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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.

Poor control of Amaranthus spp. with herbicides inhibiting acetolactate synthase (ALS) has been observed for several years in soybean fields in north-eastern Italy, but to date only a few ALS-resistant populations have been confirmed. An extensive sampling of putatively resistant Amaranthus accessions was completed in the Friuli Venezia Giulia region, across an arable land area of about 3000 km2. In total, 58 accessions were tested to confirm their resistance status, recognize the Amaranthus species, identify the mutant ALS alleles endowing the resistance and determine the efficacy of 3 pre-emergence herbicides. Most accessions resulted in cross-resistance to thifensulfuron-methyl and imazamox. Genomic DNA were extracted from single seeds with a newly developed protocol; an allele-specific PCR assay revealed the presence of the 574-leucine in 20 accessions, of the 574-methionine in 22, and of both alleles in 9 accessions. The two variants showed a different spatial distribution. All resistant populations were ascribed to A. hybridus. A. hybridus resistant to ALS herbicides is well-established in this Italian region and its resistance is due to two ALS mutant alleles. Metribuzin, clomazone and metobromuron can be used as alternative herbicides to be applied in pre-emergence and they should be integrated into the management strategies to limit the spread of 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.

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.

Sorghum halepense (L.) Pers. is a troublesome weed of maize which reproduces through seeds and rhizomes. Resistance to ACCase-inhibiting herbicides due to a target-site mutation Ile-Asn in position 2041 of the ACCase gene was confirmed in several populations in Italy. The presence of fitness costs related to the allelic variant Ile2041Asn has never been investigated in ACCase-resistant S. halepense. Three growth analyses pot experiments were performed starting from rhizome buds (Exp1 and Exp3) or seed (Exp2). Plant development was monitored throughout the life cycle through destructive samplings. The mutation responsible for resistance to ACCase inhibitors was detected in all plants included in the experiments using a Cleaved Amplified Polymorphic Sequence method. Classical growth analysis was performed for the multiple sampling experiments (Exp1 and Exp2) and allometric relationships were calculated for the different dry weight plant parts in all experiments. Allometric relationships showed that susceptible (S) and ACCase resistant (R) biotypes allocated biomass differently in the reproductive structures: S populations allocated on average 30% more biomass in the panicles, whereas R populations allocated 12% more biomass to rhizomes than S populations. This was more evident when the experiments started from rhizomes buds. Reproductive allocation and reproductive effort were higher in S biotypes and R biotypes, respectively. The higher production of rhizomes and the lower production of seeds of the R biotype can significantly impact the evolution of herbicide resistance under continuous application of herbicides with the same mode of action. A medium-long term resistance management strategy which implements appropriate soil tillage and crop rotation taking into account the different reproductive attitude of biotypes S and R should be devised.

Glyphosate-resistant biotypes of Eleusine indica (L.) Gaertn. have been detected in Asia, the Americas but not in Europe. The aim of this study was to evaluate resistance levels and possible target site resistance mechanisms of an E. indica biotype (19-1) collected from a plant nursery in Southern Italy where poor glyphosate efficacy was reported. Two dose-response experiments were conducted to evaluate the sensitivity of biotype 19-1 to glyphosate in comparison with two susceptible checks. 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequencing was performed to identify possible mutations conferring the resistance. The susceptible biotypes were completely controlled at the glyphosate recommended field dose of 360 g ae ha&minus, 1, while 50% of the plants of biotype 19-1 survived at 1440 g ae ha&minus, 1. The resistance index of biotype 19-1 ranged between 5.8 and 7.3 for the response variables of fresh weight reduction and plant survival, respectively. All the plants surviving glyphosate application and sampled for DNA analyses had the point mutation P106A. The biotype 19-1 can be confirmed as glyphosate-resistant, representing the first glyphosate-resistant population of E. indica in Europe.

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.

Long-term national European weed surveys, large scale classical phytosociological programs and camera-based documentation systems lead to results which can be documented in form of maps. Comparisons of these visual representations of relative weed positions can be used for the prediction of changing weed spectra and of plant biodiversity changes. Statistical methods connected with mapping software are used for the analysis of environmental factors and of farm managing practices influencing the occurrence of weeds. Maps produced by sensor-driven weed detection devices still differ considerably from maps produced via classical phytosociological approaches. Computer algorithms may allow the precise identification of some weeds in camera images. The present technical solutions are, however, still far from those achieved by experienced botanists. Many weed detection tools based on algorithms are not able to distinguish between closely related weeds yet. A few European countries have a long tradition of surveying weeds in major crops by traditional tools. Various software packages are employed for the analysis, documentation and visualisation of survey results. Large scale comprehensive maps including the infestation of crops over different countries are, however, often biased as not every national research group uses the same methods for the assessment of weed infestation. The ranking of the most common species seems, however, to allow comparable conclusions. The recognition of trends in spectrum changes can only be derived from long term studies as we see it. Our review reflects discussions within the Weed Mapping Working Group of the European Weed Research Society over the last ten years. We try to identify new research trends and to respond accordingly with new research projects. What we see today is a shift from traditional mapping approaches towards the use of digital devices as for example in precision farming projects. Another issue of increasing importance is the mapping of herbicide resistant biotypes.

