Resistance to Acetolactate Synthase-Inhibiting Herbicides in Annual Ryegrass (Lolium rigidum) Involves at Least Two Mechanisms

WLR1, a biotype of Lolium rigidum Gaud. that had been treated with the sulfonylurea herbicide chlorsulfuron in 7 consecutive years, was found to be resistant to both the wheat-selective and the nonselective sulfonylurea and imidazolinone herbicides. Biotype SLR31, which became cross-resistant to chlorsulfuron following treatment with the aryloxyphenoxypropionate herbicide diclofop-methyl, was resistant to the wheat-selective, but not the nonselective, sulfonylurea and imidazolinone herbicides. The concentrations of herbicide required to reduce in vitro acetolactate synthase (ALs) activity 50% with respect to control assays minus herbicide for biotype WLR1 was greater than those for susceptible biotype VLR1 by a factor of >30, >30, 7,4, and 2 for the herbicides chlorsulfuron, sulfometuron-methyl, imazapyr, imazathapyr, and imazamethabenz, respectively. ALS activity from biotype SLR31 responded in a similar manner to that of the susceptible biotype VLR1. The resistant biotypes metabolized chlorsulfuron more rapidly than the susceptible biotype. Metabolism of 50% of [phenyl-U-¹⁴C]chlorsulfuron in the culms of two-leaf seedlings required 3.7 h in biotype SLR31, 5.1 h in biotype WLR1, and 7.1 h in biotype VLR1. In all biotypes the metabolism of chlorsulfuron in the culms was more rapid than that in the leaf lamina. Resistance to ALS inhibitors in L. rigidum may involve at least two mechanisms, increased metabolism of the herbicide and/or a herbicide-insensitive ALS.     

Resistant Acetyl-CoA Carboxylase is a Mechanism of Herbicide Resistance in a Biotype of Avena sterilis ssp. ludoviciana

A biotype of Avena sterilis ssp. ludoviciana is highly resistantto a range of herbicides which inhibit a key enzyme in fattyacid synthesis, acetyl-CoA carboxylase (ACCase). Possible mechanismsof herbicide resistance were investigated in this biotype. Acetyl-CoAcarboxylase from the resistant biotype is less sensitive toinhibition by herbicides to which resistance is expressed. I₅₀values for herbicide inhibition of ACCase were 52 to 6 timesgreater in the resistant biotype than in the susceptible biotype.This was the only major difference found between the resistantand susceptible biotypes. The amount of ACCase in the meristemsof the resistant and susceptible is similar during ontogenyand no difference was found in distribution of ACCase betweenthe two biotypes. Uptake, translocation and metabolism of [¹⁴C]diclofop-methylwere not different between the two biotypes. In vivo, ACCaseactivity in the meristems of the susceptible biotype was greatlyinhibited by herbicide application whereas only 25% inhibitionoccurred in the resistant biotype. Depolarisation of plasmamembrane potential by 50 µM diclofop acid was observedin both biotypes and neither biotype showed recovery of themembrane potential following removal of the herbicide. Hence,a modified form of ACCase appears to be the major determinantof resistance in this resistant wild oat biotype. (Received February 10, 1994; Accepted March 11, 1994)     

Resistance mechanism to acetyl coenzyme A carboxylase inhibiting herbicides in Phalaris paradoxa collected in Mexican wheat fields

Background and aims

In this study, we describe the molecular, physiological and agronomic aspects involved in the resistance to acetyl coenzyme A carboxylase inhibiting herbicides (ACCase) observed in one biotype of Phalaris paradoxa from Mexico.

Methods

Dose–response Assays: The herbicide rate inhibiting plant growth of each biotype by 50% with respect to the untreated control, ED₅₀. Enzyme purification and ACCase assays to determine herbicide rate inhibiting the enzyme of each biotype by 50% with respect to the untreated control, I₅₀. Absorption and Translocation Assays with [¹⁴C]diclofop-methyl. Metabolism of diclofop-methyl and its metabolites were identified by thin-layer chromatography. Study of target site resistance mechanism at enzyme and molecular levels.

