Immobilized plant thylakoid membranes as a biosensor for herbicides

Summary Thylakoid membranes isolated from spinach leaves were used as the biological sensing material to elaborate a biosensor for the detection of small amount of the herbicides atrazine and diuron. Free and immobilized thylakoid membranes were compared for their responses to inhibition by herbicides by following the variation of the photocurrent. Immobilized thylakoid membranes were twice as sensitive to inhibition by herbicides than the native thylakoids.     

Sensitive bioassays for determining residues of sulfonylurea herbicides in soil and their availability to crop plants

Sulfonylurea herbicides are potent inhibitors of plant growth and are extremely active against a wide spectrum of weeds. They are used at very low rates (10–50 g ai/ha) and cause rapid inhibition of root and shoot growth of young plants. Routine chemical assays for detecting low levels of these compounds are difficult and there is need to develop sensitive bioassay methods for detecting their extremely low residue levels in the soil.This paper describes a simple pot bioassay method with a self watering system using turnip (Brassica rapa) seedlings as test plants for quantitative determination of sulfonylurea herbicides. Results are presented with six of these compounds whose activity was investigated in widely differing substrates. The potential availability to plants was calculated from the dose-response curves in different substrates. The dose-response relationship has been described by a specifically developed computer model. Details are also given of a direct seeded bioassay method with controlled watering system using several test species for detection of sulfonylurea herbicides. The potential uses and practical applications of both techniques are discussed.     

Regulation of photosynthetic electron transport by bicarbonate formate and herbicides in isolated broken and intact chloroplasts

In this paper, we have presented a minireview on the interaction of bicarbonate, formate and herbicides with the thylakoid membranes.The regulation of photosynthetic electron transport by bicarbonate, formate and herbicides is described. Bicarbonate, formate, and many herbicides act between the primary quinone electron acceptor Q_A and the plastoquinone pool. Many herbicides like the ureas, triazines and the phenol-type herbicides act, probably, by the displacement of the secondary quinone electron acceptor Q_B from its binding site on a Q_B-binding protein located at the acceptor side of Photosystem II. Formate appears to be an inhibitor of electron transport; this inhibition can be removed by the addition of bicarbonate. There appears to be an interaction of the herbicides with bicarbonate and/or It has been suggested that both the binding of a herbicide and the absence of bicarbonate may cause a conformational alteration of the environment of the Q_B-binding site. The alteration brought about by a herbicide decreases the affinity for another herbicide or for bicarbonate; the change caused by the absence of bicarbonate decreases the affinity for herbicides. Moreover, this change in conformation causes an inhibition of electron transport. A bicarbonate-effect in isolated intact chloroplasts is demonstrated.Paper presented at the FESPP meeting (Strasbourg, 1984)     

Inhibition of Phenylamide Hydrolysis by Bacillus sphaericus with Methylcarbamate and Organophosphorus Insecticides

The degradation of the phenylamide herbicides monolinuron, linuron, and solan by cultures of Bacillus sphaericus ATCC 12123 was inhibited by the methylcarbamate insecticides metmercapturon, aldicarb, propoxur, and carbaryl and by the organophosphorus insecticides fenthion and parathion. The extent of inhibition was largest with metmercapturon and smallest with parathion inhibition of hydrolysis of the two phenylurea herbicides was greater than of the acylanilide compound. Tests with crude enzyme preparations of aryl acylamidase derived from B. sphaericus showed that the inhibition of the hydrolysis of linuron with methylcarbamates is a competitive one. The insecticides tested did induce the enzyme, nor could they serve as its substrate.     

Comparison of experimental methods for determination of toxicity and biodegradability of xenobiotic compounds

Different methods for determining the toxicity and biodegradability of hazardous compounds evaluating their susceptibility to biological treatment were studied. Several compounds including chlorophenols and herbicides have been evaluated. Toxicity was analyzed in terms of EC₅₀ and by a simple respirometric procedure based on the OECD Method 209 and by the Microtox^® bioassay. The values of EC₅₀ obtained from respirometry were in all the cases higher than those from the Microtox^® test. The respirometric inhibition values of chlorophenols were related well with the number of chlorine atoms and their position in the aromatic ring. In general, herbicides showed lower inhibition, being alachlor the less toxic from this criterion. For determination of biodegradability an easier and faster alternative to the OECD Method 301, with a higher biomass to substrate ratio is proposed. When this test was negative, the Zahn-Wellens one was performed in order to evaluate the inherent biodegradability. In the fast test of biodegradability, 4-chlorocatechol and 4-chlorophenol showed a complete biodegradation by an unacclimated sludge upon 48 h. These results together with their low respirometric inhibition, allow concluding that these compounds could be conveniently removed in a WWTP. Alachlor, 2,4-dichlorophenol, 2,4,6-trichlorophenol and MCPA showed a partial biodegradation upon 28 days by the Zahn-Wellens inherent biodegradability test.     

