Glyphosate is easily translocated from shoots to roots and released into the rhizosphere. The objective of this study was to clarify the influence of glyphosate residues in the root tissue of glyphosate-treated weeds on wheat (Triticum aestivum L.) growth and shikimate accumulation. Foliar application to 5-leaf downy brome (Bromus tectorum L.) planted in sandy loam soil reduced wheat (‘Tubbs 06’) shoot fresh weight by 37% to 46% compared with the control when seeds were planted 0 and 1 d after applications. With Italian ryegrass [Lolium perenne L. ssp. multiflorum (Lam.) Husnot], wheat shoot fresh weight was inhibited by 20% to 34% compared with the control at 0, 1, 3, and 5 d after applications to 1.5- and 5-leaf-stage plants. Using a different wheat cultivar (‘Stephens’), shoot fresh weight was inhibited by 19% to 43% when seeds were planted 0 d after glyphosate applications to 1.5-, 2-, and 5-leaf-stage B. tectorum and L. perenne planted in sandy loam soil compared with control. In contrast, some studies using treated L. perenne and B. tectorum planted in clay loam soil resulted in increases in wheat shoot fresh weight. Lolium perenne planted in water-saturated sandy loam soil showed no differences in either shoot or root fresh weight or shikimate accumulation in shoots or roots. Compared with the control plants, shikimate accumulation in roots increased 51- to 59-fold in wheat planted in sandy loam soil that previously contained B. tectorum and 13- to 49-fold in soil that previously contained L. perenne. In both studies, glyphosate was applied at the 1.5-leaf stage, and wheat seeds were sown 0, 1, and 3 d after glyphosate applications. Thus, plant damage caused by glyphosate was associated with increased shikimate accumulation in the root tissue. Overall, crop damage caused by glyphosate residue to target plants was strongly influenced by soil type, soil water conditions, glyphosate sensitivity, target weed species identity, and weed densities.

Drift of glyphosate may cause injury to nontarget plant species. Herbicidal damage caused by glyphosate may be attributed to the herbicides that inhibit acetohydroxyacid synthase (AHAS) or acetyl CoA carboxylase (ACCase), because the time taken to show herbicidal damage and the visual injury symptoms caused by these three types of herbicides are similar. Glyphosate inhibits the biosynthesis of aromatic amino acid biosynthesis which causes accumulation of shikimate in plants. A modified, simpler method of extraction and assay for detection of shikimate in plants has been adapted from previously published procedures. With this assay, rapid accumulation of shikimate was observed in glyphosate-sensitive plants that have been treated with glyphosate. Furthermore, the accumulation of shikimate in herbicide-sensitive plants was only due to glyphosate and not due to an AHAS or an ACCase inhibitor. Therefore, high levels of shikimate in a damaged plant indicate glyphosate injury. With this assay, no accumulation of shikimate was seen in a glyphosate-resistant variety of soybean. Therefore, lack of accumulation of shikimate in a glyphosate-treated weed or crop species indicates that the plant is resistant to glyphosate.

Two rapid, nondestructive assays were developed and tested for their potential in differentiating glyphosate-resistant from glyphosate-susceptible biotypes of horseweed. In one assay, leaves of glyphosate-resistant and -susceptible corn, cotton, and soybean plants as well as glyphosate-resistant and -susceptible horseweed plants were dipped in solutions of 0, 300, 600, and 1200 mg ae L−1 glyphosate for 3 d and subsequent injury was evaluated. In the second assay, plant sensitivity to glyphosate was evaluated in vivo by incubating excised leaf disc tissue from the same plants used in the first assay in 0.7, 1.3, 2.6, 5.3, 10.6, 21.1, 42.3, and 84.5 mg ae L−1 glyphosate solutions for 16 h and measuring shikimate levels with a spectrophotometer. The leaf-dip assay differentiated between glyphosate-resistant and -susceptible crops and horseweed biotypes. The 600 mg L−1 rate of glyphosate was more consistent in differentiating resistant and susceptible plants compared with the 300 and 1,200 mg L−1 rates. The in vivo assay detected significant differences between susceptible and glyphosate-resistant plants of all species. Shikimate accumulated in a glyphosate dose-dependent manner in leaf discs from susceptible crops, but shikimate did not accumulate in leaf discs from resistant crops and levels were similar to nontreated leaf discs. Shikimate accumulated at high (≥ 21.1 mg ae L−1) concentrations of glyphosate in leaf discs from all horseweed biotypes. Shikimate accumulated at low glyphosate concentrations (≤ 10.6 mg L−1) in leaf discs from susceptible horseweed biotypes but not in resistant biotypes. Both assays were able to differentiate resistant from susceptible biotypes of horseweed and might have utility for screening other weed populations for resistance to glyphosate.

