The basis for the safening of clomazone by phorate insecticide in cotton and inhibitors of cytochrome P450s

Clomazone may be safely used in cotton to manage weeds when applied following treatments of the organophosphate insecticides phorate or disulfoton. The loss of chlorophyll and carotenoids with 6 days of 100 nM clomazone treatment of cotton seedlings was partially prevented with phorate in hydroponic solution in a rate-dependent manner. In a study to examine the timing of safening from a one-day clomazone (100 nM) treatment, maximum safening was achieved when phorate (50 μM) was applied the same day as clomazone. Phorate decreased metabolism of ¹⁴C-clomazone to polar metabolites in excised cotton shoots and shoots of intact cotton plants. Microsomal studies of corn shoots showed an NADPH-dependent/cytochrome P450 reaction was inhibited by phorate. Additional studies with corn microsomes, corn seedlings and cotton seedlings supported the basis of clomazone safening is the inhibition of toxic clomazone metabolism by P450 inhibitors.     

Comparative actions of clomazone on beta-carotene levels and growth in rice (Oryza sativa) and watergrasses (Echinochloa spp)

Seedlings of rice, early watergrass (thiobencarb-resistant and thiobencarb-susceptible biotypes, R and S, respectively), and late watergrass (thiobencarb-resistant and thiobencarb-susceptible biotypes, R and S, respectively) were hydroponically exposed to clomazone at concentrations ranging from 0.08 to 7.9 microM. Whole-plant growth (mg fresh wt) and beta-carotene concentrations (microg g(-1) fresh wt) were measured after a 7-day exposure period. For growth, the no observed effect concentrations (NOECs) were 7.9, 0.21, 0.21, 0.46 and 0.46 microM clomazone for rice, early watergrass (R), early watergrass (S), late watergrass (R) and late watergrass (S), respectively, while the concentrations causing 25% inhibition in response (IC25) were 5.6 (+/-1.6), 0.46 (+/-0.06), 0.42 (+/-0.08), 0.92 (+/-0.45) and 0.79 (+/-0.08) microM clomazone, respectively. Clomazone inhibits beta-carotene synthesis via inhibition of the non-mevalonate isoprenoid synthetic pathway. For assessment of clomazone effects, beta-carotene levels proved to be a more sensitive toxicological endpoint than growth. For rice, early watergrass (R), early watergrass (S), late watergrass (R) and late watergrass (S), the beta-carotene NOECs were 0.21, <0.08, <0.08, 0.08 and 0.46 microM clomazone respectively, while IC25 values were 0.42 (+/-0.26), 0.08 (+/-0.02), 0.08 (+/-0.02), 0.33 (+/-0.09) and 0.54 (+/-0.15) microM, respectively. No evidence was found that the thiobencarb-resistance mechanisms present in early and late watergrasses impart resistance to clomazone. Due to similar sensitivity between rice and late watergrass, use of clomazone in rice culture will require the use of a safening technique.     

稻稗对噁唑酰草胺的非靶标抗药性和对氰氟草酯的交互抗药性

为明确稻稗对噁唑酰草胺的非靶标抗药性和对氰氟草酯的交互抗药性水平,通过剂量反应试验测定辽宁新民(R_1)、黑龙江虎林(R_2)和黑龙江哈尔滨(R_3)3个稻稗种群对噁唑酰草胺的非靶标抗药性和对氰氟草酯的交互抗药性水平。结果显示,R_1、R_2、R_3在喷施细胞色素P450抑制剂杀草强后对噁唑酰草胺的抗性指数比分别为1.14、1.71、2.38,表明R_1对噁唑酰草胺的抗药性与非靶标P450的活性无关,R_2和R_3对噁唑酰草胺的抗药性可能是靶标与非靶标的共同作用。3个稻稗种群对氰氟草酯的抗性指数分别为25.73、12.79、10.29,表明3个稻稗种群都对氰氟草酯产生了交互抗药性。     

Effect of mixed function oxidase inhibitors on the toxicity of chlortoluron and isoproturon to wheat