Quantitative structure—activity relationship study on the inhibitory effect of some herbicides on the growth of soil micro-organisms

The effect of eight herbicides on the growth of six soil bacteria and three microscopic fungi, and on the amylase, cellulase and dehydrogenase activity in a Hungarian soil was determined. Herbicide decomposition rate in the soil was also assessed. Qualitative structure-activity relationship (principal component analysis, spectral mapping technique and stepwise regression) analysis showed that the overwhelming majority (about 80%) of the effect of herbicides can be explained by one background variable showing the similarity between their mode of action. The two-dimensional non-linear map of principal component loadings and spectral map characteristics suggested that the number of substituents may be important in the determination of the toxic effects. The inhibition of bacterial growth, inhibition of fungal growth, and effects on enzyme activity and decomposition rate all formed different clusters on the maps indicating that herbicides influence these processes differently. Of the nine physicochemical parameters considered, only the electron withdrawing capacity of substituents significantly influenced the biological activity of herbicides, suggesting that electrostatic interactions between the herbicide molecules and micro-organisms is important.     

Cyclohexanedione Herbicides Are Selective and Potent Inhibitors of Acetyl-CoA Carboxylase from Grasses

Biochemical studies of plant species susceptible to the cyclohexanedione herbicides, alloxydim, sethoxydim, and clethodim, have demonstrated that these selective grass herbicides inhibit acetyl-coenzyme A carboxylase, the second enzyme common to both fatty acid and flavonoid biosynthetic pathways. The K_is for the cyclohexanediones tested ranged from 0.02 to 1.95 micromolar, depending on the species. The enzyme isolated from broadleaf plants was much less sensitive to inhibition by these herbicides (K_is from 53 micromolar to 2.2 millimolar). These results may explain the mechanism of action of these herbicides and their selectivity for monocotyledonous species.     

New method for toxicity assessment in marine and brackish environments using the macroalga Gracilaria tenuistipitata (Gracilariales,Rhodophyta)

A growth inhibition test method was developed using the macroalga Gracilaria tenuistipitata as the test organism. This alga was chosen because of its high laboratory growth rates, commonly 30–40% d^–1, which are reached in salinities between 5 and 40, and its epiphyte resistance. The toxicity of a number of substances, including heavy metals, herbicides and complex wastewaters towards the alga was assayed. Anti-fouling paints were tested with a modification of the method. EC50 values for heavy metals varied between 0.05 and 17 mg l^–1 and for herbicides between 0.002 and 0.02 mg l^–l. The sensitivity to the toxicant was generally higher at low salinity. Omitting nitrogen and phosphorus additions to the test medium increased the sensitivity and a semi-static performance was possible with maintained or increased sensitivity. Preliminary tests done with a computerised photosynthesis inhibition method produced promising results.In conclusion, this is a simple, sensitive and reproducible test method for assessing the toxicity of substances, wastewaters and anti-fouling paints in brackish and marine environments.     

Caffeic acid derivatives as growth inhibitors of Setaria viridis: Structure-activity relationships and mechanisms

Weeds bring severe threats to agricultural production and most traditional herbicides cause serious pollution to the environment. Caffeic acid is a potential allelochemical of many crops. To develop novel herbicides, a series of trans-caffeic acid analogues were prepared to evaluate their growth inhibition on S. viridis and investigate structure-activity relationships. Presence of hydroxyl groups on the benzene ring greatly reduced the phytotoxicity of analogues and the double bond seemed not to be necessary. Moreover, it was found that halogenated cinnamic acid analogues showed potent inhibitory activity. 4-Fluorocinnamic acid (4-FCA) displayed the strongest growth inhibition in a concentration-dependent manner, whereas it induced significantly weaker inhibition on wheat. This selectivity could help us to develop a novel herbicide. Exposure of S. viridis to 4-FCA increased levels of H₂O₂ and catalase (CAT) in roots, suggesting that the inhibitory effect of 4-FCA might be related to oxidative stress. Thus, we have found an active lead compound for a novel herbicide derived from crop allelochemicals, which may help in the development of new environmentally safe herbicides.     

