Paul C. C. Feng

Investigations into glyphosate-resistant horseweed (Conyza canadensis): retention, uptake, translocation, and metabolism

Abstract 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. Nomenclature: Glyphosate; horseweed, Conyza canadensis (L.) Cronq. ERICA.

Droplet size affects glyphosate retention, absorption, and translocation in corn

Abstract 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. Nomenclature: Glyphosate; glyphosate-resistant corn, Zea mays L. ‘Roundup® Ready’.

Retention, uptake, and translocation of 14C-glyphosate from track-spray applications and correlation to rainfastness in velvetleaf (Abutilon theophrasti).

Abstract: 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. Nomenclature: Glyphosate, N-(phosphonomethyl)glycine. Velvetleaf, Abutilon theophrasti Medik., #3 ABUTH. Additional index words: Uptake, translocation, retention, track spray, glyphosate formulations. Abbreviations: DAT, days after treatment; GI, growth inhibition; HAT, hours after treatment.

Soil transformation of acetochlor via glutathione conjugation

Abstract Acetochlor and acetochlor S -glutathione conjugate were degraded in soil with respective half-lives of 7 days and 30 min to a similar distribution of identical metabolites. Degradation of acetochlor-glutathione conjugate appears to be initiated by γ-glutamyl transpeptidase, and the addition of γ-glutamyl transpeptidase inhibitors effectively retarded the soil degradation of both acetochlor and its glutathione conjugate. The addition of a γ-glutamyl transpeptidase inhibitor to the soil caused the accumulation of acetochlor-glutathione conjugate as a transient metabolite of acetochlor and thereby facilitated its detection. These results suggested that conjugation of acetochlor with glutathione is an initial pathway of metabolism in soil. Acetochlor-glutathione conjugate was further degraded in soil to acetochlor S -cysteine conjugate, which was shown to be a key intermediate in the formation of the major acidic metabolites of acetochlor in soil.

Nuclear magnetic resonance timecourse studies of glyphosate metabolism by microbial soil isolates

1. Triple Resonance Isotope EDited nmr spectroscopy (TRIED) has been developed to detect and examine minute levels of glyphosate metabolites in microbial soil isolates. Using stable isotopic labelling (13C and 15N), TRIED allows the simultaneous detection of multiple metabolites in crude matrices at submicrogram levels. An improvement over earlier techniques where milligrams are needed, TRIED can detect 500 ng of triply labelled compound in a crude sample (1:14,000 mass ratio) in just hours. 2. TRIED is used here to compare the kinetics and metabolic pathways of glyphosate metabolism by two strains of Ochrobactrum anthropi, LBAA and S5. Both LBAA and S5 appear to metabolize glyphosate primarily via the aminomethylphosphonate (AMPA) pathway, since no detectable levels of glycine or sarcosine are observed in the media or lysates of either microbe. The formation of N-methylAMPA is common to the metabolism of both microorganisms, but N-acetylAMPA is observed only in LBAA. N-methylacetamide is detected predominantly in media and lysates of S5, although some evidence also points to the formation of this metabolite in LBAA. 3. Results are consistent with conventional radioactive tracer studies. TRIED nmr provides more specific structural information complementary to radiolabel methods. Both nmr and radioactivity studies show S5 glyphosate metabolism to be much slower than that of LBAA.

Analysis of surfactant leaf damage using microscopy and its relation to glyphosate or deuterium oxide uptake in velvetleaf (Abutilon theophrasti)

