Trifluralin effects on carrot callus tissue

The effect of trifluralin on the growth, oxygen uptake, and adenosine phosphates level of carrot (Daucus carota L.) callus tissue was determined over a period of 18 days after subculture. The herbicide (10^?4 and 10^?5M) reduced fresh weight gain significantly; the reduction was less with lower trifluralin concentrations. Dry weight accumulation was not inhibited until after Day 6 of the test period and thereafter was reduced by all concentrations tested. Oxygen consumption was inhibited by trifluralin (10^?4 and 10^?5M) throughout the test period. Concentrations of 10^?6.10^?7, and 10^?8M produced variable effects. The response profile of O₂ consumption in the presence of dinitrophenol was different from that of trifluralin. Analysis of adenosine phosphates level gave no clear response trend. Energy charge values of 0.7 to 0.85 were obtained for untreated tissue. Trifluralin had no effect on energy charge until Day 9 (10^?4M) and after Day 15 (all concentrations).     

Comparison of trifluralin,oryzalin, pronamide,propham, and colchicine treatments on microtubules

The mode of action of trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine), oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfanilamide), pronamide(N-(1,1-dimethylpropynyl) 3,5-dichlorobenzamide), and propham (isopropyl carbanilate) on purified microtubules from pig brains and on the ultrastructure of wheat (Triticum aestivum L. “Mediterranean,” C. I. 5303) and corn (Zea mays L. “yellow dent, U. S. 13”) roots was compared with that known for colchicine. Colchicine disrupts the in vivo cortical and spindle microtubules of root cells. Like colchicine, the herbicides trifluralin, oryzalin, and pronamide caused the loss of both cortical and spindle microtubules of root cells. The rate of microtubule disappearance depended on the type of herbicide and length of exposure of roots to the herbicide. Unlike colchicine, cortical microtubules were present in propham-treated roots but they were disoriented within the cell.In vitro polymerization studies with pig brain microtubules (Sus scrofa) showed that the herbicides failed to inhibit the assembly of purified microtubular protein into microtubules and that radioactively labeled herbicides did not bind to the microtubular protein. Colchicine inhibited the polymerization of microtubular protein and readily bound to the microtubular subunits. These results indicate that the mode of action of the herbicides is not similar to that of colchicine and that the loss of microtubules from root tip cells treated with trifluralin, oryzalin, and pronamide may be caused by these herbicides interfering with synthesis of microtubular protein or metabolism of endoplasmic reticulum membranes involved in microtubule assembly. The mode of action of propham appears to be on the microtubular organizing centers rather than on microtubules per se.     

Microbial dealkylation of trifluralin in pure culture

Mixed populations of soil microorganisms were enriched in the presence of trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) and 180 pure strains were subsequently isolated. About a third were able to liberate 1.5–6% ¹⁴CO₂ from 0.15 mM [propyl-1-¹⁴C]trifluralin after growing for 21 days on a complex medium. One strain, identified as a Candida sp., showed a ¹⁴CO₂ evolution of 11%. The amount of liberated ¹⁴CO₂ could not be enhanced by adding small concentrations (<3%) of solvents to the culture, by varying the concentration of trifluralin, or by varying the composition of the complex medium. The Candida sp. was unable to cleave the aromatic ring of trifluralin or to use trifluralin as a sole source of carbon or nitrogen. Only traces (< 1%) of dealkylated trifluralin were accumulated in the culture.     

