Mutant Acetolactate Synthase Research Articles

A novel mutation Asp-2078-Glu in ACCase confers resistance to ACCase herbicides in barnyardgrass (Echinochloa crus-galli)

Multiple-herbicide resistance (MHR) in barnyardgrass (Echinochloa crus-galli) is a threat to rice production. The Ala-205-Val mutation in acetolactate synthase (ALS) conferred resistance to several ALS inhibitors in the E. crus-galli population AXXZ-2; consequently, ALS-inhibitors were unable to control this noxious weed species. In the present study, the sensitivity to acetyl-coenzyme A carboxylase (ACCase) herbicides and other herbicides having different modes of action was evaluated to determine an effective strategy for chemical weed control. Compared with that of the reportedly sensitive population JLGY-3, the AXXZ-2 population showed differential resistance to three ACCase-inhibitors (cyhalofop-butyl, fenoxaprop-P-ethyl, and pinoxaden), in addition to quinclorac and pretilachlor. A novel substitution (Asp-2078-Glu) in ACCase was detected as the main target-site resistance mechanisms in the AXXZ-2 population. Structural modeling of the mutant ACCase protein predicted that Asp-2078-Glu confers resistance to three ACCase inhibitors by reducing the binding affinity between them and the ACCase protein. To the best of our knowledge, this is the first study to report that the novel Asp-2078-Glu mutation confers resistance to several ACCase inhibitors. Target-site mutations in ALS and ACCase were detected in this MHR population. Except for quinclorac, pretilachlor, ALS inhibitors, and the three ACCase inhibitors, a number of herbicides remain effective in controlling this MHR E. crus-galli population.

Metribuzin resistance via enhanced metabolism in a multiple herbicide resistant Lolium rigidum population.

The photosystem II (PSII)-inhibiting herbicides are important for Australian farmers to control Lolium rigidum Gaud. and other weed species in trazine tolerant (TT)-canola fields. A L. rigidum population (R) collected from a TT-canola field from Western Australia showed multiple resistance to PSII, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors. The mechanisms of multiple resistance in this R population were determined. The R population showed a low-level (about 3.0-fold) resistance to the PSII-inhibiting herbicides metribuzin and atrazine. Sequencing of the psbA gene revealed no differences between the R and susceptible (S) sequences. Furthermore, [14 C]-metribuzin experiments found no significant difference in metribuzin foliar uptake and translocation between the R and S plants. However, [14 C]-metribuzin metabolism in R plants was 2.3-fold greater than in S plants. The cytochrome P450 monooxygenase inhibitor piperonyl butoxide (PBO) enhanced plant mortality response to metribuzin and atrazine in both R and S populations. In addition, multiple resistance to ALS and ACCase inhibitors are due to known resistance mutations in ALS and ACCase genes. The results demonstrate that enhanced metribuzin metabolism likely involving cytochrome P450 monooxygenase contributes to metribuzin resistance in Lolium rigidum. This is the first report of metabolic resistance to the PSII-inhibiting herbicide metribuzin in Australian Lolium rigidum. © 2020 Society of Chemical Industry.

Push It to the Limit: Identification of Novel Amino Acid Changes on the Acetolactate Synthase Enzyme of Rice That Putatively Confer High Level of Tolerance to Different Imidazolinones.

