Two populations of Palmer amaranth from New Mexico have been confirmed to be resistant to glyphosate. In the present study, the molecular basis of resistance and the mode of inheritance of resistance in those populations were investigated. Quantitative real-time polymerase chain reaction analysis indicated up to an eightfold increase in genomic EPSPS copy number in glyphosate resistant plants compared with susceptible plants. The relative genomic EPSPS copy number of resistant plants was positively correlated with the relative EPSPS cDNA expression levels. Eight hours after treatment with glyphosate, the shikimate accumulation levels in resistant plants were negatively correlated with the genomic EPSPS copy numbers. Multiple sequencing of the EPSPS cDNA of resistant plants did not reveal any glyphosate resistance-conferring mutations. The evaluation of F1, reciprocal F1, and F2 Palmer amaranth families indicated that resistance to glyphosate does not follow a single-gene segregation pattern. Results suggest that the EPSPS amplification is the primary molecular basis of resistance in glyphosate resistant populations of Palmer amaranth from New Mexico.

Tribenuron-methyl has been used widely for the last 15 yr to control white mustard in cereal crops from southern Spain. Since 2007, several cases of tribenuron-methyl resistance have been reported in wheat fields. Greenhouse and laboratory studies were conducted to characterize the mechanism of suspected tribenuron-methyl resistance in a white mustard biotype (hereafter AR16) from Malaga (southern Spain). Under greenhouse conditions, the dose (g ai ha−1) inhibiting fresh weight by 50% (ED50) was 5.20 and 0.57 for the AR16 and AR3 (known susceptible) biotypes, respectively. With the use of 14C-tribenuron-methyl, absorption and translocation from treated leaves were similar between biotypes. Thin-layer chromatography indicated foliar metabolism of tribenuron-methyl was low in both R and S biotypes. Assays on the binding affinity of tribenuron-methyl on acetolactate synthase (ALS) revealed enzyme activity was reduced by 50% (I50 value) at 638.7 and 0.23 nM for the AR16 and AR3 biotypes, respectively. This resulted in 2,777-fold greater resistance to tribenuron-methyl for the AR16 compared to AR3 biotype. Sequencing the gene encoding ALS, a proline/serine amino-acid substitution, was detected in position 197 of the A domain. Based on these results, it is concluded that tribenuron-methyl resistance in the AR16 biotype is due to a target-site mutation in the ALS enzyme, resulting in a lack of affinity to tribenuron-methyl.

Global occurrences of herbicide resistant weed populations have increased the demand for development of new herbicides targeting novel mechanisms of action. Metagenomic approaches to natural drug discovery offer potential for isolating weed suppressive compounds from microorganisms. In past research, traditional techniques entailed isolating compounds from living organisms, whereas metagenomic approaches involve extracting fragments of DNA from soil and exploring for compounds of interest produced by the transformed hosts. Several herbicidal compounds have been isolated from soil bacteria through culturing methods and have led to the development of popular herbicides, such as glufosinate. In this review, we discuss the emergence of metagenomic approaches for weed management in the context of natural product discovery using traditional culture-dependent isolation and the more recent culture-independent methods. The same techniques can be used to isolate herbicide resistance genes. Adoption of metagenomic approaches in pest management research can lead to novel control strategies in cropping and landscape systems.

As cases of resistance to herbicides escalate worldwide, there is increasing demand from growers to test for weed resistance and learn how to manage it. Scientists have developed resistance-testing protocols for numerous herbicides and weed species. Growers need immediate answers and scientists are faced with the daunting task of testing an increasingly large number of samples across a variety of species and herbicides. Quick tests have been, and continue to be, developed to address this need, although classical tests are still the norm. Newer methods involve molecular techniques. Whereas the classical whole-plant assay tests for resistance regardless of the mechanism, many quick tests are limited by specificity to an herbicide, mode of action, or mechanism of resistance. Advancing knowledge in weed biology and genomics allows for refinements in sampling and testing protocols. Thus, approaches in resistance testing continue to diversify, which can confound the less experienced. We aim to help weed science practitioners resolve questions pertaining to the testing of herbicide resistance, starting with field surveys and sampling methods, herbicide screening methods, data analysis, and, finally, interpretation. More specifically, this article discusses approaches for sampling plants for resistance confirmation assays, provides brief overviews on the biological and statistical basis for designing and analyzing dose–response tests, and discusses alternative procedures for rapid resistance confirmation, including molecular-based assays. Resistance confirmation procedures often need to be slightly modified to suit a specific situation; thus, the general requirements as well as pros and cons of quick assays and DNA-based assays are contrasted. Ultimately, weed resistance testing research, as well as resistance management decisions arising from research, needs to be practical, feasible, and grounded in science-based methods.