Localizing the action of two thiadiazolyl herbicidal derivatives

Enzymatically isolated leaf cells from navy beans (Phaseolus vulgaris L., cv. “Tuscola”) were used to study the effect of buthidazole (3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2-imidazolidinone) and tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-N,N′-dimethylurea) on photosynthesis, protein, ribonucleic acid (RNA), and lipid synthesis. The incorporation of NaH¹⁴CO₃, [¹⁴C]leucine, [¹⁴C]uracil, and [¹⁴C]acetic acid as substrates for the respective metabolic process was measured. Time-course and concentration studies included incubation periods of 30, 60, and 120 min and concentrations of 0.1, 1, 10, and 100 μM of both herbicides. Photosynthesis was very sensitive to both buthidazole and tebuthiuron and was inhibited in 30 min by 0.1 μM concentrations. RNA and lipid syntheses were inhibited 50 and 87%, respectively, by buthidazole and 42 and 64%, respectively, by tebuthiuron after 120 min at 100 μM concentration. Protein synthesis was not affected by any herbicide at any concentration or any exposure time period. The inhibitory effects of buthidazole and tebuthiuron on RNA and lipid syntheses may be involved in the ultimate herbicidal action of these herbicidal chemicals.     

Influence of the herbicides hexazinone and chlorsulfuron on the metabolism of isolated soybean leaf cells

The effects of the herbicides hexazinone [3-cyclohexyl-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4(1H,3H)-dione] and chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide) on the metabolism of enzymatically isolated leaf cells from soybean [Glycine max (L.) Merr., cv. ‘Essex’] were examined. Photosynthesis, protein, ribonucleic acid (RNA), and lipid syntheses were assayed by the incorporation of specific radioactive substrates into the isolated soybean leaf cells. These specific substrates were NaH¹⁴CO₃, [¹⁴C]leucine, [¹⁴C]uracil, and [¹⁴C]acetate, respectively. Time-course and concentration studies included incubation periods of 30, 60, and 120 min and concentrations of 0.1, 1, 10, and 100 μM of both herbicides. Photosynthesis was the most sensitive and first metabolic process inhibited by hexazinone. RNA and lipid syntheses were also inhibited significantly by hexazinone whereas the effect of this herbicide on protein synthesis was less. The most sensitive and first metabolic process inhibited by chlorsulfuron was lipid synthesis. Photosynthesis, RNA, and protein syntheses were affected significantly only by the highest concentration of this herbicide and longest exposure. Although these two herbicides may exert their herbicidal action by affecting other plant metabolic processes not examined in this study, hexazinone appears to be a strong photosynthetic inhibitor, while the herbicidal action of chlorsulfuron appeared to be related to its effects on lipid synthesis.     

Physiological responses of corn (Zea mays) to AC 243,997 in combination with valine,leucine, and isoleucine

Various physiological processes were measured in corn after treatment with AC 243,997. Neutral sugar levels in leaves increased 39% over the control 24 hr after application of AC 243,997. Protein synthesis, measured by [¹⁴C]leucine and [¹⁴C]cystine incorporation, and lipid synthesis were not inhibited 24 hr after application of 150 μM of AC 243,997, while respiration and RNA synthesis were inhibited 32 and 15%, respectively. DNA synthesis was severely inhibited (70–90%) by 150 μM of the herbicide 24 hr after application. The inhibition of DNA synthesis by AC 243,997 did not begin until 5 to 7 hr after application. Although protein synthesis rates were apparently unaffected by AC 243,997, the level of the soluble proteins decreased 40% while free amino acid levels increased 32% 24 hr after application of the herbicide. An exogenous supply of valine, leucine, and isoleucine to corn prevented the inhibition of growth and reversed the inhibition of DNA synthesis caused by AC 243,997. All three amino acids at a concentration of 1 mM were needed to provide maximum protection. The results support the hypothesis that AC 243,997 kills plants by interfering with the biosynthesis of valine, leucine, and isoleucine.     

The biochemical and physiological effects and mode of action of AC 222,293 against Alopecurus myosuroides Huds. and Avena fatua L

AC 222,293 was found to exert its mode of action through inhibition of acetolactate synthase, the first enzyme in the biosynthetic pathway of the branched chain amino acids. In vitro AC 222,293 was found to be only a very weak inhibitor of the enzyme, in contrast to the potent inhibition by the free acid, suggesting that action of the herbicide in vivo is dependent on deesterification. Enzyme preparations from susceptible Avena fatua and Alopecurus myosuroides and tolerant wheat (cultivar Fenman) were all equally sensitive with an I₅₀ of approximately 5 · 10⁻⁷ M. Inhibition of treated plants is prevented when valine, leucine, and isoleucine are supplied via the nutrient solution. Prominent secondary effects are inhibition of DNA synthesis and cell division and the action of the herbicide is characterised by cessation of leaf growth.     

