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.     

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.     

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.     

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.     

Metabolism of chlorsulfuron by plants: Biological basis for selectivity of a new herbicide for cereals

A major factor responsible for the selectivity of chlorsulfuron [2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide] (formerly DPX-4189), as a postemergence herbicide for small grains is the ability of the crop plants to metabolize the herbicide. Chlorsulfuron is the active ingredient in Du Pont “Glean” weed killer. Tolerant plants such as wheat, oats, and barley rapidly metabolize chlorsulfuron to a polar, inactive product. This metabolite has been characterized as the O-glycoside of chlorsulfuron in which the phenyl ring has undergone hydroxylation followed by conjugation with a carbohydrate moiety. Sensitive broadleaf plants show little or no metabolism of chlorsulfuron.     

Metabolism of chlorsulfuron by tolerant broadleaves

The ability of flax and black nightshade to metabolize chlorsulfuron was studied to determine if metabolism contributes to tolerance and to identify any metabolites produced. Plant leaves were treated with [¹⁴C]chlorsulfuron for a 24-hr period. The metabolites were extracted, separated by HPLC, and characterized. Mass spectral analysis and independent synthesis confirmed a major metabolite (B-1) as 2-chloro-N-{[4-(hydroxymethyl)-6-methoxy-1,3,5-triazin-2-yl]amino-carbonyl}benzenesulfonamide. A second major metabolite (B) was determined to be a carbohydrate conjugate of B-1. Plants were more tolerant to B-1 applications than to chlorsulfuron. These results suggest that metabolism may be the basis of selectivity to chlorsulfuron for tolerant broadleaf plants as well as for grasses.     

Effects of the herbicide EPTC and the protectant DDCA on incorporation and distribution of [2-14C]acetate into major lipid fractions of maize cell suspension cultures

The rapid effects of the herbicide EPTC (S-ethyl dipropylthiocarbamate) and the protectant DDCA (N,N-diallyl-2,2-dichloroacetamide) on [2-¹⁴C]acetate incorporation into lipids of maize cell cultures were studied in order to determine whether they act at similar sites of lipid synthesis. DDCA, at 0.05 mM and 0.1 mM, increased the incorporation of [2-¹⁴C]acetate into neutral lipids of a total lipid extract within 2 h. It had very little effect on the major polar lipid constituents. DDCA altered neither the distribution of label within the major lipid classes, nor turnover of the major lipids within 2 h. EPTC (0.1 mM) inhibited overall uptake of [2-¹⁴C]acetate into both neutral and polar lipids by about 30% after a 2-h incubation. The major polar lipid affected was an unidentified glycolipid. In addition to reducing the quantity of lipids synthesized, EPTC changed the lipid profile, altering the distribution of label, mainly within the neutral lipid fraction. A crude membrane fraction from maize cells contained both polar lipids and some neutral lipids. DDCA stimulated [2-¹⁴C]acetate incorporation into different lipid species. EPTC inhibited incorporation of [2-¹⁴C]acetate into both neutral and polar membrane lipids but altered significantly only its distribution into neutral lipids. DDCA (0.1 mM) given together with EPTC (0.2 mM) partially counteracted the effect of EPTC within the neutral lipid fraction. It is suggested that DDCA has a rapid effect on lipid synthesis, but it is probably not sufficient to account for the entire mode of action of the protectant.     

The mode of action of chlorsulfuron: A new herbicide for cereals

Chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesul-fonamide) is the active ingredient in DuPont “Glean” Weed Killer (formerly DPX-4189), a new herbicide for weed control in small grains as well as other uses. Continuous growth measurements of chlorsulfuron-sensitive seedlings demonstrated that the herbicide inhibits growth within 2 hr of application and by 8 hr reduces growth by 80%. This reduction in growth was closely associated with an inhibition of plant cell division. No significant effects were observed on auxin-, cytokinin-, or gibberellin-induced cell expansion. Photosynthesis, respiration, RNA synthesis, and protein synthesis were also initially unaffected under conditions where plant cell division is strongly inhibited.     

