Department of Cheamistry, Ashland University, Ashland, OH 44805 USA Email jweiden@ashland.edu
Information on allelochemical dynamics in the rhizosphere is crucial for evaluating specific hypotheses of allelopathic effects, understanding allelopathic mechanisms, and assessing the importance of allelopathic processes in plant communities. The lack of tools to obtain such information is a major barrier to investigations of allelopathy. When soils beneath suspected allelopathic plants are analyzed, allelochemical concentrations are typically low, and critics cite this as evidence that these compounds do not play a significant role in plant-plant interactions. However, static concentrations in the environment reflect the balance of input vs. output rates for a compound. Because plant roots compete with both microorganisms and other processes that remove allelochemicals from soil solution, flux rates are likely to be a key component of toxicity. This paper reports the use of polydimethylsiloxane (PDMS) sorbents to trap allelochemicals as they are released in the rhizosphere. PDMS has been used to quantify levels of the photosynthesis inhibitor sorgoleone in the undisturbed rhizosphere of sorghum-sudangrass plants. The materials used included stir bars coated with PDMS (stir bar sorptive extraction), technical grade optical fiber coated with a thin film of PDMS (matrix-solid phase microextraction), and PDMS tubing. PDMS probes were removed at 29, 55 and 88 days after planting. As analyzed by HPLC, the amount of sorgoleone recovered increased with time. Work is ongoing to optimize parameters for the use of PDMS, evaluate the potential of PDMS as a sorbent for a broad range of allelochemicals in soil, and use PDMS probes in the field.
A new methodology has been developed to monitor natural herbicides as they are released by plants in the field.
Key Words
Biomimetic extraction, polydimethylsiloxane, stir bar sorptive extraction, matrix-SPME, Sorghum bicolor x Sorghum sudanense, sorgoleone.
Introduction
Muller (1966, p. 332) summarized allelopathy’s potential importance to plant communities:
The significance of allelopathy to ecological theory is very great. Small quantities of toxins may be responsible for massive reductions in plant growth and in water or mineral absorptions and thus strongly influence microclimate. Traditional theories of competition, reaction, biomass proportions, energy flow, mineral cycling, and ecosystem organization are all liable to reevaluation where allelopathy is demonstrable.
Muller’s optimistic assessment hinges on allelopathy being, in Muller’s word, “demonstrable.” In the aftermath of what Baldwin (2003) has termed “the rodent incident,” in which haloes around Salvia shrubs in the California chaparral that Muller had attributed to allelopathy were instead shown to result from the grazing activity of small mammals, a much more critical eye was turned to the methodology used to study this phenomenon.
Among the obvious weaknesses were the compounds typically identified as suspected allelopathic agents. Often, these were phenolic acids that are widely distributed in plants and have relatively low biological activity. In many cases, compounds were not even identified and allegations of allelopathic effects rested on inhibition of plant growth or germination by concentrated foliage extracts of dubious ecological relevance. As Harper (1977, p. 369) notes, “Almost all species can, by appropriate digestion, extraction and concentration, be persuaded to yield a product that is toxic to one species or another.”
Such problems led Putnam and Tang (1986) to observe that "chemistry has been the Achilles' heel of allelopathy." However, this is no longer true with respect to the characterization of potential toxins. Several allelochemicals with toxicities rivaling synthetic herbicides are known, including α-terthienyl (Campbell et al. 1982), artemisinin (Duke et al. 1987) and sorgoleone (Nimbal et al. 1996). Unfortunately, analytical techniques for studying rhizosphere chemistry have not kept up with advances in natural product characterization. The general lack of understanding of the qualitative and quantitative dynamics of allelochemicals in the rhizosphere is perhaps the present Achilles' heel of allelopathy (Weidenhamer 1996). The need for such information has been emphasized repeatedly. Fuerst and Putnam (1983) asserted that quantifying the amount of toxin released to the environment and taken up by the target plant was crucial to proving a hypothesis of allelopathy. Radosevich and Holt (1984) concurred, arguing “research in this area must be designed specifically to prove that a toxic substance is produced and that it accumulates or persists long enough at concentrations in the environment sufficient to inhibit development of other plants.” While the Koch’s postulates approach advocated by Fuerst and Putnam (1983) is now regarded as unreasonable for a complex ecological phenomenon (Weidenhamer 1996; Baldwin 2003), demonstration of allelopathic interactions without data on allelochemical dynamics in soil will remain problematic.
Blum et al. (1999) argue that the research focus in allelopathy needs to shift to soil, and “specifically the barrier of the rhizosphere through which allelochemicals must pass.” This call is echoed in a recent review of rhizosphere chemistry. Bertin et al. (2003) note that root exudates vary greatly in chemical composition and biological effects, and emphasize the need for techniques that deepen our understanding of the chemical and biological dynamics of the rhizosphere. Experimental evidence underscores these points. Tang et al. (1987) found higher concentrations of BBT-OH relative to other thiophenes in the rhizosphere than in roots of Tagetes patula, and concluded that analyzing the rhizosphere was more important than analyzing plant tissues for studies of allelopathy. Weidenhamer and Romeo (2004) found that soil microbes rapidly convert the hydroquinone glycoside arbutin to hydroquinone and then to benzoquinone. The latter two compounds are the suspected allelopathic agents of Polygonella myriophylla, but they do not occur in free form in the plant itself. The question, then, is what methods can be used to gain insight to the chemical dynamics of the rhizosphere.