Advancements in genetically modified herbicide tolerance technology opened a new way to manage weed populations in crop fields. Since then, many important genetically modified crops that are tolerant to various herbicides have been developed and commercialized. Herbicides primarily act by disrupting key enzymes involved in essential metabolic or physiological processes associated with growth and development of plants. Most of the herbicide tolerant plants have been developed by introducing point mutations (non-GM approach) in the target site of herbicide action, due to the advantage of easier registration/release for commercial cultivation as well as wider public acceptance. Of the various herbicides, Imidazolinones are probably the most widely targeted ones for developing herbicide tolerant crops through non-GM approach. In rice, different mutant lines presenting amino acids changes in acetolactate synthase (ALS) have the ability to tolerate different Imidazolinones, including point mutations of Glycine to Glutamate in position 628, Serine to Asparagine in position 627, and a double mutation Tryptophan to Leucine in position 548/Serine to Isoleucine in position 627. The use of specific herbicides in combination of these mutant lines provides a reliable approach to eliminate weeds in the fields. However, the continuous overuse of a single herbicide multiple times in a growing season increases the potential risk of evolution of resistant weeds, which has become a major concern in agriculture worldwide. For this reason, the development of novel mutations in ALS (Os02g30630) to generate rice plants more tolerant to Imidazolinones than the available mutant rice lines is still a hot topic in plant-herbicide interaction field. Keeping that in mind, we carried out molecular docking experiments of Imidazolinone herbicides imazapic, imazapyr, imazaquin, and imazethapyr to evaluate the interaction of these molecules in the binding cavity of ALS from rice, being able to identify the most important amino acids responsible for the stability of these four herbicides. After introducing point mutations in these specific positions (one at a time) using Alanine scanning mutagenesis method and recalculating the effect in the affinity of herbicide-ALS interaction, we were able to propose novel amino acid residues (mainly Lysine in position 230 and Arginine in position 351) on the structure of ALS presenting a highest impact in the binding of Imidazolinones to ALS when compared to the already known amino acid mutations. This rational approach allows the researcher/farmer to choose the number of point mutations to be inserted in a rice cultivar, which will be dependent on the type of Imidazolinone used. To obtain a rice cultivar capable to tolerate the four Imidazolinone tested at the same time, we suggest six amino acid mutations at positions Val170, Phe180, Lys230, Arg351, Trp548, and Ser627 in the OsALS1.

Resistance evolution and mechanisms to ALS-inhibiting herbicides in Capsella bursa-pastoris populations from China

Capsella bursa-pastoris is a serious broadleaf weed in winter wheat fields in China. It has evolved high levels of resistance to acetolactate synthase (ALS) inhibiting herbicides and has caused substantial losses of wheat yield in recent years. We monitored the herbicide resistance of Capsella bursa-pastoris collected from 18 regions of Shandong Province in 2009, 2013 and 2017, respectively. Compared with the 2009 populations, the number of populations resistant to florasulam had increased in 2013 and 2017. Resistance to tribenuron-methyl increased in 2013, but decreased in 2017. The 2009 and 2013 populations developed resistance only to tribenuron-methyl, but some 2017 populations developed cross-resistance to imazethapyr and florasulam as well. Mutations in ALS (Pro-197-Thr/Ser/His/Arg/Leu/Gln) were identified in the 2009 and 2013 populations; however, two ALS mutations (Pro197 and/or Trp574) were identified in 2017 plants. Meanwhile, plants containing both point mutations (Pro197 + Trp574) were identified in the 2017 populations. This study demonstrated that target site gene mutations were the main reason for Capsella bursa-pastoris resistance to ALS-inhibiting herbicides. Although target-site mutation is the reason for resistance to ALS-inhibiting herbicides in Capsella bursa-pastoris, the resistance patterns and mutations identified have changed over time.

Spatial and temporal evolution of imidazolinone-resitant red rice in 'Clearfield' rice cultivations

Abstract: The objective of this work was to evaluate the distribution of imidazolinone-resistant (IMI-R) red rice (Oryza sativa) populations, the frequency of alleles conferring resistance to IMI, and the adoption of agronomic practices applied to red rice control, across growing seasons and production regions of the state of Rio Grande do Sul (RS), Brazil. In the experiment, 1,008 red rice populations were screened for resistance to IMI, 760 IMI-R red rice plants were genotyped for the acetolactate synthase (ALS) alleles conferring resistance to IMI, and 40 'Clearfield' rice growers were surveyed. IMI-R red rice populations were widespread throughout RS since the 2006/2007 growing season, with a higher initial frequency in the Depressão Central and Fronteira Sul production regions. The occurrence of IMI-R red rice ranged from 1.6 to 3.5 years after 'Clearfield' rice release. Gly654Glu was the most frequent ALS mutation in IMI-R red rice populations, which shows a gene flow from the most used 'Clearfield' rice cultivars to the red rice plants. Crop rotation systems and certified seed were used by only 30% of the surveyed growers of 'Clearfield' rice, with lower percentages in the production regions where IMI-R red rice appeared faster.