Distribution and degradation of dinitroaniline herbicides in an aquatic ecosystem

The accumulation potential of six, structurally related, dinitroaniline herbicides was investigated in an aquatic ecosystem. The herbicides investigated were trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine), profluralin [N-(cyclopropylmethyl)-α,α,α-trifluoro-2,6-dinitro-N-propyl-p-toluidine], dinitramine [N³,N³-diethyl-2,4-dinitro-6-(trifluoromethyl)-m-phenylenediamine], chlornidine [N,N-bis(2-chloroethyl)-2,6-dinitro-p-toluidine], fluchloralin [N-(2-chloroethyl)-2,6-dinitro-N-propyl-4-(trifluoromethyl)aniline], and butralin [4-(1,1-dimethylethyl)-N-(1-methylpropyl)-2,6-dinitrobenzenamine]. The herbicide (0.1 mg) plus 1 μCi of ¹⁴C-labeled herbicide was adsorbed on 100 g of soil (1 ppm), added to individual aquariums, and flooded with 4 liters of water. Algae, snails, and daphnia were added, and ¹⁴C in water was monitored for 30 days. Fish were added on Day 30, and all components were harvested 3 days later. Bioaccumulation ratios (concentration in organism/concentration in water) for fish depended on the amount of their exposure to sunlight: Aquariums held in the dark had higher ratios for fish (235–755) than did those exposed to sunlight (32–83). Bioaccumulation ratios in the dark for fish based on ¹⁴C from bound soil residues of butralin and profluralin were 76 and 119, respectively. Direct repeated applications of profluralin (without soil) at 4-day intervals resulted in a rapid increase, then a decrease in bioaccumulation ratios for Gambusia, but a continuous increase for catfish.     

Effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on photosynthesis and respiration of Nitella dactyl cells

Long-term experiments with dactyl cells of Nitella flexilis showed that the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) at a concentration of 1 × 10^?5M affected not only O₂ evolution in the light but also O₂ uptake in the dark. The inhibition of O₂ production was transitory, but dark respiration did not recover. DCMU induced the formation of giant mitochondria which disappeared before cell death. It was concluded that the algicidic effect of 1 × 10^?5M DCMU on N. flexilis, but not necessarily the elongation of mitochondria, was due to the inhibition of mitochondrial respiration and not of photosynthesis.     

Differential inhibition of potassium ion absorption by difenzoquat in wild oat and cereals

Over a concentration range of 5.0 × 10^?6?7.5 × 10^?4M, the selective herbicide difenzoquat (1,2-dimethyl-3,5-diphenyl-1H-pyrazolium) caused more pronounced inhibition of potassium ion (K⁺) absorption by excised seedling roots of susceptible wild oat (Avena fatua L.) compared to those of tolerant barley (Hordeum vulgare L. cv. Bonanza) or wheat (Triticum aestivum L. cv. Neepawa). At 2.5 × 10^?5M difenzoquat, the relative inhibition of K⁺ (⁸⁶Rb) absorption by wild oat root segments inceased from 30% with a 10-min uptake period to 75% with an uptake period of 90 min, whereas no inhibition at all was evident for wheat root segments even after a 90-min exposure to the herbicide. An ion efflux compartmental analysis procedure demonstrated that difenzoquat did not affect the passive permeability properties of the plasma membrane of wild oat root cells. The experimental findings indicated that difenzoquat interfered directly with the process of active ion transport across the plasma membrane of root cells.     

