Evaluation of dicamba volatilization when mixed with glyphosate using imazethapyr as a tracer.

Expansion of dicamba-resistant crops increased the frequency of off-target movement issues, especially in the midsouthern United States. Six field trials were conducted over two growing seasons with the purpose to determine the contribution of volatilization and physical suspension of particles to the off-target movement of dicamba when applied with glyphosate and imazethapyr - a non-volatile herbicide used as a tracer for physical off-target movement. Applications included dicamba at 560 g ha-1, glyphosate at 1260 g ha-1, and imazethapyr at 105 g ha-1. Applicators include glyphosate with dicamba to increase the spectrum of weed control from these applications; however, this addition increases potential for dicamba volatilization. Following application of the mixture, air samplers were placed in the field to collect dicamba and imazethapyr. Results showed there was at least 50 times more dicamba than imazethapyr detected even though the dicamba:imazethapyr ratio applied was 5.3:1. Dicamba was detected in the treated area and the off-site locations and all intervals of air sampling, ranging from 126 to 5990 ng. No more than 37.5 ng of imazethapyr was detected during the first 24-h after application (HAA) inside the treated area. Imazethapyr was only detected in 9 of the 20 sampling combinations during these experiments, and most of these detections (6) occurred during the first 24 HAA and inside the treated area. While some movement from the suspension of particles occurred based on the detection of imazethapyr in air samples, results show that most dicamba detection was due to the volatilization of the herbicide.

Cellulose biosynthesis inhibitors - a multifunctional toolbox.

In the current review, we examine the growing number of existing Cellulose Biosynthesis Inhibitors (CBIs) and based on those that have been studied with live cell imaging we group their mechanism of action. Attention is paid to the use of CBIs as tools to ask fundamental questions about cellulose biosynthesis.

Indaziflam herbicidal action: a potent cellulose biosynthesis inhibitor.

Cellulose biosynthesis is a common feature of land plants. Therefore, cellulose biosynthesis inhibitors (CBIs) have a potentially broad-acting herbicidal mode of action and are also useful tools in decoding fundamental aspects of cellulose biosynthesis. Here, we characterize the herbicide indaziflam as a CBI and provide insight into its inhibitory mechanism. Indaziflam-treated seedlings exhibited the CBI-like symptomologies of radial swelling and ectopic lignification. Furthermore, indaziflam inhibited the production of cellulose within <1 h of treatment and in a dose-dependent manner. Unlike the CBI isoxaben, indaziflam had strong CBI activity in both a monocotylonous plant (Poa annua) and a dicotyledonous plant (Arabidopsis [Arabidopsis thaliana]). Arabidopsis mutants resistant to known CBIs isoxaben or quinoxyphen were not cross resistant to indaziflam, suggesting a different molecular target for indaziflam. To explore this further, we monitored the distribution and mobility of fluorescently labeled CELLULOSE SYNTHASE A (CESA) proteins in living cells of Arabidopsis during indaziflam exposure. Indaziflam caused a reduction in the velocity of YELLOW FLUORESCENT PROTEIN:CESA6 particles at the plasma membrane focal plane compared with controls. Microtubule morphology and motility were not altered after indaziflam treatment. In the hypocotyl expansion zone, indaziflam caused an atypical increase in the density of plasma membrane-localized CESA particles. Interestingly, this was accompanied by a cellulose synthase interacting1-independent reduction in the normal coincidence rate between microtubules and CESA particles. As a CBI, for which there is little evidence of evolved weed resistance, indaziflam represents an important addition to the action mechanisms available for weed management.

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.

Presence of the HPPD Inhibitor Sensitive 1 Gene and ALSS653N Mutation in Weedy Oryza sativa Sensitive to Benzobicyclon.

Benzobicyclon has shown varying results in controlling weedy rice, including those with imidazolinone (IMI) resistance. Tolerance to benzobicyclon in cultivated japonica rice, but not indica or aus-like cultivars, is conferred by a fully functional HPPD Inhibitor Sensitive 1 (HIS1) gene. Herein, a diagnostic Kompetitive Allele Specific PCR (KASP) assay was developed to predict the HIS1 genotype of weedy rice plants from 37 accessions and correlated to their response to benzobicyclon in the field. Two-thirds of the 693 weedy rice plants screened were tolerant to benzobicyclon (371 g ai ha-1, SC formulation) at 30 days after treatment (DAT). Thirty-four percent of plants were homozygous for the HIS1 allele and 98% of these plants exhibited field tolerance. However, the his1 genotype did not always correlate with field data. Only 52% of his1 plants were considered sensitive, indicating that the single nucleotide polymorphisms (SNPs) chosen in the KASP assay are not a reliable tool in predicting his1 homozygous plants. In an additional experiment, 86% of the 344 plants with at least one copy of the ALSS653N trait harbored a HIS1 allele, suggesting fields infested with IMI herbicide-resistant weedy rice are unlikely to be controlled with benzobicyclon.

Biochemical and physiological flexibility accompanies reduced cellulose biosynthesis in Brachypodium cesa1 S830N.