Greenhouse and laboratory studies were conducted to confirm and quantify glyphosate resistance, quantify pyrithiobac resistance, and investigate interaction between flumiclorac and glyphosate mixtures on control of Palmer amaranth from Mississippi. The GR50 (herbicide dose required to cause a 50% reduction in plant growth) values for two glyphosate-resistant biotypes, C1B1 and T4B1, and a glyphosate-susceptible (GS) biotype were 1.52, 1.3, and 0.09 kg ae ha−1 glyphosate, respectively. This indicated that the C1B1 and T4B1 biotypes were 17- and 14-fold resistant to glyphosate, respectively, compared with the GS biotype. The C1B1 and T4B1 biotypes were also resistant to pyrithiobac, an acetolactate synthase (ALS) inhibitor, with GR50 values of 0.06 and 0.07 kg ai ha−1, respectively. This indicated that the C1B1 and T4B1 biotypes were 7- and 8-fold, respectively, more resistant to pyrithiobac compared with the GS biotype, which had a GR50 value of 0.009 kg ha−1. Flumiclorac was antagonistic to glyphosate by reducing glyphosate translocation. The C1B1 and T4B1 absorbed less glyphosate 48 h after treatment (HAT) compared with the GS biotype. The majority of the translocated glyphosate accumulated in the shoot above the treated leaf (that contains the apical meristem) in the GS biotype and in the shoot below the treated leaf in the resistant biotypes, C1B1 and T4B1, by 48 HAT. The C1B1 biotype accumulated negligible shikimate levels, whereas the T4B1 and GS biotypes recorded elevated levels of shikimate. Metabolism of glyphosate to aminomethylphosphonic acid was not detected in either of the resistant biotypes or the susceptible GS biotype. The above results confirm multiple resistance to glyphosate and pyrithiobac in Palmer amaranth biotypes from Mississippi and indicate that resistance to glyphosate is partly due to reduced absorption and translocation of glyphosate.

A threefold glyphosate tolerance was identified in two Italian ryegrass populations, T1 and T2, from Mississippi. Laboratory experiments were conducted to characterize the mechanism of glyphosate tolerance in these populations. The T1 population absorbed less 14C-glyphosate (43% of applied) compared to the susceptible (S) population (59% of applied) at 48 h after treatment (HAT). The T2 population absorbed 14C-glyphosate at levels (56% of applied at 48 HAT) that were similar to both T1 and S populations, but tended to be more comparable to the S population. The amount of 14C-glyphosate that remained in the treated leaf was significantly higher in both T1 (67% of absorbed) and T2 (65% of absorbed) populations compared to the S population (45% of absorbed) at 48 HAT. The amount of 14C-glyphosate that moved out of treated leaf to shoot and root was lower in both T1 (25% of absorbed in shoot and 9% of absorbed in root) and T2 (25% of absorbed in shoot and 11% of absorbed in root) populations compared to the S population (40% of absorbed in shoot and 16% of absorbed in root) at 48 HAT. There were no differences in epicuticular wax mass among the three populations. Treating a single leaf with glyphosate solution at the field use rate (0.84 kg ae ha−1) as 10 1-µl droplets killed the S plant but not the T1 and T2 plants (33 and 55% shoot fresh-weight reduction, respectively). Shikimic acid accumulated rapidly at higher levels in glyphosate-treated leaf segments of the S population compared to the T1 population up to 100 µM glyphosate. However, above 500 µM glyphosate, the levels of shikimate were similar in both the S and T1 populations. Furthermore, shikimic acid content was three- to sixfold more in whole plants of the S population treated with 0.22 kg ae ha−1 glyphosate compared to the T1 and T2 populations. No degradation of glyphosate to aminomethylphosphonic acid was detected among the tolerant and susceptible populations. These results indicate that tolerance to glyphosate in the T1 population is partly due to reduced absorption and translocation of glyphosate and in the T2 population it is partly due to reduced translocation of glyphosate.

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.