Synthetic auxin herbicides are commonly used in forage, pasture, range, and turfgrass settings for dicotyledonous weed control. Aminocyclopyrachlor (AMCP) is a newly developed pyrimidine carboxylic acid with a chemical structure and mode of action similar to the pyridine carboxylic acids—aminopyralid, clopyralid, and picloram. Injury to sensitive dicotyledonous plants has been observed following exposure to monocotyledonous plant material previously treated with pyridine compounds. The absorption, translocation, and metabolism of AMCP has been documented in susceptible broadleaf weeds; however, no information is available, to our knowledge, regarding AMCP fate in tolerant Poaceae, which may serve as the vector for off-target plant injury. Based on this premise, research was conducted to characterize absorption, translocation, and metabolism of AMCP in tall fescue. 14C-AMCP was applied to single tiller tall fescue plant foliage under controlled laboratory conditions at North Carolina State University (Raleigh, NC). Radiation was quantified in leaf wash, treated leaf, foliage, crown, roots, and root exudates at 3, 12, 24, 48, 96, and 192 h after treatment (HAT). 14C-AMCP was rapidly absorbed by tall fescue, reaching 38 and 68% at 3 and 48 HAT, respectively. Translocation of 14C-AMCP was limited to the foliage, which reached maximum translocation (34%) at 96 HAT. Most of the recovered 14C-AMCP remained in the leaf wash, treated leaf, or foliage, whereas minimal radiation was detected in the crown, roots, or root exudates throughout the 192-h period. No AMCP metabolism was observed in tall fescue through the 192 HAT. These data suggest AMCP applied to tall fescue can remain bioavailable, and mishandling treated plant material could result in off-target injury.

Exposure of soybean to dicamba can result in leaf malformation and sometimes yield loss, but it is unclear how yield components are affected by exposure to low quantities of this herbicide. The objectives were to characterize soybean injury and quantify changes in seed yield and yield components of soybean plants exposed to dicamba, and determine if seed yield loss can be estimated from visual injury ratings. Nine dicamba rates (0, 0.06, 0.23, 0.57, 1.1, 2.3, 4.5, 9.1, and 22.7 g ae ha−1) were applied at three growth stages (V2 – two trifoliates, V5-five trifoliates, or R2-full flowering soybean) to Beck's brand ‘342NRR’ soybean planted near Lafayette, IN, in 2009 and 2010 and near Fowler, IN, in 2009. Visually estimated soybean injury of 20% at the V2, V5, or R2 timing was 0.676 to 0.937 g ha−1 dicamba at 14 d after treatment (DAT) and 0.359 to 1.37 g ha−1 dicamba at 28 DAT. Seed yield was reduced by 5% from 0.042 to 0.528 g ha−1 dicamba and a 10% reduction was caused by 0.169 to 1.1 g ha−1 dicamba. The number of seeds m−2, pods m−2, reproductive nodes m−2, and nodes m−2 were the most sensitive yield components. Path analysis indicated that dicamba reduced seeds m−2, pods m−2, reproductive nodes m−2, and nodes m−2 which were the main causes of seed yield loss from dicamba exposure. The correlation of seed yield loss and visual soybean injury was significant (P < 0.0001) for both the V2 treatment timing (R2 = 0.92) and the V5 and R2 treatment timings (R2 = 0.91). Early-season injury rating of 8% at the V2 treatment and 2% at the V5 or R2 treatments caused 10% or more yield loss.