Target-Site Resistance Mechanisms to Tribenuron-methyl and Cross-resistance Patterns to ALS-inhibiting Herbicides of Catchweed Bedstraw (Galium aparine) with Different ALS Mutations

AbstractCatchweed bedstraw (Galium aparine L.) is a problematic dicot weed that occurs in major winter wheat (Triticum aestivum L.) fields in China. Tribenuron-methyl has been widely used to control broadleaf weeds since 1988 in China. However, overuse has led to the resistance evolution of G. aparine to tribenuron-methyl. In this study, 20 G. aparine populations collected from Shandong and Henan provinces were used to determine tribenuron-methyl resistance and target-site resistance mechanisms. In dose–response experiments, 12 G. aparine populations showed different resistance levels (2.92 to 842.41-fold) to tribenuron-methyl compared with the susceptible population. Five different acetolactate synthase (ALS) mutations (Pro-197-Leu, Pro-197-Ser, Pro-197-His, Asp-376-Glu, and Trp-574-Leu) were detected in different resistant populations. Individuals heterozygous for Pro-197-Ser and Trp-574-Leu mutations were also observed in a resistant population (HN6). In addition, pHB4 (Pro-197-Ser), pHB7 (Pro-197-His), pHB8 (Pro-197-Leu), pHB5 (Asp-376-Glu), and pHB3 (Trp-574-Leu) subpopulations individually homozygous for specific ALS mutations were generated to evaluate the cross-resistance to ALS-inhibiting herbicides. The pHB4, pHB7, pHB8, pHB5, and pHB3 subpopulations all were resistant to sulfonylurea, pyrazosulfuron-ethyl, triazolopyrimidine, flumetsulam, sulfonylamino-carbonyl-triazolinone, flucarbazone-sodium, pyrimidinyl thiobenzoate, pyribenzoxim, and the imidazolinone imazethapyr. These results indicated the diversity of the resistance-conferring ALS mutations in G. aparine, and all these mutations resulted in broad cross-resistance to five kinds of ALS-inhibiting herbicides.

Discovery of mutations in Chenopodium quinoa Willd through EMS mutagenesis and mutation screening using pre-selection phenotypic data and next-generation sequencing

AbstractQuinoa (Chenopodium quinoaWilld) is a dicotyledonous annual species belonging to the family Amaranthaceae, which is nutritionally well balanced in terms of its oil, protein and carbohydrate content. Targeting-induced local lesions in genomes (the TILLING strategy) was employed to find mutations in acetolactate synthase (AHAS) genes in a mutant quinoa population. TheAHASgenes were targeted because they are common enzyme target sites for five herbicide groups. Ethyl methane sulfonate (EMS) was used to induce mutations in theAHASgenes; it was found that 2% EMS allowed a mutation frequency of one mutation every 203 kilobases to be established. In the mutant population created, a screening strategy using pre-selection phenotypic data and next-generation sequencing (NGS) allowed identification of a mutation that alters the amino acid composition of this species (nucleotide 1231 codon GTT→ATT, Val→Ile); however, this mutation did not result in herbicide resistance. The current work shows that TILLING combined with the high-throughput of NGS technologies and an overlapping pool design provides an efficient and economical method for detecting induced mutations in pools of individuals.

Dinitroaniline herbicide resistance in a multiple-resistant Lolium rigidum population.

