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The invasive weed Amaranthus palmeri is spreading throughout Spain, with Catalonia being one of the most affected regions. For this species, acetolactate synthase-inhibiting herbicide-resistant populations have been reported, and now glyphosate resistance is also suspected. Glyphosate targets and inhibits the enzyme 5-enolpyruvylshikimate3-phosphate synthase (EPSPS), but A. palmeri has evolved different resistant mechanisms leading to plant survival. One of the most effective is the EPSPS overexpression due to copy number variation (CNV). Gene copies accumulate within the EPSPS cassette that is an extrachromosomal circular DNA displaying unique structural polymorphisms. This study aims to determine the response to glyphosate of a suspected resistant population collected from a roadside and investigate the resistance mechanism involved. The herbicide bioassay confirmed that 40% of the plants survived glyphosate applied at 540 g a.i. ha 1 . No known mutations endowing glyphosate resistance were found at EPSPS amongst confirmed resistant plants, while in most of them (70%) specific molecular markers revealed the presence of the EPSPS cassette. All these results indicate that this population is glyphosate resistant and it is very likely that the EPSPS gene CNV is the main resistance mechanism. This is the first case of glyphosate resistance in A. palmeri in Europe whose introduction is likely due to importation of contaminated seed with glyphosate-resistant Palmer amaranth from the Americas. This introduction poses a significant danger to summer crops in our continent. This work has been supported by the Spanish State Research Agency,Spain (AEI) and the European Regional Development Fund, EU (ERDF)through the projects AGL2017-83325-C4-2-R and PID2020-113229RB-C42. 2021-2024. Joel Torra acknowledges support from the SpanishMinistry of Science, Innovation, and Universities (grant Ramon y CajalRYC2018-023866-I). This project has received funding from the European Union's H2020 research and innovation program under MarieSklodowska-Curie grant agreement No 801586. The authors are gratefulto Alison Garside for revising the English text. The authors also thank twoanonymous reviewers for their helpful comments on the draft of themanuscript.

Lolium spp. are troublesome weeds mainly found in winter cereal crops worldwide, including Europe. In recent years resistant mechanisms have been evolved to several important herbicides. In this study we investigated the mechanisms responsible for conferring glyphosate resistance in some Lolium spp. populations. A holistic approach was used, based on dose-response experiments, determination of shikimic acid concentration in plant leaf tissue, as well as molecular analyses. More specifically, in three Lolium spp. populations the existence of a mutation in the Pro-106 codon of the 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) gene was investigated as well as the relative transcript levels of four ABC-transporter genes were monitored at three time points after glyphosate application. The results demonstrated that glyphosate resistance is a multifactor phenomenon. Relative transcript levels of the ABC-transporter genes were abundant at very early time points after glyphosate treatments. Dose-response experiments and shikimate analyses were in accordance with the findings of the quantitative PCR (qPCR) analyses. We suggest that relative expression ratio of ABC-transporter genes can be a useful tool to rapidly identify Lolium spp. populations resistant to glyphosate.

BACKGROUND Before 2010, Amaranthus tuberculatus (Moq.) J. D. Sauer was barely known to farmers and stakeholders in Italy. Since then, several populations resistant to acetolactate synthase (ALS)‐inhibiting herbicides have been collected. In most populations, a known target site resistance‐endowing mutation was found, a Trp to Leu substitution at position 574 of the ALS gene, but it was unclear whether they had evolved resistance independently or not. The aims of the work were (i) to elucidate the population structure of Italian ALS‐resistant A. tuberculatus populations, and (ii) to analyze the ALS haplotypes of the various populations to determine whether resistance arose multiple times independently. RESULTS In order to determine the population structure of eight A. tuberculatus populations, eight previously described microsatellite loci were used. Two ancestors were found: three populations derived from one, and five from the other. In the 4‐kb ALS region of the genome, including the 2‐kb coding region, 389 single nucleotide polymorphisms were found. In silico haplotype estimation was used to reconstruct the sequence of three distinct haplotypes carrying the Trp574Leu mutation. In addition, no mutation was found in 83% of plants of a single population. CONCLUSIONS (i) Resistance must have arisen independently at least three times; (ii) at least one population was already resistant to ALS inhibitors when introduced in Italy; (iii) a single haplotype with a Trp574Leu mutation was shared among six populations, probably because of broad seed dispersal; and (iv) one population likely evolved nontarget site ALS inhibitors resistance. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., The same mutant ALS haplotype was found in five A. tuberculatus populations in Italy. Another population had the same resistance‐endowing mutation, but a different haplotype. A seventh population had no mutations.

