Regina G. Belz

A Novel Laboratory Screening Bioassay for Crop Seedling Allelopathy

Crops that control weeds by root exudation of allelochemicals are receiving increased attention, and there are efforts to breed allelopathic cultivars in several crops. The genetic improvement of allelopathic traits is based upon parental germ plasm with high allelopathic activity. Identification of allelopathic germplasm is done in laboratory screening bioassays, but experimental protocols are limited. We developed a fast and reliable laboratory screening bioassay for grain crops that includes dose–response considerations as an integral part of the experimental design. The bioassay was conducted in hydroponic culture, and a range of experiments with 2-(3H)-benzoxazolinone (BOA), an allelochemical of several grain crops, was carried out to define the basic protocol. Because of its sensitivity to BOA, Sinapis alba L. was selected as the receiver species. BOA affected growth (fresh weight and length of shoot and root), enzyme activities (ascorbate peroxidase, catalase, glutathione S-transferase, peroxidase, phenylalanine ammonia-lyase), and chlorophyll fluorescence, whereby root length was the most reliable response parameter. BOA sensitivity was dependent on nutrients for all parameters measured, and, thus, no nutrients were added. A set of experiments with Secale cereale L. and Triticum aestivum L. as donor species was carried out to optimize the protocol. Light and pH were eliminated as primary causes for the observed inhibition. The proposed bioassay has several methodological advantages over current bioassays.

Hormesis in mixtures — Can it be predicted?

Binary mixture studies are well established for mixtures of pollutants, pesticides, or allelochemicals and sound statistical methods are available to evaluate the results in relation to reference models. The majority of mixture studies are conducted to investigate the effect of one compound on the inhibitory action of another. However, since stimulatory responses to low concentrations of chemicals are gaining increased attention and improved statistical models are available to describe this phenomenon of hormesis, scientists are challenged by the question of what will happen in the low concentration range when all or some of the chemicals in a mixture induce hormesis? Can the mixture effects still be predicted and can the size and concentration range of hormesis be predicted? The present study focused on binary mixtures with one or both compounds inducing hormesis and evaluated six data sets of root length of Lactuca sativa L. and areal growth of Lemna minor L., where substantial and reproducible hormetic responses to allelochemicals and herbicides have been found. Results showed that the concentration giving maximal growth stimulatory effects (M) and the concentration where the hormetic effect had vanished (LDS) could be predicted by the most-used reference model of concentration addition (CA), if the growth inhibitory concentrations (EC50) followed CA. In cases of deviations from CA at EC50, the maximum concentration M and the LDS concentration followed the same deviation patterns, which were described by curved isobole models. Thus, low concentration mixture effects as well as the concentration range of hormesis can be predicted applying available statistical models, if both mixture partners induce hormesis. Using monotonic concentration-response models instead of biphasic concentration-response models for the prediction of joint effects, thus ignoring hormesis, slightly overestimated the deviation from CA at EC20 and EC50, but did not alter the general conclusion of the mixture study in terms of deviation from the reference model. Mixture effects on the maximum stimulatory response were tested against the hypothesis of a linear change with mixture ratio by constructing 95% prediction intervals based on the single concentration-response curves. Four out of the six data sets evaluated followed the model of linear interpolation reasonably well, which suggested that the size of the hormetic growth stimulation can be roughly predicted in mixtures from knowledge of the concentration-response relationships of the individual chemicals.

Dose-response-a challenge for allelopathy?

The response of an organism to a chemical depends, among other things, on the dose. Nonlinear dose-response relationships occur across a broad range of research fields, and are a well established tool to describe the basic mechanisms of phytotoxicity. The responses of plants to allelochemicals as biosynthesized phytotoxins, relate as well to nonlinearity and, thus, allelopathic effects can be adequately quantified by nonlinear mathematical modeling. The current paper applies the concept of nonlinearity to assorted aspects of allelopathy within several bioassays and reveals their analysis by nonlinear regression models. Procedures for a valid comparison of effective doses between different allelopathic interactions are presented for both, inhibitory and stimulatory effects. The dose-response applications measure and compare the responses produced by pure allelochemicals [scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one); DIBOA (2,4-dihydroxy-2H-1,4-benzoxaxin-3(4H)-one); BOA (benzoxazolin-2(3H)-one); MBOA (6-methoxy-benzoxazolin-2(3H)-one)], involved in allelopathy of grain crops, to demonstrate how some general principles of dose responses also relate to allelopathy. Hereupon, dose-response applications with living donor plants demonstrate the validity of these principles for density-dependent phytotoxicity of allelochemicals produced and released by living plants (Avena sativa L., Secale cereale L., Triticum L. spp.), and reveal the use of such experiments for initial considerations about basic principles of allelopathy. Results confirm that nonlinearity applies to allelopathy, and the study of allelopathic effects in dose-response experiments allows for new and challenging insights into allelopathic interactions.

