PROBING THE PLANT CELL WALL WITH HERBICIDES: A CHEMICAL GENETICS APPROACH

The primary cell wall is a highly organized multi-layered matrix of polysaccharides (cellulose, hemi-cellulose, and pectin). The ability of the rigid cell wall to sufficiently loosen to allow growth is a complex process that differs considerably between grasses monocots and dicots. Cellulose is the major structural component required for anisotropic cell expansion and is synthesized by CELLULOSE SYNTHASE A (CesA) proteins. Here, our objectives were two-fold: 1) dissect cell walls and cellulose biosynthesis in dicots and grasses using chemical biology and reverse genetic approaches 2) characterize and classify the inhibitory mechanisms of cellulose biosynthesis inhibitors (CBIs). A reverse genetics TILLING experiment was conducted to study CesAs in the model grass Brachypodium (Bd). New mutant alleles of BdCesA1 and BdCesA3 were identified and characterized. On average, Bdcesa1S830N and Bdcesa3P986S mutants had 15% and 8% less cellulose than wild type plants, respectively. No obvious vegetative growth phenotypes were detected in mutants. However, at reproduction, inflorescence stems of cesa1S830N were 62% shorter than that of the wild type while cesa3P986S mutants were 20% longer. To classify CBIs, time-lapse confocal microscopy data were used to categorize CBIs based on how they disrupted the normal tracking and localization of fluorescently labeled CesAs. Furthermore, biochemical and confocal microscopy data were used to characterize the putative CBI, indaziflam. Three different inhibitory mechanisms were discovered within the CBI mode of action. Next, CBIs were used as molecular probes to study grass cell walls. However, grasses were found to be inherently tolerant to isoxaben and other CesA targeting CBIs. Isoxaben-tolerance was investigated but could not be explained by target and non-target site mechanisms. Thus, it was hypothesized mixed linkage glucans (MLGs), a unique grass cell wall polysaccharide, have cell wall strengthening characteristic and may partially compensate for reduced cellulose content. Bdcslf6 mutants deficient in MLGs were 2.1 times more susceptible to isoxaben than wild type plants indicating MLGs do have a structural role in expanding cells, but likely cannot explain tolerance. These data, collectively, support a conclusion that the non-cellulosic fraction of grass primary cell walls has more load-bearing capacity than dicot cell walls

[\u3csup\u3e14\u3c/sup\u3eC] Glucose Cell Wall Incorporation Assay for the Estimation of Cellulose Biosynthesis

Cellulose is synthesized by Cellulose Synthase A proteins at the plasma membrane using the substrate UDP glucose. Herein, we provide a detailed method for measuring the incorporation of radiolabeled glucose into the cellulose fraction of the cell wall. In this method Arabidopsis seedlings are treated for 2 h with a cellulose biosynthesis inhibitor in the presence of radiolabeled glucose, and are subsequently boiled in acetic-nitric acid to solubilize non-cellulosic material. The radiolabeled glucose detected in the insoluble fraction indicates the amount of cellulose synthesized during the experimental timeframe. The short-term nature of this method is a useful tool in determining if inhibition of cellulose biosynthesis is the herbicides primary mode of action

Confirmation of S-metolachlor resistance in Palmer amaranth (Amaranthus palmeri)

AbstractS-Metolachlor is commonly used by soybean and cotton growers, especially with POST treatments for overlapping residuals, to obtain season-long control of glyphosate- and acetolactate synthase (ALS)–resistant Palmer amaranth. In Crittenden County, AR, reports of Palmer amaranth escapes following S-metolachlor treatment were first noted at field sites near Crawfordsville and Marion in 2016. Field and greenhouse experiments were conducted to confirm S-metolachlor resistance and to test for cross-resistance to other very-long-chain fatty acid (VLCFA)–inhibiting herbicides in Palmer amaranth accessions from Crawfordsville and Marion. Palmer amaranth control in the field (soil <3% organic matter) 14 d after treatment (DAT) was ≥94% with a 1× rate of acetochlor (1,472 g ai ha–1; emulsifiable concentrate formulation) and dimethenamid-P (631 g ai ha–1). However, S-metolachlor at 1,064 g ai ha–1 provided only 76% control, which was not significantly different from the 1/2× and 1/4× rates of dimethenamid-P and acetochlor (66% to 85%). In the greenhouse, Palmer amaranth accessions from Marion and Crawfordsville were 9.8 and 8.3 times more resistant to S-metolachlor compared with two susceptible accessions based on LD50 values obtained from dose–response experiments. Two-thirds and 1.5 times S-metolachlor at 1,064 g ha–1 were the estimated rates required to obtain 90% mortality of the Crawfordsville and Marion accessions, respectively. Data collected from the field and greenhouse confirm that these accessions have evolved a low level of resistance to S-metolachlor. In an agar-based assay, the level of resistance in the Marion accession was significantly reduced in the presence of a glutathione S-transferase (GST) inhibitor, suggesting that GSTs are the probable resistance mechanism. With respect to other VLCFA-inhibiting herbicides, Marion and Crawfordsville accessions were not cross-resistant to acetochlor, dimethenamid-P, or pyroxasulfone. However, both accessions, based on LD50 values obtained from greenhouse dose–response experiments, exhibited reduced sensitivity (1.5- to 3.6-fold) to the tested VLCFA-inhibiting herbicides

