Is hormesis an underestimated factor in the development of herbicide resistance?

The growing impact of herbicide resistant weeds increasingly affects weed management and the delay of resistance evolution has become a major task of chemical weed control. Hormesis and, thus, the phenomenon that low doses of herbicides can boost weed growth could be of importance in this regard since the recommended field rate may represent a low dose for weeds that have evolved resistance to the applied herbicide and, thus, a potential hormetic dose. Applying the field rate may thus not only directly select resistant biotypes, it may also indirectly promote the success and spread of resistant biotypes via hormesis. Nevertheless, hormetic effects in resistant weeds are hitherto merely randomly observed and, thus, a clear quantitative basis to judge the significance of hormesis for resistance evolution is lacking. Therefore, this study aimed at quantifying the degree and frequency of herbicide hormesis in sensitive and resistant weed species in order to provide a first indication of whether the phenomenon deserves consideration as a potential factor contributing to the development of herbicide resistance. In germination assays complete dose-response experiments were conducted with sensitive and resistant biotypes of Matricaria inodora (ALS-target-site resistant; treated with iodosulfuron-methyl-sodium/mesosulfuron-methyl), Eleusine indica (glyphosate­resistant; treated with glyphosate), and Chenopodium album (triazine/triazinone-target-site resistant; treated with terbuthylazine). After 10 days of cultivation under controlled conditions plant growth was analyzed by measuring shoot/root length and mass. Results indicated that herbicide hormesis occurred on average with a total frequency of 29% in sensitive/resistant biotypes with an average growth increase of 53% occurring typically within a dose zone exceeding 350fold. Hormetic effects occurred, however, very variable and only for specific endpoints and not plant growth in general. If such a variable stimulation of specific traits will translate to resistance relevant growth promotion under more practical conditions is uncertain. None-the-less, for a full understanding of the development of herbicide resistance, hormetic effects should be considered as a potential factor in resistance evolution. Keywords: Biphasic, growth stimulation, herbicides, target-site resistance Ist Hormesis ein unterschätzter Faktor bei der Entwicklung von Herbizidresistenz? Zusammenfassung Der wachsende Einfluss der Herbizidresistenz in Unkräutern erschwert zunehmend das Unkrautmanagement und das Hinauszögern der Resistenzentwicklung ist zu einem wichtigen Aspekt der chemischen Unkrautkontrolle geworden. Hormesis und damit das Phänomen, dass niedrige Dosierungen von Herbiziden das Unkrautwachstum fördern können, könnte in diesem Zusammenhang von Bedeutung sein, da die empfohlene Aufwandmenge für resistente Unkräuter eine niedrige Dosis und somit eine hormetische Dosis darstellen kann. Eine Applikation der empfohlenen Aufwandmenge könnte somit nicht nur direkt resistente Biotypen selektieren, sondern die Ausbreitung von resistenten Biotypen durch Hormesis indirekt fördern. Bisher wurden hormetische Effekte bei resistenten Unkräutern allerdings nur zufällig beobachtet, sodass die Datenbasis nicht ausreicht, um die Relevanz hormetischer Effekte für die Resistenzentwicklung abzuschätzen. Ziel dieser Studie war es deshalb, das Ausmaß und das Auftreten von Hormesis in sensitiven und resistenten Unkautbiotypen zu untersuchen, um einen Hinweis darauf zu erhalten, ob Hormesis als potentieller Mechanismus zur Resistenzentwicklung beitragen könnte. In Keimtests wurden deshalb Dosis-Wirkungsversuche durchgeführt mit sensitiven und resistenten Biotypen von Matricaria inodora (ALS-Target-Site Resistenz; Behandlung mit Iodosulfuron-methyl-Natrium/Mesosulfuron-methyl), Eleusine indica (Glyphosat­resistent; Behandlung mit Glyphosat) und Chenopodium album (Triazin/Triazinon-Target-Site Resistenz; Behandlung mit Terbuthylazin). Nach 10 Tagen Versuchsdauer wurden die Länge und das Gewicht von Spross und Wurzel als Wirkungsparameter erhoben. Die Ergebnisse zeigten, dass hormetische Effekte sowohl beim sensitiven, als auch beim resistenten Biotyp im Durchschnitt mit einer Frequenz von 29% auftrat bei einer durchschnittlichen Wachstumsstimulierung von 53% und einem hormetischen Dosisbereich von durch­schnittlich über 350fach. Hormetische Effekte zeigten jedoch eine hohe Variabilität und waren nur jeweils für spezifische Wirkungsparameter zu beobachten und nicht das Pflanzenwachstum im Allgemeinen. Ob die beobachtete variable und spezifische Stimulierung einzelner Wachstumsparameter unter Praxisbedingungen zur Resistenzentwicklung beitragen kann ist fraglich. Für ein vollständiges Verständnis der Entwicklung von Herbizidresistenz sollten hormetische Effekte dennoch als ein potenzieller Faktor der Resistenzevolution in Erwägung gezogen werden. Stichwörter: Biphasisch, Herbizide, Wachstumsstimulierung, Wirkortresisten