A population of oriental mustard from Port Broughton in South Australia was reported as not being controlled by 2,4-D. Dose response experiments determined this population was resistant to both 2,4-D and MCPA, requiring greater than 20 times more herbicide for equivalent control compared to a known susceptible population (from Roseworthy, South Australia) and a population resistant only to the acetohydroxyacid synthase (AHAS)-inhibiting herbicides (from Tumby Bay, South Australia). The Port Broughton population was also found to be resistant to three chemical groups that inhibit AHAS; however, the level of resistance was lower than the known acetolactate synthase–resistant population from Tumby Bay. Herbicides from other modes of action were able to control the Port Broughton population. Assays of isolated AHAS from the Port Broughton population showed high levels of resistance to the sulfonylurea and sulfonamide herbicide groups, but not to the imidazolinone herbicides. A single nucleotide change in the AHAS gene that predicted a Pro to Ser substitution at position 197 in the protein was identified in the Port Broughton population. This population of oriental mustard has evolved multiple resistance to AHAS-inhibiting herbicides (AHAS inhibitors) and auxinic herbicides, through a mutation in AHAS and a second nontarget-site mechanism. Whether the same mechanism provides resistance to both AHAS inhibitors and auxinic herbicides remains to be determined. Multiple resistance to auxinic herbicides and AHAS inhibitors in the Port Broughton population will make control of this population more difficult.

Annual bluegrass is commonly controlled by acetolactate synthase (ALS)-inhibiting herbicides in managed turfgrass. An annual bluegrass population with suspected resistance to ALS-inhibiting herbicides was collected from Grand National Golf Course in Opelika, AL (GN population). Subsequent testing confirmed resistance of the GN population to foramsulfuron, trifloxysulfuron, bispyribac-sodium (bispyribac), and imazaquin when compared to a susceptible population collected locally at Auburn University (AU population). Sequencing of the ALS gene revealed a point mutation resulting in an amino acid substitution at Trp574. Cloning of the ALS gene surrounding the Trp574 region yielded two distinct ALS gene sequences: one producing Trp574 and one producing Leu574. Trp574 to Leu has been previously correlated with resistance to ALS-inhibiting herbicides. Both AU and GN gene sequences contained other similar silent and missense mutations. This research confirms resistance of annual bluegrass to ALS-inhibiting herbicides with Trp574 to Leu amino acid substitution being the most likely mode of resistance based on past literature.

Kochia is a troublesome weed throughout the western United States. Although glyphosate effectively controls kochia, poor control was observed in several no-till fields in Kansas. The objectives of this research were to evaluate kochia populations response to glyphosate and examine the mechanism that causes differential response to glyphosate. Glyphosate was applied at 0, 54, 109, 218, 435, 870, 1305, 1740, 3480, and 5220 g ae ha−1 on 10 kochia populations. In general, kochia populations differed in their response to glyphosate. At 21 d after treatment, injury from glyphosate applied at 870 g ha−1 range from 4 to 91%. In addition, glyphosate rate required to cause 50% visible injury (GR50) ranged from 470 to 2149 g ha−1. Differences in glyphosate absorption and translocation and kochia mineral content were not sufficient to explain differential kochia response to glyphosate.

Common lambsquarters tolerance to glyphosate is problematic because of the species' widespread distribution, competitive ability with many crop species, the widespread use of glyphosate in agriculture, and the weed's potential to develop decreased sensitivity to multiple herbicide sites of action. The mechanism that confers common lambsquarters tolerance to glyphosate is not known. Therefore, we conducted experiments to determine the mechanism of tolerance to glyphosate in an accession of common lambsquarters from Indiana relative to a sensitive accession from Wisconsin. The ED50 (the effective dose that reduced shoot mass 50% relative to nontreated plants) value for the tolerant accession (1.6 kg ae ha−1 ± 0.4 standard error of the mean [SEM]) was eightfold greater than the ED50 for the sensitive accession (0.2 kg ae ha−1 ± 0.2 SEM) 28 d after treatment. The glyphosate target-site (EPSPS) DNA sequence at proline 106, shikimate accumulation as an estimate of EPSPS sensitivity, and EPSPS protein abundance did not differ between accessions. Absorption of 14C-glyphosate was slightly greater in the tolerant accession than it was in the sensitive accession at 48 and 72 h after treatment (HAT). However, the tolerant accession translocated a smaller percentage of absorbed 14C-glyphosate to the tissue above the treated leaf, which included the shoot apical meristem, at 24, 48, and 72 HAT (P ≤ 0.05, 0.01, and 0.10, respectively). These results suggest an important role of reduced translocation in conferring tolerance of common lambsquarters to glyphosate.