The sustainability of rice cropping systems is jeopardized by the large number and variety of populations of polyploid Echinochloa spp. resistant to ALS inhibitors. Better knowledge of the Echinochloa species present in Italian rice fields and the study of ALS genes involved in target-site resistance could significantly contribute to a better understanding of resistance evolution and management. Using a CAPS-rbcL molecular marker, two species, E. crus-galli (L.) P. Beauv. and E. oryzicola (Vasinger) Vasing., were identified as the most common species in rice in Italy. Mutations involved in ALS inhibitor resistance in the different species were identified and associated with the ALS homoeologs. The relative expression of the ALS gene copies was evaluated. Molecular characterization led to the identification of three ALS genes in E. crus-galli and two in E. oryzicola. The two species also carried different point mutations conferring resistance: Ala122Asn in E. crus-galli and Trp574Leu in E. oryzicola. Mutations were carried in the same gene copy (ALS1), which was significantly more expressed than the other copies (ALS2 and ALS3) in both species. These results explain the high resistance level of these populations and why mutations in the other ALS copies are not involved in herbicide resistance.

The sustainability of rice cropping systems is jeopardized by the large number and variety of populations of polyploid Echinochloa spp. resistant to ALS inhibitors. Better knowledge of the Echinochloa species present in Italian rice fields and the study of ALS genes involved in target-site resistance could significantly contribute to a better understanding of resistance evolution and management. Using a CAPS-rbcL molecular marker, two species, E. crus-galli (L.) P. Beauv. and E. oryzicola (Vasinger) Vasing., were identified as the most common species in rice in Italy. Mutations involved in ALS inhibitor resistance in the different species were identified and associated with the ALS homoeologs. The relative expression of the ALS gene copies was evaluated. Molecular characterization led to the identification of three ALS genes in E. crus-galli and two in E. oryzicola. The two species also carried different point mutations conferring resistance: Ala122Asn in E. crus-galli and Trp574Leu in E. oryzicola. Mutations were carried in the same gene copy (ALS1), which was significantly more expressed than the other copies (ALS2 and ALS3) in both species. These results explain the high resistance level of these populations and why mutations in the other ALS copies are not involved in herbicide resistance.

Yellow nutsedge (C. esculentus) is a perennial geophyte and invasive weed which is very difficult to control in rice and other irrigated row crops. Acetolactate synthase (ALS) inhibitors are the most commonly used herbicides to control sedges in rice. Failure to control C. esculentus was recently reported in a rice field in north-western Italy. The resistance status of this C. esculentus population was determined through a whole-plant bioassay. The mechanism underlying the resistance was elucidated, and the available chemical and non-chemical control options were discussed. The population proved to be resistant to halosulfuron and azimsulfuron at the recommended field rate. The ALS trancripts amplified from resistant and susceptible plants revealed the presence of a Pro197-to-Arg amino acid substitution in resistant plants, indicating that the resistance mechanism is target-site mediated. This is the first confirmation of herbicide resistance in C. esculentus in Europe. Resistance management should be based on an integrated approach, through the combination of diversified cultural and agronomic practices that can limit its spread and propagation through tubers.

Background Worldwide feralization of crop species into agricultural weeds threatens global food security. Weedy rice is a feral form of rice that infests paddies worldwide and aggressively outcompetes cultivated varieties. Despite increasing attention in recent years, a comprehensive understanding of the origins of weedy crop relatives and how a universal feralization process acts at the genomic and molecular level to allow the rapid adaptation to weediness are still yet to be explored. Results We use whole-genome sequencing to examine the origin and adaptation of 524 global weedy rice samples representing all major regions of rice cultivation. Weed populations have evolved multiple times from cultivated rice, and a strikingly high proportion of contemporary Asian weed strains can be traced to a few Green Revolution cultivars that were widely grown in the late twentieth century. Latin American weedy rice stands out in having originated through extensive hybridization. Selection scans indicate that most genomic regions underlying weedy adaptations do not overlap with domestication targets of selection, suggesting that feralization occurs largely through changes at loci unrelated to domestication. Conclusions This is the first investigation to provide detailed genomic characterizations of weedy rice on a global scale, and the results reveal diverse genetic mechanisms underlying worldwide convergent rice feralization.