Results

In this work, it has been studied the whole-plant response of Phalaris paradoxa biotypes from Mexico resistant (R) and susceptible (S) to ACCase-inhibiting herbicides: aryloxyphenoxypropionate (APP), cyclohexanedione (CHD) and phenylpyrazoline (PPZ), and the mechanism behind their resistance were studied. To analyse the resistance mechanism, the enzyme ACCase activity was investigated. Results from biochemical assays indicated a target-site resistance as the cause of reduced susceptibility to ACCase inhibitors. The absorption, translocation and metabolism were similar between R and S biotypes. A point mutation never described before was detected within the triplet of glycine at the amino acid position 2096 (referring to EMBL accession no. AJ310767) and resulted in the triplet of serine. This new mutation could explain the loss of affinity for the ACCase-inhibiting herbicides.

Conclusions

We found a new mutation, which had never been described before. This mutation was detected within the triplet of glycine at the amino acid position 2096. This new mutation confers cross-resistance to three different chemical groups of ACCase-inhibiting herbicides.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum) : II. Chlorsulfuron Resistance Involves a Wheat-Like Detoxification System

Lolium rigidum Gaud. biotype SLR31 is resistant to the herbicide diclofop-methyl and cross-resistant to several sulfonylurea herbicides. Wheat and the cross-resistant ryegrass exhibit similar patterns of resistance to sulfonylurea herbicides, suggesting that the mechanism of resistance may be similar. Cross-resistant ryegrass is also resistant to the wheat-selective imidazolinone herbicide imazamethabenz. The cross-resistant biotype SLR31 metabolized [phenyl-U-¹⁴C]chlorsulfuron at a faster rate than a biotype which is susceptible to both diclofop-methyl and chlorsulfuron. A third biotype which is resistant to diclofop-methyl but not to chlorsulfuron metabolized chlorsulfuron at the same rate as the susceptible biotype. The increased metabolism of chlorsulfuron observed in the cross-resistant biotype is, therefore, correlated with the patterns of resistance observed in these L. rigidum biotypes. During high performance liquid chromatography analysis the major metabolite of chlorsulfuron in both susceptible and cross-resistant ryegrass coeluted with the major metabolite produced in wheat. The major product is clearly different from the major product in the tolerant dicot species, flax (Linium usitatissimum). The elution pattern of metabolites of chlorsulfuron was the same for both the susceptible and cross-resistant ryegrass but the cross-resistant ryegrass metabolized chlorsulfuron more rapidly. The investigation of the dose response to sulfonylurea herbicides at the whole plant level and the study of the metabolism of chlorsulfuron provide two independent sets of data which both suggest that the resistance to chlorsulfuron in cross-resistant ryegrass biotype SLR31 involves a wheat-like detoxification system.     