Growth responses of wheat plants to combined treatments of four mixed function oxidase (MFO) inhibitors and chlortoluron were determined. Analysis of interactions showed that piperonyl-butoxide and especially ABT (1-aminobenzotriazole) increased the toxicity of chlortoluron. Metyrapone and 2,4-dichlorophenoxypropyne were phytotoxic and did not exert any clear interaction. ABT also increased the toxicity of isoproturon to wheat. Our results suggest that ABT Strongly inhibits the breakdown of chlortoluron and isoproturon in wheat. Since ABT is known to act as a suicide substrate for plant cytochrome P-450, wheat enzymes involved in the metabolism of these two herbicides are likely to belong to this class. It thus appears that compounds designed to inhibit plant cytochrome P-450 enzymes may interact with herbicide metabolism and are potential synergists of herbicide activity.     

Enhanced effect of thiobencarb on bispyribac-sodium control of Echinochloa phyllopogon (Stapf ) Koss. in California rice (Oryza sativa L.)

Bispyribac-sodium {sodium 2,6-bis[(4,6-dimethoxy-2-pyrimidinyl)oxy] benzoate} has recently been introduced to California where it effectively controls Echinochloa spp. in rice ( Oryza sativa L.). However, biotypes of early watergrass ( Echinochloa oryzoides (Ard.) Fritsch) and late watergrass ( E. phyllopogon (Stapf ) Koss.) have evolved resistance to this herbicide. In 2001 and 2002, greenhouse and field experiments evaluated interactions between thiobencarb { S -[(4-chlorophenyl) methyl] diethylcarbamothioate} and bispyribac-sodium on resistant (R) and susceptible (S) late watergrass in California rice. Synergism was assessed using Colby's test and regression analysis. In the greenhouse, thiobencarb at 4480 and 5333 g ai ha⁻¹ synergistically reduced bispyribac-sodium GR₅₀ values on the R and S biotypes by 50–70% without increasing toxicity to rice. Synergism was also observed on S late watergrass in the field when 10 g ai ha⁻¹ bispyribac-sodium was mixed with 1120–2240 g ai ha⁻¹ thiobencarb. These effects could be related to interactions between thiocarbamates and enzymes in Phase I reactions of herbicide metabolism. This synergism results in better control at lower rates allowing a reduction in weed control costs, the herbicide load on the environment and a lower selection pressure towards resistant weed biotypes.     

Quinclorac resistance: a concerted hormonal and enzymatic effort in Echinochloa phyllopogon

BACKGROUND: Quinclorac (3,7-dichloro-quinoline-carboxylic acid) is a selective herbicide widely used to control annual grasses and certain broadleaf weeds. Echinochloa phyllopogon (Stapf) Koss. is the most noxious grass weed in California rice fields and has evolved resistance to multiple herbicides with different modes of action. A quinclorac-resistant (R) E. phyllopogon biotype found in a Sacramento Valley rice field where quinclorac has never been applied was investigated. RESULTS: Resistant to susceptible (S) GR₅₀ (herbicide rate for 50% growth reduction) ratios ranged from 6 to 17. The cytochrome P450 inhibitor malathion (200 mg L⁻¹) caused R plants to become as quinclorac susceptible as S plants. Quinclorac rapidly (6 HAT) stimulated ethylene formation in S plants, but only marginally in R plants. Malathion pretreatment did not reduce ethylene formation by quinclorac-treated S and R plants. Activity of β-cyanoalanine synthase (β-CAS) in tissue extracts was 2-3-fold greater in R than in S plants, and incubation of shoot extracts with 1 mM malathion reduced β-CAS activity by 40% in both biotypes. CONCLUSION: Resistance to quinclorac in R E. phyllopogon involved at least two mechanisms: (a) insensitivity along the response pathway whereby quinclorac induces ethylene production; (b) enhanced β-CAS activity, which should enable greater HCN detoxification following quinclorac stimulation of ethylene biosynthesis. This unveils new resistance mechanisms for this multiple-resistant biotype widely spread throughout California rice fields. Copyright © 2011 Society of Chemical Industry     

