Genetic diversity of sulfonylurea-resistant and -susceptible Monochoria vaginalis populations in Japan

Monochoria vaginalis is one of the most serious weeds of rice fields in Asia. The species is predominantly selfing. To reveal the potential for multiple mutational events, outcrossing and gene flow in the sulfonylurea‐resistant (SU‐R) M. vaginalis populations, we investigated (i) if each SU‐R population was a single SU‐R biotype or a mixture of several SU‐R biotypes using restriction analysis or direct sequencing of acetolacatate synthase (ALS) genes and (ii) genetic diversity of SU‐R and ‐susceptible (S) populations using amplified fragment length polymorphism (AFLP) analysis. Nineteen or 20 individuals were sampled from four SU‐R and five SU‐S populations respectively. Amino acid substitutions conferring resistance in the SU‐R populations were Pro₁₉₇Ser in the ALS1 or ALS3, or Asp₃₇₆Glu in the ALS1 and each SU‐R population was composed of a single SU‐R biotype. In cluster analysis each SU‐R individual formed a cluster, whereas the individuals from a SU‐S population belonged to different clusters. Some SU‐R populations showed polymorphic AFLP loci. The results indicated that these SU‐R biotypes emerged from a single mutational event and any gene flow of SU‐R genes from adjacent populations did not occur. A low level of outcrossing and recombinations of SU‐R genes occurred within some SU‐R populations of M. vaginalis.     

抗苄嘧磺隆雨久花乙酰乳酸合成酶突变的研究

本研究采用cDNA末端快速扩增技术(RACE)结合RT-PCR方法克隆抗苄嘧磺隆雨久花生物型和敏感性雨久花生物型乙酰乳酸合成酶(ALS)基因cDNA序列,并对测序结果进行比对分析。结果表明:与敏感性的雨久花ALS相比,公主岭(GZL)抗性生物型中第197位脯氨酸突变为组氨酸,第556位亮氨酸突变为苯丙氨酸;柳河(LH)抗性生物型中第358位天冬酰胺突变为天冬氨酸;磐石市(PS)抗性生物型中第525位缬氨酸突变为异亮氨酸。分析表明,高度保守区Domain A的第197位氨基酸残基的突变可能是导致公主岭稻区雨久花产生抗药性的主要原因之一,而其他抗性生物型抗性产生的原因有待进一步研究。     

Seasonal changes in the germination of buried seeds of Monochoria vaginalis

This study investigates the seasonal variation of germination ability of buried seeds of Monochoria vaginalis (Burm.f.) Presl var. plantaginea Solms. The field-collected seeds were buried in a flooded or an upland field and then exhumed monthly. The exhumed seeds were germinated under four temperature regimes. The seeds exhumed from the flooded soil were dormant at the beginning of burial and proceeded into a conditional dormancy/non-dormancy/conditional dormancy cycle throughout the remaining period of the experiment. The seeds exhumed monthly from the non-flooded soil exhibited an annual dormant cycle, which is dormancy/conditional dormancy/non-dormancy/conditional dormancy/dormancy. At day and night temperatures of 25/20 °C, the exhumed seeds from both the flooded and the upland soil resembled each other in terms of seasonal variation of the germination percentage. In September and October, more seeds exhumed from upland soil failed to germinate under higher temperature than from flooded soil. Strictly avoiding exposure to light during seed exhuming and seed testing prevented the seeds from germinating. A short exposure of the exhumed seeds to light during preparation promoted dark germination when the seeds were at the non-dormant stage. The potential implications of our results for weed management strategies in rice production are discussed.     