Low dose responses of different glyphosate formulations on plants

Although glyphosate clearly has real and potential commercial uses as a growth regulator at low doses, its main commercial significance has been as an herbicide. An important prerequisite for low dose applications gaining significance is a high efficiency and reliability of effects. This, however, seems to be a major constraint, especially regarding the approach of increasing yield by glyphosate hormesis. Glyphosate is marketed in various formulations, but potential disparities in low dose responses are unknown. Therefore, this study evaluated the expression and reliability of hormetic effects of different glyphosate formulations as a possible means for glyphosate hormesis to be more reliably and sustainably be achieved. Four commercial products sold in Germany (Glyfos, Glyfos Supreme, Glyfos Dakar, and Roundup Speed) were evaluated in germination assays with Lactuca sativa. Experiments were conducted as dose-response assays and evaluated for root length and shikimic acid production. In bioassays with exposure of seeds, none of the commercial formulations induced hormesis, while all formulations showed a similar hormetic effect if methanol pre-treated seedlings were exposed. Evaluating the reliability of the observed hormetic effect showed that the effect could only be reproduced in one out of three repeats independent of the formulation used. Hence, results indicated that in controlled bioassays, the induction of hormesis by glyphosate is independent of the formulation used and requires a preconditioning, although this does not ensure a hormetic effect. Therefore, the reliability of glyphosate hormesis may remain a major constraint for potential practical uses of this phenomenon despite new formulations claiming a safer response.Keywords: Crop enhancement, dose-response, growth stimulation, hormesis, pelargonic acidSubletale Wirkungen verschiedener Glyphosat-Formulierungen auf PflanzenObwohl Glyphosat in niedrigen Dosierungen ein wachstumsregulatorisches Potenzial besitzt, ist seine kommerzielle Bedeutung weitgehend auf den Einsatz als Herbizid beschränkt. Eine wichtige Voraussetzung zur Nutzung des Potenzials niedriger Dosierungen, ist eine hohe Effizienz und Zuverlässigkeit der Wirkung. Dies scheint jedoch vor allem hinsichtlich der Nutzung hormetischer Effekte von Glyphosat zur Ertragssteigerung problematisch zu sein. Da Glyphosat in verschiedensten Formulierungen vertrieben wird und bisher keine Erkenntnisse über mögliche Unterschiede in der hormetischen Wirkung vorliegen, wurden in dieser Studie die Expression und die Zuverlässigkeit hormetischer Effekte verschiedener Glyphosat-Formulierungen untersucht. Vier kommerzielle Produkte, die in Deutschland vertrieben werden, wurden dazu in Keimtests mit Lactuca sativa geprüft (Glyfos, Glyfos Supreme, Glyfos Dakar und Roundup Speed). Die Versuche wurden als Dosis-Wirkungsversuche durchgeführt und Auswirkungen auf die Wurzellänge und den Shikimisäuregehalt wurden erhoben. In Versuchen mit Samen ergab sich bei allen Produkten regelmäßig keine Hormesis, während bei Behandlung von in Methanol vorgekeimten Keimlingen alle Produkte einen ähnlichen Hormesiseffekt zeigten. Dieser konnte allerdings unabhängig vom eingesetzten Produkt nur in einem von drei Versuchen reproduziert werden. Dies zeigt, dass Glyphosat Hormesis im Biotest mit L. sativa weitgehend unabhängig vom eingesetzten Produkt und nur nach Präkonditionierung auftritt, obgleich letzteres keine Garantie für das Auftreten von Hormesis gewährt. Die Zuverlässigkeit der hormetischen Wirkung wird deshalb trotz vielfältiger Glyphosat-Formulierungen vermutlich auch in Zukunft ein wichtiger Hemmfaktor für einen möglichen praktischen Einsatz dieses Phänomens bleiben.Stichwörter: Dosis-Wirkungsbeziehung, Hormesis, Kulturpflanzenförderung, Pelargonsäure, Wachstumsstimulatio