Spontaneous ultraweak photon emission from rice (Oryza sativa L.) and paddy weeds treated with a sulfonylurea herbicide

All living organisms spontaneously generate ultraweak photon emissions, which originate from biochemical reactions in cells. Current research uses the ultraweak photon emissions from organisms as a novel tool to investigate the physiological states of plants. In this study, we found ultraweak photon emissions from leaf segments of rice and several paddy weed species treated with a sulfonylurea herbicide. There is a definite difference in photon emissions among plant species, and rice (Oryza sativa), barnyardgrass (Echinochloa crus-galli) and Cyperus serotinus showed extremely strong enhancement of photon emissions. Photon emissions from these three species treated with sulfonylurea herbicide were suppressed when the leaf segments were treated with the cytochrome P450 monooxygenase (P450) inhibitors, piperonyl butoxide and malathion. These results suggest that P450 inhibitors affect the ultraweak photon emissions from plants.     

Herbicide resistance in transgenic plants with mammalian P450 monooxygenase genes

Transgenic potato and rice plants were generated by the introduction of human P450 species, CYP1A1, CYP2B6, CYP2C9 and CYP2C19, which metabolized a number of herbicides, insecticides and industrial chemicals. The transgenic potato plant T1977 co-expressing CYP1A1, CYP2B6 and CYP2C19 genes showed remarkable cross-resistance to several herbicides with different structures and modes of action due to metabolism of these herbicides by the P450 species expressed. The transgenic rice plant 2C9-57R2 expressing CYP2C9 gene showed resistance to sulfonylureas, and the transgenic rice plant 2C19-12R1 expressing CYP2C19 gene showed cross-resistance to certain herbicides with different structures and modes of action. These transgenic plants appear to be useful for herbicide resistance as well as phytoremediation of environmental contaminants.     

Resistance to diclofop-methyl in two Lolium spp. populations from Italy: studies on the mechanism of resistance

The mechanisms of herbicide resistance were investigated in two diclofop-methyl-resistant Lolium spp. populations from central Italy, Roma '94 and Tuscania '97. These two populations were compared with two susceptible Italian populations (Vetralla '94, Tarquinia '97) and a resistant and a susceptible population from Australia, SLR31 and VLR1. The activity of acetyl Co-A carboxylase (ACCase) extracted from susceptible (S) or resistant (R) individuals from the Italian populations was inhibited by both aryloxyphenoxypropanoate (diclofop acid and fluazifop acid) and cyclohexanedione (sethoxydim) herbicides. Diclofop-methyl was rapidly de-esterified to diclofop acid at a similar rate in both R and S populations. In all populations, diclofop acid was subsequently degraded to other metabolites. The rate of degradation of diclofop acid was not significantly faster in R than in S populations; however, diclofop acid was degraded more completely in Roma '94 and Tuscania '97 compared with the S populations. Application of the mixed-function oxidase inhibitor 1-aminobenzotriazole (ABT) significantly enhanced diclofop-methyl toxicity towards both R populations, but not in S populations. However, enhanced herbicide metabolism does not completely account for the measured resistance level. A mechanism other than an altered ACCase and enhanced herbicide metabolism appears to be responsible for resistance to diclofop-methyl in Roma '94 and Tuscania '97.     

Mode of action of naphthalic anhydride as a safener for the herbicide AC 263222 in maize