Sulfonylurea herbicide-resistant Monochoria vaginalis in Korean rice culture

Nine Monochoria vaginalis Pres1 accessions from Chonnam province, Korea were tested for resistance to the sulfonylurea herbicide, imazosulfuron, in whole-plant response bioassay. All accessions were confirmed resistant (R) to imazosulfuron. The GR50 (imazosulfuron concentration that reduced shoot dry weight by 50%) values of R accessions were 1112-3172 (accession #9) times higher than that of the standard susceptible (S) accession. Accession #9 exhibited cross-resistance to other sulfonylurea herbicides, bensulfuron-methyl, cyclosulfamuron and pyrazosulfuron-ethyl, but not to the imidazolinone herbicides, imazapyr and imazaquin. The R biotype could be controlled by other herbicides with different modes of action, such as mefenacet and pyrazolate, applied to soil at recommended rates. Foliar-applied herbicides, 2,4-D and bentazone, also controlled both the R and S biotypes. Sulfonylurea-based mixtures, except ethoxysulfuron plus fentrazamide, did not control resistant M. vaginalis. Rice yield was reduced 70% by resistant M. vaginalis that escaped pyrazosulfuron-ethyl plus molinate, compared with hand weeding in direct-seeded rice culture. In contrast, rice yield was reduced 44% by resistant M. vaginalis that survived the pyrazosulfuron-ethyl plus molinate treatment, compared with pyrazolate plus butachlor in transplanted rice culture. In vitro acetolactate synthase (ALS) activity of the R biotype was 183, 35, 130 and 31 times more resistant to imazosulfuron, bensulfuron-methyl, cyclosulfamuron and pyrazosulfuron-ethyl, respectively, than the S biotype. Imidazolinone herbicides, imazapyr and imazaquin had similar effect on in vitro ALS activity of the R and S biotypes. The in vivo ALS activity of the R biotype was also less affected than the S biotype by the sulfonylurea herbicides imazosulfuron and pyrazosulfuron-ethyl. Results of in vitro and in vivo ALS assays indicate that the resistance mechanism of M. vaginalis to sulfonylurea herbicides may be due, in part, to an alteration in the target enzyme, ALS. Since the level of resistance in the enzyme assay was much lower than that in the whole-plant assay, other mechanisms of resistance, such as herbicide metabolism, may be involved.     

Occurrence of sulfonylurea resistance in Sagittaria trifolia,a basal monocot species,based on target‐site and non‐target‐site resistance

Sagittaria trifolia L. is one of the most serious weeds in paddy fields in Japan. Since the late 1990s, severe infestations of S. trifolia have occurred following applications of sulfonylurea herbicides in Akita prefecture. In this study, two accessions of S. trifolia, R1 and R2, were collected from paddy fields with severe infestations and their resistance profiles were determined in comparison to a susceptible accession, S1. R1 and R2 were highly resistant to bensulfuron‐methyl. R1 was also highly resistant to pyrazosulfuron‐ethyl, but R2 was susceptible. Relative to S1, R1 had an amino acid substitution at the Pro₁₉₇ residue of acetolactate synthase (ALS), a well‐known mutation that confers sulfonylurea resistance, suggesting that R1 has a target‐site‐based resistance (TSR) mechanism. The sequence of the ALS gene in R2 was identical to that in S1. A Southern blot analysis indicated that there was only one copy of the ALS gene in S1 and R2. These results suggest that R2 has a non‐target‐site‐based resistance (NTSR) mechanism. R2 was moderately resistant to imazosulfuron but susceptible to thifensulfuron‐methyl. R2 and S1 were susceptible to pretilachlor, benfuresate, MCPA‐ethyl and bentazon. The results reveal the occurrence of two sulfonylurea‐resistant biotypes of S. trifolia that show different mechanisms of cross‐resistance to sulfonylureas related to TSR in R1 and NTSR in R2.     

Biology and mechanisms of sulfonylurea resistance in Schoenoplectiella juncoides,a noxious sedge in the rice paddy fields of Japan

Schoenoplectiella juncoides is a noxious sedge weed in rice paddy fields that has evolved resistance to sulfonylurea (SU) herbicides. The molecular basis of resistance is amino acid substitutions at Pro₁₉₇, Trp₅₇₄ or Asp₃₇₆ in the acetolactate synthase (ALS) enzyme, which is the target of SUs. Schoenoplectiella juncoides has two ALS genes and resistant plants have point mutations that cause amino acid substitutions in either encoded protein. Single‐nucleotide substitutions at the codon for Pro₁₉₇ in the ALS genes can cause six types of amino acid substitutions and all of these substitutions have been found in both ALS genes among Japanese SU‐resistant biotypes. Whole‐plant herbicide responses differ among the amino acid substitution types. Furthermore, analyses of ALS activity in plant extracts show that the extracts’ responses to herbicides differ, depending on which ALS gene is mutated. The activity responses of the ALS extracts to the SU, imazosulfuron, showed double‐sigmoid curves with plateaus of ~30% inhibition for Pro₁₉₇ substitutions in ALS1 and ~70% for Pro₁₉₇ substitutions in ALS2. This indicates that ALS1 and ALS2 contribute to the responses with a proportion of 7:3. The double‐sigmoid curves can be reconstructed to show the responses of the resistant and susceptible enzymes separately by regression analysis. The resistance levels of the separate ALS1 or ALS2 mutated enzyme are highly correlated with the whole‐plant responses, with a relationship that the former is the square of the latter. This could provide a quantitative insight into the physiological basis of resistance.     