Synthesis of potential herbicides designed to uncouple photophosphorylation

In a search for novel herbicides we attempted to make uncouplers of photophosphorylation. Good herbicides were discovered, but not all were good uncouplers and we present evidence which supports the view that their primary action is through inhibition of carotenoid biosynthesis. This paper describes the synthesis and in vivo and in vitro activities of 21 compounds of the series. The best herbicide was active enough to justify extensive field testing.     

Studies on synthetic hybrids of british species of Senecio: I Senecio viscosus L. × S. vulgaris L.

Summary

Four aquatic hyphomycetes and one terrestrial fungus were examined for their responses to the phenoxy herbicides (±)-MCPP and 2,4-D as both single and binary preparations with respect to hyphal extension, sporulation and respiration. Hyphal extension of all species was unaffected at concentrations less than 100 mg l^?1. At higher concentrations there was a variable inhibitory response to the herbicides but no clear pattern was observed between the five fungi. The binary herbicide mixture had a weak synergistic effect on inhibition of growth rate. For the four aquatic hyphomycetes sporulation was reduced at several herbicide concentrations, but there was no consistent reduction over the experimental period. Flagellospora curvula and Clavariopsis aquatica showed increased sporulation at 100 and 1000 mg l^?1 for only some herbicide combinations. The respiration rates of the fungi varied with species and herbicide concentration and ranged between stimulation at 100 mg l^?1 to inhibition at 4000 mg l^?1. The results indicate that the five fungi are not likely to be severely effected by the phenoxy herbicides at concentrations normally occurring in the field. The possible effects of these herbicides on nutrient cycling are briefly discussed.     

Response of multiple herbicide resistant strain of diazotrophic cyanobacterium, Anabaena variabilis, exposed to atrazine and DCMU

Effect of two photosynthetic inhibitor herbicides, atrazine (both purified and formulated) and [3-(3,4-dichlorophenyl)-1,1-dimethyl urea] (DCMU), on the growth, macromolecular contents, heterocyst frequency, photosynthetic O2 evolution and dark O2 uptake of wild type and multiple herbicide resistant (MHR) strain of diazotrophic cyanobacterium A. variabilis was studied. Cyanobacterial strains showed gradual inhibition in growth with increasing dosage of herbicides. Both wild type and MHR strain tolerated < 6.0 mg L(-1) of atrazine (purified), < 2.0 mg L(-1) of atrazine (formulated) and < 0.4 mg L(-1) of DCMU indicating similar level of herbicide tolerance. Atrazine (pure) (8.0 mg L(-1)) and 4.0 mg L(-1) of atrazine (formulated) were growth inhibitory concentrations (lethal) for both wild type and MHR strain indicating formulated atrazine was more toxic than the purified form. Comparatively lower concentrations of DCMU were found to be lethal for wild type and MHR strain, respectively. Thus, between the two herbicides tested DCMU was more growth toxic than atrazine. At sublethal dosages of herbicides, photosynthetic O2 evolution showed highest inhibition followed by chlorophyll a, phycobhiliproteins and heterocyst differentiation as compared to carotenoid, protein and respiratory O2 uptake.     

Simulation and experimental analysis of the influence of herbicides on soil nitrification

The effect of 35 herbicides on the nitrification process was tested both by experiment, and by simulation of possible mechanisms of inhibition in a mathematical model. The model consists of nine equations with six coordinated constant and seven measurable parameters (or initial values), depending on the specific soil. The only free parameters are the initial values of the oxidative enzyme systems, and the parameters which determine the course of possible inhibition effects. For the majority of the herbicides, the inhibitory effects on the NH₄ ⁺ or NO₂ ^- oxidation were found negligible in the range of practical application. Hypotheses of a completely reversible or partially reversible inhibition of the oxidase systems gave the best correspondence between the model and reality, while an alteration of the growth parameters of the nitrifying populations in the model (death rate, proliferation rate, initial kill) due to the application of herbicides led to strong contrasts between simulated and experimental curves. Significant inhibitory effects became evident only when the hydrogen ion concentration in the soil fell below pH 7. Results with several herbicides indicated that the process of NO₂ ^- oxidation was more sensitive than that of NH₄ ⁺ oxidation. With a number of herbicides, an accumulation of NO₂ ^- ions was noticed during the course of soil percolation. In consideration of the buffering capacity, the model is applicable to other soils.     