The pre-emergence dinitroaniline herbicides (such as trifluralin and pendimethalin) are vital to Australian no-till farming systems. A Lolium rigidum population collected from the Western Australian grain belt with a 12-year trifluralin use history was characterised for resistance to dinitroaniline, acetyl CoA carboxylase (ACCase)- and acetolactate synthase (ALS)-inhibiting herbicides. Target-site resistance mechanisms were investigated. This L. rigidum population exhibited 32-fold resistance to trifluralin, as compared with the susceptible population. It also displayed 12- to 30-fold cross-resistance to other dinitroaniline herbicides (pendimethalin, ethalfluralin and oryzalin). In addition, this population showed multiple resistance to commonly used post-emergence ACCase- and ALS-inhibiting herbicides. Two target-site α-tubulin gene mutations (Val-202-Phe and Thr-239-Ile) previously documented in other dinitroaniline-resistant weed species were identified, and some known target-site mutations in ACCase (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg) and ALS (Pro-197-Gln/Ser) were found in the same population. An agar-based Petri dish screening method was established for the rapid diagnosis of resistance to dinitroaniline herbicides. Evolution of target-site resistance to both pre- and post-emergence herbicides was confirmed in a single L. rigidum population. The α-tubulin mutations Val-202-Phe and Thr-239-Ile, documented here for the first time in L. rigidum, are likely to be responsible for dinitroaniline resistance in this population. Early detection of dinitroaniline herbicide resistance and integrated weed management strategies are needed to maintain the effectiveness of dinitroaniline herbicides. © 2017 Society of Chemical Industry.

Independent Evolution of Acetolactate Synthase–inhibiting Herbicide Resistance in WeedySorghumPopulations across Common Geographic Regions

Traditional breeding has been used to develop grain sorghum germplasm that is tolerant to acetolactate synthase (ALS)-inhibiting herbicides (Inzen Technology, DuPont). Inzen sorghum carries a double mutation in the ALS gene (Val560Ile and Trp574Leu), which confers high level of tolerance to ALS-inhibiting herbicides. Overreliance on ALS-inhibiting herbicides for weed control during the 1990s resulted in the evolution of ALS inhibitor–resistant shattercane populations in Nebraska. According to a survey conducted in 2013, ALS inhibitor–resistant weedySorghumpopulations persist in Nebraska. The objectives of this research were to determine whether the ALS mutations present in Inzen sorghum were present in the ALS inhibitor–resistant shattercane and johnsongrass populations detected in Nebraska and northern Kansas, and whether these populations evolved ALS resistance independently. Primers specific to the Val560and Trp574region of the ALS gene were used to screen the populations with PCR. The Trp574Leu mutation was present in one ALS inhibitor–resistant johnsongrass population. The Val560Ile was detected in three ALS inhibitor–resistant shattercane, one susceptible shattercane, one ALS inhibitor–resistant johnsongrass, and one susceptible johnsongrass population. Moreover, Val560Ile was present in resistant and/or susceptible individuals within johnsongrass and shattercane populations that were segregating for ALS resistance, indicating that by itself the Val560Ile mutation does not confer resistance to ALS-inhibiting herbicides. None of the populations presented both mutations simultaneously, as does Inzen sorghum. A shattercane population containing the Ser653Thr mutation was also detected. This research indicates that the ALS mutations present in Inzen sorghum already exist individually in weedy sorghum populations. Moreover, our results present strong evidence that ALS resistance in these populations evolved independently. Thus, widespread overreliance on ALS-inhibiting herbicides prior to adoption of glyphosate-tolerant crops in the 1990s exerted sufficient selective pressure on shattercane and johnsongrass populations for resistance to evolve multiple times in the Midwest. Finally, a survey of the 5′ portion of the ALS gene in more diverse wild and weedySorghumspecies was hampered by limited coverage in genomic resequencing surveys, suggesting that refined PCR-based methods will be needed to assess SNP variation in this gene region, which includes the Ala122, Pro197, and Ala205codons known to confer ALS resistance in other species.