In hydroponic experiments, seed-dressing with the herbicide safener 1,8-naphthalic anhydride (NA), significantly enhanced the tolerance of maize, (Zea mays L., cv. Monarque) to the imidazolinone herbicide, AC 263222, (2-[4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl]-5-methylnicotinic acid). Uptake, distribution and metabolism studies where [¹⁴C]AC 263222 was applied through the roots of hydroponically grown maize plants showed that NA treatment reduced the translocation of radiolabel from root to shoot tissue and accelerated the degradation of this herbicide to a hydroxylated metabolite. Reductions in the lipophilicity and, therefore, mobility of this compound following hydroxylation may account for NA-induced retention of radiolabel in the root system. Hydroxylation of AC 263222 suggested that NA may stimulate the activity of enzymes involved in oxidative herbicide metabolism, such as the cytochrome P₄₅₀ mono-oxygenases. In agreement with this theory, the cytochrome P₄₅₀ inhibitor, 1-aminobenzotriazole (ABT), synergized AC 263222 activity and inhibited its hyroxylation in vivo. NA seed-dressing enhanced the total cytochrome P₄₅₀ and b₅ content of microsomes prepared from etiolated maize shoots. Isolated microsomes catalyzed AC 263222 hydroxylation in vitro. This activity possessed the characteristics of a cytochrome P₄₅₀ mono-oxygenase, being NADPH-dependent and susceptible to inhibition by ABT. Activity was stimulated four-fold following NA seed treatment. Differential NA enhancement of AC 263222 hydroxylase and the cytochrome P₄₅₀-dependent cinnamic acid-4-hydroxylase (CA4H) activity, suggested that separate P₄₅₀ isozymes were responsible for each activity. These results indicate that the protective effects of NA result from enhancement of AC 263222 hydroxylation and concomitant reduction in herbicide translocation. This may be attributed to the stimulation of a microsomal cytochrome P₄₅₀ system. © 1998 SCI.     

The behavior of clomazone in the soil environment

BACKGROUND: Clomazone is a herbicide used to control broadleaf weeds and grasses. Clomazone use in agriculturally important crops and forests for weed control has increased and is a potential water contaminant given its high water solubility (1100 µg mL^?1). Soil sorption is an environmental fate parameter that may limit its movement to water systems. The authors used model rice and forest soils of California to test clomazone sorption affinity, capacity, desorption, interaction with soil organic matter and behavior with black carbon. RESULTS: Sorption of clomazone to the major organic matter fraction of soil, humic acid (HA) (K_d = 29–87 L kg^?1), was greater than to whole soils (K_d = 2.3–11 L kg^?1). Increased isotherm non‐linearity was observed for the whole soils (N = 0.831–0.893) when compared with the humic acids (N = 0.954–0.999). Desorption isotherm results showed hysteresis, which was greatest at the lowest solution concentration of 0.067 µg mL^?1 for all whole soils and HA extracts. Aliphatic carbon content appeared to contribute to increased isotherm linearity. CONCLUSION: The results indicate that clomazone does not sorb appreciably to sandy or clay soils. Its sorption affinity and capacity is greater in humic acid, and consequently clomazone has difficulty desorbing from soil organic matter. Sorption appears to follow processes explained by the dual‐mode model, the presence of fire residues (black carbon) and a recently proposed sorption mechanism. Copyright © 2009 Society of Chemical Industry     

Biotransformation of clomazone in rice (Oryza sativa) and early watergrass (Echinochloa oryzoides)

Rice (Oryza sativa), a relatively tolerant species, and early watergrass (Echinochloa oryzoides; EWG), a relatively susceptible species, were exposed to ¹⁴C-labeled clomazone to determine accumulation, biotransformation, and mass balance. On a total mass basis, rice absorbed more clomazone than EWG (p < 0.05), but on a nmol/g basis, there was no significant difference between the two species (p > 0.05). Rice contained more extractable ¹⁴C residues (7.7 ± 0.5 vs. 4.8 ± 0.5 nmol in rice vs. EWG, respectively; p < 0.5), but the concentration in EWG was significantly higher (4.2 ± 0.5 vs. 1.8 ± 0.1 nmol/g in EWG vs. rice, respectively; p < 0.01). More metabolized residue was measured in EWG compared to rice (84.1% vs. 67.9%; p < 0.01). Both species produced hydroxylated forms, β-d-glucoside conjugates, and several other unidentified polar metabolites, but EWG generally produced higher metabolite concentrations. The concentration of the suspected active metabolite, 5-ketoclomazone, was significantly higher in EWG vs. rice (21 ± 2 vs. 5.7 ± 0.5 pmol/g, respectively; p < 0.01). Differences in sensitivity to clomazone between rice and EWG appear to be due to differential metabolism, but in this case the more susceptible EWG qualitatively and quantitatively metabolized more clomazone than the more tolerant rice. This is consistent with the action of a metabolically activated herbicide. This metabolic difference could be exploited to develop herbicide safeners for use with clomazone.     