A mechanism of chlorotoluron resistance in Lolium rigidum

Many biotypes of Lolium rigidum Gaud, (annual ryegrass) have developed resistance to herbicides; however, few have developed resistance to phenylurea herbicides. Two biotypes with different histories of herbicide selection pressure were six to eight times less sensitive to the phenylurea herbicide, chlorotoluron, than a susceptible biotype. Resistance was not due to differences in the herbicide target site as oxygen evolution by thylakoids isolated from resistant and susceptible biotypes was similarly inhibited by diuron and chlorotoluron. There was no difference in the uptake and distribution of chlorotoluron into resistant and susceptible plants. There was a twofold greater rate of chlorotoluron detoxification in resistant plants with N-demethylation being a major detoxification reaction. Resistant plants treated with a 3-h pulse of 120 M chlorotoluron recovered net carbon fixation after 42 h, half the time taken by susceptible plants. The mixed-function oxidase inhibitor 1-aminobenzotriazole (70 M) intensified the effects of chlorotoluron in resistant plants when applied in combination with the herbicide for 7 d. 1-Aminobenzotriazole also inhibited the metabolism of chlorotoluron in both resistant and susceptible plants. The cytochrome P-₄₅₀ inhibitor, piperonyl butoxide piperonyl butoxide, interacted with chlorotoluron when applied to plants growing in soil. Chlorotoluron applied with reduced plant dry weight to a greater extent than chlorotoluron alone. It appears, therefore, that enhanced detoxification is the major mechanism of resistance to chlorotoluron in the resistant biotypes studied.Abbreviations ABT 1-aminobenzotriazole - VLR1 Victorian L. rigidum biotype 1 — herbicide susceptible - VLR69 Victorian L. rigidum biotype 69 — herbicide resistant - WLR2 Western Australian L. rigidum biotype 2 — herbicide resistant M.W.M.B, was supported by an Australian Postgraduate Research Award and a supplementary scholarship from the Grains Research and Development Corporation. We are very grateful to Dr. E. Ebert, Ciba Geigy, Basal, Switzerland for providing [¹⁴C]chlorotoluron and standards of chlorotoluron metabolites. We express our gratitude to Dr. John Huppatz of the CSIRO Division of Plant Industry for providing ABT. We also thank Ciba Geigy Australia for providing technical-grade chlorotoluron and formulated phenylurea herbicides.     

Investigations of mechanisms of resistance to bipyridyl herbicides in Arctotheca calendula (L.) Levyns

The mechanism of resistance to diquat and paraquat was investigated in a bipyridyl-herbicide-resistant biotype of Arctotheca calendula (L.) Levyns. No differences were observed in the interactions of these herbicides with Photo-system I, the active site, in thylakoids isolated from resistant and susceptible biotypes. Likewise, absorption of herbicide through the cuticle and gross translocation were identical in plants of the two biotypes. Foliar application of either 25 g ha⁻¹ diquat or 200 g ha⁻¹ paraquat rapidly inhibited CO₂-dependent O₂ evolution of leaf segments of the susceptible biotype. O₂ evolution of leaf segments of the resistant biotype was less affected by these treatments. Fluorescence imaging was used to observe visually, as fluorescence quenching, the penetration of herbicide to the active site. These experiments demonstrated that diquat appears at the active site more slowly in the resistant biotype compared to the susceptible biotype. HCO₃-dependent O₂ evolution of thin leaf slices was less inhibited by diquat in the resistant biotype than in the susceptible biotype. The mechanism of resistance to the bipyridyl herbicides in this biotype of A. calendula is not a result of changes at the active site, decreased herbicide absorption or decreased translocation, but appears to be due to reduced herbicide penetration to the active site.     

Double herbicide-resistant biotypes of wild oat (<Emphasis Type="Italic">Avena fatua</Emphasis>) display characteristic metabolic fingerprints before and after applying ACCase- and ALS-inhibitors

Plant herbicides inhibit specific enzymes of biosynthetic metabolism, such as acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS). Herbicide resistance can be caused by point mutations at the binding domains, catalytic sites and other regions within multimeric enzymes. Direct-injection electrospray mass spectrometry was used for high-throughput metabolic fingerprinting for finding significant differences among biotypes in response to herbicide application. A Mexican biotype of wild oat (Avena fatua) that displays multiple resistances to ACCase- and ALS-inhibiting herbicides was characterized. The dose–response test showed that the double-resistant biotype had a resistance index of 3.58 for pinoxaden and 3.53 for mesosulfuron-methyl. Resistance was accompanied by characteristic mutations at the site of action: an I-1781-L substitution occurred in the ACCase enzyme and an S-653-N mutation was identified within the ALS enzyme. Other mutations were also detected in the genes of the Mexican biotypes. The ionomic fingerprint showed that the multiple-resistant biotype had a markedly different metabolic pattern under control conditions and that this difference was accentuated after herbicide treatment. This demonstrates that single changes of amino acid sequences can produce several holistic modifications in the metabolism of resistant plants compared to susceptible plants. We conclude that in addition to genetic resistance, additional mechanisms of metabolic adaptation and detoxification can occur in multiple-resistant weed plants.     