Cross-resistance patterns to acetolactate synthase (ALS)-inhibiting herbicides of flixweed (Descurainia sophia L.) conferred by different combinations of ALS isozymes with a Pro-197-Thr mutation or a novel Trp-574-Leu mutation

Acetolactate synthase (ALS) is the common target of ALS-inhibiting herbicides, and target-site ALS mutations are the main mechanism of resistance to ALS-inhibiting herbicides. In this study, ALS1 and ALS2 genes with full lengths of 2004bp and 1998bp respectively were cloned in individual plants of susceptible (S) or resistant (R) flixweed (Descurainia sophia L.) populations. Two ALS mutations of Pro-197-Thr and/or Trp-574-Leu were identified in plants of three R biotypes (HB24, HB30 and HB42). In order to investigate the function of ALS isozymes in ALS-inhibiting herbicide resistance, pHB24 (a Pro-197-Thr mutation in ALS1 and a wild type ALS2), pHB42 (a Trp-574-Leu mutation in ALS1 and a wild type ALS2) and pHB30 (a Trp-574-Leu mutation in ALS1 and a Pro-197-Thr mutation in ALS2) subpopulations individually homozygous for different ALS mutations were generated. Individuals of pHB30 had mutations in each isozyme of ALS and had higher resistance than pHB24 and pHB42 populations containing mutations in only one ALS isozyme. Moreover, the pHB24 had resistance to SU, TP and SCT herbicides, whereas pHB24 and pHB42 had resistance to these classes of herbicides as well as IMI and PTB herbicides. The sensitivity of isolated ALS enzyme to inhibition by herbicides in these populations correlated with whole plant resistance levels. Therefore, reduced ALS sensitivity resulting from the mutations in ALS was responsible for resistance to ALS-inhibiting herbicides in flixweed.

Target-site and non-target-site based resistance to the herbicide tribenuron-methyl in flixweed (Descurainia sophia L.).

BackgroundFlixweed (Descurainia sophia L.) is a troublesome and widespread broadleaf weed in winter fields in China, and has evolved high level resistance to acetolactate synthase (ALS)-inhibiting sulfonylurea herbicide tribenuron-methyl.ResultsWe identified a resistant flixweed population (N11) exhibiting 116.3-fold resistance to tribenuron-methyl relative to the susceptible population (SD8). Target-site ALS gene mutation Pro-197-Thr was identified in resistant plants. Moreover, the resistance can be reversed to 28.7-fold by the cytochrome P450 inhibitor malathion. The RNA-Sequencing was employed to identify candidate genes involved in non-target-site metabolic resistance in this population. Total 26 differentially expressed contigs were identified and eight of them (four P450s, one ABC transporter, three glycosyltransferase) verified by qRT-PCR. Consistent over-expression of the two contigs homology to CYP96A13 and ABCC1 transporter, respectively, were further qRT-PCR validated using additional plants from the resistant and susceptible populations.ConclusionsTribenuron-methyl resistance in flixweed is controlled by target-site ALS mutation and non-target-site based mechanisms. Two genes, CYP96A13 and ABCC1 transporter, could play an important role in metabolic resistance to tribenuron-methyl in the resistant flixweed population and justify further functional studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2915-8) contains supplementary material, which is available to authorized users.