This paper reviews recent experiments demonstrating the use of polydimethylsiloxane-based materials to monitor sorgoleone in the rhizosphere of greenhouse-grown sorghum-sudangrass hybrid plants (Sorghum bicolor x S. sudanense) over a three month period (Weidenhamer 2005).
Methods
Sorghum and sorghum-sudangrass are known to exude sorgoleone, a potent inhibitor of photosystem II, from their roots (Czarnota et al. 2003). The experimental sorghum-sudangrass hybrid ‘High Sugar’ (Browning Seed Co., Plainview, TX) was selected on the basis of a preliminary screening study of several sorghum and sorghum-sudangrass hybrid cultivars as one which produced the largest quantities of sorgoleone. Plants were grown in a greenhouse in 3.8 L polyethylene nursery pots containing sandy loam soil. The materials used included stir bars coated with PDMS (stir bar sorptive extraction), technical grade optical fiber coated with a thin film of PDMS (matrix-solid phase microextraction), and PDMS tubing. Several non-PDMS materials (polyurethane foam plugs, C18 disks, Tenax disks, and carbonaceous resin capsules) were also included for comparison. At planting, one type of PDMS extractant was buried per pot, centered at a depth of 5 cm. PDMS probes were removed at harvest. Four sorghum-sudangrass plants were maintained per pot. There were three replicates of each treatment per harvest, and PDMS probes in unplanted pots served as background controls. PDMS materials were extracted with 250-500 μL acetonitrile and analyzed by high performance liquid chromatography (HPLC) following methods of Czarnota et al. (2003).
Results
All PDMS materials were found to retain sorgoleone, and the amounts found increased over time (Fig. 1). The presence of sorgoleone in the PDMS extracts was verified by HPLC-mass spectrometry. PDMS tubing (four 10-cm strands of 0.30 mm ID x 0.64 mm OD tubing were used per pot) retained the most sorgoleone. The non-PDMS materials proved to be problematic to work with. The capsules, foam plugs and disks were all penetrated by sorghum roots to greater or lesser extent (Fig. 2). Analyses based on these materials are therefore suspect, given that any sorgoleone recovered might be from root fragments that could not be removed rather than sorgoleone adsorbed from the rhizosphere by these extractants. Root penetration was not a problem with any of the PDMS materials.
Conclusions
PDMS sorbents provide a new tool for obtaining information on allelochemical dynamics in the rhizosphere. Procedures for soil extraction of allelochemicals seek to recover all of a certain fraction of a compound present in the soil at a certain point in time (e.g. soil solution, reversibly/irreversibly adsorbed) (Blum et al. 1999). In contrast, PDMS traps function in a manner more analogous to plant roots by sorbing compounds from soil solution or through direct contact with root exudates. In principle, allelochemicals sorbed by PDMS traps should be potentially available to roots of other plants. For this reason, the term “biomimetic extraction” is proposed to describe this technique.
The strong performance of PDMS tubing and PDMS-coated optical fiber is noteworthy also because both materials are less expensive than the commercially available PDMS-coated stir bars. Because of its rigidity, optical fiber can be directly inserted into the soil with minimal disturbance. PDMS tubing could either be buried or also inserted directly into the soil if a stiff wire is inserted into the tubing. These materials are therefore potentially useful for non-destructive sampling of the rhizosphere in large-scale greenhouse and field studies.
Further work is clearly needed to elucidate the stability of sorbed compounds over time, how broad a range of compounds can be effectively trapped by these sorbents, and what forms of PDMS are most advantageous for field studies. It should be noted that the demonstration that fluxes of allelochemicals can be measured in the rhizosphere will not prove that allelopathic interactions are occurring. Yet it is also true that demonstration of allelopathic interactions without data on allelochemical dynamics in soil will remain problematic. PDMS-based materials provide a new tool for obtaining this information, and thereby helping to assess the importance of allelopathic processes in plant communities.
Acknowledgements
Sorghum and sorghum-sudangrass seeds were a gift of Browning Seed Co. (Plainview, TX). Gerstel, Inc. supported this research by waiving the license fee for its Twister technology. Acquisition of the Agilent HPLC was supported by the National Science Foundation, DUE-9952552. Jeanne Durkalski assisted with greenhouse operations. Warren A. Dick of the Ohio Agricultural Research and Development Center – Ohio State University provided greenhouse space for this study. The HPLC-MS analyses were performed by K. Green-Church and the staff at the Ohio State Mass Spectrometry and Proteomics Facility. The mass spectrometer used in this study was purchased by a grant from the Hayes Investment Fund of the Ohio Board of Regents.
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