Effects of dimethazone (FMC 57020) on chloroplast development: 1. Ultrastructural effects in cowpea (Vigna unguiculata L.) primary leaves

The effects of dimethazone [FMC 57020; 2-(2-chlorophenyl)methyl-4,4-dimethyl-3-isoxalidinone] on the growth and ultrastructure of cowpea (Vigna unguiculata L.) were examined. Seeds were imbibed in 0.5 mM dimethazone for 1 day and grown for 4 to 5 subsequent days in darkness without the herbicide. The herbicide stunted etiolated hypocotyl growth and retarded greening under 150 μmol · m⁻² · sec⁻¹ white light. No effects of dimethazone on the in vivo absorption spectrum of the etiolated primary leaf was detected. The herbicide caused some reduction and disorganization of prothylakoids in etiplasts. After 3 hr of white light chlorophyll accumulation was greatly reduced in treated leaves and ultrastructural development of the chloroplasts of herbicide-treated tissues appeared to be retarded. Pronounced thylakoid disruption was noticed in some cells after 12 hr, was more common after 24 hr, and was found in all cells by 72 hr. Maximally affected plastids lacked thylakoids, had irregular envelopes, and contained numerous vesicles.     

Growth regulator inhibition of tobacco callus growth

The activity of two groups of growth regulators, substituted dinitroanilines and nitrophenylhydrazines, were evaluated in a tobacco (Nicotiana tabacum L. “X-73”) callus tissue bioassay. Molar concentrations required to inhibit fresh weight gain by 50% (I₅₀) was determined by using linear regression analysis on data obtained by testing a range of five concentrations of each chemical. All chemicals tested were inhibitory to callus tissue grown in the dark. Cell division seemed to be the primary activity inhibited. The most active of the dinitroaniline series was α,α,α-trifluoro-2,6-dinitro-N-ethyl-N-2′,6′-dichlorobenzyl-p-toluidine (I) (I₅₀ = 1.5 × 10^?10M). I and two other N-(o-halobenzyl) dinitroanilines were more active than α,α,α-trifluoro-2,6-dinitro-N-ethyl-N-2′-chloro-6′-fluorobenzyl-p-toluidine (IV), which is being developed commercially for suppression of axillary buds in tobacco. The two most active nitrophenylhydrazines tested were 1,1-dimethyl-2-(2′,6′-dinitro-3′-n-propylamino-α,α,α-trifluoro-p-tolyl)hydrazine (XVIII) and 3′,5′-dinitro-p-(2,2-diethylhydrazino)-N-methoxy-N-methylbenzamide (XIX) (I₅₀ values of 7.9 × 10^?9 and 9.3 × 10^?9M, respectively). Factors such as electronic distribution, steric hindrance, and lipid solubility were considered to influence the biological activity of the compounds tested.     

Cytological studies of dinitroaniline-resistant Eleusine

Ultrastructural studies of primary roots (goosegrass) from dinitroaniline-resistant (R) and susceptible (S) biotypes of Eleusine indica (L). Gaertnr. establish a possible cytological basis for trifluralin resistance. Although the S biotype has a normal ultrastructure when grown in water, exposure to trifluralin solutions (between 10⁻⁸ and 10⁻⁵M) for 24 h results in a swelling of the root tip, typical of herbicides that affect microtubule production. The loss of spindle microtubules in the S biotype results in a mitosis arrested at prometaphase and the loss of cortical microtubules results in the formation of isodiametric cells in the zone of elongation. Nuclear membranes reform around the chromosomes in arrested prometaphase, producing abnormal, polymorphic nuclei. The mitotic index is increased in the S biotype after trifluralin treatment because many of the cells are arrested in prometaphase. The root tips of R biotypes are not swollen by even 10⁻⁵M trifluralin treatment. Trifluralin does not markedly affect cell division in the R biotype nor are the mitotic irregularities noted in the S biotype after treatment. However, even when the R biotype is not exposed to trifluralin, the microtubules are less abundant than in the S biotype and frequently cell walls are oriented abnormally or are incompletely formed. The level of resistance exhibited by the R biotype, the apparent difference in microtubule number and function between the two biotypes, and the lack of effect on the microtubules at high trifluralin concentrations indicate a site-of-action mutation.     