STIMULATION VERSUS INHIBITION—BIOACTIVITY OF PARTHENIN, A PHYTOCHEMICAL FROM PARTHENIUM HYSTEROPHORUS L.

Parthenium hysterophorus L. is an invasive weed that biosynthesizes several phytochemicals. The sesquiterpene lactone parthenin receives most attention regarding allelopathy of the plant or potential herbicidal properties. Since parthenin exhibits dose-dependent phytotoxicity with low dose stimulation, this study investigated the occurrence and temporal features of parthenin hormesis in Sinapis arvensis L. sprayed with parthenin under semi-natural conditions. Dose/response studies showed that the occurrence and the magnitude of hormesis depended on climatic conditions and the parameter measured. Within the tested dose range, stimulatory responses were only observed under less-stressful conditions and were most pronounced for leaf area growth [138 % of control; 13 days after treatment (DAT)]. Temporal assessment of leaf area development showed that doses causing a stimulatory response at the end of the experiment (< 0.42 ± 0.04 kg/ha; 13 DAT) were initially inhibitory up to ED50 values (2 DAT). This clearly demonstrated an overcompensatory response. Inhibition of leaf area at 13 DAT reached ED50 values on average at 0.62 ± 0.12 kg/ha, and S. arvensis was completely inhibited at doses exceeding 1.81 ± 0.56 kg/ha (ED90). Based on these findings, implications of parthenin hormesis are discussed with respect to allelopathy of P. hysterophorus and exploitation of growth stimulatory responses in agriculture.

Modeling effective dosages in hormetic dose-response studies.

Background Two hormetic modifications of a monotonically decreasing log-logistic dose-response function are most often used to model stimulatory effects of low dosages of a toxicant in plant biology. As just one of these empirical models is yet properly parameterized to allow inference about quantities of interest, this study contributes the parameterized functions for the second hormetic model and compares the estimates of effective dosages between both models based on 23 hormetic data sets. Based on this, the impact on effective dosage estimations was evaluated, especially in case of a substantially inferior fit by one of the two models. Methodology/Principal Findings The data sets evaluated described the hormetic responses of four different test plant species exposed to 15 different chemical stressors in two different experimental dose-response test designs. Out of the 23 data sets, one could not be described by any of the two models, 14 could be better described by one of the two models, and eight could be equally described by both models. In cases of misspecification by any of the two models, the differences between effective dosages estimates (0–1768%) greatly exceeded the differences observed when both models provided a satisfactory fit (0–26%). This suggests that the conclusions drawn depending on the model used may diverge considerably when using an improper hormetic model especially regarding effective dosages quantifying hormesis. Conclusions/Significance The study showed that hormetic dose responses can take on many shapes and that this diversity can not be captured by a single model without risking considerable misinterpretation. However, the two empirical models considered in this paper together provide a powerful means to model, prove, and now also to quantify a wide range of hormetic responses by reparameterization. Despite this, they should not be applied uncritically, but after statistical and graphical assessment of their adequacy.

Plants Release Precursors of Histone Deacetylase Inhibitors to Suppress Growth of Competitors

Chemical compounds in plant root exudates influence the growth of neighboring plants by interfering with their chromatin configuration and gene expression. To secure their access to water, light, and nutrients, many plant species have developed allelopathic strategies to suppress competitors. To this end, they release into the rhizosphere phytotoxic substances that inhibit the germination and growth of neighbors. Despite the importance of allelopathy in shaping natural plant communities and for agricultural production, the underlying molecular mechanisms are largely unknown. Here, we report that allelochemicals derived from the common class of cyclic hydroxamic acid root exudates directly affect the chromatin-modifying machinery in Arabidopsis thaliana. These allelochemicals inhibit histone deacetylases both in vitro and in vivo and exert their activity through locus-specific alterations of histone acetylation and associated gene expression. Our multilevel analysis collectively shows how plant-plant interactions interfere with a fundamental cellular process, histone acetylation, by targeting an evolutionarily highly conserved class of enzymes.

BIOCONTROL OF WEEDS WITH ALLELOPATHY: CONVENTIONAL AND TRANSGENIC APPROACHES

Growing highly allelopathic crops has the potential to significantly reduce our reliance on synthetic herbicides for weed management. Specific phytotoxins have been found in allelopathic rice, wheat, and rye varieties, but this information has not been used in breeding varieties that can be marketed on the basis of their weed management properties. Although such a conventional approach is viable, transgenic strategies may be better. For example, genes encoding enzymes of the highly potent phytotoxin sorgoleone in Sorghum spp. might be transgenically manipulated to enhance the allelopathic properties of sorghum crops. This potent phytotoxin is exclusively synthesized and secreted by root hairs. The sorgoleone pathway has been elucidated and putative genes encoding them have been identified and partially verified.