Application of glyphosate in combination with planting soybeans in narrow rows is an effective practice for management of weeds in glyphosate-resistant soybean. Farmers in Michigan reported higher levels of Sclerotinia stem rot (caused by Sclerotinia sclerotiorum) in fields of glyphosate-resistant soybean. Studies were conducted to determine if glyphosate or shading reduced the defense response of glyphosate-resistant soybean to S. sclerotiorum. Glyphosate caused shikimate accumulation in glyphosate-susceptible cultivar GL2415 but not in glyphosate-resistant cultivar GL2600RR. Ethylacetate extracts containing the plant defense compound glyceollin inhibited S. sclerotiorum hyphae in a rate-dependent manner. Glyphosate had no effect on either baseline or induced levels of glyceollin in glyphosate-resistant soybean, indicating that glyphosate did not impair plant defense responses to S. sclerotiorum. Shade levels of 60 and 90% in the greenhouse did not inhibit the induction of glyceollin synthesis. Glyphosate herbicide and shading did not affect the glyphosate-resistant soybean defense response to S. sclerotiorum.

Field trials were conducted in 2001 at the Tobacco Research Station near Oxford, NC, and in 2002 at the Lower Coastal Plains Research Station near Kinston, NC, to determine tobacco yield, injury, and shikimic acid accumulation in response to simulated glyphosate drift. Glyphosate was applied to 12- to 13-cm-high tobacco ‘K326’ early postemergence at 0, 9, 18, 35, 70, 140, 280, 560, and 1,120 (1×) g ai/ha. Crop injury was rated 7 and 35 d after treatment (DAT) and shikimic acid accumulation in leaves at 7 DAT, tobacco yield, and leaf grade index (whole-plant index of harvest interval leaf value) were also assessed. Shikimic acid accumulation and injury symptoms increased similarly as glyphosate rate increased. Glyphosate rates of 140 g/ha (0.125 of recommended rate) or higher resulted in significant crop injury, reduced tobacco yield, and decreased leaf grade index. Shikimic acid accumulation at 7 DAT was inversely related to tobacco yield. Shikimic acid accumulation was found to be an effective diagnostic tool to determine glyphosate drift in tobacco; however, in-season data are needed to correlate shikimic acid accumulation with yield loss.

Two rapid, nondestructive assays were developed and tested for their potential in differentiating glyphosate-resistant from glyphosate-susceptible biotypes of horseweed. In one assay, leaves of glyphosate-resistant and -susceptible corn, cotton, and soybean plants, as well as glyphosate-resistant and -susceptible horseweed plants, were dipped in solutions of 0, 300, 600, and 1,200 mg ae L−1 glyphosate for 3 d, and subsequent injury was evaluated. In the second assay, plant sensitivity to glyphosate was evaluated in vivo by incubating excised leaf disc tissue from the same plants used in the first assay in 0.7, 1.3, 2.6, 5.3, 10.6, 21.1, 42.3, and 84.5 mg ae L−1 glyphosate solutions for 16 h and measuring shikimate levels with a spectrophotometer. The leaf dip assay differentiated between glyphosate-resistant and -susceptible crops and horseweed biotypes. The 600 mg L−1 rate of glyphosate was more consistent in differentiating resistant and susceptible plants compared with the 300 and 1,200 mg L−1 rates. The in vivo assay detected significant differences between susceptible and glyphosate-resistant plants of all species. Shikimate accumulated in a glyphosate dose–dependent manner in leaf discs from susceptible crops, but shikimate did not accumulate in leaf discs from resistant crops, and levels were similar to nontreated leaf discs. Shikimate accumulated at high (≥ 21.1 mg ae L−1) concentrations of glyphosate in leaf discs from all horseweed biotypes. Shikimate accumulated at low glyphosate concentrations (≤ 10.6 mg L−1) in leaf discs from susceptible horseweed biotypes but not in resistant biotypes. Both assays were able to differentiate resistant from susceptible biotypes of horseweed and could have utility for screening other weed populations for resistance to glyphosate.

Experiments were conducted to examine the utility of a spectrophometric leaf disc assay for detecting shikimate accumulation after glyphosate application in sunflower, proso millet, and wheat. The assay was conducted on both greenhouse- and field-grown plants. Glyphosate was applied at five rates ranging from 840 to 53 g ae ha−1. Shikimate accumulation data were generated at 1, 4, 7, and 14 d after application (DAA). Sunflower accumulated shikimate more rapidly and at lower glyphosate rates than the other two species. At 14 DAA, glyphosate at the two highest rates remained detectable in all three species. Plants receiving lower glyphosate doses (210, 105, and 53 g ae ha−1) had begun to grow out of the injury, or at least the shikimate levels in the plants were no longer significantly different than that present in the untreated controls. This spectrophotometric assay is both rapid and simple, with respect to other means of detecting shikimate, and it can be used to detect glyphosate drift. For it to be used by crop managers, samples from potentially drift-affected crops should be taken as soon as possible after the suspected drift event or immediately after the appearance of glyphosate injury.