Mechanism of the rice hull‐induced germination of Monochoria vaginalis seeds in darkness

The seeds of Monochoria vaginalis require light to germinate. However, they are able to germinate in darkness in the presence of rice hulls. The mechanism of this phenomenon was investigated in this study, in which only the unsterilized seeds of Monochoria were induced to germinate by the presence of a rice hull extract. The rice hull extract was able to be replaced by either a mixture of amino acids and phosphate or a yeast extract. The culture media in which germination was observed always showed turbidity, indicating the propagation of bacteria, which were identified as Bacillus sp., Pedobacter sp., Pantoea sp. and Sphingomonas sp. Treatment with the individual bacterial species or combinations of these bacteria induced Monochoria seed germination when added to the media containing the rice hull extract. It was concluded that the rice hull extract accelerates the propagation of bacteria, which in turn digest the seed coat of Monochoria, facilitating its germination.     

Pollination of chasmogamous flowers and the effects of light and emergence time on chasmogamy and cleistogamy in Monochoria vaginalis

Monochoria vaginalis is one of the most serious weeds of rice fields in Asia. The species has both chasmogamous (CH) and cleistogamous (CL) flowers on individual plants. The objective of this study is to clarify the reproductive characteristics that affect the selfing rate of M. vaginalis. An emasculation experiment revealed that the CH flowers emasculated just after flower opening produced seeds. The seed production in the flowers emasculated just after flower opening was less than that of the flowers emasculated and hand‐pollinated with pollen from another plant. These results indicate that, although self‐pollination before anthesis occurs, cross‐pollination also can produce seeds. An experimental manipulation of light and the emergence time of the plants revealed that more than half of the flowers produced were CL flowers and the CH/CL ratio decreased with reduced light availability. The plants that emerged later produced as many CH flowers as those that emerged earlier. There was no significant difference between the CH and CL flowers in the seed number per fruit and the seed mass. These results suggest that M. vaginalis has reproductive characteristics that make the selfing rate high; however, outcrossing also can occur. Under favorable light intensity conditions, such as on the edges of paddy fields or in fallow fields, M. vaginalis will produce more CH flowers and will have a higher outcrossing rate than in shaded conditions.     

Characterization of acetolactate synthase from sulfonylurea herbicide‐resistant Schoenoplectus juncoides

Ten accessions of sulfonylurea‐resistant Schoenoplectus juncoides were collected from paddy fields in Japan. In order to characterize acetolactate synthase from sulfonylurea‐resistant S. juncoides, acetolactate synthase amino acid substitutions, whole‐plant growth inhibition and acetolactate synthase enzyme inhibition were examined. Schoenoplectus juncoides has two acetolactate synthase genes (ALS1 and ALS2). The sulfonylurea‐resistant accessions harbored amino acid substitutions at Pro197 or Trp574 in either ALS1 or ALS2 (the amino acid number is standardized to the Arabidopsis thaliana sequence). The whole plants of all the sulfonylurea‐resistant accessions showed resistance to imazosulfuron. The resistance level depended on the altered amino acid residues in acetolactate synthase. The acetolactate synthase enzyme that was partially purified from all the sulfonylurea‐resistant accessions was less sensitive to imazosulfuron, compared to the susceptible accession, suggesting that the resistance is related to the altered acetolactate synthase enzyme. In addition, the concentration–response inhibition of acetolactate synthase activity by imazosulfuron in the sulfonylurea‐resistant accessions was remarkably different with the presence of an amino acid substitution in either ALS1 or ALS2. Furthermore, the concentration–response inhibition of acetolactate synthase activity in the sulfonylurea‐resistant accessions with a P197S, P197T or W574L mutation showed a double‐sigmoid curve. The regression analysis of enzyme inhibition suggested that the abundance ratio of ALS1 to ALS2 enzymes was approximately 70:30%, with a range of ±15%. Taken together, these results suggest that the resistance of sulfonylurea‐resistant accessions of S. juncoides is related to altered acetolactate synthase in either ALS1 or ALS2, although the abundance of the altered acetolactate synthase in the plants is different among the sulfonylurea‐resistant accessions.     