Annual ryegrass species (Lolium spp.) infest cereal crops worldwide. Ryegrass populations with multiple resistance to the acetyl coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors are an increasing problem in several European countries. We investigated the resistance pattern and level of resistance in ryegrass populations collected in Denmark, Greece and Italy and studied the diversity of mechanisms endowing resistance, both target-site and metabolism based. All populations showed high resistance indexes (RI) to the ALS inhibitors, iodosufuron-methyl-sodium + mesosulfuron-methyl (RI from 8 to 70), whereas the responses to the two ACCase inhibitors, clodinafop-propargyl and pinoxaden, differed. The Greek and Italian populations were moderately to highly resistant to clodinafop (RI > 8) and showed low to moderate resistance to pinoxaden (RI ranged from 3 to 13) except for one Italian population. In contrast, the Danish Lolium populations showed low to moderate resistance to clodinafop (RI ranged from 2 to 7) and only one population was resistant to pinoxaden. Different mutant ACCase alleles (Leu1781, Cys2027, Asn2041, Val2041, Gly2078, Arg2088, Ala2096) and ALS alleles (Gly122, Ala197, Gln197, Leu197, Ser197, Thr197, Val205, Asn376, Glu376, Leu574) endowing resistance were detected in the Greek and Italian populations. In several plants, no mutated ALS and ACCase alleles were found showing a great heterogeneity within and among the Greek and Italian populations. Conversely, no mutant ACCase alleles were identified in the four Danish populations and only one mutant ALS allele (Leu574) was detected in two Danish populations. The expression level of nitronate monooxygenase (NMO), glutathione S-transferase (GST) and cytochrome P450s (CYP72A1 and CYP72A2) varied broadly among populations and individual plants within the populations. Constitutive up-regulation of GST, CYP72A1 and CYP72A2 was detected in resistant plants respect to susceptible plants in one Danish and one Italian population. It appears that the mechanisms underlying resistance are rather complex and diversified among Lolium spp. populations from the three countries, coevolution of both target-site resistance and metabolic based herbicide resistance appears to be a common feature in Denmark and Italy. This must be considered and carefully evaluated in adopting resistance management strategies to control Lolium spp. in cereal crops.

Amaranthus palmeri S. Watson (Amaranthaceae Juss.) is a dioecious noxious weed, native to the Americas, which infests summer crops. It causes high crop losses, and rapidly evolves resistance to herbicides. In Europe, A. palmeri was recorded mostly as a casual alien, but in 2018 it was reported infesting a soybean field in Italy, and the next year two more populations were found in the same area. Experiments were conducted on these three populations to evaluate the resistance to ALS-inhibiting herbicides, to determine the main resistance mechanisms involved and assess the efficacy of alternative herbicides with different sites of action than ALS. The three populations were confirmed cross-resistant to ALS-inhibiting herbicides (thifensulfuron-methyl and imazamox). Gene sequencing identified a Trp to Leu substitution at position 574 of ALS gene in resistant plants, proving that the main resistance mechanism for the three populations is target-site related. The presence of other resistance mechanisms cannot be excluded. Metobromuron, metribuzin and glyphosate are still effective on these populations.

Background Several soybean fields in Italy were found to be infested by multiple species of Amaranthus spp. not adequately controlled by acetolactate (ALS) inhibitor herbicides. The objectives of this research were (i) to create a simplified botanical key to identify weedy amaranths; (ii) to determine the number and type of sites of action the accession are resistant to, i.e. resistance pattern; and (iii) to determine the main resistance mechanisms involved d) to evaluate the efficacy of herbicides with different site of action. Results An easy-to-use botanical key was devised and successfully used in the infested sites and results were confirmed through a species-specific molecular marker. Amaranthus retroflexus L. (redrood pigweed) was found in three sites; plants with Asp376 Glu substitution at the ALS gene were resistant to thifensulfuron-methyl. Amaranthus tuberculatus (Moq.) J.D.Sauer (waterhemp) and Amaranthus hybridus L. (smooth pigweed) accessions were cross-resistant to thifensulfuron-methyl and imazamox; most ALS-resistant plants had a point mutation at position 574. One A. hybridus accession had the substitution Trp574 Met, new for Amaranthus genus. All ALS-resistant accessions were controlled by glyphosate and metribuzin. A. retroflexus accessions were controlled by bentazon, instead an A. hybridus and some A. tuberculatus accession were not. Conclusions The simplified botanical key proposed herein could be a useful tool for farmers and weed scientists to reliably identify Amaranthus species in the field. The main resistance mechanism in the three Amaranthus species is target-site mediated. This is the first evidence of ALS-resistant A. tuberculatus outside its native North American range. © 2019 Society of Chemical Industry.