Molecular basis of multiple resistance to herbicides inhibiting acetyl-CoA carboxylase and acetolactate synthase in American sloughgrass (Beckmannia syzigachne) from China

American sloughgrass (Beckmannia syzigachne Steud.) is a problematic grass that is widely distributed in wheat and oilseed rape fields in China. The herbicides fenoxaprop-P-ethyl and mesosulfuron-methyl failed to control B. syzigachne JCWJ-R populations collected from a wheat field in Jiangsu Province. Dose-response experiments showed that JCWJ-R was resistant to the acetyl-CoA carboxylase (ACCase) inhibitors fenoxaprop-P-ethyl (33.8-fold), haloxyfop-R-methyl (12.7-fold), clethodim (7.8-fold) and pinoxaden (11.6-fold), and to the acetolactate synthase (ALS) inhibitors mesosulfuron-methyl (15.9-fold), pyroxsulam (17.6-fold), flucarbazone-Na (10.7-fold) and imazethapyr (7-fold). Resistance to ALS inhibitors was due to a Pro-197-Ser mutation in the ALS gene and resistance to ACCase inhibitors was due to an Ile-1781-Leu mutation in the ACCase gene. A derived cleaved amplified polymorphic sequence method was developed to detect the ALS mutation in B. syzigachne. This was combined with a previously established method to detect Ile-1781-Leu, and the mutation frequency and homozygous mutation rates in the JCWJ-R population were determined. The evolution of multiple resistance to ACCase and ALS inhibitors in this B. syzigachne population indicated that alternative methods should be developed to control resistant weeds.

Resistance Mechanisms to an Acetolactate Synthase (ALS) Inhibitor in Water Starwort (Myosoton aquaticum) Populations from China

Overreliance on tribenuron has resulted in resistance evolution in water starwort. This study investigates the resistance mechanisms to tribenuron in water starwort populations from China. The cytochrome P450 monooxygenase (P450) inhibitor malathion increased tribenuron sensitivity in all populations. The decrease in the amount of herbicide dose that causes 50% growth reduction (GR50) for the sensitive (S) population JS24 and the resistant (R) populations JS16 and JS17 were 2.3-, 2.5-, and 4.1-fold, respectively. However, the GR50 values for the R populations were still much higher than those of the S population. This observation indicates that P450-mediated enhanced metabolism is one mechanism for resistance in water starwort. The glutathione-S-transferase (GST) activity could be induced by tribenuron for all tested populations. In particular, the GST activity of JS16 is inherently greater and is more rapidly induced than that of JS17 or JS24. Resistance attributed to mutant acetolactate synthase (ALS) alleles was identified by sequence analysis for each population. Pro197Ser substitution was detected in JS16 and JS17. Molecular markers were also developed to rapidly identify resistance as well as individuals carrying the specific Pro197Ser mutation in water starwort populations. The resistance patterns experiment revealed that the R populations exhibited different levels of resistance to pyrithiobac sodium salt, florasulam, pyroxsulam, and flucarbazone-Na; however, R populations were sensitive to imazethapyr, fluroxypyr-meptyl, 2,4-D butylate, isoproturon, and diflufenican. This study establishes that either one or at least two resistance mechanisms are involved in herbicide resistance in water starwort. Moreover, these mechanisms might contribute to the different levels of resistance to tribenuron among water starwort populations.

English

The plasmids pULGU1 and pEmuGN were introduced by biolistics in embryogenic cell suspensions of pearl millet, Pennisetum glaucum . The plasmid pULGU1 contained the mutant acetolactate synthase (ALS) gene of Arabidopsis thaliana , responsible for resistance to the chlorsulfuron herbicide. The plasmid pEmuGN carried the reporter β-glucuronidase (GUS) gene of Escherichia coli . Two months after bombardment of the cells, transformed calli were selected on Murashige and Skoog medium containing 30 nM chlorsulfruron, a completely concentration inhibitory for non-transformed cells. A concentration of 200 nM chlorsufuron did not affect the growth of these selected resistant calli. Genomic DNA of resistant cells presented an electrophoretic pattern revealing the integration of the mutant ALS gene into the genome of Pennisetum glaucum . Expression of the GUS reporter gene was revealed by histochemical assay of the blue coloration of the calli in the presence of the substrate Xgluc (5-bromo-4-chloro-3-indolyl-β-D-Glucuronic acid). The presence of GUS gene was furthermore confirmed, by southern blot hybridization of non-radioactive probes, labelled with digoxigenin, on genomic DNA extracted from selected chlorsulfuron-resistant calli. GUS gene activity observed in all selected calli was high during the first 6 months after bombardment and then decreased. Pearl millet plants were regenerated from cell lines derived from chlorsulfuron resistant calli. Southern blot hybridization of non-radioactive probes with genomic DNA extracted from these regenerated plants showed the presence of the GUS and ALS transgenes, confirming the stable transformation of these plants. Key words : Pennisetum glaucum , stable transformation, chlorsulfuron (ALS), β-glucuronidase (GUS), CaMV35S and Emu promoters.

Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.

Genetic basis, evolutionary origin and spread of resistance to herbicides inhibiting acetolactate synthase in common groundsel (Senecio vulgaris).

Following control failure by herbicides inhibiting acetolactate synthase (ALS) in French wheat fields and vineyards, we aimed to confirm resistance evolution and investigate the evolutionary origin and spread of resistance in the tetraploid species Senecio vulgaris (common groundsel), a widespread, highly mobile weed. Sequencing of two ALS homeologues in S. vulgaris enabled the first identification and characterisation of ALS-based resistance in this species. Cross-resistance patterns associated with Leu-197 and Ser-197 ALS1 were established using eight herbicides. Sequencing and genotyping showed that ALS-based resistance evolved by multiple, independent appearances of mutant ALS1 and ALS2 alleles followed by spread. Spread of a mutant ALS1 allele issued from one particular appearance event was observed over 60 km. Independent resistance appearance events and easy seed dispersion are the most likely reasons for populations of S. vulgaris containing different mutant ALS alleles. Accumulation of different alleles probably due to sexual reproduction was observed in the same plant. Mutant ALS alleles and possibly other mechanisms cause resistance to ALS inhibitors in S. vulgaris. Management strategies should aim at limiting S. vulgaris establishment and seed set. Considering the mobility of this species, control coordination at a regional level is clearly necessary if resistance spread is to be contained.

Occurrence, genetic control and evolution of non-target-site based resistance to herbicides inhibiting acetolactate synthase (ALS) in the dicot weed Papaver rhoeas

Non-target-site resistance (NTSR) to herbicides is a major issue for the chemical control of weeds. Whilst predominant in grass weeds, NTSR remains largely uninvestigated in dicot weeds. We investigated the occurrence, inheritance and genetic control of NTSR to acetolactate synthase (ALS) inhibitors in Papaver rhoeas (corn poppy) using progenies from plants with potential NTSR to the imidazolinone herbicide imazamox. NTSR to imazamox was inherited from parents over two successive generations. NTSR to tritosulfuron (a sulfonylurea) was observed in F1 generations and inherited in F2 generations. NTSR to florasulam (a triazolopyrimidine) emerged in F2 generations. Our findings suggest NTSR was polygenic and gradually built-up by accumulation over generations of loci with moderate individual effects in single plants. We also demonstrated that ALS alleles conferring herbicide resistance can co-exist with NTSR loci in P. rhoeas plants. Previous research focussed on TSR in P. rhoeas, which most likely caused underestimation of NTSR significance in this species. This may also apply to other dicot species. From our data, resistance to ALS inhibitors in P. rhoeas appears complex, and involves well-known mutant ALS alleles and a set of unknown NTSR loci that confer resistance to ALS inhibitors from different chemical families.