Mechanisms of herbicide resistance were studied in a quizalofop–ethyl-resistant barnyardgrass biotype. Acetyl-coenzyme A carboxylase (ACCase) sensitivity to quizalofop-p-ethyl was measured by high-performance liquid chromatography and the trend in ACCase gene expression over time was determined using real-time polymerase chain reaction. The results showed that an insensitive ACCase was present in Geqiushan resistant plants (R), with a resistance index of 106. The basal ACCase activities in Geqiushan R and Geqiushan susceptible plants (S) were similar, at 1.20 and 1.17 ng malonyl-CoA min−1 µg−1 extract protein, respectively. Basal ACCase gene expression in Geqiushan R was similar to that in Geqiushan S. The relative expression of ACCase gene decreased after spraying quizalofop–ethyl at 60 g ai ha−1 in Geqiushan S, whereas it was almost not changed in Geqiushan R. From these results we concluded that plastid ACCase sensitivity change might be responsible for the resistance and gene overexpression does not play a role in this resistance.

Annual bluegrass is a problematic turfgrass weed. Methiozolin is a new, currently unregistered herbicide that selectively controls annual bluegrass in desirable turfgrasses. Studies were conducted to evaluate and compare annual bluegrass control from PRE-applied methiozolin as influenced by rate and soil type and from POST-applied methiozolin as influenced by rate, soil type, annual bluegrass growth stage, and treatment placement. Studies were also conducted to evaluate foliar and root absorption and subsequent translocation of methiozolin by annual bluegrass using radio-tracer techniques. PRE-applied methiozolin controlled annual bluegrass > 99%. POST-applied methiozolin resulted in < 80% control regardless of foliar versus root exposure. POST applications are more effective at higher rates and smaller growth stages. Foliar-plus-soil methiozolin application trended to result in the best control, compared to foliar-only and soil-only applications. Absorption and translocation data indicate that methiozolin is absorbed by both leaves and roots and moderately translocates upward in the plant toward the leaf tip with little to no basipetal translocation. Because control is limited from a single methiozolin application (as observed in POST experiments), successful field application of methiozolin requires multiple and timely applications directed toward the roots and/or foliage of annual bluegrass.

Out of 1,343 mutant lines of rice mutated by sodium azide from the parental Japonica-type variety ‘Tainung 67’ (TNG67), a paraquat-susceptible line 1192 and a paraquat-tolerant line 72 were selected using whole seedlings at the four-leaf stage and leaf segments at the tillering stage as test materials. Further selection from progenies of these two mutant lines yielded the susceptible 1192-11 (S) and tolerant 72-16 (T), which were studied herein. Chlorophyll fluorescence, electrolyte leakage, and lipid peroxidation were measured for leaf segments of rice following treatment with 0.1 mM paraquat. A comparison of these responses among the three rice lines (TNG67, 72-16, and 1192-11), revealed a higher tolerance to paraquat in the tolerant mutant line 72-16 and the parental variety TNG67 than in the susceptible mutant 1192-11. Analysis of the antioxidative system in paraquat-treated leaf segments showed that the reduced form of glutathione (GSH) and the ratio of GSH to total glutathione increased by 3.5-fold within 6 h after treatment (HAT) and up to 5-fold 9 HAT in the T line, as compared with the S line. In view of the high activities of both dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in paraquat-treated leaves of TNG67 and the T line, the antioxidative effect of the ascorbate–glutathione cycle is hereby proposed to play an essential role in paraquat tolerance of rice. Pretreatment of rice segments with spermine enhanced DHAR and GR activities as well as paraquat tolerance of the S line. These results suggest that the activity of ascorbate–glutathione cycle induced by spermine is involved in rice tolerance to this herbicide. Although kinetics studies showed no significant difference among the three rice lines in paraquat inhibition of GR, a lower affinity of enzyme to substrate (Km) in TNG67 and the T line and a higher maximal reaction rate (Vmax) in the T line for the oxidized glutathione substrate (GSSG) were detected. These observations further implicate the importance of glutathione reductase in paraquat tolerance of rice.