Morphological observation on development of juvenile seedlings of Monochoria vaginalis establishing on a flooded paddy soil surface

The development of juvenile seedlings of Monochoria vaginalis (Burm.f.) Kunth var. vaginalis concerning its hypocotyl hairs was investigated morphologically at establishing on 1 mm flooded paddy soil surface and in distilled water. Prime germination of M. vaginalis seeds sown after breaking dormancy was found on a flooded paddy soil surface and in distilled water at 6 h after seeding (HAS). Elongation of cotyledons and hypocotyl hairs of juvenile seedlings appeared at 12 HAS on a paddy soil surface, while lengths of hypocotyl hairs were almost the same as longitudinal length of seeds and cotyledons formed arch on the soil surface at 18 HAS. Seminal roots began to emerge and seedlings adhered completely to the soil surface by hypocotyl hairs, which were longer in length than seminal roots at 24 HAS. At 36 HAS, seed chaffs completely separated from the soil surface, as cotyledons had elongated from standing up, and lengths of seminal roots became longer than those of hypocotyl hairs. Emergence of first leaves and crown roots was simultaneously observed at 48 HAS. Elongations of cotyledons, seminal roots and hypocotyl hairs were completely terminated at 96, 120 HAS and 120 HAS respectively, and the elongation of 2 to 4 crown roots was observed at 120 HAS. Almost half the number of hypocotyl hairs putrefied at 240 HAS. In distilled water, hypocotyl hairs elongated to their longest at 36 HAS earlier than those on a paddy soil surface. It was concluded that hypocotyl hairs supported a strongly physical mechanism in establishing M. vaginalis seedlings on a paddy soil surface.     

Cross‐resistance of Echinochloa species to acetolactate synthase inhibitor herbicides

This study was conducted to evaluate the cross‐resistance of acetolactate synthase (ALS) inhibitors with different chemistries, specifically azimsulfuron (sulfonylurea), penoxsulam (triazolopyrimidine sulfonanilide) and bispyribac‐sodium (pyrimidinyl thio benzoate), in Echinochloa oryzicola and Echinochloa crus‐galli that had been collected in South Korea and to investigate their herbicide resistance mechanism. Both Echinochloa spp. showed cross‐resistance to the ALS inhibitors belonging to the above three different chemistries. In a whole plant assay with herbicides alone, the resistant/susceptible ratios for azimsulfuron, penoxsulam and bispyribac‐sodium were 12.6, 28.1 and 1.9 in E. oryzicola and 21.1, 13.7 and 1.8 in E. crus‐galli, respectively. An in vitro ALS enzyme assay with herbicides showed that the I ₅₀‐values of the resistant accessions were approximately two‐to‐three times higher than the susceptible accessions, with no statistical difference, suggesting that the difference in ALS sensitivity cannot explain ALS inhibitor resistance in Echinochloa spp. for azimsulfuron, penoxsulam and bispyribac‐sodium. A whole plant assay with fenitrothion showed that the GR ₅₀‐values significantly decreased in both the resistant E. oryzicola and E. crus‐galli accessions when azimsulfuron, penoxsulam and bispyribac‐sodium were applied with the P450 inhibitor, while no significant decrease was observed in the susceptible accessions when the P450 inhibitor was used. Thus, these results suggest that ALS inhibitor cross‐resistance for azimsulfuron, penoxsulam and bispyribac‐sodium is related to enhanced herbicide metabolism.     

Selection for resistance to the herbicide imazethapyr in somaclones of soyabean

Cultivars of soyabean [ Glycine max (L.) Merr.] resistant to the herbicide imazethapyr were identified by suspending the roots of 5-day-old seedlings in nutrient culture containing 2.5 mg a.i. L^–1 imazethapyr and then comparing the inhibitory effect on root length and shoot dry weight. The four most resistant cultivars were subsequently screened as regenerating tissue cultures in a medium containing 2.0 mg a.i. L^–l imazethapyr to select somaclonal cells with increased resistance. Surviving portions of cultures were regenerated to give shoots, the plants isolated, allowed to flower and seed. These progeny were then used for further seed multiplication and seedlings from this latter generation were exposed to imazethapyr in vivo and callus and cell suspension cultures derived from these seedlings were exposed to imazethapyr in vitro . A reduction in the inhibitory effect of the imazethapyr was noted in the somaclone seedlings and tissue cultures. However, measurement of acetolactate synthase (ALS) activity showed no differences among the parent cultivars and in the selected somaclones in this trait.     