An Attempt to Detect siRNA-Mediated Genomic DNA Modification by Artificially Induced Mismatch siRNA in Arabidopsis

Although tremendous progress has been made in recent years in identifying molecular mechanisms of small interfering RNA (siRNA) functions in higher plants, the possibility of direct interaction between genomic DNA and siRNA remains an enigma. Such an interaction was proposed in the ‘RNA cache’ hypothesis, in which a mutant allele is restored based on template-directed gene conversion. To test this hypothesis, we generated transgenic Arabidopsis thaliana plants conditionally expressing a hairpin dsRNA construct of a mutated acetolactate synthase (mALS) gene coding sequence, which confers chlorsulfuron resistance, in the presence of dexamethasone (DEX). In the transgenic plants, suppression of the endogenous ALS mRNA expression as well as 21-nt mALS siRNA expression was detected after DEX treatment. After screening >100,000 progeny of the mALS siRNA-induced plants, no chlorsulfuron-resistant progeny were obtained. Further experiments using transgenic calli also showed that DEX-induced expression of mALS siRNA did not affect the number of chlorsulfuron-resistant calli. No trace of cytosine methylation of the genomic ALS region corresponding to the dsRNA region was observed in the DEX-treated calli. These results do not necessarily disprove the ‘RNA cache’ hypothesis, but indicate that an RNAi machinery for ALS mRNA suppression does not alter the ALS locus, either genetically or epigenetically.

Multiple Resistance to Herbicides from Four Site-of-Action Groups in Waterhemp (Amaranthus tuberculatus)

In 2006 and 2007, farmers from two counties in Illinois reported failure to control waterhemp with glyphosate. Subsequent onsite field experiments revealed that the populations might be resistant to multiple herbicides. Greenhouse experiments therefore were conducted to confirm glyphosate resistance, and to test for multiple resistance to other herbicides, including atrazine, acifluorfen, lactofen, and imazamox. In glyphosate dose-response experiments, both populations responded similarly to a previously characterized glyphosate-resistant population (MO1). Both Illinois populations also demonstrated high frequencies of resistance to the acetolactate synthase (ALS) inhibitor, imazamox. Additionally, one of the populations demonstrated high frequencies of resistance to both atrazine and the protoporphyrinogen oxidase (PPO) inhibitor, lactofen. Furthermore, using combinations of sequential and tank-mix herbicide applications, individual plants resistant to herbicides spanning all four site-of-action groups were identified from one population. Molecular experiments were performed to provide an initial characterization of the resistance mechanisms and to provide confirmation of the presence of multiple resistance traits within the two populations. Both populations contained the W574L ALS mutation and the ΔG210 PPO mutation, previously shown to confer resistance to ALS and PPO inhibitors, respectively. Atrazine resistance in both populations is suspected to be metabolism-based, because a triazine target-site mutation was not identified. A P106S EPSPS mutation, previously reported to confer glyphosate resistance, was identified in one population. This mutation was identified in both resistant and sensitive plants from the population; however, and so more research is needed to determine the glyphosate-resistance mechanism(s). This is the first known case of a weed population in the United States possessing multiple resistance to herbicides from four site-of-action groups.

Mechanisms of resistance to acetolactate synthase‐inhibiting herbicides in populations of Apera spica‐venti from the Czech Republic

This study investigates the mechanisms of resistance to acetolactate synthase-inhibiting herbicides in populations of Apera spica-venti (L.) P.B. from the Czech Republic. The proportion of resistance due to mutant acetolactate synthase (ALS) alleles was estimated by genotyping individuals from each of three populations for the eight ALS mutations known to confer resistance. Four resistance-conferring ALS mutations were identified: Pro-197-Ala, Pro-197-Thr, Trp-574-Leu and previously unreported Trp-574-Met substitution. Two populations (R1, R3) have amino acid substitution at positions Pro-197 and Trp-574. Individuals from the R3 population had two different resistance alleles. In the R2 population, only the resistant Trp-574-Met substitution was detected. Ten other single point mutations were identified, but these were not related to resistance. The cytochrome malathion decreased chlorsulfuron resistance in the resistant populations that were examined. Although malathion increased mortality, the GR50 values were too high to conclude that non-target-based mechanism was the main one for the resistance in Apera spica-venti populations tested in this study. Individuals of Apera spica-venti populations tested in this study possess the target-site ALS resistance mutation and an additional so far unknown resistance mechanism(s).