A comparison of effects of several herbicides on photoautotrophic,photomixotrophic and heterotrophic cultured tobacco cells and seedlings

The effects of herbicides with different primary modes of action were examined on the growth of photoautotrophic, photomixotrophic, and heterotrophic cultures of tobacco cells. These responses were compared with those of tobacco seedlings to the same herbicides. Herbicides, which primarily inhibit or disturb photosynthetic processes, suppressed the growth of photoautotrophic cells most strongly, as compared to photomixotrophic and heterotrophic cells (atrazine, diuron, paraquat). Herbicides having a primary mode of action other than the inhibition of photosynthetic processes, suppressed the growth of all types of cultured cells at similar concentrations (2,4-D, diphenamid, glyphosate, dinoseb, sodium chlorate, bialaphos, DTP), but the photoautotrophic cells were still the most sensitive to all kinds of herbicides except sodium chlorate. Furthermore, photoautotrophic cells responded to most of the herbicides as did the seedlings, with the exception of glyphosate and diphenamid. The possibility of photoautotrophically cultured cells as a model system to study the effects of herbicides are discussed.Abbreviation bialaphos (2-amino-4-methylphosphinobutyryl)alanylalanine sodium salt - diuron 3-(3,4-dichloro-phenyl)-1,1-dimethyl-urea - 2,4-D 2,4-dichlorophenoxy-acetic acid - DTP 1,3-dimethyl-4-(2,4-dichlorobenzoyl)-5-hydoxy-pyrazolate - dinoseb 2-secbutyl-4,6-dinitrophenol     

A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen

In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.     

The effect of herbicides,plant growth regulators and other compounds on phenylalanine ammonia-lyase activity

The in vitro and in vivo effects of a number of herbicides and plant growth regulators on phenylalanine ammonia-lyase (PAL) activity were investigated. The most elective in vitro inhibitors were product analogs, t-cinnamic and p-coumaric acids, and carbonyl reagents, hydroxylamine and nitromethane. Application of the herbicides diuron, dalapon, amiben, and chloropropham, to plants resulted in a decrease in the intracellular concn of PAL. The inhibitory effect of alachlor was found to be dose-responsive and somewhat specific. A correlation between PAL inhibition and herbicidal activity was observed for hydroxylamine. The cytokinin, pyranyl benzyladenine, (PBA) increased PAL activity in pigweed. The possibility of developing herbicides acting through PAL inhibition is discussed.     

Behaviour of Herbicides in Dicotyledonous Roots Transformed by Agrobacterium rhizogenes: I. SELECTIVITY

Mugnier, J. 1988. Behaviour of herbicides in dicotyledonousroots transformed by Agrobacterium rhizogenes. I. Selectivity.—J.exp. Bot. 39: 1045–1056. The effect of various herbicides on the growth of dicotyledonousroots transformed by the root-inducing transferred DNA of Agrobacteriumrhizogenes was studied. When a compound affected the growthof different root species differentially, the difference mightbe attributed to root uptake and metabolism of the herbicide.In general, metabolism of the herbicide led to inactivation(clopyralid, linuron, phenmedipham), but in certain instances,the change resulted in activation (quizalofop-ethyl). Visibleeffects on the root morphology were observed: dinitroanilinesand certain carbamates led to remarkable swelling of the roottips; norflurazon and diflufenican were effective bleachingagents in greening root cultures in Murashige and Skoog medium,whereas the presence of sucrose in the medium antagonized theeffect of triazine herbicides. Growth inhibition by sulphonylureascan be antagonized by addition of valine and leucine; asulaminhibition was antagonized by addition of folic acid but glyphosateinhibition was not significantly reversed by aromatic aminoacids. Bipyridinium and diphenyl ether herbicides, with certainexceptions, have rapid and devastating phytotoxic effects onroot growth. The phytotoxic effects of the herbicides on transformedroot growth is discussed with particular reference to theirmode of action in intact plants. Key words: Arobacterium rhizogenes, herbicides, root organ cultures     

Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf Weed, Kochia scoparia

Selection of kochia (Kochia scoparia) biotypes resistant to the sulfonylurea herbicide chlorsulfuron has occurred through the continued use of this herbicide in monoculture cereal-growing areas in the United States. The apparent sulfonylurea resistance observed in kochia was confirmed in greenhouse tests. Fresh and dry weight accumulation in the resistant kochia was 2- to >350-fold higher in the presence of four sulfonylurea herbicides as compared to the susceptible biotype. Acetolactate synthase (ALS) activity isolated from sulfonylurea-resistant kochia was less sensitive to inhibition by three classes of ALS-inhibiting herbicides, sulfonylureas, imidazolinones, and sulfonanilides. The decrease in ALS sensitivity to inhibition (as measured by the ratio of resistant I₅₀ to susceptible I₅₀) was 5- to 28-fold, 2- to 6-fold, and 20-fold for sulfonylurea herbicides, imidazolinone herbicides, and a sulfonanilide herbicide, respectively. No differences were observed in the ALS-specific activities or the rates of [¹⁴C]chlorsulfuron uptake, translocation, and metabolism between susceptible and resistant kochia biotypes. The K_m values for pyruvate using ALS from susceptible and resistant kochia were 2.13 and 1.74 mm, respectively. Based on these results, the mechanism of sulfonylurea resistance in this kochia biotype is due solely to a less sulfonylurea-sensitive ALS enzyme.     

Mode of action of Photosystem II herbicides studied by thermoluminescence

The mode of action of chemically different herbicides (ureas, pyridazinones, phenylcarbamates, triazines, hydroxyquinolines, hydroxybenzonitriles and dinitrophenols) on photosynthetic electron transport was investigated by measurements of oxygen evolution and thermoluminescence. Depending on the particular herbicide used the thermoluminescence band related to Q (the primary acceptor of Photosystem II) appears at +5, 0 or −14°C. It was shown that these three different peak positions can be ascribed to various redox states of Q, the shifts being due to the binding of herbicides to the chloroplast membrane. Both displacement experiments and additive inhibition of herbicide pairs measured by thermoluminescence and oxygen evolution suggested that the sites of action of these herbicides are on the same protein. However, herbicide treatment of trypsinized chloroplasts showed that there were three different binding sites on the same protein, in agreement with the classification of herbicides into three groups based on thermoluminescence measurements. Our results suggest that the primary and secondary acceptors of Photosystem II (Q and B, respectively) are in close proximity and form a common complex with the herbicide-binding protein within the chloroplast membrane.     

Mediation of Herbicide Effects by Hormone Interactions

Chemical manipulation of the phytohormone system involves the use of herbicides for weed control in modern crop production. In the latter case, only compounds interacting with the auxin system have gained practical importance. Auxin herbicides mimic the overdose effects of indole-3-acetic acid (IAA), the principal natural auxin in higher plants. With their ability to control, particularly, dicotyledonous weeds in cereal crops, the synthetic auxins have been among the most successful herbicides used in agriculture. A newly discovered sequential hormone interaction plays a decisive role in their mode of action. The induction of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase in ethylene biosynthesis is the primary target process, following auxin herbicide signalling. Although the exact molecular target site has yet to be identified, it appears likely to be at the level of auxin receptor(s) for perception or signalling, leading ultimately to species- and organ-specific de novo enzyme synthesis. In sensitive dicots, ethylene causes epinastic growth and tissue swelling. Ethylene also triggers the biosynthesis of abscisic acid (ABA), mainly through the stimulated cleavage of xanthophylls to xanthoxal, catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). ABA mediates stomatal closure which limits photosynthetic activity and biomass production, accompanied by an overproduction of reactive oxygen species. Growth inhibition, senescence and tissue decay are the consequences. Recent results suggest that ethylene-triggered ABA is not restricted to the action of auxin herbicides. It may function as a module in the signalling of a variety of stimuli leading to plant growth regulation. An additional phenomenon is caused by the auxin herbicide quinclorac which also controls grass weeds. Here, quinclorac induces the accumulation of phytotoxic levels of cyanide, a co-product of ethylene, which ultimately derives from herbicide-induced ACC synthase activity in the tissue. Phytotropins are a further group of hormone-related compounds which are used as herbicides. They inhibit polar auxin transport by interacting with a regulatory protein, the NPA-binding protein, of the auxin efflux carrier. This causes an abnormal accumulation of IAA and applied synthetic auxins in plant meristems. Growth inhibition, loss of tropic responses and, in combination with auxin herbicides, synergistic effects are the consequences.