Genetic diversity of the annual weed Monochoria vaginalis in southern China detected by random amplified polymorphic DNA and inter-simple sequence repeat analyses

Two molecular genetic screening techniques, RAPD (random amplified polymorphic DNAs) and ISSR (inter-simple sequence repeats), were applied to detect the level and pattern of genetic diversity of Monochoria vaginalis, a common weed of rice fields, in seven populations from southern China. Among these populations, 116 bands were amplified by 18 RAPD primers, of which 34 bands (29.31%) were polymorphic, and 14 ISSR primers produced 111 bands with 87 polymorphic bands (78.38%). Within each population, a relatively low level of genetic diversity was detected by both RAPD and ISSR analyses, with a mean genetic diversity (H) of 0.0348 and 0.0551 respectively. Analysis of molecular variance of the data from the RAPD and ISSR markers detected that the majority of total genetic variation existed among populations (73.50% and 76.70% respectively) and only minor genetic variation within populations (26.50% and 23.30% respectively). Cluster analysis divided the seven populations into two groups, indicating that the genetic relationships among populations have relatively low correlation with their geographical distribution (Mantel test; r = 0.45 and 0.48 respectively). Our results indicated that both RAPD and ISSR markers were effective and reliable for accurately assessing the degree of genetic variation of M. vaginalis. Comparing the two techniques, ISSR markers were more efficient than the RAPD assay. The Mantel test gave r = 0.16, suggesting no correlation between these two molecular markers.     

Molecular basis of diverse responses to acetolactate synthase-inhibiting herbicides in sulfonylurea-resistant biotypes of Schoenoplectus juncoides

Sulfonylurea-resistant biotypes of Schoenoplectus juncoides were collected from Nakafurano, Shiwa, Matsuyama, and Yurihonjyo in Japan. All of the four biotypes showed resistance to bensulfuron-methyl and thifensulfuron-methyl in whole-plant experiments. The growth of the Nakafurano, Shiwa, and Matsuyama biotypes was inhibited by imazaquin-ammonium and bispyribac-sodium, whereas the Yurihonjyo biotype grew normally after treatment with these herbicides. The herbicide concentration required to inhibit the acetolactate synthase (ALS) enzyme by 50% (I₅₀), obtained using in vivo ALS assays, indicated that the four biotypes were > 10-fold more resistant to thifensulfuron-methyl than a susceptible biotype. The Nakafurano, Shiwa, and Matsuyama biotypes exhibited no or little resistance to imazaquin-ammonium, whereas the Yurihonjyo biotype exhibited 6700-fold resistance to the herbicide. The Nakafurano and Shiwa biotypes exhibited no resistance to bispyribac-sodium, but the Matsuyama biotype exhibited 21-fold resistance and the Yurihonjyo biotype exhibited 260-fold resistance to the herbicide. Two S. juncoides ALS genes (ALS1 and ALS2) were isolated and each was found to contain one intron and to encode an ALS protein of 645 amino acids. Sequencing of the ALS genes revealed an amino acid substitution at Pro₁₉₇ in either encoded protein (ALS1 or ALS2) in the biotypes from Nakafurano (Pro₁₉₇ → Ser₁₉₇), Shiwa (Pro₁₉₇ → His₁₉₇), and Matsuyama (Pro₁₉₇ → Leu₁₉₇). The ALS2 of the biotype from Yurihonjyo was found to contain a Trp₅₇₄ → Leu₅₇₄ substitution. The relationships between the responses to ALS-inhibiting herbicides and the amino acid substitutions, which are consistent with previous reports in other plants, indicate that the substitutions at Pro₁₉₇ and Trp₅₇₄ are the basis of the resistance to sulfonylureas in these S. juncoides biotypes.     

Molecular characterisation of resistance to ALS-inhibiting herbicides in Hordeum leporinum biotypes

BACKGROUND: The acetolactate synthase (ALS)-inhibiting herbicide sulfosulfuron is registered in Australia for the selective control of Hordeum leporinum Link. in wheat crops. This herbicide failed to control H. leporinum on two farms in Western Australia on its first use. This study aimed to determine the level of resistance of three H. leporinum biotypes, identify the biochemical and molecular basis and develop molecular markers for diagnostic analysis of the resistance. RESULTS: Dose-response studies revealed very high level (>340-fold) resistance to the sulfonylurea herbicides sulfosulfuron and sulfometuron. In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to herbicide inhibition. This altered ALS sensitivity in the resistant biotypes was found to be due to a mutation in the ALS gene resulting in amino acid proline to serine substitution at position 197. In addition, two- to threefold higher ALS activities were consistently found in the resistant biotypes, compared with the known susceptible biotype. Two cleaved amplified polymorphic sequence (CAPS) markers were developed for diagnostic testing of the resistant populations. CONCLUSION: This study established the first documented case of evolved ALS inhibitor resistance in H. leporinum and revealed that the molecular basis of resistance is due to a Pro to Ser mutation in the ALS gene.     

