Avena fatua (wild oat) populations with resistance (R) to one or more herbicides have been described in numerous cropping systems worldwide. We previously reported that the R3 and R4 wild oat populations from Montana, USA, were resistant to four herbicides representing three different modes of action: tralkoxydim [acetyl-CoA carboxylase (ACCase] inhibitor), imazamethabenz and flucarbazone [acetolactate synthase (ALS) inhibitors] and difenzoquat (growth inhibitor). We now quantify resistance levels of these populations to triallate [very long chain fatty acid (VLCFA) biosynthesis inhibitor], pinoxaden (ACCase inhibitor) and paraquat (photosystem I inhibitor). Glasshouse doseresponse experiments showed that, compared with the means of two susceptible (S) populations, the R3 and R4 populations were 17.5-and 18.1-fold more resistant to triallate, 3.6-and 3.7-fold more resistant to pinoxaden, respectively, and 3.2-fold (R3) more resistant to paraquat. Pre-treatment of R plants with the cytochrome P450 inhibitor malathion partially reversed the resistance phenotype for flucarbazone (both populations), imazamethabenz (R4), difenzoquat (R4) and pinoxaden (R3), but not for tralkoxydim, fenoxaprop-P-ethyl or triallate. Target site point mutations known to confer resistance to ALS or ACCase inhibitors were not detected via DNA sequencing and allele-specific PCR assays in R plants, suggesting the involvement of non-target site resistance mechanism(s) for these herbicides. Together, our results complete the initial characterisation of wild oat populations that are resistant to seven (R3) or six (R4) herbicides from five or four mode of action families respectively.

Th e evolution of weed biotypes resistant to multiple herbicide modes of action, here termed multiple herbicide resistance, is a growing problem around the world. We investigated two multiple herbicide resistant (MHR) wild oat (Avena fatua L.) populations from Montana and hypothesized that they would exhibit fi tness costs compared with two herbicide-susceptible (HS) populations. Dose-response tests showed that the MHR populations were resistant to difenzoquat (a membrane disruptor), imazamethabenz (an acetolactate synthase [ALS] inhibitor), fl ucarbazone (an ALS inhibitor), and tralkoxydim (an acetyl-CoA carboxylase inhibitor). In greenhouse studies, we assessed diff erences between MHR and HS populations in seed germination, photosynthetic parameters, plant growth, and reproduction. Seeds of one HS population germinated more at cold temperature (4.9°C) and less at high temperature (29.6°C) compared with the other populations. Plants of this HS population also had lower stomatal conductance (23%), intercellular CO 2 concentration (7.5%), and transpiration (15.3%) than the other populations, but there were no diff erences in photosynthetic rates between any populations. Also, there were no diff erences in relative growth rate among all HS and MHR populations. Th e MHR populations initiated seed production several days sooner than the HS populations; however, HS populations produced 67% more tillers, and one HS population ultimately produced 43% more seeds than the MHR populations, indicating a potential fi tness cost of resistance. With the exception of seed production diff erences, our results do not indicate a consistent fi tness cost. More research is needed in fi eld settings and with resource competition to further evaluate fi tness costs in MHR populations.

Although the individual and collective contributions of these DEGs and proteins to MHR remain to be determined, our results support the idea that intensive herbicide use has selected for MHR populations with altered, constitutively regulated patterns of gene expression that are similar to those in abiotic stress-tolerant plants. © 2017 Society of Chemical Industry.

Extensive herbicide usage has led to the evolution of resistant weed populations that cause substantial crop yield losses and increase production costs. The multiple herbicide resistant (MHR) Avena fatua L. populations utilized in this study are resistant to members of all selective herbicide families, across five modes of action, available for A. fatua control in U.S. small grain production, and thus pose significant agronomic and economic threats. Resistance to ALS and ACCase inhibitors is not conferred by target site mutations, indicating that non-target site resistance mechanisms are involved. To investigate the potential involvement of glutathione-related enzymes in the MHR phenotype, we used a combination of proteomic, biochemical, and immunological approaches to compare their constitutive activities in herbicide susceptible (HS1 and HS2) and MHR (MHR3 and MHR4) A. fatua plants. Proteomic analysis identified three tau and one phi glutathione S-transferases (GSTs) present at higher levels in MHR compared to HS plants, while immunoassays revealed elevated levels of lambda, phi, and tau GSTs. GST specific activity towards 1-chloro-2,4-dinitrobenzene was 1.2-fold higher in MHR4 than in HS1 plants and 1.3- and 1.2-fold higher in MHR3 than in HS1 and HS2 plants, respectively. However, GST specific activities towards fenoxaprop-P-ethyl and imazamethabenz-methyl were not different between untreated MHR and HS plants. Dehydroascorbate reductase specific activity was 1.4-fold higher in MHR than HS plants. Pretreatment with the GST inhibitor NBD-Cl did not affect MHR sensitivity to fenoxaprop-P-ethyl application, while the herbicide safener and GST inducer mefenpyr reduced the efficacy of low doses of fenoxaprop-P-ethyl on MHR4 but not MHR3 plants. Mefenpyr treatment also partially reduced the efficacy of thiencarbazone-methyl or mesosulfuron-methyl on MHR3 or MHR4 plants, respectively. Overall, the GSTs described here are not directly involved in enhanced rates of fenoxaprop-P-ethyl or imazamethabenz-methyl metabolism in MHR A. fatua. Instead, we propose that the constitutively elevated GST proteins and related enzymes in MHR plants are representative of a larger, more global suite of abiotic stress-related changes.