Occurrence of a herbicide-resistant acetyl-coenzyme A carboxylase mutant in annual ryegrass (Lolium rigidum) selected by sethoxydim

The spectrum of herbicide resistance was determined in an annual ryegrass (Lolium rigidum Gaud.) biotype (SLR 3) that had been exposed to the grass herbicide sethoxydim, an inhibitor of the plastidic enzyme acetylcoenzyme A carboxylase (ACCase, EC 6.4.1.2), for three consecutive years. This biotype has an 18-fold resistance to sethoxydim and enhanced resistance to other cyclohexanedione herbicides compared with a susceptible biotype (VLR 1). The resistant biotype also has a 47- to >300-fold cross-resistance to the aryloxyphenoxypropanoate herbicides which share ACCase as a target site. No resistance is evident to herbicide with a target site different from ACCase. The absorption of [4-¹⁴C]sethoxydim, the rate of metabolic degradation and the nature of the herbicide metabolites are similar in the resistant and susceptible biotypes. While the total activity of the herbicide target enzyme ACCase is similar in extracts from the two biotypes, the kinetics of herbicide inhibition differ. The concentrations of sethoxydim and tralkoxydim required to inhibit the activity of ACCase by 50% are 7.8 and >9.5 times higher, respectively, in the resistant biotype. The activity of ACCase from the resistant biotype was also less sensitive to aryloxyphenoxypropanode herbicides than the susceptible biotype. The spectrum of resistance at the whole-plant level is correlated with resistance at the ACCase level and confirms that a less sensitive form of the target enzyme endows resistance in biotype SLR 3.Abbreviations ACCase acetyl-coenzyme A carboxylase - AOPP aryloxyphenoxypropanoate - CHD cyclohexanedione - GR₅₀ dose giving 50% reduction of growth - IG₅₀ dose giving 50% reduction of germination - LD₅₀ lethal dose 50 This work was partially supported by The Grains Research and Development Corporation of Australia through a grant to Dr. R. Knight, Department of Plant Science, Waite Agricultural Research Institute. The encouragement and generous support of Dr. R. Knight is gratefully acknowledged.     

Differential Light Responses of Photosynthesis by Triazine-resistant and Triazine-susceptible Senecio vulgaris Biotypes

Studies were conducted to determine a physiological basis for competitive differences between Senecio vulgaris L. biotypes which are either resistant or susceptible to triazine herbicides. Net carbon fixation of intact leaves of mature plants was higher at all light intensities in the susceptible biotype than in the resistant biotype. Quantum yields measured under identical conditions for each biotype were 20% lower in the resistant than in the susceptible biotype. Oxygen evolution in continuous light measured in stroma-free chloroplasts was also higher at all light intensities in the susceptible biotype than in the resistant biotype. Oxygen evolution in response to flashing light was measured in stroma-free chloroplasts of both biotypes. The steady-state yield per flash of resistant chloroplasts was less than 20% that of susceptible chloroplasts. Susceptible chloroplasts displayed oscillations in oxygen yield per flash typically observed in normal chloroplasts, whereas the pattern of oscillations in resistant chloroplasts was noticeably damped. It is suggested that modification of the herbicide binding site which confers s-triazine resistance may also affect the oxidizing side of photosystem II, making photochemical electron transport much less efficient. This alteration has resulted in a lowered capacity for net carbon fixation and lower quantum yields in whole plants of the resistant type.     