Evaluation of the suppression ability of rice (Oryza sativa) on Monochoria vaginalis by measuring photosynthetic photon flux density below rice canopy

Field experiments involving eight cultivars were conducted in 1998 and 16 cultivars in 1999 to study the ability of rice (Oryza sativa L.) to suppress Monochoria vaginalis (Burm. f ) Kunth through light competition. Dry weights of M. vaginalis shoots in early season culture exceeded those in normal season culture of any rice cultivars. The relative photosynthetic photon flux density (R‐PPFD), which was calculated as the ratio of the photosynthetic photon flux density (PPFD) below the rice canopy to that measured above the rice canopy, varied according to rice cultivar. A strong linear correlation was observed between the mean R‐PPFD at 29–35 days after transplanting (DAT) (r² = 0.80; p < 0.01 in 1998; r² = 0.63, p < 0.001; and r² = 0.93, p < 0.001 in 1999), or 36–42 DAT (r² = 0.66, p < 0.05 in 1998; r² = 0.72, p < 0.001; and r² = 0.97, p < 0.001 in 1999), and the dry weight of M. vaginalis shoots at approximately 60 DAT. Data from the three experiments could be pooled into one regression line because intercepts and regression coefficients were not significantly different. The r² values of the combined regression were highest when R‐PPFD was expressed as the mean of measurements taken during 14 days (from 29 to 42 DAT; r² = 0.81, p < 0.001). The shortest period for measuring mean R‐PPFD in order to obtain a meaningful relationship with M. vaginalis shoot dry weight was 7 days (from 29 to 35 DAT; r² = 0.78, p < 0.001). For that same period, relationships between M. vaginalis shoot dry weight at 60 DAT and rice tiller number or leaf area index (LAI) at ground level were weak. However, there were negative relationships between M. vaginalis shoot dry weights at 60 DAT and rice LAI measured 20 cm above the ground, plant heights or rice shoot dry weight, but these coefficients of determination were smaller than those calculated by R‐PPFD for the same period. Thus, the ability of rice to suppress M. vaginalis can be evaluated more accurately by measuring mean R‐PPFD below the rice canopy for 7 days (from 29 to 35 DAT) than by measuring rice LAI, plant height and shoot dry weight.     

杂草对乙酰乳酸合成酶抑制剂抗药性研究进展

乙酰乳酸合成酶(ALS)抑制剂类除草剂已经成为一类广泛使用的除草剂。综述了杂草对ALS抑制剂类除草剂抗药性的产生与发展、抗性机理、抗性基因应用等方面的研究进展。其抗性产生机理主要有杂草对除草剂代谢能力增强、ALS基因突变导致对除草剂敏感性降低和ALS含量提高等。     

Conyza albida: a new biotype with ALS inhibitor resistance

Summary A biotype of Conyza albida resistant to imazapyr was discovered on a farm in the province of Seville, Spain, on land that had been continuously treated with this herbicide. This is the first reported occurrence of target site resistance to acetolactate synthase (ALS)-inhibiting herbicides in C. albida . In order to characterize this resistant biotype, dose–response experiments, absorption and translocation assays, metabolism studies, ALS activity assays and control with alternative herbicides were performed. Dose–response experiments revealed a marked difference between resistant (R) and susceptible (S) biotypes with a resistance factor [ED₅₀(R)/ED₅₀(S)] of 300. Cross-resistance existed with amidosulfuron, imazethapyr and nicosulfuron. Control of both biotypes using alternative herbicides was good using chlorsulfuron, triasulfuron, diuron, simazine, glyphosate and glufosinate. The rest of the herbicides tested did not provide good control for either biotype. There were no differences in absorption and translocation between the two biotypes, the maximum absorption reached about 15%, and most of the radioactivity taken up remained in the treated leaf. The metabolism pattern was similar and revealed that both biotypes may form polar metabolites with similar retention time (R_f). The effect of several ALS inhibitors on ALS (target site) activity measured in leaf extracts from both biotypes was investigated. Only with imazapyr and imazethapyr did the R biotype show a higher level of resistance than the S biotype [I₅₀ (R)/I₅₀(S) value of 4.0 and 3.7 respectively]. These data suggest that the resistance to imazapyr found in the R biotype of C. albida results primarily from an altered target site.     

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.