distilled water (hereafter referred to as water) for 72 h and then filtered. The filtrate was described as full-strength (FS) WSL. Appropriate amounts of water were added to the FS WSL to obtain 50, 25, and 12.5% (v/v, WSL/water) WSL. In addition, 50 g of wheat straw was burned, and WSL of different strengths (FS, 50, 25, and 12.5%) were prepared by soaking the burned wheat straw in water as described above.
Soil Amendments
The soil (90 g) was amended with 40 mL of FS, 50, 25, and 12.5% WSL of unburned and burned wheat straw. Soil that was amended with 40 mL water served as the control.
Soil (90 g) was amended with 0.25, 0.50, and 1 g of activated charcoal (Sigma, USA) and 40 mL FS, 50, 25, and 12.5% WSL. Soil that was amended with 0.25, 0.50, and 1 g of activated charcoal and 40 mL of water served as control.
Appropriate amounts of ammonium nitrate (NH4NO3) were dissolved in water to obtain final concentrations of 0.25, 0.50, 0.75, and 1 mM of N fertilizer. The soil (90 g) was amended with 20 mL of 0.50, 1, 1.5, and 2 mM of fertilizer and 20 mL of FS, 50, 25, and 12.5% WSL. Soil that was amended with 0.50, 1, 1.5, and 2 mM of fertilizer and 20 mL of water served as the control for the respective treatments.
Growth Experiments
Perennial ryegrass was selected as an assay species because (i) it is often found in wheat fields, and (ii) it has been shown that wheat has the potential to suppress the root growth of perennial ryegrass (unpublished, 1999). Soil (90 g) as described above (soil 1, 2, and 3), was placed in 9-cm petri dishes, and 15 perennial ryegrass seeds were sown on the soil surface. The experiment had four replications and was repeated once (identified as Exp. 1 and 2). Data on the root and shoot length of wheat and perennial ryegrass were recorded after 10 d, and the longest root and shoot of each seedling was measured. The average environmental conditions were light regimes of 12.3 µmol photons m-2s-1 and a temperature of 22°C.
Data Analyses
Data were subjected to analyses of variance and linear regression. The analyses of variance and lack-of-fit tests for the linear regressions were used to check whether the linear regressions were adequate to describe the variation in data. (Weisberg, 1985).
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Results and discussion
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In general, soil that was amended with different amounts of WSL suppressed the root growth of perennial ryegrass (Fig. 1)
. The shoot growth of perennial ryegrass, however, was not affected by soil amendment. These results show the inhibitory effects of WSL on the root growth of perennial ryegrass. Further, the fact that straw leachate was prepared in an aqueous medium explains the root suppression of perennial ryegrass in the field where natural precipitation and irrigation are common. Even if the seeds of weed species were able to germinate due to adequate soil moisture, their further establishment would be restricted because of phytotoxic interference by the WSL.
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Fig. 1 Effect of soil amended with different concentrations 12.5, 25, 50, and 100% (full strength) of wheat straw (unburned and burned) leachate on root growth of perennial ryegrass. Experiments were replicated four times and repeated once
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The paracelsus (Streibig et al., 1993) axiom is very much applied to the concentration-dependent dose responses. The English translation of the German axiom is that all chemicals are poisonous—it is a question of dose. It is important to select more than three levels of concentrations of phytotoxic material to know whether the dose response relationship is properly explained by a linear regression. Any conclusion drawn with one or two concentrations of phytotoxic material is not enough in a biological system. In the present study, we have selected different levels of straw leachates to study the growth responses. Furthermore, it is important to select a range of concentrations that are close to the natural agroecosystem. The amount of wheat straw used in the present study is relevant in terms of incorporating wheat straw in Danish arable fields. However, it is difficult to predict what concentration of leachate will be added in the field setting. This is because of an uneven distribution of straw in the field as well as a varied site specific climatic and edaphic conditions. Phytotoxic material (e.g., wheat straw) is often distributed unevenly in nature, and there are pockets with varying amounts of phytotoxic material in the same field (Liebl and Worsham, 1983).