Empirical observations generally indicate a shifting and decreased Lolium spp. susceptibility to glyphosate in Italy. This is likely due to the long history of glyphosate use and to the sub-lethal doses commonly used. There is, therefore, a need to determine the variability of response of Lolium spp. to glyphosate and identify the optimum field dose. To perform a sensitivity analysis on Lolium spp. populations in an agriculture area, collection sites were mainly chosen where glyphosate had not been applied intensely. Known glyphosate-resistant or in-shifting populations were included. Two outdoor dose-response pot experiments, including eleven doses of glyphosate, were conducted. The dose to control at least 93%&ndash, 95% of susceptible Lolium spp. was around 450 g a.e. ha-1. However, to preserve its efficacy in the long term, it would be desirable not to have survivors, and this was reached at a glyphosate dose of 560 &plusmn, 88 g a.e. ha-1. Taking into account the variability of response among populations, it was established that the optimal dose of glyphosate to control Lolium spp. in Italy up to the stage BBCH 21 has to be at least 700 g a.e. ha-1. As a consequence, it is recommended to increase the label recommended field rate for Lolium spp. control in Italy to a minimum of 720 g a.e. ha-1.

Empirical observations generally indicate a shifting and decreased Lolium spp. susceptibility to glyphosate in Italy. This is likely due to the long history of glyphosate use and to the sub-lethal doses commonly used. There is, therefore, a need to determine the variability of response of Lolium spp. to glyphosate and identify the optimum field dose. To perform a sensitivity analysis on Lolium spp. populations in an agriculture area, collection sites were mainly chosen where glyphosate had not been applied intensely. Known glyphosate-resistant or in-shifting populations were included. Two outdoor dose-response pot experiments, including eleven doses of glyphosate, were conducted. The dose to control at least 93%–95% of susceptible Lolium spp. was around 450 g a.e. ha−1. However, to preserve its efficacy in the long term, it would be desirable not to have survivors, and this was reached at a glyphosate dose of 560 ± 88 g a.e. ha−1. Taking into account the variability of response among populations, it was established that the optimal dose of glyphosate to control Lolium spp. in Italy up to the stage BBCH 21 has to be at least 700 g a.e. ha−1. As a consequence, it is recommended to increase the label recommended field rate for Lolium spp. control in Italy to a minimum of 720 g a.e. ha−1.

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.

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.

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...

Integrated Pest Management (IPM) aims to promote physical and biological regulation strategies that help farmers contain populations of pests (pathogens, animal pests and weeds) and to finally reduce the reliance on pesticides. It is based on the holistic combination of multiple management measures rather than on the sum of single methods, each of them having only small effects on pests reduction. Thus, to analyse the interactions between IPM measures and to evaluate the sustainability of their implementation, we require an approach considering the whole cropping system (CS), i.e. a functional entity whose complexity is more than the sum of its parts. A network of European experiments at the CS level was set up recently, and aimed at sharing data and expertise to enhance knowledge of IPM. Comparison of existing methodologies highlighted a diversity of CS designs and experimental layouts. We deduced that the concept of CS itself was viewed differently among scientists, and this affected experimental protocols. Other differences were related to the research context and objectives. Some experiments aimed to explore very innovative strategies and generated knowledge on both their effects on the agroecosystem and their ability to satisfy a set of performance targets, while others aimed to provide quickly adoptable solutions for local farmers in line with the current socio-economic constraints. In some research programmes, the experiment was part of the CS design process — and tested CS were regularly revised based on an continuous improvement loop — while in other cases CS were kept stable across years so as to enable the evaluation of their long-term cumulative effects. A critical aspect contributing to the diversity among CS experiments was the distinction between a factorial design of experimental CS and systemic approaches: factorial experiments allowed quantification of the effects of each IPM component regardless of the consistency between components defining the CS. In contrast, systemic approaches focused on the overall evaluation of CS designed with consideration of their consistency, hence maximising their ability to meet the objectives. Because CS experiments represent a huge investment in terms of economics and time, preliminary reflections of the relevance of the experimental strategy is of critical importance.

Italian ryegrass populations investigated in this study were harvested in an alfalfa-based cropping system. In that system, the agronomic practices and chemical weed management, based on the use of aryloxyphenoxy-propionates herbicides (i.e., quizalofop ethyl ester), were optimized to obtain a dual seed–forage production. Five of seven populations tested were confirmed resistant to quizalofop ethyl ester with resistance indexes ranging from 4.5 to >209. Both target- and nontarget-site resistance mechanisms were most likely involved. Three allelic variants were detected (Ile-1781–Leu, Trp-2027–Cys, and Ile-2041–Asn) in four resistant populations, whereas no known mutations were found in one resistant population. The herbicide treatment on Italian ryegrass plants at different phenological stages suggested that to control regrowth, it is necessary to use two to fives times the herbicide dose suitable for younger plants. This situation is encountered in fields when Italian ryegrass plants need to be controlled to maximize the alfalfa seed production, and it is comparable to using a sublethal herbicide dose, leading to the selection of herbicide-resistant biotypes. In such a situation, the cropping system is not sustainable, and integrated weed management should be implemented to deplete the soil weed seed bank and prevent new weed seed production.

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.

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

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.