Propanil activity, uptake and metabolism in resistant Echinochloa spp. biotypes

Field resistance of Echinochloa spp. to propanil has been previously reported in Costa Rica, Colombia and Arkansas (USA). In this study, the mechanism of resistance was investigated in three resistant (R) and three susceptible (S) biotypes. The shoot fresh weight reduction in pot-grown plants from a post-emergence spray of propanil at 2.44 kg a.i. ha⁻¹ on biotypes R/S from Costa Rica, Colombia and Arkansas was 35/98%, 25/79% and 20/82% respectively. In vitro chlorophyll fluorescence data from leaf tissue incubated in propanil showed that photosynthesis was inhibited in all biotypes, indicating that the propanil-binding site and enzyme were not altered. After transfer to herbicide-free solution, photosynthesis recovered only in resistant biotypes, indicating that the mechanism of resistance was caused by enhanced metabolism of the herbicide. Simultaneous treatment with fenitrothion, an aryl acylamidase inhibitor, prevented the recovery of photosynthesis in leaf tissue in two resistant biotypes. In contrast, the cytochrome P450 mono-oxygenase inhibitor, 1-aminobenzotriazole, did not prevent recovery from propanil in leaf tissue. Application of ¹⁴C-propanil to the second leaf of intact Echinochloa plants showed that c . 90% of the radioactivity remained in the treated leaf for up to 72 h after application. No major differences in translocation between R and S biotype plants were found. TLC analysis of tissue extracts from the treated leaves showed substantially less radioactivity associated with propanil, present after 72 h in rice or in the three R biotypes, compared with S biotypes.     

Photolytic versus microbial degradation of clomazone in a flooded California rice field soil

BACKGROUND: Clomazone is a popular herbicide used on California rice fields and exhibits rapid anaerobic microbial degradation (t_1/2 = 7.9 days). To test the potential of direct and indirect photolytic degradation as a cofactor in the overall degradation rate, sacrificial time‐series microcosms were amended with water, non‐sterilized soil + water and sterilized soil + water. Clomazone was added to each microcosm, which was then exposed to natural and artificial sunlight over 35 days. Water and acetonitrile extracts were analyzed for clomazone and metabolites via LC/MS/MS. RESULTS: The calculated pseudo‐first‐order degradation rate constants (k) were k_water = 0–0.005 ± 0.003 day^?1, k_sterile = 0–0.005 ± 0.003 day^?1 and k_non?sterile = 0.010 ± 0.002–0.044 ± 0.007 day^?1, depending on light type. The formation of ring‐open clomazone, a microbial metabolite, correlated with clomazone degradation. Trace amounts of 5‐hydroxyclomazone (m/z = 256 → 125), aromatic hydroxyclomazone (m/z = 256 → 141) and an unknown product (m/z = 268 → 125) were observed. CONCLUSIONS: The photolytic degradation rate depends on both light type and the quality of the chromophores that induce indirect photolysis. Microbial degradation was found to be sensitive to temperature fluctuations. Overall, microbes are shown to be more detrimental to the environmental fate of clomazone than photolysis. Copyright © 2012 Society of Chemical Industry     

A molecular assay for the proactive detection of target site-based resistance to herbicides inhibiting acetolactate synthase in Alopecurus myosuroides

Acetolactate synthase (ALS) inhibitors are the most resistance‐prone herbicide group. Rapid resistance diagnosis is thus of importance for their optimal use. We formulate rules to use the derived cleaved amplified polymorphic sequence method to develop molecular tools detecting a change at a given codon, the nature of which is unknown. We applied them to Alopecurus myosuroides (black grass) to develop assays targeting ALS codons A122, P197, A205, W574 and S653 that are crucial for herbicide sensitivity. These assays detected W574L or P197T, or both substitutions, in most plants analysed from a field where ALS inhibitors failed after 3 years of use. Similar assays can easily be set up for any species. Given the rapidity of selection for resistance to ALS inhibitors, these assays should be very useful in proactive herbicide resistance diagnosis.     

