Development of a rapid detection assay for acetolactate synthase inhibitors resistance in three Amaranthus weed species through loop-mediated isothermal amplification.

BACKGROUND: The early detection of herbicide resistance in weeds is a key factor to avoid herbicide waste and improve agriculture sustainability. The present study aimed to develop and validate an allele-specific loop-mediated isothermal amplification (AS-LAMP) assay for the quick on-site detection of the resistance-endowing point mutation Trp-574-Leu in the acetolactate synthase (ALS) gene in three widely diffused Amaranthus weed species: Amaranthus retroflexus, Amaranthus hybridus and Amaranthus tuberculatus. RESULTS: The AS-LAMP protocol was developed on wild-type and ALS-mutant plants of the three species and revealed that the amplification approach with only the primer set specific for the mutant allele (574-Leu) was the most promising. The validation and estimation of the AS-LAMP performance evaluated by comparing the results with those of the molecular marker (cleaved amplified polymorphic sequences) indicated that, although the sensitivity and specificity were relatively high in all species (overall 100 and > 65%, respectively), precision was high for A. hybridus L. and A. retroflexus L. (75 and 79%, respectively), but quite low for A. tuberculatus (Moq.) J. D. Sauer (59%). The LAMP assay was also effective on crude genomic DNA extraction, allowing the quick detection of mutant plants in field situation (on site resistance detection). CONCLUSION: The proposed AS-LAMP method has proven to be a promising technique for rapid detection of resistance as a result of Trp-574-Leu on the two monoecious weedy Amaranthus species but resulted less effective in the genetically variable dioecious species A. tuberculatus. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Can allele-specific loop-mediated isothermal amplification be used for rapid detection of target-site herbicide resistance in Lolium spp.?

BACKGROUND: Herbicide resistance is one of the threats to modern agriculture and its early detection is one of the most effective components for sustainable resistance management strategies. Many techniques have been used for target-site-resistance detection. Allele-Specific Loop-Mediated Isothermal Amplification (AS-LAMP) was evaluated as a possible rapid diagnostic method for acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibiting herbicides resistance in Lolium spp. RESULTS: AS-LAMP protocols were set up for the most frequent mutations responsible for herbicide resistance to ALS (positions 197, 376 and 574) and ACCase (positions 1781, 2041 and 2078) inhibitors in previously characterized and genotyped Lolium spp. POPULATIONS: A validation step on new putative resistant populations gave the overview of a possible use of this tool for herbicide resistance diagnosis in Lolium spp. Regarding the ACCase inhibitor pinoxaden, in more than 65% of the analysed plants, the LAMP assay and genotyping were in keeping, whereas the results were not consistent when ALS inhibitors resistance was considered. Limitations on the use of this technique for herbicide resistance detection in the allogamous Lolium spp. are discussed. CONCLUSIONS: The LAMP method used for the detection of target-site resistance in weed species could be applicable with target genes that do not have high genetic variability, such as ACCase gene in Lolium spp.

Diversity and Spread of Acetolactate Synthase Allelic Variants at Position 574 Endowing Resistance in Amaranthus hybridus in Italy.

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.

Target-Site Mutations and Expression of ALS Gene Copies Vary According to Echinochloa Species.

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.

Sensitivity Analysis of Italian Lolium spp. to Glyphosate in Agricultural Environments.

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.

Diversified Resistance Mechanisms in Multi-Resistant Lolium spp. in Three European Countries.

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.

A New Ala-122-Asn Amino Acid Change Confers Decreased Fitness to ALS-Resistant Echinochloa crus-galli.

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.

Occurrence of Different Resistance Mechanisms to Acetolactate Synthase Inhibitors in European Sorghum halepense.

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 metabolism of P450 enzymes.

iMAR: An Interactive Web-Based Application for Mapping Herbicide Resistant Weeds.

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.

Protocols for Robust Herbicide Resistance Testing in Different Weed Species.

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.

Allelic variation of the ACCase gene and response to ACCase-inhibiting herbicides in pinoxaden-resistant Lolium spp.

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.