Uptake and translocation of glyphosate in three commercial formulations were examined in velvetleaf, a dicotyledonous weed that is commonly treated with glyphosate. The formulations included Roundup® (MON 35085), Roundup Ultra, and Touchdown® as sold in Canada. A minimal amount of 14C-glyphosate was spiked into a lethal rate of each formulation, and the short-term (3 to 72 h) uptake into the treated leaf and subsequent translocation into the plant were measured. Time-course studies showed very rapid uptake and translocation of glyphosate in the Ultra formulation. In comparison, the uptake and translocation of glyphosate in Touchdown was much slower but continued throughout the 72-h period. Glyphosate in the Roundup formulation showed intermediate uptake and translocation. Tissue necrosis at the application sites of Ultra and Roundup was visible within 24 h after treatment. Examinations using stereo and fluorescence microscopy revealed extensive cell death and tissue disruption. Tissue necrosis from Ultra and Roundup was also observed in blank formulations containing no glyphosate and therefore was likely caused by the surfactants. In contrast, the application sites of Touchdown produced little to no leaf damage. Our results demonstrated a direct correlation between tissue necrosis and rapid rates of glyphosate uptake and translocation.

The mechanism of glyphosate resistance in horseweed was investigated. Eleven biotypes of putative sensitive (S) and resistant (R) horseweed were obtained from regions across the United States and examined for foliar retention, absorption, translocation, and metabolism of glyphosate. Initial studies used spray application of 14C-glyphosate to simulate field application. When S and R biotypes were compared in the absence of toxicity at a sublethal dose, we observed comparable retention and absorption but reduced root translocation in the R biotypes. S and R biotypes from Delaware were further examined at field use rates and results confirmed similar retention and absorption but reduced root translocation in the R biotypes. Application of 14C-glyphosate to a single leaf demonstrated reduced export out of the treated leaf and lower glyphosate import into other leaves, the roots, and the crown in R relative to S biotypes. Examination of the treated leaf by autoradiography showed that glyphosate loading into the apoplast and phloem was delayed and reduced in the R biotype. Our results consistently showed a strong correlation between impaired glyphosate translocation and resistance. Tissues from both S and R biotypes showed elevated levels of shikimate suggesting that 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) remained sensitive to glyphosate. Analysis of tissue shikimate levels demonstrated reduced efficiency in EPSPS inhibition in the R biotypes. Our results suggest that resistance is likely due to altered cellular distribution that impaired phloem loading and plastidic import of glyphosate resulting in reduced overall translocation as well as inhibition of EPSPS.

The evolution of glyphosate resistance in weedy species places an environmentally benign herbicide in peril. The first report of a dicot plant with evolved glyphosate resistance was horseweed, which occurred in 2001. Since then, several species have evolved glyphosate resistance and genomic information about nontarget resistance mechanisms in any of them ranges from none to little. Here, we report a study combining iGentifier transcriptome analysis, cDNA sequencing, and a heterologous microarray analysis to explore potential molecular and transcriptomic mechanisms of nontarget glyphosate resistance of horseweed. The results indicate that similar molecular mechanisms might exist for nontarget herbicide resistance across multiple resistant plants from different locations, even though resistance among these resistant plants likely evolved independently and available evidence suggests resistance has evolved at least four separate times. In addition, both the microarray and sequence analyses identified non–target-site resistance candidate genes for follow-on functional genomics analysis.

The significance of glyphosate and the appearance of glyphosate-resistant weeds have raised concerns about glyphosate sustainability. Resistance-prevention strategies, however, should first consider the mechanisms for resistance. For example, target-site resistance can provide virtual immunity, ensuring that every herbicide application successfully selects for resistance. However, metabolism and exclusion mechanisms provide lower magnitudes of resistance and are dependent on dosage. This discussion proposes that the relative risk of weed resistance is most highly correlated to mode of action (MOA), due to the respective principal mechanism for resistance. The development of data correlating agronomic practices with weed resistance vs. herbicide/MOA choices will be critical to the design of effective prevention strategies. Because resistance to glyphosate in weeds is typically of a low magnitude, using a high-dose strategy should minimize the potential for the selection of resistance and thus help to make the use of glyphosate sustainable. Maximizing weed control is the key to successful agronomic practice with limited weed resistance.