Herbicides have facilitated weed management but their incorrect use can lead to environmental contamination. Reducing herbicide use by limiting their application to a band along the crop row can decrease their environmental impact. Three field experiments were conducted in North-eastern Italy to evaluate herbicide band application systems integrated with inter-row hoeing for silage maize. Post-emergence herbicide band application (sprayed area 50% of total field, herbicide dose 50% of that recommended, application with an inter-row cultivator prototype) was compared with pre-emergence band application (sprayed area 33% of total field, herbicide dose 33% of that recommended, application with a seeder) and pre-emergence broadcast application (sprayed area 100% of total field, full recommended herbicide dose, application with a boom sprayer) that is standard management for maize. Weed density and composition were evaluated before and after post-emergence herbicide application and at crop harvest. Crop yield was also recorded. Weed density in untreated areas ranged between 5 and 15 plants m&minus, 2 in the different experiments. Optimal weed control and good yields were achieved without significant differences between all treatments. Herbicide band application can provide optimal weed control in silage maize, at the same time allowing a relevant reduction of herbicide input.

International audience; AbstractHerbicide resistance is a major weed control issue that threatens the sustainability of rice cropping systems. Its epidemiology at large scale is largely unknown. Several rice weed species have evolved resistant populations in Italy, including multiple resistant ones. The study objectives were to analyze the impact in Italian rice fields of major agronomic factors on the epidemiology of herbicide resistance and to generate a large-scale resistance risk map. The Italian Herbicide Resistance Working Group database was used to generate herbicide resistance maps. The distribution of resistant weed populations resulted as not homogeneous in the area studied, with two pockets where resistance had not been detected. To verify the situation, random sampling was done in the pockets where resistance had never been reported. Based on data from 230 Italian municipalities, three different statistics, stepwise discriminant analysis, stepwise logistic regression, and neural network, were used to correlate resistance distribution in the main Italian rice growing area with seeding type, rotation rate, and soil texture. Through the integration of complaint monitoring, mapping, and neural network analyses, we prove that a high risk of resistance evolution is associated with traditional rice cropping systems with intense monoculture rates and where water-seeding is widespread. This is the first study that determines the degree of association between herbicide resistance and a few important predictors at large scale. It also demonstrates that resistance is present in areas where it had never been reported through extensive complaint monitoring. However, these resistant populations cause medium-low density infestations, likely not alarming rice farmers. This highlights the importance of integrated agronomic techniques at cropping system level to prevent the diffusion and impact of herbicide resistance or limit it to an acceptable level. The identification of concise, yet informative, agronomic predictors of herbicide resistance diffusion can significantly facilitate effective management and improve sustainability.

Gene mutations conferring herbicide resistance may cause pleiotropic effects on plant fitness. Knowledge of these effects is important for managing the evolution of herbicide-resistant weeds. An Echinochloa crus-galli population resistant to acetolactate synthase (ALS) herbicides was collected in a maize field in north-eastern Italy and the cross-resistance pattern, resistance mechanism and fitness costs associated to mutant-resistant plants under field conditions in the presence or absence of intra-specific competition were determined. The study reports for the first time the Ala-122-Asn amino-acid change in the ALS gene that confers high levels of cross-resistance to all ALS inhibitors tested. Results of 3-year growth analysis showed that mutant resistant E. crus-galli plants had a delayed development in comparison with susceptible plants and this was registered in both competitive (3, 7, and 20 plants m-2) and non-competitive (spaced plants) situations. The number of panicles produced by resistant plants was also lower (about 40% fewer panicles) than susceptible plants under no-intraspecific competition. Instead, with the increasing competition level, the difference in panicle production at harvest time decreased until it became negligible at 20 plants m-2. Evaluation of total dry biomass as well as biomass allocation in vegetative parts did not highlight any difference between resistant and susceptible plants. Instead, panicle dry weight was higher in susceptible plants indicating that they allocated more biomass than resistant ones to the reproductive organs, especially in no-competition and in competition situations at lower plant densities. The different fitness between resistant and susceptible phenotypes suggests that keeping the infestation density as low as possible can increase the reproduction success of the susceptible phenotype and therefore contribute to lowering the ratio between resistant and susceptible alleles. If adequately embedded in a medium or long-term integrated weed management strategy, the presence of R plants with a fitness penalty provides an opportunity to minimize or reverse herbicide resistance evolution through the implementation of integrated weed management, i.e., all possible control tools available.