Rapid effects of a thiocarbamate herbicide and its dichloroacetamide protectant on macromolecular syntheses and glutathione levels in maize cell cultures

The rapid effects of the thiocarbamate herbicide S-ethyl dipropyl thiocarbamate (EPTC) and the herbicide protectant N,N-diallyl-2,2-dichloroacetamide (DDCA) on macromolecular syntheses and glutathione (GSH) levels in maize cell cultures were studied to determine whether stimulation of GSH could be the primary mechanism of action of DDCA. EPTC (0.5 and 1 mM) reduced incorporation of radioactive precursors within 1 hr after treatment, and affected incorporation of [³H]acetate into lipids more than incorporation of [³H]adenosine into acid-precipitable nucleic acids, or [¹⁴C]protein hydrolysate into protein. [¹⁴C]EPTC was rapidly biotransformed within 8 hr by maize cell suspensions. Measureable decreases in GSH levels following treatment with 1 mM EPTC occurred after 15 hr. DDCA stimulated incorporation of [³H]acetate into lipids within 4 hr but did not affect incorporation of [¹⁴C]protein hydrolysate into protein or [³H]adenosine incorporation into nucleic acids. Measureable increases in GSH following DDCA treatment began after 12 hr. Treatment with EPTC and DDCA in combination inhibited incorporation of [³H]acetate into lipids less than EPTC given alone. Increases in GSH levels could be observed following pretreatments with glutathione precursors, but no protectant activity could be detected, in contrast to treatments with DDCA. It is suggested that DDCA has an initial rapid effect on lipid metabolism followed by a slower effect involving increases in cellular GSH.     

Interference of benomyl and methyl-2-benzimidazolecarbamate (MBC) with DNA metabolism and nuclear divisions in Fusarium oxysporum

Methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl) severely decreased DNA synthesis when applied at 3.5 × 10^?6M during the G1 phase of germinating conidia of Fusarium oxysporum; nuclear divisions were completely inhibited at a fungicide concentration of 10 × 10^?6M. The same concentration applied only after the S phase also completely inhibited the nuclear divisions. This dual interference of benomyl with DNA formation and mitosis might be related to a disturbed phosphorus metabolism.     

Herbicide and inhibitor effects on cell leakage in suspension cultures of tobacco (Nicotiana tabacum)

Effects of selected herbicides and respiratory inhibitors on leakage from tobacco (Nicotiana tabacum) cell suspension cultures were studied. Leakage was monitored by quantitation of fluorescein dye released from preloaded cells and by measuring conductivity changes in the suspension medium. The herbicides ioxynil, Barban, 2,4,5-T, MCPB, and PCP (10^?6 to 10^?4M) caused leakage of fluorescein dye and electrolytes within 2 hr of exposure to the herbicides. Potassium cyanide and 2,4-DNP caused appreciable leakage at the same concentrations. Similar responses were induced by anaerobiosis. Atrazine, metolachlor, paraquat, and nitrofen did not induce leakage when tested at concentrations of 10^?6 to 10^?4M.     

Absorption,translocation, and metabolism of ethalfluralin and trifluralin in Solanum spp

Seedlings of Solanum scabrum Mill. and Solanum ptycanthum Dun. were treated with [¹⁴C]ethalfluralin (N-ethyl-α,α,α-trifluoro-N-(methylallyl)-2,6-dinitro-p-toluidine) and [¹⁴C]trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) supplied in nutrient solution to determine the basis for differences in response by these two species to these two herbicides. Plants of S. scabrum absorbed more [¹⁴C]ethalfluralin and [¹⁴C]trifluralin than plants of S. ptycanthum. During the first 24 h, S. scabrum seedlings, but not S. ptycanthum seedlings absorbed more [¹⁴C]ethalfluralin than did plants treated with [¹⁴C]trifluralin. More [¹⁴C]ethalfluralin than [¹⁴C]trifluralin was found in the shoots of plants of both species. Seventy-two hours after treatment with [¹⁴C]herbicides, the conversion to water-soluble metabolites was greater for [¹⁴C]ethalfluralin than for [¹⁴C]trifluralin. In the shoots of plants from both species an average of nearly 55% of the ¹⁴C recovered was found in the water-soluble fraction following [¹⁴C]ethalfluralin treatment whereas an average of only 40% was found in the water-soluble fraction following [¹⁴C]trifluralin treatment.     

