Investigating resistance and the rapid response to glyphosate in giant ragweed (Ambrosia trifida L.)
Date
2016
Authors
Van Horn, Christopher R., author
Westra, Phil, advisor
Reddy, Anireddy, advisor
Argueso, Cris, committee member
Gaines, Todd, committee member
Leach, Jan, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
The introduction of glyphosate-resistant crops along with widespread multiple in-season applications of glyphosate as part of weed management strategies that fail to address long-term weed control have provided the perfect scenario to foster the recent boom in glyphosate-resistant weeds. In order to implement best strategies to manage glyphosate-resistant weeds, it is important to understand the mechanism of resistance. Glyphosate targets and inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which prevents the synthesis of aromatic amino acids. We have investigated the mechanism of glyphosate resistance using several geographically diverse giant ragweed populations. From these populations we have characterized three phenotypic responses to glyphosate treatment: susceptible (GS), resistant slow response (GR slow), and resistant rapid response (GR rapid). Glyphosate resistance in giant ragweed (Ambrosia trifida L.) was first discovered in 2004 and the rapid response biotype was first identified in 2008. The molecular basis for resistance and the rapid response remains unknown. Our objective is to analyze glyphosate resistance and the rapid response in giant ragweed using physiological and molecular techniques. In whole-plant dose-response experiments conducted under greenhouse conditions, the glyphosate ED50 values (the effective dose that reduced shoot mass 50% relative to non-treated plants) of GR rapid biotypes were 4.2- and 2.3-fold greater than the ED50 values of GS biotypes and GR slow biotypes were 3.3- and 3.6-fold greater than the ED50 values of GS biotypes. The effective concentration that increased shikimate accumulation 50% relative to non-treated leaf tissue (EC50) was 4.1- and 3.9-fold higher (P < 0.05) for GR rapid and GR slow biotypes, respectively, than GS biotypes based on values averaged across accessions of the same biotype. However, at high glyphosate concentrations (1,000 to 2,000 μM), shikimate accumulation in the GR biotypes was similar to or greater than the GS biotypes. EPSPS sequence analysis showed no nucleotide mutation at a position where mutations are known to confer resistance in multiple species. Genomic copy number analysis found no evidence of target gene amplification. Whole plant physiology experiments suggest the rapid response is carbon dependent and both Phenylalanine and Tyrosine play a role in the stimulation of the rapid response. Excised leaf discs show H2O2 accumulation in mature leaf tissue of the GR rapid response biotype within 30 minutes after glyphosate application. Transcriptomic analysis of GR rapid and GS giant ragweed has identified significant differentially expressed genes that may be linked to glyphosate resistance and/or the rapid response. RNA-seq data was validated through qRT-PCR analysis. Six candidate genes were analyzed using qRT-PCR, two of which were selected for further expression analysis in an F2 population segregating for resistance. The final two candidate genes, osmotin 34 and S-adenosylmethionine synthetase 3, did not show a similar segregation pattern as seen in the F2 individuals. The large amount of RNA-seq data collected will continue to be used to determine key marker genes that could provide further insight into the glyphosate resistance mechanism present in this species. These initial results provide a framework for the future of giant ragweed glyphosate resistance research, which becomes increasingly important as the use of glyphosate-resistant crops develops world-wide. With this research, we can continue to work toward sustainable forms of herbicide weed management.