Effect of pyridazinone herbicides on lipid metabolism in groundnut (Arachis hypogaea) leaves

The effect of five substituted pyridazinones (pyrazon, San 133-410H, San 9774, norflurazon, and San 6706) on lipid metabolism in groundnut (Arachis hypogaea) leaves was investigated under nonphotosynthetic conditions. In experiments with leaf disks, the uptake of [1-¹⁴C]acetate, [³²P]orthophosphate, and [³⁵S]sulfate was significantly inhibited by these herbicides and the magnitude of inhibition varied, depending on the substituents. When the incorporation of these precursors into lipids was measured and expressed as percentage of total uptake, no effect was observed in the case of [1-¹⁴C]acetate but there was significant inhibition in the incorporation of the other two precursors, suggesting that pyridazinones interfere with the metabolism of the phospholipids and the sulfolipid. None of these compounds affected the uptake of [methyl-¹⁴C]choline but all inhibited its incorporation into phosphatidylcholine indicating that phosphatidylcholine metabolism is vulnerable to pyridazinones. The fatty acid synthetase of isolated chloroplasts assayed in the absence of light was inhibited 20–50% by the pyridazinones at 0.1–0.5 mM concentrations. San 9774 showed the most potent inhibition. In addition, the pyridazinone herbicides significantly inhibited sn-glycerol-3-phosphate acyltransferase(s) in both chloroplast and microsomal fractions but showed no effect on phosphatidic acid phosphatase. The magnitude of inhibition of fatty acid synthetase and acyltransferase(s) is related to the nature of the substituent groups on the herbicide. Trifluorophenyl substitution at position 2 or amino substitution at position 5 of the pyridazinone molecule caused the maximum inhibitory effect.     

Uptake, translocation and fate of 2,4-D and chlorsulfuron in Silene vulgaris (Moench) Garcke

Experiments were conducted to examine the up take, translocation and metabolism by S. vulgaris of two distinctly different herbicides: 2,4-D, a phenoxyalkanoic acid with growth regulator activity to which this species exhibits complete tolerance, and chlorsulfuron, a sul-fonylurea to which S. vulgaris is highly sensitive. Despite their structural dissimilarities 2,4-D and chlorsulfuron was readily absorbed by S. vulgaris with 65 and 69%, respectively, of the applied dosage being absorbed within 72 hours after treatment. Approximately 35% of the 2,4-D and 10% of the chlorsulfuron label was translocated out of the treated leaf after 72 hours. Neither herbicide accumulated in the terminal bud. Seventy-two hours after treatment 63% of the recovered ¹⁴C remained as unaltered 2,4-D in S. vulgaris, while in tomato, a 2,4-D sensitive species, 65% of the recovered ¹⁴C remained as intact herbicide. In S. vulgaris approximately 86% of the radioactivity remained as intact chlorsulfuron 72 hours after treatment compared to 12% in the tolerant wheat. The tolerance of S. vulgaris to 2,4-D could not be accounted for by limited absorption, translocation nor metabolic degradation of the herbicide. The sensitivity of S. vulgaris to chlorsulfuron would appear to be related to the inability of this species to metabolize the herbicide molecule.     

Field persistence of chlorsulfuron and DPX-L5300 in relation to rotational crops

A pot bioassay procedure, based on root growth of pre-germinated maize was used to study residual phytotoxicity of chlorsulfuron and DPX-L5300 methyl-([4-methoxy-6-methyl-1,3,5-triazin-2-yl(methyl) carbamoyl]sulphamoyl)benzoate under field conditions. The results indicate that residual bioactivity of both herbicides, applied either pre-or post-emergence at 5, 10, 20 and 40 g a.i. ha^?1, was increased with increasing rate of application. Chlorsulfuron persisted longer than DPX-L5300, and both herbicides, when applied pre-emergence, persisted longer than when applied post-emergence. Pot bioassay did not detect any residues eight months after either application. Maize and sunflower, planted as rotational field crops eight months after pre-emergence application, were not injured by either herbicide. Also, these crops were not affected when planted four months after post-emergence application of any of the DPX-L5300 rates or 5 or 10 g a.i. ha^?1 of chlorsulfuron, but their fresh weight was significantly reduced where 20 or 40 g a.i. ha^?1 of chlorsulfuron were applied.     

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.     

The mode of action of chlorsulfuron: The lack of direct inhibition of plant DNA synthesis

Chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzene-sulfonamide), the active ingredient in DuPont Glean Weed Killer, has been proposed to act by inhibiting plant cell division. In order to further define the mode of action of this new herbicide, studies were made of the effects of chlorsulfuron on processes associated with plant DNA synthesis. No inhibitory effects were observed on DNA synthesis in isolated plant nuclei, and the enzymes DNA polymerase and thymidine kinase. Nucleoside precursors of DNA were not effective in lessening chlorsulfuron inhibition of thymidine incorporation into DNA of corn root tips. These results indicate that chlorsulfuron does not inhibit plant cell division by a direct inhibition of DNA synthesis.     