Graminicide resistance in a blackgrass (Alopecurus myosuroides) population correlates with insensitivity of acetyl-CoA carboxylase

The appearance of biotypes of the annual grass weed black‐grass (Alopecurus myosuroides L. Huds), which are resistant to certain graminicides, is the most significant example of acquired resistance to herbicides seen so far in European agriculture. An investigation was perfomed into the basis of the specific cross‐resistance to cyclohexanedione (CHD) and aryloxyphenoxypropionoic acid (AOPP) herbicides in the ‘Notts A1’ population of A. myosuroides, which survived treatment of fields with recommended rates of AOPP herbicides. In comparison with the wild‐type ‘Rothamsted’ population, the resistant biotype showed over 100‐fold resistance to these herbicides in a hydroponic growth system. Biosynthesis of fatty acids and activity of crude extracts of acetyl‐CoA carboxylase (ACCase) were commensurately less sensitive to these herbicides in Notts A1 compared with the Rothamsted biotype. These data are consistent with the hypothesis that the highly resistant population has arisen through selection of a mutant ACCase which is much less sensitive to the AOPP and CHD graminicides. Rapidly growing cell suspension cultures established from the Notts A1 population also showed high resistance indices for CHD or AOPP herbicides compared with cultures from the Rothamsted biotype. Fatty acid biosynthesis and ACCase activity in the cell suspensions were similarly sensitive towards the graminicides to those in the foliar tissue counterparts of the resistant and sensitive populations. Moreover, purification of the main (chloroplast) isoform of acetyl‐CoA carboxylase showed that this enzyme from the Notts A1 population was over 200‐fold less sensitive towards the AOPP herbicide, quizalofop, than the equivalent isoform from the Rothamsted population. These data again fully supported the proposal that resistance in the Notts biotype is due to an insensitive acetyl‐CoA carboxylase isoform. Overall, cell suspensions were also demonstrated to be excellent tools for further investigation of the molecular basis of the high level herbicide resistance which is prone to occur in A. myosuroides.     

Triazine Resistance in Senecio vulgaris Parental and Nearly Isonuclear Backcrossed Biotypes Is Correlated with Reduced Productivity

Isonuclear triazine-susceptible and triazine-resistant Senecio vulgaris L. biotypes were developed by making reciprocal crosses between susceptible and resistant biotypes to obtain F₁ hybrids and backcrossing the hybrids to the appropriate pollen parent. The electrophoretic isozyme patterns of the enzyme aconitase obtained from leaf extracts of triazine-susceptible parental (S) and backcrossed (S×R_BC6) biotypes, and triazine-resistant parental (R) and backcrossed (R×S_BC6) biotypes verified that the biotypes had the expected nuclear genomes. Atrazine inhibition of chloroplast whole chain electron transport from water to methyl viologen was measured to verify susceptibility or resistance to triazine herbicides. The photosynthetic rate and biomass accumulation of greenhouse grown susceptible and resistant S. vulgaris biotypes were measured 28, 35, 42, 50, 57, and 64 days after planting to determine the effect of altered chloroplast function. S and S×R_BC6 biotypes had CO₂ assimilation rates of 16.2 and 16.6 micromoles CO₂ per square meter per second, respectively, and I₅₀ values (herbicide concentration producing 50% inhibition) of about 0.49 micromolar atrazine. The corresponding values for the R and R×S_BC6 biotypes were 14.7 and 14.6 micromoles CO₂ per square meter per second with I₅₀ values of 65.0 micromolar atrazine. The S biotype was larger and more productive than the R biotype at all harvests. At the harvest 57 days after planting, mean shoot dry weight was 33.2 and 8.7 grams for the S and R biotypes, respectively. The growth effect associated with chloroplast differences was shown in comparisons of the S biotype with the R×S_BC6 biotype and of the S×R_BC6 biotype with the R biotype. The R×S_BC6 biotype had 72% of the shoot dry weight of the S biotype while the R biotype had 55% of the shoot dry weight of the S×R_BC6 biotype. The R×S_BC6 and R biotypes produced about 73 and 62% of the leaf area of the S and S×R_BC6 biotypes, respectively. Relative growth rate was similar in biotypes with the same nuclear genome; however, instantaneous unit leaf rate was higher in the S compared to the R×S_BC6 biotype and in the S×R_BC6 compared to the R biotype. At 57 days after planting, the cumulative leaf area duration (i.e. photosynthetic opportunity) of the R×S_BC6 and R biotypes was 86 and 66% of that of the S and S×R_BC6 biotypes, respectively. Our data indicate that impaired chloroplast function in triazine resistant S. vulgaris biotypes limits growth and productivity at the whole plant level.     