Low toxin doses change plant size distribution in dense populations – Glyphosate exposed Hordeum vulgare as a greenhouse case study

Numerous intentionally released toxins persist in agricultural or natural environments at low concentrations. Such low toxin doses are regularly associated with hormesis, i.e., growth stimulation, and they are suspected to affect mortality and within-population plant size distribution in dense plant stands. However, it is not known whether all these low-dose effects exist when plants grow in soil. We exposed barley to a range of low glyphosate doses and let the plants grow in dense stands for several weeks in soil. Six experiments were done that contained altogether 10,260 seedlings in 572 pots. We evaluated if the changes in average biomass and shoot length occur at the same concentrations as do the effects on slow- and fast-growing individuals, if seed size or early vigor explains variation in the response to glyphosate, and if low toxin doses change within-population mortality. Plant biomass, length and survival of subpopulations changed at doses that did not affect mean biomass. Effects of early vigor faded early, but differences in seed size and particularly vegetative growth had impacts: fast-growing plants hardly showed hormesis, whereas hormesis was particularly strong among slow-growing individuals. Compared to the population mean, glyphosate effects started at lower doses among slow-growing individuals and at higher doses among fast-growing individuals. Several times higher doses were needed before the fast-growing individuals showed the same toxicity as most of the population. Low toxin doses regularly enhanced the growth of the smallest individuals, which reduced size variation within populations and was associated with a higher number of surviving plants. Indeed, in one experiment self-thinning was not observed at low doses that stimulated the growth of slow-growing plants. As glyphosate levels in this study match those observed in agricultural fields and natural environments, we conclude that even low-levels of agro-environmental contamination are likely to shape phenotypic response, which might lead to adaptation and cascading ecological impacts.Peer reviewe

Transgenerational effects of herbicide hormesis in PSII target-site-resistant Chenopodium album L.