Comparative effects of three isoxazole-urea herbicidal derivatives on the metabolism of enzymatically isolated soybean leaf cells*

The effects of the herbicide isouron and of its plant degradation products designated as metabolite l {N-[5-(1,1-dimethylethyl)-3-isoxazolyl]-N-methylurea} and metabolite 2 {N-[5-(1,1-dimethylethyl)-3-isoxazolyl]-urea} on the metabolism of enzymatically isolated leaf cells of soybean [Glycine max (L.) Merr., cv. Essex] were compared under laboratory conditions. Photosynthesis, protein synthesis, ribonucleic acid synthesis, and lipid synthesis were assayed by the incorporation of NaH¹⁴CO₃, [¹⁴C]-leucine, [¹⁴C]-uracil, and [¹⁴C]-acetate, respectively, into the isolated cells. Time-course and concentration studies included incubation periods of 30, 60, and 120 min and concentrations of 0.1, 1, 10 and 100 μM of the three herbicides. The urea derivative of isouron (metabolite 2) was the least active of the three compounds. The activity of the mono-methylated derivative of isouron (metabolite 1) was comparable to that of isouron and the sensitivity of the four processes to both chemicals decreased in the order: photosynthesis > ribonucleic acid synthesis > lipid synthesis > protein synthesis. The concentration of isouron that caused a 50% inhibition of photosynthesis of the isolated soybean leaf cells was calculated at 0.51 μM. The effects of isouron and metabolite 1 on photosynthesis, lipid and RNA synthesis appeared to be independent of incubation lime as maximal inhibition occurred within 30 min. Inhibition of protein synthesis by both chemicals was time-dependent, increasing in magnitude with concomitant increases in incubation time.     

Localization of metabolic sites of action of herbicides

Time- and concentration-course studies were conducted to determine the effect of thirteen herbicides on photosynthesis, respiration, RNA synthesis, protein synthesis, and lipid synthesis using isolated single leaf cells. Each herbicide was from a different chemical class. Appropriate ¹⁴C-substrates and product purification procedures were used for each process prior to liquid scintillation counting. The most sensitive metabolic site of inhibition was photosynthesis for atrazine, bromacil, dichlobenil, monuron, and paraquat; RNA synthesis for dalapon and dinoseb; protein synthesis for chlorpropham; and lipid synthesis for CDAA, chloramben, 2,4-D, EPTC, and trifluralin. However, with several herbicides, one or more process was almost as sensitive as the one mentioned above. All herbicides inhibited more than one process, and the most sensitive site of inhibition may not be the same process that was inhibited the greatest at the maximum concentration and maximum exposure time used. Therefore, a concept of metabolic sites of action, rather than a primary site of action, appears to be more meaningful for herbicides.     

Effects of herbicidal carbamates on mitochondria and chloroplasts

At concentrations near 2 × 10^?4M, barban, chlorpropham, and phenmedipham are inhibitors of the electron transfer in potato and mung bean mitochondria. The inhibition seems to be localized in the flavoprotein region. It affects preferentially the exogenous NADH dehydrogenation, in potato mitochondria (I₅₀, 10^?4M). Succinate dehydrogenation is less inhibited. At noninhibiting concentrations, the studied carbamates cannot uncouple the oxidative phosphorylations. Photosynthesis is completely inhibited by 2.10^?7M phenmedipham, 5 × 10^?5M barban, and 2 × 10^?4M chlorpropham. The inhibition takes place at the PS II level. Moreover, barban and chlorpropham are uncouplers of the photophosphorylations for concentrations between 5 × 10^?5 and 5 × 10^?4M. The effects observed on mitochondrial respiration can also be found on respiration of Acer cultured cells. The effects on isolated chloroplast photosynthesis are also observed for slightly higher concentrations on cultured Chlorella and on pea and oat leaf fragments.