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

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

Movement of paraquat in resistant and susceptible biotypes of Erigeron philadelphicus and E. canadensis

Paraquat (1,1'-dimethyl-4,4'-bipyridinium) resistant biotypes of Erigeron philadelphicus and E. canadensis , from fields where paraquat had been used for weed control, showed more than 100 times higher resistance than the susceptible biotype of both plants. Excised leaves of the susceptible biotypes wilted when supplied with more than 5 μ M paraquatat at the cut ends, but those from the resistant biotypes did not wilt even at 500 μ M. Autoradiographs indicated that (¹⁴CH₃)-paraquat taken up through the cut ends was rapidly distributed through the vascular system in leaves of the susceptible biotype, but was barely translocated in leaves of the resistant biotype. The amount of paraquat taken up during 48 h in the resistant biotype was 0.5% of that in the susceptible biotype in light. This difference in paraquat movement may be correlated with paraquat resistance in Erigeron.     

The Effect of Light and Temperature on Competition between Atrazine Susceptible and ResistantBrassica rapa

Biotypes ofBrassica rapasusceptible (S) and resistant (R) toatrazine were grown in competitive replacement series in allpossible combinations of two light levels and three temperatureregimes in controlled growth cabinets. Photosystem II functionwas investigated in all conditions by fluorescence-inductiontechniques. There were no significant differences in the dryweight of the two biotypes when grown in pure stands. In purestands both biotypes produced more biomass under the high lightlevel. Under high light both biotypes yielded more biomass athigh temperature; in low light they did so at medium temperature.Under high light conditions at high and medium temperaturesthe susceptible biotype had a greater photon yield and relativecompetitive ability than the resistant due to the greater vulnerabilityof triazine-resistant biotypes to photoinhibition. However,surprisingly, the resistant biotype was the better competitor,and had a higher photon yield, in the high light/low temperatureregime. In low light no photoinhibition was expected and indeedthere were no significant differences in any fluorescence parametersbetween the resistant and susceptible biotypes. Nevertheless,there were differences in the whole plant performance; the susceptiblebiotype was a better competitor at low and medium temperatures,but the resistant biotype was better at high temperature. Relativelysmall variations in both light and temperature, well withinthe range encountered during British summer time, can have largeeffects on the relative competitiveness of triazine R and Sbiotypes in this species with implications for the spread ofresistance genes through semi-natural communities. In lightof predicted climate changes, interactions between climate andresistance should be studied across a wider range of herbicidetypes and weed species.Copyright 1997 Annals of Botany Company Brassica rapa; chlorophyll fluorescence; competition; light; navew; temperature; triazine resistance     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum) : III. On the Mechanism of Resistance to Diclofop-Methyl