Studies were conducted to determine the growth, fecundity, and competitive ability of an acetyl-CoA carboxylase (ACCase)–inhibitor resistant (R) sterile wild oat biotype compared with a susceptible (S) biotype. Seed germination studies indicated that there were no differences in seed germination and seedling vigor between R and S biotypes at any temperature regime. R and S biotypes were grown under noncompetitive and competitive arrangement in the greenhouse. Under noncompetitive greenhouse conditions, growth of the R biotype was similar to that of the S biotype on the basis of plant height, canopy area, and plant biomass. Seed production and weight of R and S plants were also at the same levels. Furthermore, relative competitiveness among the R and S sterile wild oat biotypes was investigated by means of replacement series experiments. The R and S biotypes were compared under seven mixture proportions (6 : 0, 5 : 1, 4 : 2, 3 : 3, 2 : 4, 1 : 5, and 0 : 6). No significant differences in competitive ability were observed between R and S biotypes on the basis of plant height, canopy area, or plant biomass. In most cases, relative crowding coefficient (RCC) values at 20, 60, and 100 d after transplanting (DAT) were close to one, indicating equal competitiveness between the R and S biotypes of wild oat used in this competitive study. However, in some cases, the RCC value was 1.31 for plant height, evident of a slight competitive advantage for the R biotype at 100 DAT. In general, ACCase-inhibitor R and S sterile wild oat biotypes were equally competitive, clearly without any growth penalty for R plants in either noncompetitive or competitive conditions.

Glyphosate-resistant (GR) weeds, including giant ragweed, are among the most challenging weeds for growers to control in cotton. A field study was conducted in 2011 and 2012 to determine the competitiveness of giant ragweed with densities of 0, 0.1, 0.2, 0.4, 0.8, or 1.6 plants m−1 of row. Early in the growing season, giant ragweed competition with densities of at least 0.8 plants m−1 row reduced cotton height compared with the weed-free control. Based on node above white flower (NAWF) and node above cracked boll (NACB) data, a delay in cotton maturity was observed for treatments with giant ragweed present at a density of 1.6 m−1 of cotton row for NAWF and 0.8 m−1 or 1.6 m−1 of row for NACB. Lint yield losses of 50% were estimated for cotton with rows growing along side of giant ragweed at a density of 0.26 plants m−1 row. Cotton in rows located 140 cm away from giant ragweed required an estimated 1.85 plants m−1 row to reduce yield by 50%. These data suggest that giant ragweed sphere of influence was at least 1 m wide. Cotton fiber quality was not affected by giant ragweed at any density. Giant ragweed is a highly competitive weed in cotton, even at low densities, and efforts should be implemented to control giant ragweed early in the season to prevent cotton yield loss.

Methiozolin controls annual bluegrass in creeping bentgrass but application timing and temperature could influence efficacy in turf. In field experiments, sequential methiozolin applications totaling 3.36 kg ai ha−1 provided excellent (> 90%) annual bluegrass control at 8 wk after initial treatment when treatments were initiated in February/March or May but programs totaling 0.84 and 1.68 kg ha−1 provided poor control (< 70%) at both timings. Methiozolin at all rates caused minimal turf injury (< 8%) but creeping bentgrass was only injured from February/March applications. In growth chamber experiments, creeping bentgrass injury from methiozolin at 10 C was 2 and 4 times greater than at 20 C and 30 C, respectively, while annual bluegrass injury was similar across temperatures. In laboratory experiments, annual bluegrass had more foliar absorption of 14C-methiozolin than creeping bentgrass at 30/25 C (day/night), compared to 15/10 C, but translocation was similar at both temperatures as > 90% of absorbed 14C remained in the treated leaf after 72 h. Annual bluegrass distributed and recovered more radioactivity to shoots from root-applied 14C-methiozolin than creeping bentgrass while both species had about 2 times more distribution to shoots at 30/25 C than 15/10 C. Metabolites were not detected in annual bluegrass or creeping bentgrass at 1, 3, or 7 d after treatment when grown at 15/10 C or 30/25 C suggesting uptake and translocation contributes to methiozolin selectivity in turfgrass.