Die Exposition von Pflanzen gegenüber moderatem Umweltstress ist eine wichtige Quelle für evolutionäre Ver­änderungen aufgrund von genetischen und phänotypischen Reaktionen. Da Herbizid-Hormesis als die wachs­tumsstimu­lierende Wirkung niedriger Herbizid-Dosierungen ebenfalls einen moderaten Stress für exponierte Pflan­zen darstellt, sind generationsübergreifende Auswirkungen denkbar. Insbesondere bei herbizidresistenten Unkräu­tern, die bei praxisüblichen Aufwandmengen in ihrer reproduktiven Fitness hormetisch gefördert wer­den, könnte dieser Aspekt zur Evolution von Herbizidresistenz beitragen. Im Gegensatz zu Insekten und Bakte­rien liegen Erkenntnisse eines solchen generationsübergreifenden Einflusses von Hormesis bei Pflanzen bisher nicht vor. In der vorliegenden Studie wurden PSII Target-Site resistente Chenopodium album Pflanzen mit verschiedenen Dosierungen des PSII-Inhibitors Metamitron behandelt und bis zur Samenreife kultiviert (Parentalgeneration P). Die resultierenden F1-Generationen ausgewählter P-Vorbehandlungen wurden erneut in einem Dosis-Wir­kungsversuch mit Metamitron behandelt. Dabei zeigte sich ein signifikanter Einfluss der P-Vorbehandlung auf die Reaktion der F1-Nachkommen. Die Sensitivität von F1-Pflanzen bei P-Vorbehandlung mit ultra-niedri­gen und inhibierenden Dosierungen war teilweise höher als bei F1-Pflanzen unbehandelter P-Pflanzen. Eine P-Vor­behandlung mit hormetischen Dosierungen führte dagegen zu einer signifikant geringeren Sensitivität bei F1-Pflanzen. Die Ergebnisse deuten darauf hin, dass Herbizid-Hormesis generationsübergreifende Reaktionen hinsichtlich Herbizidsensitivität induziert, deren Ausprägung jedoch in Abhängigkeit der Dosierung der P-Vorbehandlung variiert. Grundsätzlich könnte dies evolutionäre Veränderungen bei Unkräutern fördern.Plant exposure to moderate environmental stress is an important source of evolutionary change due to genetic and phenotypic responses. Since herbicide hormesis, the stimulatory effect of low herbicide doses, also repre­sents a moderate stress for exposed plants, transgenerational effects may be possible. Especially in herbicide-resistant weeds whose reproductive fitness is promoted at practical application rates, this aspect could contrib­ute to the evolution of herbicide resistance. In contrast to insects and bacteria, findings of such a transgenera­tional influence of hormesis in plants are absent. In this study, PSII target-site-resistant Chenopodium album was treated with various doses of the PSII-inhibitor metamitron and cultured to maturity (parental generation P). The F1-generations of selected P-pretreatments were re-treated in a dose-response trial with metamitron. This resulted in a significant influence of the P-pre­treatment on the sensitivity of the F1-generation. The sensitivity of F1-plants to P-pretreatments with ultra-low and inhibitory doses was partly higher than of F1-plants of untreated P-plants. Instead, P-pretreatments with hormetic doses resulted in a significantly lower sensitivity of F1-plants. The results suggest that herbicide hormesis induces transgenerational responses in herbicide sensitivity, but its severity varies with the dose of P-pretreatment. In principle, this could promote evolutionary changes in weeds

Realistic low-doses of two emerging contaminants change size distribution of an annual flowering plant population