The inheritance, fitness, and control of glyphosate-resistant giant ragweed

Giant ragweed is one the most competitive and problematic weeds in corn and soybean fields in the Midwest. Giant ragweed is difficult to control because of an early and extended germination period, rapid growth rate, inherent tolerance to herbicides, and has now evolved resistance to glyphosate. The main goal in this study was to determine the fate of glyphosate resistance in a giant ragweed population. Our objectives were to determine the inheritance of glyphosate resistance, fitness, and control of a glyphosate-resistant (GR) giant ragweed biotype from Indiana. In a glyphosate dose response experiment, the LD 50 values of GR, glyphosate-susceptible (GS), R[female]S[male] F 1, and S[female]R[male] F1 plants at 21 DAT were 3,142 g, 73 g, 2,181 g, and 774 g ae ha-1, respectively. At 400 g ae ha-1 of glyphosate, the backcross progeny of GS x S[female]R[male] F1 segregated according to our null hypothesis of 1 R: 1 S for mortality at 21 DAT, but not for necrosis at 3 DAT. The results indicated glyphosate resistance is manifested by a single major, semi-dominant to dominant gene, depending on the maternal parent, and is transferable through pollen and/or seed. Also, our results demonstrated that rapid necrosis did associated with plant survival at 21 DAT, but the continuous phenotypic variation observed suggested resistance is influenced by environmental conditions, epistatic interactions, or by additional minor genes. In the absence of glyphosate, GR and GS plants had a similar growth pattern, but during reproduction, GR plants flowered earlier and produced 25% less seed than GS plants. In the presence of glyphosate at 840 g followed by 2,520 g ae ha-1, plants with the GR trait were the superior biotype and required additional methods of control, rather than glyphosate alone. Tank mixtures of glyphosate plus fomesafen controlled 15 cm tall GR plants ≤65%; however, reduced rates of fomesafen tank mixed with glyphosate or glufosinate at the label field use rate did provide ≥90% control of 20 to 30 cm tall GS giant ragweed. Our results demonstrate control of GR giant ragweed will not be easy and the fate of glyphosate resistance in giant ragweed will continue to persist, and could potentially spread rapidly via pollen and/or seed mediated gene flow in our current glyphosate-dominated agricultural ecosystem

Recurrent Selection with Sub-Lethal Doses of Mesotrione Reduces Sensitivity in Amaranthus palmeri

Amaranthus palmeri, ranked as the most prolific and troublesome weed in North America, has evolved resistance to several herbicide sites of action. Repeated use of any one herbicide, especially at lower than recommended doses, can lead to evolution of weed resistance, and, therefore, a better understanding of the process of resistance evolution is essential for the management of A. palmeri and other difficult-to-control weed species. Amaranthus palmeri rapidly developed resistance to 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors such as mesotrione. The objective of this study was to test the potential for low-dose applications of mesotrione to select for reduced susceptibility over multiple generations in an A. palmeri population collected from an agricultural field in 2001. F0 plants from the population were initially treated with sub-lethal mesotrione rates and evaluated for survival three weeks after treatment. All F0 plants were controlled at the 1× rate (x = 105 g ai ha−1). However, 2.5% of the F0 plants survived the 0.5× treatment. The recurrent selection process using plants surviving various mesotrione rates was continued until the F4 generation was reached. Based on the GR50 values, the sensitivity index was determined to be 1.7 for the F4 generation. Compared to F0, HPPD gene expression level in the F3 population increased. Results indicate that after several rounds of recurrent selection, the successive generations of A. palmeri became less responsive to mesotrione, which may explain the reduced sensitivity of this weed to HPPD-inhibiting herbicides. The results have significance in light of the recently released soybean and soon to be released cotton varieties with resistance to HPPD inhibitors

Dicamba air concentrations in eastern Arkansas and impact on soybean

Damage to non–dicamba resistant (non-DR) soybean [Glycine max (L.) Merr.] has been frequent in geographies where dicamba-resistant (DR) soybean and cotton (Gossypium hirsutum L.) have been grown and sprayed with the herbicide in recent years. Off-target movement field trials were conducted in northwest Arkansas to determine the relationship between dicamba concentration in the air and the extent of symptomology on non-DR soybean. Additionally, the frequency and concentration of dicamba in air samples at two locations in eastern Arkansas and environmental conditions that impacted the detection of the herbicide in air samples were evaluated. Treatment applications included dicamba at 560 g ae ha−1 (1X rate), glyphosate at 860 g ae ha−1, and particle drift retardant at 1% v/v applied to 0.37-ha fields with varying degrees of vegetation. The relationship between dicamba concentration in air samples and non-DR soybean response to the herbicide was more predictive with visible injury (generalized R 2 = 0.82) than height reduction (generalized R 2 = 0.43). The predicted dicamba air concentration resulting in 10% injury to soybean was 1.60 ng m−3 d−1 for a single exposure. The predicted concentration from a single exposure to dicamba resulting in a 10% height reduction was 3.78 ng m−3 d−1. Dicamba was frequently detected in eastern Arkansas, and daily detections above 1.60 ng m−3 occurred 17 times in the period sampled. The maximum concentration of dicamba recorded was 7.96 ng m−3 d−1, while dicamba concentrations at Marianna and Keiser, AR, were ≥1 ng m−3 d−1 in six samples collected in 2020 and 22 samples in 2021. Dicamba was detected consistently in air samples collected, indicating high usage in the region and the potential for soybean damage over an extended period. More research is needed to quantify the plant absorption rate of volatile dicamba and to evaluate the impact of multiple exposures of gaseous dicamba on non-targeted plant species