Musk thistle is an invasive weed that is widely distributed throughout much of North America, including grasslands in temperate climates of the midwest USA. A series of laboratory and greenhouse experiments were conducted to determine the effect of various environmental factors on germination of musk thistle seeds. In temperature-fluctuation experiments, seed germination was greater than 65% in both alternating (30/20 C) and constant (20 or 25 C) temperature regimes with an 8-h day but less (33%) in warmer regimes (35/20 C). Germination of musk thistle seeds was 37% in alternating temperature regimes of 30/20 C in total darkness, but less than 67% in pots in the greenhouse. Differences of 10 and 15 C between day and night temperatures resulted in 91 and 75% maximum germination of musk thistle, respectively. Increasingly dryer soils reduced germination of musk thistle seeds from 35% (−0.03 MPa) to 0% (−1.2 MPa), whereas saline soils (> 80 mM) reduced maximum germination to less than 10%. Musk thistle seeds collected from populations in a bare-ground area had 96% germination, which was greater than that of seeds collected from populations growing in a perennial grass pasture (71%). A residence time (i.e., period that seeds remained on the parent plant) of 9 to 12 wk after capitulum maturity resulted in seeds germinating more quickly than those dispersed earlier. Overall, reduced light levels, cool and fluctuating temperatures, and amount of time seeds remained in residence are some of the most important factors that contribute to germination of musk thistle seeds. Information on germination dynamics of musk thistle seeds provides an understanding of the interactions that affect this process and underscores the importance of timely management strategies in temperate grasslands.

A tall waterhemp population from Missisippi was suspected to be resistant to glyphosate. Glyphosate dose response experiments resulted in GR50 (dose required to reduce plant growth by 50%) values of 1.28 and 0.28 kg ae ha−1 glyphosate for the glyphosate-resistant (GR) and -susceptible (GS) populations, respectively, indicating a five-fold resistance. The absorption pattern of 14C-glyphosate between the GR and GS populations was similar up to 24 h after treatment (HAT). Thereafter, the susceptible population absorbed more glyphosate (55 and 49% of applied) compared to the resistant population (41 and 40% of applied) by 48 and 72 HAT, respectively. Treatment of a single leaf in individual plants with glyphosate at 0.84 kg ha−1, in the form of 10 1-µl droplets, provided greater control (85 vs. 29%) and shoot fresh weight reduction (73 vs. 34% of nontreated control) of the GS plants compared to the GR plants, possibly indicating a reduced movement of glyphosate in the GR plants. The amount of 14C-glyphosate that translocated out of the treated leaves of GR plants (20% of absorbed at 24 HAT and 23% of absorbed at 48 HAT) was significantly lower than the GS plants (31% of absorbed at 24 HAT and 32% of absorbed at 48 HAT). A potential difference in shikimate accumulation between GR and GS populations at different concentrations of glyphosate was also studied in vitro. The IC50 (glyphosate concentration required to cause shikimate accumulation at 50% of peak levels measured) values for the GR and GS populations were 480 and 140 µM of glyphosate, respectively, resulting in more shikimate accumulation in the GS than the GR population. Sequence analysis of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the target site of glyphosate, from GR and GS plants identified a consistent single nucleotide polymorphism (T/C, thymine/cytosine) between GR/GS plants, resulting in a proline to serine amino acid substitution at position 106 in the GR population. The GR and GS plants contained equal genomic copy number of EPSPS, which was positively correlated with EPSPS gene expression. Thus, glyphosate resistance in the tall waterhemp population from Mississippi is due to both altered target site and nontarget site mechanisms. This is the first report of an altered EPSPS-based resistance in a dicot weed species that has evolved resistance to glyphosate.

Florida hydrilla populations have shown an alarming increase in resistance to fluridone, an herbicide used extensively for controlling invasive US hydrilla populations. A rapid PCR and sequencing method was developed to identify and screen hydrilla genomic DNA for three previously identified phytoene desaturase (pds) gene mutations that confer resistance to fluridone. Ninety hydrilla accessions were screened for fluridone resistant genotypes including 46 accessions from the US and 44 accessions from 15 other countries. In Florida, hydrilla from five of nine sites tested was heterozygous for wild-type and herbicide-resistant alleles. Additionally, a new resistant population was identified from Lake Seminole in Georgia, the first genetically confirmed strain of resistant hydrilla outside of Florida. All resistance-conferring mutations were located on the same homologous haplotype of US dioecious hydrilla. All other hydrilla samples tested possessed only wild type alleles, including monoecious strains that had been exposed to fluridone. Management implications are discussed.