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.

Four Hungarian and two Italian Sorghum halepense populations harvested in maize fields were investigated to elucidate the levels and mechanisms underlying acetolactate synthase (ALS) inhibitors resistance. The two Italian populations were highly cross-resistant to all ALS inhibitors tested, and the variant ALS allele Leu574 was identified in most of the plants; no differences were observed when the plants were treated with herbicide plus malathion. This suggests that the main resistance mechanism is target-site mediated. The Hungarian populations proved to be controlled by imazamox, while they were resistant to sulfonylureas and bispyribac-Na. All Hungarian populations, but not all plants of population 12–49H, presented the variant allele Glu376. This is the first documented occurrence of the Asp-376-Glu substitution in S. halepense. ALS enzyme bioassay and treatment with malathion confirmed that at least in plants of two populations the resistance is very likely due to both target-site and enhanced metab...

Phalaris paradoxa is a competitive grass commonly found in durum wheat crops of central and southern Italy. Among the 85 populations screened from 1998 to 2008 for resistance to ACCase-inhibitors and graminicide sulfonylureas, 17 resulted as being resistant to at least one ACCase inhibitor while none of the populations showed resistance to sulfonylureas. ACCase resistance in hood canary-grass seems to be spreading rather slowly in Italy. Out of the 17 populations, seven were characterised through outdoor dose-response pot experiments to investigate resistance levels and cross-resistance patterns to ACCase-inhibitors and multiple resistance to other mode of action. Molecular bases of resistance to the recently introduced DEN herbicide pinoxaden were also investigated. Six populations were confirmed to be ACCase-resistant with various cross-resistance patterns. Two populations were resistant to all tested ACCase herbicides, with pinoxaden resistance indexes (RI) based on survival ranging from 22 to 50. The two populations have been molecularly characterised for resistance to pinoxaden. A single point-mutation in the ACCase gene was identified in each population, causing the amino-acid substitutions of Ile 1781 Val and Asp 2078 Gly in 0478L and 0025, respectively. The results suggest that resistance of P. paradoxa to pinoxaden is due to an altered target site and different mutations cause different resistance levels. The biological characteristics of the species, mainly self-pollinated, and the absence of multiple resistance allow herbicides with different modes of action to be used for controlling ACCase-resistant populations. Chemical tools should be carefully used within integrated weed management strategies.

International audience; We investigated the diversity of mechanisms conferring resistance to herbicides inhibiting acetolactate synthase (ALS) in corn poppy (Papaver rhoeas L.) and the processes underlying the selection for resistance. Six mutant ALS alleles, Arg197, His197, Leu197, Ser197, Thr197 and Leu574 were identified in five Italian populations. Different alleles were found in a same population or a same plant. Comparison of individual plant phenotype (herbicide sensitivity) and genotype (amino-acid substitution(s) at codon 197) showed that all mutant ALS alleles conferred dominant resistance to the field rate of the sulfonylurea tribenuron and moderate or no resistance to the field rate of the triazolopyrimidine florasulam. Depending on the allele, dominant or partially dominant resistance to the field rate of the imidazolinone imazamox was observed. Putative non-target-site resistance mechanisms were also likely present in the populations investigated. The derived Cleaved Amplified Polymorphic Sequence assays targeting ALS codons crucial for herbicide sensitivity developed in this work will facilitate the detection of resistance due to mutant ALS alleles. Nucleotide variation around codon 197 indicated that mutant ALS alleles evolved by multiple, independent appearances. Resistance to ALS inhibitors in P. rhoeas clearly evolved by redundant evolution of a set of mutant ALS alleles and likely of non-target-site mechanisms.