HHCB [1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta(g)-2-benzopyran] and 4-tert-octylphenol [4-(1,1,3,3-tetramethylbutyl)phenol] are widely used emerging contaminants that have the potential to cause adverse effects in the environment. The purpose of this study was to observe if and how environmentally realistic concentrations of these contaminants alter growth in plant populations. It was hypothesized that within an exposed Gypsophila elegans Bieb (annual baby's breath) population especially fast-growing seedlings are impaired even when the population mean is unaffected, and small doses can cause hormesis and, thus, an increase in shoot or root length. In a dose-response experiment, an experimental population of G. elegans was established (total 15.600 seeds, 50 seeds per replicate, 24 replicates per concentration, 5.2 seedlings/cm(2)) and exposed to 12 doses of HHCB or 4-tert-octylphenol. After five days, shoot and root length values were measured and population averages, as well as slow- and fast-growing subpopulations, were compared with unexposed controls. Growth responses were predominantly monophasic. HHCB seemed to selectively inhibit both root and shoot elongation among slow- and fast-growing individuals, while 4-tert-octylphenol selectively inhibited both root and shoot elongation of mainly fast-growing seedlings. The ED50 values (dose causing 50% inhibition) revealed that the slow-growing seedlings were more sensitive and fast-growing seedlings less sensitive than the average of all individuals. Although there was toxicant specific variation between the effects, selective toxicity was consistently found among both slow- and fast-growing plants starting already at concentrations of 0.0067 mu M, that are usually considered to be harmless. This study indicates that these contaminants can change size distribution of a plant population at low concentrations in the nM/mu M range.HHCB [1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta(g)-2-benzopyran] and 4-tert-octylphenol [4-(1,1,3,3-tetramethylbutyl)phenol] are widely used emerging contaminants that have the potential to cause adverse effects in the environment. The purpose of this study was to observe if and how environmentally realistic concentrations of these contaminants alter growth in plant populations. It was hypothesized that within an exposed Gypsophila elegans Bieb (annual baby's breath) population especially fast-growing seedlings are impaired even when the population mean is unaffected, and small doses can cause hormesis and, thus, an increase in shoot or root length. In a dose-response experiment, an experimental population of G. elegans was established (total 15.600 seeds, 50 seeds per replicate, 24 replicates per concentration, 5.2 seedlings/cm(2)) and exposed to 12 doses of HHCB or 4-tert-octylphenol. After five days, shoot and root length values were measured and population averages, as well as slow- and fast-growing subpopulations, were compared with unexposed controls. Growth responses were predominantly monophasic. HHCB seemed to selectively inhibit both root and shoot elongation among slow- and fast-growing individuals, while 4-tert-octylphenol selectively inhibited both root and shoot elongation of mainly fast-growing seedlings. The ED50 values (dose causing 50% inhibition) revealed that the slow-growing seedlings were more sensitive and fast-growing seedlings less sensitive than the average of all individuals. Although there was toxicant specific variation between the effects, selective toxicity was consistently found among both slow- and fast-growing plants starting already at concentrations of 0.0067 mu M, that are usually considered to be harmless. This study indicates that these contaminants can change size distribution of a plant population at low concentrations in the nM/mu M range.Peer reviewe

Resistenzausprägung von hetero- und homozygot resistenten Genotypen eines Acker-Fuchsschwanz-Biotypen mit Target-Site Resistenz (Haplotyp Leu1781) in Dosis-Wirkungsversuchen mit Clethodim und Cycloxydim

Anhand von Dosis-Wirkungsversuchen und molekularbiologischen Analysen wurde die Reaktion der Gesamtpopulation eines Biotyps von Acker-Fuchsschwanz (Alopecurus myosuroides) mit Target-Site Resistenz gegen ACCase-Inhibitoren (Ile1781-Leu) auf die Wirkstoffe Clethodim und Cycloxydim mit der Reaktion der homozygoten (reinerbigen) und der heterozygoten (mischerbigen) Teilpopulation verglichen. Um die beobachtete Reaktion in die einer homo- und heterozygoten Teilpopulation aufzuschlüsseln, wurden individuelle Pflanzen bezüglich des Haplotyps Leu1781 mittels SNP-Analytik (Pyrosequencing) differenziert. Für die Gesamtpopulation ergaben sich signifikant unterschiedliche Resistenzfaktoren (RF) von 8 für Clethodim und 153 für Cycloxydim. Nach Korrelation der Phäno- und Genotypen ergab sich für die heterozygote Teilpopulation ein RF von 6 für Clethodim und 118 für Cycloxydim und für die homozygote Teilpopulation ein RF von 10 für Clethodim und 136 für Cycloxydim. Obwohl sich die RF der homo- und heterozygoten Teilpopulationen nicht signifikant unterschieden, zeigten die geringeren RF der heterozygoten Teilpopulationen den deutlichen Trend einer verringerten Resistenzausprägung. Lässt sich dieser Trend in weiteren Versuchen bestätigen, so kann angenommen werden, dass die Selektion von Resistenz in einer Feld-Population neben dem eingesetzten Wirkstoff auch von der Frequenz der Genotypen abhängig ist. Die beobachteten Unterschiede zwischen der Reaktion von Acker-Fuchsschwanz mit dem Haplotyp Leu1781 auf die Wirkstoffe Clethodim und Cycloxydim verdeutlichen zudem die Bedeutung der stets individuell zu betrachtenden Konstellation von Resistenzmechanismus, Genotyp und Wirkstoff für die Selektion und Ausbreitung von Resistenz. Stichwörter: Alopecurus myosuroides, Dosis-Wirkungsversuche, Pyrosequenzierung, Target-Site Resistenz Degree of resistance of hetero- and homozygous resistant genotypes of a target-site resistant blackgrass biotype (haplotype Leu1781) in dose-response experiments with clethodim and cycloxydim Abstract In dose-response experiments and by molecular analyses the reaction of a black grass biotype (Alopecurus myosuroides) with target site resistance to ACCase inhibitors (Ile1781-Leu) was examined for the response to clethodim and cycloxydim and compared to the responses of the homozygous and heterozygous sub-populations. To decipher the population into the homo- and heterozygous sub-populations, individual plants of the resistant biotype were analyzed for the haplotype Leu1781 by means of SNP analytics (pyrosequencing). For the entire population, significant different resistance factors (RF) of 8 and 153 resulted for clethodim and cycloxydim, respectively. For the heterozygous sub-population a RF of 6 was estimated for clethodim and 118 for cycloxydim. For the homozygous sub-population a RF of 10 for clethodim and 136 for cycloxydim was estimated. The RF between the homo- and heterozygous sub-populations for each herbicide were, however, not significantly different. Despite this, a tendency of the heterozygous sub-population being less resistant was indicated. Therefore, it is hypothesized that the selection of resistance depends not only on the herbicide used, but on the frequency of the genotypes in a field population. Furthermore, the significant different reaction of black grass with the haplotype 1781 to both tested herbicides reflects the meaning of the individual constellation of active ingredient, resistance mechanisms and genotype for selection and spread of resistance. Keywords: Alopecurus myosuroides, dose-response, pyrosequencing, target-site resistanc