Implications of delayed Echinochloa spp. germination and duration of competition for integrated weed management in water-seeded rice

Summary Co-ordinating herbicide applications with the suppressive ability of the crop has the potential to improve weed control and optimize herbicide use in water-seeded rice. However, the successful integration of herbicide applications and crop development depends on the timing and duration of competition between rice and weeds. The critical period of competition between rice and Echinochloa species was examined in field and glasshouse experiments from 1996 to 2000. In 1999 and 2000, Echinochloa species seeded 30 days after rice in field experiments did not survive and rice yields were not reduced when plots were kept weed-free for 30 days or longer. In a basin experiment conducted in 1998, E. phyllopogon seeded with the crop was unaffected by light competition alone but the relative importance of shading by the crop increased when E. phyllopogon was seeded after rice. Management strategies that delay the germination or growth of Echinochloa species might confer a competitive advantage to rice and reduce the need for herbicide applications. However, yields in the field experiments were reduced by at least 18% after only 30 days of competition in both years, suggesting that it may be difficult to integrate currently available herbicides with crop growth in water-seeded rice.     

Isolation and functional characterization in yeast of CYP72A18, a rice cytochrome P450 that catalyzes (ω-1)-hydroxylation of the herbicide pelargonic acid

Cytochrome P450 proteins play important roles in plant herbicide selectivity. Here, we demonstrate metabolism of the herbicide pelargonic acid by CYP72A18, a novel cytochrome P450 isolated from the rice Oryza sativa L. cv. Nipponbare. The CYP72A18 cDNA was cloned from rice and heterologously expressed in Saccharomyces cerevisiae AH22 cells from the alcohol dehydrogenase (ADH1) promoter. Microsomes isolated from recombinant yeast cells contained the CYP72A18, which was found to catalyze the (ω-1)-hydroxylation of the herbicide pelargonic acid. We also show that (ω-1)-hydroxypelargonic acid has reduced herbicide activity against rice seedlings. Based on these results, we suggest that CYP72A18 participates in the detoxification of the herbicide pelargonic acid in rice plants.     

Cross‐resistance patterns to acetyl coenzyme A carboxylase (ACCase) inhibitors associated with different ACCase mutations in Beckmannia syzigachne

Beckmannia syzigachne (American sloughgrass) is a competitive grass weed found in China. Fenoxaprop‐P‐ethyl is widely used for control of this species in China. Resistance to fenoxaprop‐P‐ethyl in B. syzigachne has been reported to be conferred by an isoleucine(Ile)‐1781‐leucine(Leu) substitution in the gene encoding the herbicide target, acetyl‐CoA carboxylase (ACCase). In this study, three mutations were detected by derived cleaved amplified polymorphic sequence (dCAPS) method in fenoxaprop‐P‐ethyl‐resistant B. syzigachne populations: Ile‐1781‐Leu in population JCWL‐R, Ile‐2041‐Asn in JCJT‐R and Gly‐2096‐Ala in JYJD‐R. The data indicated they were genetically homogeneous (homozygous mutant) at the ACCase locus. The use of cytochrome P450 inhibitors was shown to slightly reduce the GR₅₀ value of fenoxaprop‐P‐ethyl‐resistant populations, from which we inferred a combination of target‐site resistance (TSR) and non‐target‐site resistance (NTSR) was involved in fenoxaprop‐P‐ethyl‐resistance. We characterised the cross‐resistance patterns to ACCase inhibitors in B. syzigachne. The plants in the JCWL‐R population were highly resistant to all tested APPs (aryloxyphen‐oxypropionates), sethoxydim and pinoxaden, and moderately resistant to clethodim. The plants in the JCJT‐R population were highly resistant to fluazifop‐P‐butyl, clodinafop‐propargyl, cyhalofop‐butyl, metamifop and pinoxaden; moderately resistant to haloxyfop‐R‐methyl, quizalofop‐P‐ethyl and sethoxydim; and sensitive to clethodim. The plants in the JYJD‐R population were highly resistant to clodinafop‐propargyl, metamifop and pinoxaden; moderately resistant to haloxyfop‐R‐methyl, cyhalofop‐butyl, quizalofop‐P‐ethyl, fluazifop‐P‐butyl and sethoxydim; and sensitive to clethodim. If resistance to ACCase inhibitors is present in B. syzigachne populations in the field, then our results indicate that clethodim should be used. While we demonstrated the cross‐resistance patterns of TSR resulting from three mutations in B. syzigachne, we also demonstrated that NTSR plays a role in resistance, which will complicate weed management.     