Ecological theory predicts that fitness costs of herbicide resistance should lead to the reduced relative abundance of resistant populations upon the cessation of herbicide use. This greenhouse research investigated the potential fitness costs of two multiple herbicide resistant (MHR) wild oat (Avena fatua) populations, an economically important weed that affects cereal and pulse crop production in the Northern Great Plains of North America. We compared the competitive ability of two MHR and two herbicide susceptible (HS) A. fatua populations along a gradient of biotic and abiotic stresses The biotic stress was imposed by three levels of wheat (Triticum aestivum) competition (0, 4, and 8 individuals pot−1) and an abiotic stress by three nitrogen (N) fertilization rates (0, 50 and 100 kg N ha−1). Data were analyzed with linear mixed-effects models and results showed that the biomass of all A. fatua populations decreased with increasing T. aestivum competition at all N rates. Similarly, A. fatua relative growth rate (RGR) decreased with increasing T. aestivum competition at the medium and high N rates but there was no response with 0 N. There were no differences between the levels of biomass or RGR of HS and MHR populations in response to T. aestivum competition. Overall, the results indicate that MHR does not confer growth-related fitness costs in these A. fatua populations, and that their relative abundance will not be diminished with respect to HS populations in the absence of herbicide treatment.

Plants are routinely confronted with numerous biotic and abiotic stressors, and in response have evolved highly effective strategies of systemic acquired resistance (SAR) and systemic acquired acclimation (SAA), respectively. A much more evolutionarily recent abiotic stress is the application of herbicides to control weedy plants, and their intensive use has selected for resistant weed populations that cause substantial crop yield losses and increase production costs. Non-target site resistance (NTSR) to herbicides is rapidly increasing worldwide and is associated with alterations in generalized stress defense networks. This work investigated protein post-translational modifications associated with NTSR in multiple herbicide resistant (MHR) Avena fatua, and their commonalities with those of SAR and SAA. We used proteomic, biochemical, and immunological approaches to compare constitutive protein profiles in MHR and herbicide susceptible (HS) A. fatua populations. Phosphoproteome and redox proteome surveys showed that post-translational modifications of proteins with functions in core cellular processes were reduced in MHR plants, while those involved in xenobiotic and stress response, reactive oxygen species detoxification and redox maintenance, heat shock response, and intracellular signaling were elevated in MHR as compared to HS plants. More specifically, MHR plants contained constitutively elevated levels of three protein kinases including the lectin S-receptor-like serine/threonine-protein kinase LecRK2, a well-characterized component of SAR. Analyses of superoxide dismutase enzyme activity and protein levels did not reveal constitutive differences between MHR and HS plants. The overall results support the idea that herbicide stress is perceived similarly to other abiotic stresses, and that A. fatua NTSR shares analogous features with SAR and SAA. We speculate that MHR A. fatua's previous exposure to sublethal herbicide doses, as well as earlier evolution under a diversity of abiotic and biotic stressors, has led to a heightened state of stress preparedness that includes NTSR to a number of unrelated herbicides.

SummaryExtensive herbicide usage has led to the evolution of resistant weed populations that cause substantial crop yield losses and increase production costs. The multiple herbicide-resistant (MHR) Avena fatua populations utilised in this study are resistant to members of all selective herbicide families, across five modes of action, available for A. fatua control in US small grain production, and thus pose significant agronomic and economic threats. Resistance to acetolactate synthase and acetylCoA carboxylase inhibitors is not conferred by known target site mutations, indicating that non-target site resistance (NTSR) mechanisms are involved. Understanding the inheritance of NTS MHR is of upmost importance for continued agricultural productivity in the face of the rapid increase in resistant weed populations worldwide. As few studies have examined the inheritance of NTSR in autogamous weeds, we investigated the inheritance and genetic control of NTSR in the highly autogamous, allohexaploid species A. fatua. We found that NTSR in MHR A. fatua is controlled by three separate, closely-linked nuclear genes for flucarbazone-sodium, imazamethabenz-methyl and pinoxaden. The single-gene NTSR inheritance patterns reported here contrast with other examples in allogamous species and illustrate the diversity of evolutionary responses to strong selection.

Choline monooxygenase (CMO) is the first regulatory enzyme in the biosynthetic pathway for glycine betaine, an effective osmoprotectant in Kochia scoparia, a highly drought-and salt-tolerant species. In seedlings, CMO transcript levels are rapidly increased in response to both NaCl and osmotic stress treatments. The mRNA level in shoots was substantially higher than in roots. The rapid induction seen in whole plants was in contrast to the apparent downregulation observed in suspension-cultured K. scoparia cells in response to the same salt stress. Treatment with exogenous abscisic acid (ABA) or fluridone shows that CMO induction proceeds via an ABA-independent signal transduction pathway. Examination of the CMO upstream regulatory region reveals a number of stress response-related elements, some of which may be involved in the stress tolerance shown by this species.