Commercial formulations of glyphosate were compared for surfactant leaf damage and glyphosate uptake. The formulations (Roundup™ Ultra, Roundup™, and Touchdown™ were diluted with water to 12.5 g AI litre−1 and applied as 1-μl drops to the first leaf adaxial surface. Tissues at application sites were examined by light, fluorescence and scanning electron microscopy. At 24 h after treatment, tissue necrosis was clearly visible with Ultra and Roundup™, but not with Touchdown™. The application sites of Ultra and Roundup™, but not with Touchdown™. The application sites of Ultra and Roundup™ demonstrated a well-demarcated zone of injury showing extensive rupturing of cell membranes in both epidermal and mesophyll cells. Studies using blank formulations without glyphosate confirmed that tissue damage was caused by the surfactant formulants. Diluted formulations (12.5 g AI litre−1) spiked with a minimum of [14C-glyphosate were applied similarly. Time-course studies showed the fastest uptake with Ultra, followed by Roundup™ and Touchdown™. Mobilization of glyphosate away from the treated leaf was proportional to uptake. The use of a deuterium NMR method demonstrated that pretreatment of leaves with glyphosate formulations facilitated subsequent leaf loading of deuterium oxide. The extent of the latter correlated with leaf loading of glyphosate in formulations. These results indicate that the role of the surfactant is to overcome the leaf cuticle and membrane barriers to facilitate glyphosate entry into the leaf. © 1999 Society of Chemical Industry

In vitro oxidation of alachlor by liver microsomal enzymes from rats, mice, and monkeys

Abstract Incubation of alachlor with microsomal enzyme preparations from rats, mice, and monkeys produced four major metabolites. These metabolites arose from benzylic hydroxylation(s) at one or both arylethyl side chains and N -dealkylation of alachlor. Kinetic studies demonstrated similar rates of alachlor oxidation by liver microsomal enzymes of mice, monkeys, and male rats. The rate of oxidation by female rat liver microsomes was significantly less than that observed by male rats. A slight induction of liver microsomal oxygenase activity was observed after chronic oral dosing of male rats with alachlor.

Identification of mercapturic acid pathway metabolites of alachlor formed by liver and kidney homogenates of rats, mice, and monkeys

Abstract Incubation of alachlor in the presence of glutathione (GSH) with the cytosolic fraction from rat, mouse, and monkey livers produced the GSH conjugate of alachlor as the initial metabolite. The conjugation occurred through thiol displacement of the chlorine atom of alachlor and was catalyzed by glutathione S -transferases (GST). Species and gender differences were observed in the activity of liver cytosolic GST for alachlor in rats, mice, and monkeys. Prior exposure of male rats to acute and chronic doses of alachlor resulted in a 1.5- to 2-fold increase in their liver GST activity. Further enzymatic conversion of the alachlor-GSH conjugate was studied with kidney cell-free preparations of rats and monkeys, which readily degraded the alachlor-GSH conjugate through the mercapturic acid pathway to the corresponding cysteinylglycine, cysteine, and N -acetylcysteine conjugates of alachlor.

Effect of glyphosate spray droplets on leaf cytology in velvetleaf (Abutilon theophrasti)

Abstract 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. Nomenclature: Glyphosate; velvetleaf, Abutilon theophrasti Medicus ABUTH.

Improving hybrid seed production in corn with glyphosate‐mediated male sterility

BACKGROUND Hybrid corn varieties exhibit benefits associated with heterosis and account for most of the corn acreage in the USA. Hybrid seed corn is produced by crossing a female parent which is male-sterile and therefore incapable of self-pollination with a male parent as the pollen donor. The majority of hybrid seed corn is produced by mechanical detasseling which involves physically removing the tassel, a process that is laborious and costly. RESULTS Glyphosate-resistant corn was developed via expression of a glyphosate insensitive 5-enolpyruvyl-shikimate 3-phosphate synthase enzyme (CP4-EPSPS). Experimentation with molecular expression elements resulted in selective reduction of CP4-EPSPS expression in male reproductive tissues. The resulting plant demonstrated sterile tassel following glyphosate application with little to no injury to the rest of the plant. Using (14)C-glyphosate as a marker, we also examined the translocation of glyphosate to the tassel via spray application in a track sprayer to simulate field application. The results allowed optimization of spray parameters such as dose, spray timing and target to maximize tassel delivery of glyphosate for efficient sterilization. CONCLUSION The Roundup hybridization system (RHS) is a novel process for hybrid seed production based on glyphosate-mediated male sterility. RHS replaces mechanical detasseling with glyphosate spray and greatly simplifies the process of hybrid seed corn production.