Characterisation of Avena fatua populations with resistance to multiple herbicides

Avena fatua (wild oat) populations with resistance (R) to one or more herbicides have been described in numerous cropping systems worldwide. We previously reported that the R3 and R4 wild oat populations from Montana, USA, were resistant to four herbicides representing three different modes of action: tralkoxydim [acetyl‐CoA carboxylase (ACCase] inhibitor), imazamethabenz and flucarbazone [acetolactate synthase (ALS) inhibitors] and difenzoquat (growth inhibitor). We now quantify resistance levels of these populations to triallate [very long chain fatty acid (VLCFA) biosynthesis inhibitor], pinoxaden (ACCase inhibitor) and paraquat (photosystem I inhibitor). Glasshouse dose–response experiments showed that, compared with the means of two susceptible (S) populations, the R3 and R4 populations were 17.5‐ and 18.1‐fold more resistant to triallate, 3.6‐ and 3.7‐fold more resistant to pinoxaden, respectively, and 3.2‐fold (R3) more resistant to paraquat. Pre‐treatment of R plants with the cytochrome P450 inhibitor malathion partially reversed the resistance phenotype for flucarbazone (both populations), imazamethabenz (R4), difenzoquat (R4) and pinoxaden (R3), but not for tralkoxydim, fenoxaprop‐P‐ethyl or triallate. Target site point mutations known to confer resistance to ALS or ACCase inhibitors were not detected via DNA sequencing and allele‐specific PCR assays in R plants, suggesting the involvement of non‐target site resistance mechanism(s) for these herbicides. Together, our results complete the initial characterisation of wild oat populations that are resistant to seven (R3) or six (R4) herbicides from five or four mode of action families respectively.     

Mechanism of resistance to bensulfuron-methyl in Alisma plantago-aquatica biotypes from Portuguese rice paddy fields

Two Alisma plantago‐aquatica biotypes resistant to bensulfuron‐methyl were detected in rice paddy fields in Portugal’s Mondego (biotype T) and Tagus and Sorraia (biotype Q) River valleys. The fields had been treated with bensulfuron‐methyl‐based herbicide mixtures for 4–6 years. In order to characterize the resistant (R) biotypes, dose–response experiments, absorption and translocation assays, metabolism studies and acetolactate synthase (ALS) activity assays were performed. There were marked differences between R and susceptible (S) biotypes, with a resistance index (ED₅₀R/S) of 500 and 6.25 for biotypes Q and T respectively. Cross‐resistance to azimsulfuron, cinosulfuron and ethoxysulfuron, but not to metsulfuron‐methyl, imazethapyr, bentazone, propanil and MCPA was demonstrated. No differences in the absorption and translocation of ¹⁴C‐bensulfuron‐methyl were found between the biotypes studied. Maximum absorption attained 1.12, 2.02 and 2.56 nmol g⁻¹ dry weight after 96 h incubation with herbicide, for S, Q and T biotypes respectively. Most of the radioactivity taken up by the roots was translocated to shoots. Bensulfuron‐methyl metabolism in shoots was similar in all biotypes. The R biotypes displayed a higher level of ALS activity than the S biotype, both in the presence and absence of herbicide and the resistance indices (IC₅₀R/S) were 20 197 and 10 for biotypes Q and T respectively. These data confirm for the first time that resistance to bensulfuron‐methyl in A. plantago‐aquatica is target‐site‐based. In practice, to control target site R biotypes, it would be preferable to use mixtures of ALS inhibitors with herbicides with other modes of action.