Mechanism of atrazine resistance in atrazine- and HPPD inhibitor-resistant Palmer amaranth (Amaranthus palmeri S. Wats.) from Nebraska

Palmer amaranth (Amaranthus palmeri S. Wats.) is one of the most problematic weed species in agronomic crops in the United States. A Palmer amaranth biotype multiple-resistant to atrazine and 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors was reported in a seed corn production field in Nebraska. Rapid detoxification mediated by cytochrome P450 monooxygenases and increased HPPD gene expression were reported as the mechanisms of mesotrione resistance in atrazine- and HPPD inhibitor-resistant Palmer amaranth biotype from Nebraska; however, the mechanism of atrazine resistance is unknown. The objectives of this study were to investigate target site or non-target site based mechanisms conferring atrazine resistance in Palmer amaranth from Nebraska. 14C-atrazine absorption and translocation studies revealed that reduced atrazine absorption or translocation were not involved as one of the mechanisms of atrazine resistance. Instead, greater 14C-atrazine absorption and recovery in treated leaves were observed in resistant compared with susceptible Palmer amaranth. No known mutations including Ser264Gly substitution in the psbA gene causing target site based atrazine resistance were observed. However, the parent 14C-atrazine was metabolized rapidly <4 h after treatment in resistant plants, conferring enhanced atrazine metabolism as the mechanism of resistance.

Prevalence and Mechanism of Atrazine Resistance in Waterhemp (Amaranthus tuberculatus) from Nebraska

Resistance to atrazine (a photosystem II [PSII] inhibitor) is prevalent in waterhemp [Amaranthus tuberculatus(Moq.) J. D. Sauer] across the U.S. Midwest. Previous research suggests that target-site mutation or rapid metabolism of atrazine mediated by glutathioneS-transferase (GST) conjugation confers resistance inA. tuberculatusfrom Illinois. The distribution and mechanism of resistance to atrazine inA. tuberculatuspopulations from Nebraska (NE) are unknown. In this research we (1) evaluated the response and frequency of resistance in NEA. tuberculatusto soil-applied PSII (metribuzin and atrazine) and protoporphyrinogen oxidase (sulfentrazone) inhibitors, as well as POST-applied atrazine; and (2) determined the mechanism of atrazine resistance in NEA. tuberculatus. The chloroplasticpsbAgene, coding for a D1 protein (the target site of atrazine) was sequenced in 85 plants representing 27 populations ofA. tuberculatus. Furthermore, 24 plants selected randomly from four atrazine-resistant (AR) populations were used to determine the metabolism of atrazine via GST conjugation. Results from the soil-applied herbicide evaluation suggest that metribuzin (0.56 kg ai ha−1) and sulfentrazone (0.28 kg ai ha−1) were effective onA. tuberculatusmanagement. PRE and POST screenings against atrazine in the greenhouse indicate that atrazine (1.345 kg ai ha−1) was not effective on 39% and 73% of theA. tuberculatuspopulations evaluated (total of 109 and 85 populations, respectively), suggesting the prevalence of atrazine resistance inA. tuberculatusin NE. Sequence analysis of thepsbAgene found no known point mutations conferring atrazine resistance. However, the AR plants conjugated atrazine via GST activity faster than the known atrazine-susceptibleA. tuberculatus. Overall, the outcome of this study demonstrates the predominance of metabolism-based resistance to atrazine inA. tuberculatusfrom NE, which may predispose this species to evolve resistance to other herbicides. The use of integrated management strategies forA. tuberculatusis crucial for the control of this troublesome species.

The genetic intractability of Symbiodinium microadriaticum to standard algal transformation methods

Modern transformation and genome editing techniques have shown great success across a broad variety of organisms. However, no study of successfully applied genome editing has been reported in a dinoflagellate despite the first genetic transformation of Symbiodinium being published about 20 years ago. Using an array of different available transformation techniques, we attempted to transform Symbiodinium microadriaticum (CCMP2467), a dinoflagellate symbiont of reef-building corals, with the view to performing subsequent CRISPR-Cas9 mediated genome editing. Plasmid vectors designed for nuclear transformation containing the chloramphenicol resistance gene under the control of the CaMV p35S promoter as well as several putative endogenous promoters were used to test a variety of transformation techniques including biolistics, electroporation and silicon carbide whiskers. Chloroplast-targeted transformation were attempted using an engineered Symbiodinium chloroplast minicircle encoding a modified PsbA protein that confers atrazine resistance. We report that we have been unable to confer chloramphenicol or atrazine resistance to Symbiodinium microadriaticum strain CCMP2467.

Physiological and Molecular Characterization of Atrazine Resistance in a Wild Radish (Raphanus raphanistrum) Population

This study documents the physiology and genetics of evolved atrazine resistance in a wild radish population from Western Australia. Plant response to atrazine treatment confirmed a high level of resistance in population WARR5. At 0.25 kg atrazine/ha, all plants from a susceptible population were killed, whereas resistant WARR5 was unaffected at the highest dose tested (4 kg atrazine/ha). Leaf photosynthesis in susceptible plants was inhibited after 1 kg atrazine/ha treatment, whereas leaf photosynthesis in WARR5 plants was unaffected. Furthermore, atrazine resistance was maternally inherited. Sequencing of apsbAgene fragment in resistant WARR5 and susceptible plants revealed a single point mutation resulting in a coding change from Ser264to Gly of the D1 protein in resistant plants. We are confident that this mutation is the basis of resistance to the photosystem II inhibitors in this wild radish population.