Compared with the control, soil amended with different amounts of burned straw leachate had no effect on either the root (Fig. 1) or shoot growth of perennial ryegrass. Most of the organic compounds present in wheat straw are likely to be destroyed after burning. The observed elimination of the root growth inhibition of perennial ryegrass in soil amended with burned straw leachate could be due to the absence of organic compounds, which were present in the leachates prepared from unburned wheat straw. To further confirm the involvement of organic compounds in the root growth inhibition of perennial ryegrass by WSL, we used activated charcoal, which adsorbs organic molecules, to separate the phytotoxic effects from other interference (Wardle and Nilsson, 1997; Inderjit and Foy, 1999). In general, the addition of different amounts of charcoal to soil amended with WSL eliminated the leachate effects on the root growth of perennial ryegrass (Fig. 2A, 2B) . The significant affect on the root growth of perennial ryegrass was observed in soil amended with FS WSL and lower amounts of charcoal (Fig. 2A, 2B). This can be explained due to the concentration-dependent nature of phytotoxins. This means that every interaction between charcoal and leachate and the test for lack of fit was significant. It is likely that amount of organic compounds that were contributed by FS WSL was not completely sorbed by the amount of added charcoal.
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Fig. 2 Effect of soil amended with four concentrations 12.5, 25, 50, and 100% (full strength) of wheat straw leachate (WSL) and three concentrations (0.25, 0.50, and 1 g) of charcoal on root growth of perennial ryegrass for (A) Experiment 1 and (B) Experiment 2. Soil amended only with charcoal served as the control. Experiments were replicated four times and repeated once
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In the present study, different levels of N fertilizer were added in soil amended with WSL. In general, the root growth of perennial ryegrass was still suppressed even though the amended soils were supplemented with lower levels of N fertilizers, e.g., 0.25 and 0.50 mM (Fig. 3A, 3B)
. There was a highly significant interaction between leachate and N, and a test of the lack of fit of the linear regression was significant because of nonlinearity. Less influence on the root growth of perennial ryegrass was recorded when the amended soils were supplemented with higher amounts of 0.50 and 1 mM of N fertilizer (Fig. 3A, 3B). While a significant promotion in the shoot growth of perennial ryegrass was observed in the amended soils that were supplemented with N fertilizer, no affect on the shoot growth was recorded when the experiment was repeated. Any conclusion on the promotion or lack of promotion in the shoot growth of perennial ryegrass in amended soils that were supplemented with N fertilizer can not be argued because two experiments were carried out and gave different results. We did not measure the microbial activity, but higher microbial activity due to N fertilizer is likely (Novak et al., 1995). The higher microbial activity might have influenced the qualitative and quantitative availability of organic compounds, which could have resulted in a significant influence on the seedling growth.
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Fig. 3 Effect of soil amended with four concentrations 12.5, 25, 50, and 100% (full strength) of wheat straw leachate (WSL) and five concentrations (0, 0.25, 0.50, 0.75, and 1 mM) of N fertilizer on root growth of perennial ryegrass for (A) Experiment 1 and (B) Experiment 2. Soil amended with 0 mM of N fertilizer served as the control. Experiments were replicated four times and repeated once
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Conclusion
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The present study indicates the phytotoxic nature of WSL and the possible involvement of organic molecules in the growth inhibition of perennial ryegrass. However, no laboratory study can predict the possibility of allelopathy occurring in nature (Blum, 1999). We can make some controlled experiments under simulated field conditions, which can help in deciding whether further experimentation or field study on phytotoxicity is justified. It is important how researchers interpret their data. For example, in the present study, we can not argue the phytotoxic potential of wheat straw in nature because there may be several interference mechanisms (resource competition, allelopathy, nutrient immobilization, mycorrhizae, and soil microbial ecology) that may interact and influence plant growth (Inderjit and Del Moral, 1997). What we can interpret from the present study is the phytotoxic action of wheat straw to the root growth of perennial ryegrass under given conditions.
We feel that experiments carried out under controlled conditions can never confirm allelopathy to be operational in nature (Blum, 1999). We have avoided using the term allelopathy in the discussion and used the term phytotoxicity instead because the terms are often misused (Romeo and Weidenhamer, 1998). To designate the growth inhibition due to chemicals, the term allelopathy should not be used until data are available on (i) the natural release of compounds from the aggressive plant, (ii) the concentration and persistence of these compounds in the environment, and (iii) the direct involvement of these compounds with the inhibition of the target plant, which can be demonstrated by uptake studies. However, the term phytotoxicity can be used.
Received for publication November 20, 1999.
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