Annual ryegrass (Lolium rigidum) biotype SLR 31 is resistant to the postemergent graminicide methyl-2-[4-(2,4-dichlorophenoxy)phenoxy]-propanoate (diclofop-methyl). Uptake of [¹⁴C](U-phenyl)diclofop-methyl and root/shoot distribution of radioactivity in susceptible and resistant plants were similar. In both biotypes, diclofop-methyl was rapidly demethylated to the biocidal metabolite diclofop acid which, in turn, was metabolized to ester and aryl-O-sugar conjugates. Susceptible plants accumulated 5 to 15% more radioactivity in dicloflop acid than did resistant plants. Resistant plants had a slightly greater capacity to form nonbiocidal sugar conjugates. Despite these differences, resistant plants retained 20% of ¹⁴C in the biocidal metabolite diclofop acid 192 hours after treatment, whereas susceptible plants, which were close to death, retained 30% in diclofop acid. The small differences in the pool sizes of the active and inactive metabolites are by themselves unlikely to account for a 30-fold difference in sensitivity to the herbicide at the whole plant level. Similar high-pressure liquid chromatography elution patterns of conjugates from both susceptible and resistant biotypes indicated that the mechanisms and the products of catabolism in the biotypes are similar. It is suggested that metabolism of diclofop-methyl by the resistant biotype does not alone explain resistance observed at the whole-plant level. Diclofop acid reduced the electrochemical potential of membranes in etiolated coleoptiles of both biotypes; 50% depolarization required 1 to 4 μm diclofop acid. After removal of diclofop acid, membranes from the resistant biotype recovered polarity, whereas membranes from the susceptible biotype did not. Internal concentrations of diclofop acid 4 h after exposing plants to herbicide were estimated to be 36 to 39 micromolar in a membrane fraction and 16 to 17 micromolar in a soluble fraction. Such concentrations should be sufficient to fully depolarize membranes. It is postulated that differences in the ability of membranes to recover from depolarization are correlated with the resistance response of biotype SLR 31.     

Physiological tests for early detection of rigid ryegrass (Lolium rigidum Goud.) resistance to fenoxaprop-P

The study aimed to determine the usefulness of isothermal calorimetry and FT-Raman spectroscopy for the early evaluation of rigid ryegrass resistance to fenoxaprop-P ethyl (active ingredient one of aryloxyphenoxypropionate herbicides). The calorimetric measurements were done on the 4-day-old seedlings of susceptible and resistant biotypes of rigid ryegrass (Lolium rigidum Goud.) for 72 h, at 20 °C. It was observed that the specific thermal power–timecurves of the susceptible and resistant biotypes growing on water (control) were qualitatively similar. Herbicides changed the shape of the specific thermal power–time curves of both biotypes. Furthermore, the total specific thermal energy was significantly higher for the seedlings of resistant biotype, growing both on water or herbicide, as compared to the susceptible ones. The analysis of the seedlings’ endosperm, conducted using FT-Raman spectroscopy, showed a weaker intensity of the bands in the spectra derived from the resistant biotype. Differences in the specific thermal power–time curves and FT-Raman spectra between susceptible and resistant biotypes growing on water indicate that the sensitive and resistant biotypes are metabolically and chemically different already in the early stages of the seedling growth. We conclude that isothermal calorimetry and FT-Raman spectroscopy are efficient tools for the early detection of rigid ryegrass resistance to fenoxaprop-P ethyl.     

Glyphosate,paraquat and ACCase multiple herbicide resistance evolved in a <Emphasis Type="Italic">Lolium rigidum</Emphasis> biotype

Glyphosate is the world’s most widely used herbicide. A potential substitute for glyphosate in some use patterns is the herbicide paraquat. Following many years of successful use, neither glyphosate nor paraquat could control a biotype of the widespread annual ryegrass (Lolium rigidum), and here the world’s first case of multiple resistance to glyphosate and paraquat is confirmed. Dose–response experiments established that the glyphosate rate causing 50% mortality (LD₅₀) for the resistant (R) biotype is 14 times greater than for the susceptible (S) biotype. Similarly, the paraquat LD₅₀ for the R biotype is 32 times greater than for the S biotype. Thus, based on the LD₅₀ R/S ratio, this R biotype of L. rigidum is 14-fold resistant to glyphosate and 32-fold resistant to paraquat. This R biotype also has evolved resistance to the acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides. The mechanism of paraquat resistance in this biotype was determined as restricted paraquat translocation. Resistance to ACCase-inhibiting herbicides was determined as due to an insensitive ACCase. Two mechanisms endowing glyphosate resistance were established: firstly, a point mutation in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, resulting in an amino acid substitution of proline to alanine at position 106; secondly, reduced glyphosate translocation was found in this R biotype, indicating a co-occurrence of two distinct glyphosate resistance mechanisms within the R population. In total, this R biotype displays at least four co-existing resistance mechanisms, endowing multiple resistance to glyphosate, paraquat and ACCase herbicides. This alarming case in the history of herbicide resistance evolution represents a serious challenge for the sustainable use of the precious agrochemical resources such as glyphosate and paraquat.     