The effect of droplet size on retention, absorption, and translocation of 14C-glyphosate was studied in glyphosate-resistant corn. Fine, medium, and coarse spray droplets were studied using a track-sprayer equipped with commercially available nozzles. Glyphosate-resistant corn was used to obtain measurements at field use rates in the absence of phytotoxicity. Spray retention on corn leaves was calculated based on recovered glyphosate per leaf area, and retention was higher with application of fine droplets (47%) than with application of coarse (38%) and medium (37%) droplets. Absorption in corn leaves was directly correlated with droplet size and reached a plateau 1 d after treatment (DAT) for all droplet sizes. Based on glyphosate recovered 3 DAT, coarse droplets showed the highest absorption (49%), followed by medium (35%) and fine (30%) droplets. Percentage of translocation also increased with droplet size, and translocation was primarily toward strong sink tissues such as roots and young leaves. Our results show that large droplets have slightly reduced retention in corn but have increased absorption resulting in increased translocation of glyphosate to growing sink tissues.

Three commercial formulations of glyphosate were spiked with 14C-glyphosate and applied via a spray nozzle to velvetleaf plants. The use of 14C-glyphosate as a marker caused minimal alteration to formulation properties, and the use of spray application simulated field practices. Formulation retention, calculated based on maximum plant-leaf area, showed that only 27 to 33% of the available area retained the spray. The small differences in retention among the formulations suggest that they contribute little to differences in efficacy. Following spray application, plants were harvested at various times to measure the levels of glyphosate uptake into the plant and translocation into roots. Significant differences were observed among the formulations in the rate of glyphosate uptake. The most efficient formulation absorbed about one third of the dose by 24 hr after treatment. Root translocation of glyphosate was approximately proportional to uptake and accounted for less than one third of the absorbed dose. The relationship between uptake and rainfastness was examined in greenhouse studies with simulated rainfall at various times after glyphosate application. A direct correlation was observed between rainfastness with the speed and quantity of glyphosate uptake.

Leaf cytoarchitecture was evaluated by light microscopy and scanning electron microscopy, and cell viability was monitored by fluorescence after treatment of velvetleaf with defined concentrations and droplet sizes of formulated glyphosate and blended tallowamine surfactant. In response to droplets of formulated glyphosate larger than in field sprays but useful for studying structural change, we observe that the leaf epidermis thins and flattens within 1.5 h, the epidermal, mesophyll, and vascular cells at the contact site exhibit localized cytolysis by 6 h, and cytolysis and pycnosis remain restricted to the contact site at 24 h. Using endogeneous fluorescence as a marker for nonviable cells, it was determined that cellular changes are directly correlated with droplet size and that the changes are minimal after exposure to spray sizes and concentrations of formulated glyphosate and blended tallowamine typically used in the field. The results show that, at field use concentrations, the effect of formulated glyphosate and blended tallowamine on leaf cytoarchitecture is modest and localized but sufficient to allow herbicide entry.

Various fluorescent stains and vital dyes have been used to identify dead cells in animal tissues and celi lines. In plants, fluorescein diacetate and propidium iodide have been used to label nuclei and to identify necrotic cells in plant protoplasts and 4,6-diamidino-2-phenylindole (DAPI) has been used to mark senescing cells in sections of roots. However, these dyes may be problematic when used with intact plant tissue with well-developed cells walls which may impede dye penetration. Endogenous fluorescence has been used to identify dead cells in intact and sectioned plant tissues. Published procedures typically employ ultraviolet (UV) excitation wavelengths of 340-380 nm and emission wavelengths of 400- 425 nm, thus requiring a UV filter set.