BACKGROUND: The polyploid weed Schoenoplectus mucronatus (L.) Palla has evolved target-site resistance to ALS-inhibiting herbicides in Italian rice crops. Molecular and genetic characterisation of the resistance mechanism is relevant to the evolution and management of herbicide resistance. The authors aimed (a) to study the organisation of the target-site loci in two field-selected S. mucronatus populations with different cross-resistance patterns, (b) to identify the mutations endowing resistance to ALS inhibitors and determine the role of these mutations by using transgenesis and (c) to analyse the implications for the management of the S. mucronatus populations. RESULTS: Two complete ALS genes (ALS1 and ALS2) having an intron and a third partial intronless ALS gene (ALS3) were identified. The presence of multiple ALS genes was confirmed by Southern blot analyses, and ALS loci were characterised by examining cytosine methylation. In S. mucronatus leaves, the transcripts of ALS1, ALS2 and ALS3 were detected. Two mutations endowing resistance (Pro197 to His and Trp574 to Leu) were found in both resistant populations, but at different frequencies. Tobacco plants transformed with the two resistant alleles indicated that the Pro197-to-His substitution conferred resistance to SU and TP herbicides, while the allele with the Trp574-to-Leu substitution conferred cross-resistance to SU, TP, IMI and PTB herbicides. CONCLUSION:Schoenoplectus mucronatus has multiple ALS genes characterised by methylated sites that can influence the expression profile. The two mutated alleles proved to be responsible for ALS resistance. At population level, the resistance pattern depends on the frequency of various resistant genotypes, and this influences the efficacy of various ALS-inhibiting herbicides. Copyright © 2009 Society of Chemical Industry

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

The introduction in Italy of Clearfield® rice cultivars carrying imidazolinone-resistant traits provides an efficient option to control red rice, a conspecific weed of cultivated rice. However, despite the promulgation of specific guidelines for Clearfield® technology management, imazamox red rice survivors have been reported by farmers. Forty-two fields were monitored in 2010 and 2011 throughout the Piedmont and Lombardy regions and field cases were recorded of herbicides use and agronomic practices. Whole-plant sensitivity to imazamox was assessed and the resistance mechanism was determined by molecular analysis. Twenty-six red rice populations out of 42 were imazamox-resistant and plants of all the resistant populations possess a Ser to Asn substitution at locus 653 of the ALS gene determining the target-site resistance. Farmers frequently grow Clearfield® varieties for more than two consecutive years so increasing the selection pressure exerted by imazamox and favoring the evolution of resistant red rice. To maintain the sustainability of this new technology, a proper management based on crop rotation, utilization of certified seeds and strict control of red rice escapes has to be implemented. More generally, all stakeholders must increase their awareness that the selection pressure exerted by ALS inhibitors in rice cropping system should be reduced.

BACKGROUND: The repeated use of acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides to control grass weeds has selected for resistance in Lolium spp. populations in Italy. The efficacy of pinoxaden, a recently marketed phenylpyrazoline herbicide, is of concern where resistance to ACCase inhibitors has already been ascertained. ACCase mutations associated with pinoxaden resistance were investigated, and the cross-resistance pattern to clodinafop, haloxyfop, sethoxydim, clethodim and pinoxaden was established on homo/heterozygous plants for four mutant ACCase alleles. RESULTS: Seven different mutant ACCase alleles (1781-Leu, 1999-Leu, 2041-Asn, 2041-Val, 2078-Gly, 2088-Arg and 2096-Ala) and 13 combinations with two types of mutation were detected in the pinoxaden-resistant plants. The 1781-Leu allele appears to confer a dominant resistance to pinoxaden, clodinafop, haloxyfop, sethoxydim and clethodim at 60 g AI ha−1. The 2041-Asn and 2041-Val alleles are associated with dominant or partially dominant resistance to FOPs, no substantial resistance to DIMs and a moderate resistance to pinoxaden. The 2088-Arg allele endows a partially dominant resistance to clodinafop, sethoxydim and most likely to pinoxaden. In addition, non-target-site resistance mechanisms seem to be involved in pinoxaden resistance. CONCLUSION: Almost all the ACCase mutations selected in the field by other ACCase inhibitors are likely to confer resistance to pinoxaden. Although pinoxaden is sometimes able to control FOP-resistant populations, it should not be considered as a sustainable ACCase resistance management tool. The presence of non-ACCase-based resistance mechanisms that could confer resistance to herbicides with different modes of action further complicates the resistance management strategies. Copyright © 2011 Society of Chemical Industry