A root bioassay procedure for the determination of chlorsulfuron, diclofop acid and sethoxydim residues in soils

Measurement of the root lengths of pre-ger-minated oat seedlings (Avena sativa L. var. Sioux) grown in the dark in treated soils was used to assay residues of diclofop acid (2-[4-(2,4-dichloro-phenoxy)phenoxy]propionate) and sethoxydim (2-[1-(ethoxyimino)-butyl]-5-[2-(ethylthio)-propy]-3-hydroxy-2-cyclohexene-1-one). Similar measurements involving maize seedlings (Zea Mays L. var. Sunny Vee) were also used to determine residues of the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbony]benzenesulfonamide) in soils. The procedure appeared to be reproducible with residues of chlorsulfuron, diclofop acid and sethoxydim being detectable at amounts of 0.001, 0.2 and 0.05 μg g^?1 respectively.     

Study of the enhancement of herbicide activity and rainfastness by an organosilicone adjuvant utilizing radiolabelled herbicide and adjuvant

‘Sylgard® 309’ organosilicone surfactant is a very effective adjuvant for broadleaf weed control with a number of herbicides. It is also effective in providing rainfastness lo these post-emergence herbicide applications. To elucidate the basis for herbicide activity enhancement and rainfastness, the absorption of [¹⁴C]acifluorfen, [¹⁴C]bentazone and [¹⁴C]‘Sylgard 309’ were studied. Non-ionic surfactants and crop oil concentrates were used as adjuvants with [¹⁴C]acifluorfen and [¹⁴C]bentazone, respectively, for purposes of comparison. Maximum absorption of [¹⁴C]acifluorfen and [¹⁴C]bentazone was obtained within 15 min after herbicide application with the organosilicone, versus ≥ 24 h with the convenlional adjuvants. [¹⁴C]-Organosilicone absorption closely paralleled that of the [¹⁴C]-herbicides. The organosilicone appears to exert its action by increasing greatly herbicide absorption. The enhancement effect did not appear to be a function of reduced surface tension. Rainfastness appeared to be a result of greatly accelerated herbicide penetration through the leaf cuticle in the presence of the organosilicone.     

The action of herbicides on fatty acid biosynthesis and elongation in barley and cucumber

BACKGROUND: Herbicides that affect lipid metabolism have been used commercially for many years. Here, napropamide, diphenamid, dimethachlor and cafenstrole are compared; these have all been classified by the Herbicide Resistance Action Committee (HRAC) as K₃ herbicides and inhibitors of cell division and/or synthesis of very‐long‐chain fatty acids (VLCFAs). In addition, spiro‐decanedione A and pinoxaden dione are compared as inhibitors of lipid synthesis through inhibition of acetyl‐CoA carboxylase (ACCase). RESULTS: Whereas the chloracetamide dimethachlor and the carboxyamide cafenstrole potently inhibited VLCFA synthesis in both barley and cucumber, the acetamides napropamide and diphenamid which are also classified as K₃ herbicides and likewise the unclassified herbicide cinmethylin did not. The graminicide pinoxaden dione inhibited de novo fatty acid synthesis in barley, but not in cucumber, and correspondingly inhibited the plastid form of maize ACCase much more than the cytosolic form (IC₅₀ values of 0.1 and 17 µM ). By contrast, spiro‐decanedione A exhibited herbicidal effects not only on grasses but also on broad leaves, strongly inhibited maize cytosolic ACCase and inhibited synthesis of VLCFAs in cucumber. CONCLUSIONS: The acetamides napropamide and diphenamid, which do not inhibit VLCFA synthesis, should be classified separately from K₃ herbicides that do. Pinoxaden dione and spiro‐decanedione A represent new classes of chemicals acting on plant lipid synthesis. Copyright © 2010 Society of Chemical Industry     