Here, we present a study into the mechanisms of primary cell wall cellulose formation in grasses, using the model cereal grass Brachypodium distachyon. The exon found adjacent to the BdCESA1 glycosyltransferase QXXRW motif was targeted using Targeting Induced Local Lesions in Genomes (TILLING) and sequencing candidate amplicons in multiple parallel reactions (SCAMPRing) leading to the identification of the Bdcesa1 S830N allele. Plants carrying this missense mutation exhibited a significant reduction in crystalline cellulose content in tissues that rely on the primary cell wall for biomechanical support. However, Bdcesa1 S830N plants failed to exhibit the predicted reduction in plant height. In a mechanism unavailable to eudicotyledons, B. distachyon plants homozygous for the Bdcesa1 S830N allele appear to overcome the loss of internode expansion anatomically by increasing the number of nodes along the stem. Stem biomechanics were resultantly compromised in Bdcesa1 S830N . The Bdcesa1 S830N missense mutation did not interfere with BdCESA1 gene expression. However, molecular dynamic simulations of the CELLULOSE SYNTHASE A (CESA) structure with modelled membrane interactions illustrated that Bdcesa1 S830N exhibited structural changes in the translated gene product responsible for reduced cellulose biosynthesis. Molecular dynamic simulations showed that substituting S830N resulted in a stabilizing shift in the flexibility of the class specific region arm of the core catalytic domain of CESA, revealing the importance of this motion to protein function.

Grass cell walls have a role in the inherent tolerance of grasses to the cellulose biosynthesis inhibitor isoxaben.

BACKGROUND: Cellulose biosynthesis inhibitors (CBIs) are pre-emergence herbicides that inhibit anisotropic cell expansion resulting in a severely swollen and stunted growth phenotype. Resistance to group 21 CBIs, such as isoxaben, is conferred by missense mutations in CELLOSE SYNTHASE A (CesA) genes required for primary cell wall synthesis, concluding that this is their in vivo target. RESULTS: Herein, we show that grasses exhibit tolerance to group 21 CBIs and explore the mechanism of tolerance to isoxaben in the grass Brachypodium distachyon (L.). Comparative genomics failed to identify synonymous point mutations that have been found to confer isoxaben resistance in the dicot Arabidopsis thaliana (L.). Brachypodium did not metabolize 14 C-isoxaben. We next explored the role of grass-specific non-cellulosic cell wall components, specifically the hemicellulose polysaccharide mix linkage glucans (MLG), as a potential tolerance mechanism by compensating for the loss of cellulose during cell elongation. A partial-transcriptional knockdown T-DNA insertion was found in a key MLG synthesis gene, Cellulose synthase-like F6 (CslF6) and this mutant was found to be 2.1 times more sensitive to isoxaben than wild-type plants. CONCLUSION: These data suggest that the composition and compensatory response of grass cell walls may be a factor in conferring tolerance to group 21 CBIs. © 2017 Society of Chemical Industry.

Acetobixan, an inhibitor of cellulose synthesis identified by microbial bioprospecting.

In plants, cellulose biosynthesis is an essential process for anisotropic growth and therefore is an ideal target for inhibition. Based on the documented utility of small-molecule inhibitors to dissect complex cellular processes we identified a cellulose biosynthesis inhibitor (CBI), named acetobixan, by bio-prospecting among compounds secreted by endophytic microorganisms. Acetobixan was identified using a drug-gene interaction screen to sift through hundreds of endophytic microbial secretions for one that caused synergistic reduction in root expansion of the leaky AtcesA6prc1-1 mutant. We then mined this microbial secretion for compounds that were differentially abundant compared with Bacilli that failed to mimic CBI action to isolate a lead pharmacophore. Analogs of this lead compound were screened for CBI activity, and the most potent analog was named acetobixan. In living Arabidopsis cells visualized by confocal microscopy, acetobixan treatment caused CESA particles localized at the plasma membrane (PM) to rapidly re-localize to cytoplasmic vesicles. Acetobixan inhibited 14C-Glc uptake into crystalline cellulose. Moreover, cortical microtubule dynamics were not disrupted by acetobixan, suggesting specific activity towards cellulose synthesis. Previous CBI resistant mutants such as ixr1-2, ixr2-1 or aegeus were not cross resistant to acetobixan indicating that acetobixan targets a different aspect of cellulose biosynthesis.

Chemical genetics to examine cellulose biosynthesis.

Long-term efforts to decode plant cellulose biosynthesis via molecular genetics and biochemical strategies are being enhanced by the ever-expanding scale of omics technologies. An alternative approach to consider are the prospects for inducing change in plant metabolism using exogenously supplied chemical ligands. Cellulose biosynthesis inhibitors (CBIs) have been identified among known herbicides, during diverse combinatorial chemical libraries screens, and natural chemical screens from microbial agents. In this review, we summarize the current knowledge of the inhibitory effects of CBIs and further group them by how they influence fluorescently tagged cellulose synthase A proteins. Additional attention is paid to the continuing development of the CBI toolbox to explore the cell biology and genetic mechanisms underpinning effector molecule activity.