Summary An acetolactate synthase (ALS)-resistant Amaranthus retroflexus biotype was collected in a soyabean crop after repeated exposure to imazethapyr and thifensulfuron-methyl in north-eastern Italy. Studies were conducted to characterise the resistance status and determine alternative post-emergence herbicides for controlling this biotype. Whole-plant bioassay revealed that the GR50 values were 1898- and 293-fold higher than those observed for the biotype susceptible to imazethapyr and imazamox respectively. The biotype also displayed high cross-resistance to sulfonylureas. Molecular analysis demonstrated that a single nucleotide substitution had occurred in domain B (TGG to TTG at position 574), conferring a change from the amino acid tryptophan to leucine in the resistant biotype. However, herbicides with other modes of action (PSII, 4-HPPD and PPO inhibitors) provided excellent control. The GR50 ratios for metribuzin, terbuthylazine and mesotrione were close to 1 and treatments with fomesafen gave 100% control of both susceptible and resistant biotypes at the recommended field dose. This study documents the first case of an imidazolinone and ALS-resistant biotype in European crops and identifies the post-emergence herbicide options available for managing this troublesome weed in soyabean crops. Alternative management strategies are also discussed.

Herbicides are the major weed control tool in most cropping systems worldwide. However, the high reliance on herbicides has led to environmental issues as well as to the evolution of herbicide-resistant biotypes. Resistance is a major concern in modern agriculture and early detection of resistant biotypes is therefore crucial for its management and prevention. In this context, a timely update of resistance biotypes distribution is fundamental to devise and implement efficient resistance management strategies. Here we present an innovative web-based application called iMAR (interactive MApping of Resistance) for the mapping of herbicide resistant biotypes. It is based on open source software tools and translates into maps the data reported in the GIRE (Italian herbicide resistance working group) database of herbicide resistance at national level. iMAR allows an automatic, easy and cost-effective updating of the maps a nd provides two different systems, "static" and "dynamic". In the first one, the user choices are guided by a hierarchical tree menu, whereas the latter is more flexible and includes a multiple choice criteria (type of resistance, weed species, region, cropping systems) that permits customized maps to be created. The generated information can be useful to various stakeholders who are involved in weed resistance management: farmers, advisors, national and local decision makers as well as the agrochemical industry. iMAR is freely available, and the system has the potential to handle large datasets and to be used for other purposes with geographical implications, such as the mapping of invasive plants or pests.

ALS gene from susceptible and field-selected Papaver rhoeas populations resistant to ALS-inhibitor herbicides was studied. The full-length cDNA and genomic sequence coding for acetolactate synthase (ALS) of a susceptible population of poppy was cloned and sequenced. Some peculiarities in poppy ALS gene were identified aligning the sequences in GenBank. Southern analysis using a 800 bp fragment of ALS showed that poppy possesses a single copy of the gene. Partial ALS genomic DNAs from nine poppy populations resistant to ALS-inhibitor herbicides and four susceptible populations, collected in central and southern Italy where resistance to ALS-inhibitors is increasing due to repeated use of sulfonylureas, were amplified and sequenced. Comparison of the coding sequences identified three independent point mutations leading to different amino acid substitutions in the deduced polypeptide sequence. The three point mutations, all at proline 197 (CCT) (based on Arabidopsis numbering) in the conserved domain A of the gene, included a change of Pro to His (CAT), to Thr (ACT) or to Ser (TCT). These mutations cause similar cross-resistance patterns. Analysis of the progeny of two crosses between resistant (R) and susceptible (S) biotypes indicated that resistance is inherited as a dominant monogenic trait, although seed dormancy may interfere with a correct segregation of R and S biotypes in the progeny. These are the first mutations completely characterised in poppy ALS gene that confer resistance to ALS-inhibitor herbicides.