Interspecies Variability of Plant Hormesis by the Antiauxin PCIB in a Laboratory Bioassay

Chemical hormesis constitutes an alternative possible use of herbicidal agents for crop enhancement that is, however, compromised by the apparent variability of this low-dose stimulation phenomenon. Studies demonstrating the variability are rare and, therefore, this study investigated the interspecies variability of growth stimulation induced by the auxin-inhibitor PCIB [2-(p-chlorophenoxy)-2-methylpropionic acid] to determine if hormesis is generalizable enough and sufficiently stable between species/cultivars for practical use or which implications may have to be taken into account. In 85 complete dose–response bioassays with 23 cultivars of five species, the variability of PCIB effects was evaluated. The expression of PCIB hormesis proved to depend on the species/cultivar tested, ranging from a cultivar-dependent hormetic efficacy and an occasional lack of hormesis, to a complete lack of hormetic effectiveness in certain species/cultivars. Therefore, frequency estimations, as well as the pattern of dose-dependent variability of dose–response quantities, may inevitably depend on the biological model(s) used and, thus, apply only to the specific conditions for characterization. Comparing the frequency distribution of effective doses demonstrated a risk of a previously hormetic dose causing a loss of hormesis or inhibitory effects in another species/cultivar. Therefore, selecting a dose that will induce hormesis in every species/cultivar is unrealistic. This may limit the window for practical applications to stimulants with negligible varietal differences, to cultivar selective treatments, and/or to cultivars that enable a beneficial long-term use. Hence, efficient crop enhancement by chemical hormesis needs not only a good stimulant, but also a species/cultivar able to convert a specific low-dose treatment into an economic benefit.

Statistical modeling of the hormetic dose zone and the toxic potency completes the quantitative description of hormetic dose responses.

Quantifying the characteristics of hormesis provides valuable insights into this low-dose phenomenon and helps to display and capture its variability. A prerequisite to do so is a statistical procedure allowing quantification of general hormetic features, namely the maximum stimulatory response, the dose range of hormesis, and the distance from the maximum stimulation to the dose where hormesis disappears. Applying extensions of a hormetic dose-response model that is well-established in plant biology provides a direct estimation of several quantities, except the hormetic dose range. Another dose range that is difficult to model directly is the distance between the dose where hormesis disappears and the dose giving 50% inhibition, known as toxic potency. The present study presents 2 further model extensions allowing for a direct quantification of the hormetic dose range and the toxic potency. Based on this, a 4-step mathematical modeling approach is demonstrated to quantify various dose-response quantities, to compare these quantities among treatments, and to interrelate hormesis features. Practical challenges are exemplified, and possible remedies are identified. The software code to perform the analysis is provided as Supplemental Data to simplify adoption of the modeling procedure. Because numerous patterns of hormesis are observed in various sciences, it is clear that the proposed approach cannot cope with all patterns; however, it should be possible to analyze a great range of hormesis patterns.

Low doses of six toxicants change plant size distribution in dense populations of Lactuca sativa

Toxicants are known to have negligible or stimulatory, i.e. hormetic, effects at low doses below those that decrease the mean response of a plant population. Our earlier observations indicated that at such low toxicant doses the growth of very fast- and slow-growing seedlings is selectively altered, even if the population mean remains constant. Currently, it is not known how common these selective low-dose effects are, whether they are similar among fast- and slow-growing seedlings, and whether they occur concurrently with hormetic effects. We tested the response of Lactuca sativa in complete dose-response experiments to six different toxicants at doses that did not decrease population mean and beyond. The tested toxicants were IAA, parthenin, HHCB, 4-tert-octylphenol, glyphosate, and pelargonic acid. Each experiment consisted of 14,400-16,800 seedlings, 12-14 concentrations, 24 replicates per concentration and 50 germinated seeds per replicate. We analyzed the commonness of selective low-dose effects and explored if toxic effects and hormetic stimulation among fast- and slow-growing individuals occurred at the same concentrations as they occur at the population level. Irrespective of the observed response pattern and toxicant, selective low-dose effects were found. Toxin effects among fast-growing individuals usually started at higher doses compared to the population mean, while the opposite was found among slow-growing individuals. Very low toxin exposures tended to homogenize plant populations due to selective effects, while higher, but still hormetic doses tended to heterogenize plant populations. Although the extent of observed size segregation varied with the specific toxin tested, we conclude that a dose-dependent alteration in size distribution of a plant population may generally apply for many toxin exposures.