A Conserved Mechanism for Hormesis in Molecular Systems

Hormesis refers to dose-response phenomena where low dose treatments elicit a response that is opposite the response observed at higher doses. Hormetic dose-response relationships have been observed throughout all of biology, but the underlying determinants of many reported hormetic dose-responses have not been identified. In this report, we describe a conserved mechanism for hormesis on the molecular level where low dose treatments enhance a response that becomes reduced at higher doses. The hormetic mechanism relies on the ability of protein homo-multimers to simultaneously interact with a substrate and a competitor on different subunits at low doses of competitor. In this case, hormesis can be observed if simultaneous binding of substrate and competitor enhances a response of the homo-multimer. We characterized this mechanism of hormesis in binding experiments that analyzed the interaction of homotrimeric proliferating cell nuclear antigen (PCNA) with uracil DNA glycosylase (UNG2) and a fluorescein-labeled peptide. Additionally, the basic features of this molecular mechanism appear to be conserved with at least two enzymes that are stimulated by low doses of inhibitor: dimeric BRAF and octameric glutamine synthetase 2 (GS2). Identifying such molecular mechanisms of hormesis may help explain specific hormetic responses of cells and organisms treated with exogenous compounds

Does the root to shoot ratio show a hormetic response to stress? An ecological and environmental perspective

Root/shoot (R/S) ratio is an important index for assessing plant health, and has received increased attention in the last decades as a sensitive indicator of plant stress induced by chemical or physical agents. The R/S ratio has been discussed in the context of ecological theory and its potential importance in ecological succession, where species follow different strategies for above-ground growth for light or below-ground competition for water and nutrients. We present evidence showing the R/S ratio follows a biphasic dose–response relationship under stress, typical of hormesis. The R/S ratio in response to stress has been widely compared among species and ecological succession classes. It is constrained by a variety of factors such as ontogeny. Furthermore, the current literature lacks dose–response studies incorporating the full dose–response continuum, hence limiting scientific understanding and possible valuable application. The data presented provide an important perspective for new-generation studies that can advance current ecological understanding and improve carbon storage estimates by R/S ratio considerations. Hormetic response of the R/S ratio can have an important role in forestry for producing seedlings with desired characteristics to achieve maximum health/productivity and resilience under plantation conditions

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