Evaluation of resistance in Cyperus difformis populations to ALS inhibiting herbicides

Due to the intensification of rice cultivation in Spain and the repeated use of herbicides as an effective tool to control weeds, one important fact is the appearance of resistant populations of weeds, which are no longer controlled effectively at field doses. Therefore, it is necessary to increase doses to such an extent as to produce phytotoxicity in the rice crop. In paddy fields in Badajoz (Spain), a few years ago, populations of Cyperus difformis resistant to bensulfuron-methyl appeared. In order to characterize this resistance, whole plant assays were carried out in fourteen populations, eight of them originating from treated Spanish paddy fields. The results were expressed as resistance factor (RF) = ED50(R)/ED50(S). Two populations of Cyperus difformis, resistant (R) and tolerant (T) to bensulfuron-methyl, displayed resistance factors greater than 30 and 12.5, respectively. Furthermore, in order to evaluate the existence of a possible cross-resistance, the recommended field doses of 3 other herbicides (imazamox, ethoxysulfuron and bentazone + MCPA) were applied to the plants. All populations were susceptible to both imazamox and bentazone + MCPA and two populations showed cross-resistance to ethoxysulfuron. ALS activity was assayed in vitro in four biotypes, the R/S ratios of the I50 values calculated indicate that resistance involves a reduction in the sensitivity of the ALS mainly in the R population (biotype 4) to bensulfuron-methyl and ethoxysulfuron, displaying R/S ratios of 37500 and 142857, respectively, which may be attributed to inherent differences in the ALS protein itself and/or in the stability of the enzyme.     

Inheritance and mechanism of resistance to herbicides inhibiting acetolactate synthase in Sonchus oleraceus L.

A biotype of Sonchus oleraceus L. (Compositae) has developed resistance to herbicides inhibiting acetolactate synthase (ALS) following field selection with chlorsulfuron for 8 consecutive years. The aim of this study was to determine the inheritance and mechanism of resistance in this biotype. Determination of ALS activity and inhibition kinetics revealed that K_m and V_max did not vary greatly between the resistant and susceptible biotypes. ALS extracted from the resistant biotype was resistant to five ALS-inhibiting herbicides in an in vitro assay. ALS activity from the resistant biotype was 14 19, 2, 3 and 3 times more resistant to inhibition by chlorsulfuron, sulfometuron, imazethapyr, imazapyr and flumetsulam, respectively, than the susceptible biotype. Hybrids between the resistant and a susceptible biotype were produced, and inheritance was followed through the F₁, F₂ and F₃ generations. F₁ hybrids displayed a uniform intermediate level of resistance between resistant and susceptible parents. Three distinct phenotypes, resistant, intermediate and susceptible, were identified in the F₂ generation following chlorsulfuron application. A segregation ratio of 121 was observed, indicative of the action of a single, nuclear, incompletely dominant gene. F₃ families, derived from intermediate F₂ individuals, segregated in a similar manner. Resistance to herbicides inhibiting ALS in this biotype of S. oleraceus is due to the effect of a single gene coding for a resistant form of the target enzyme, ALS.     

褐飞虱生物型研究进展:致害性变异的遗传机制

能够“克服”水稻抗性的褐飞虱生物型的出现 ,是目前水稻育种及害虫防治工作中面临的重大难题。为探明褐飞虱生物型的形成机制 ,许多学者致力于生物型致害性的遗传学研究 ,然而得出的结论却不尽一致。该文综述了有关褐飞虱生物型研究中争论较多的 3个方面 ,即生物型致害性的稳定性 ,致害性的遗传机制 ,以及生物型的形成机制 ,并介绍了最新的研究进展 ,展望了该领域的研究方向