Studies on the mechanism of action of cymoxanil in Phytophthora infestans

Colony growth and germ tube emergence of sporangia and encysted zoospores of Phytophthora infestans were highly sensitive to cymoxanil (ED₅₀ 0.5–1.5 μg/ml), whereas differentiation of sporangia and zoospore release were insensitive at concentrations up to 100 μg/ml. Treated sporangia did not show distorted germ tubes. Oxygen consumption for glucose oxidation by germinating sporangia and zoospore motility were not inhibited at concentrations up to 100 μg/ml. Cymoxanil hardly affected the uptake of radiolabeled precursors of DNA, RNA, and protein at concentrations up to 100 μg/ml. Incorporation of [¹⁴C]phenylalanine into protein was completely insensitive. RNA synthesis as measured by [³H]uridine incorporation was differentially inhibited in the various developmental stages of the fungus. Inhibition did not occur at differentiation of sporangia, whereas at cyst and sporangial germination and mycelial growth this process was inhibited 20–45% at a concentration of 100 μg cymoxanil/ml. Endogenous RNA polymerase activity of isolated nuclei was not inhibited by cymoxanil. DNA synthesis as measured by [methyl-³H]thymidine incorporation was inhibited 20–80% at the various stages of development at cymoxanil concentrations higher than 10 μg/ml. Metalaxyl, a specific inhibitor of ribosomal RNA synthesis, inhibited [³H]uridine incorporation 40–60% at all developmental stages. The data suggest that although DNA synthesis is affected more than RNA synthesis, inhibition of both biosynthetic processes is a secondary effect. The primary mode of action of cymoxanil thus remains unknown.     

Pesticide inhibition of growth and macromolecular synthesis in the cellular slime mold Dictyostelium discoideum

The effects of two pesticides, dieldrin and captan, upon the growth and macromolecular syntheses of the vegetative cells of Dictyostelium discoideum strain Ax-2 were investigated. Dieldrin at a concentration of 5 μg/ml inhibited growth as well as the synthesis of RNA, DNA, and protein, while as little as 1 μg/ml of captan produced the same effects. After a 1-hr exposure to either pesticide, all macromolecular syntheses ceased. Within a period of 5 to 10 hr the amoebae began to shrink, and eventually some lysis occurred. Lysis was most pronounced in cells incubated with captan. When the amoebae were grown in the presence of 5 μg/ml of either pesticide and then washed and resuspended in fresh medium, the effects on growth were annulled. No growth inhibition was observed when 0.05 M cysteine was added prior to the addition of 5 μg/ml of captan. Further experimentation to study possible degradation effects of these two synthetic pesticides upon RNA and protein molecules showed that breakdown of these macromolecules into TCA-soluble units did not occur. Preliminary studies have also shown that [2-¹⁴C]uracil and [¹⁴C]amino acids are taken up in their respective pools in the presence of captan or dieldrin.     

Biological Activity of Two Stereoisomers of the N-Thienyl Chloroacetamide Herbicide Dimethenamid

Due to the presence of an asymmetrically substituted C atom, dimethenamid [2-chloro-N-(2,4-dimethyl-3-thienyl)-N-(2-methoxy-1-methylethyl)acetamide], a recently introduced N-thienyl chloroacetamide herbicide, exists as two stereoisomers (S and R) having differing herbicidal activities as demonstrated with a selection of weeds and Lemna minor. The activity of the two isomers was investigated in greater detail with the green alga Scenedesmus acutus and compared to that of alachlor [2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide]. As with alachlor, the S isomer (5 μM ) strongly inhibited algal growth and fatty acid desaturation while the R isomer had no effect. In short-term experiments (up to 5·5 h), the S isomer and alachlor (100 μM ) inhibited [¹⁴C]acetate uptake and its incorporation into fatty acids in the same manner, while the R isomer did not. Incorporation of [¹⁴C]acetate into a non-lipid fraction of the algae was strongly inhibited by alachlor and the S isomer (100 μM ) and only slightly by the R isomer. A 50% inhibition of incorporation of [¹⁴C]oleic acid into the same non-lipid fraction was attained with less than 10^-7 M of the S isomer while 10^-5 M of the R form of dimethenamid achieved only a 40% inhibition. The same stereospecificity of the compound on growth, fatty acid desaturation, acetate uptake and oleic acid incorporation provides strong evidence that dimethenamid may act upon a primary, specific target in lipid metabolism. Furthermore, the comparable biological activities of dimethenamid and alachlor indicate that this target is common to both N-phenyl and N-thienyl chloroacetamide herbicides. © 1997 SCI.     

Mode of action of triarimol in Ustilago maydis

Triarimol (2 μg/ml) strongly inhibited multiplication of Ustilago maydis sporidia after one doubling, but growth continued and sporidia became abnormally large, branched and multicellular. Oxidation of glucose or acetate was not affected, and only slight limitations occurred in DNA, RNA and protein syntheses. The toxicant did not inhibit triglyceride synthesis but markedly increased the quantity and altered the quality of free fatty acids. Incorporation of [¹⁴C]acetate into ergosterol and an unidentified sterol was inhibited more than 90%, but incorporation into two other unidentified sterols was almost unaffected. Inhibition in the sterol biosynthetic pathway at a point preceeding ergosterol is regarded as a primary site of triarimol action in U. maydis.