Summary: | <i>Conyza sumatrensis</i> was reported to be associated with 20 cases of herbicide resistance worldwide, with a recent report of multiple drug resistance to paraquat, glyphosate, and chlorimuron in Brazil. In Paraguay, there were no reports of cases of resistance for this species; however, in 2017, researchers began identifying biotypes with resistance to paraquat, glyphosate, and chlorimuron, which is the focus of the present study. The goal of this study was to investigate the case of multiple resistance of <i>C. sumatrensis</i> to paraquat, glyphosate, and chlorimuron and to monitor the resistant biotypes in the departments of Canindeyú and Alto Paraná. Seeds were collected from sites where plants survived after herbicide application in the 2017/18 and 2018/19 seasons. After screening, biotypes were selected for the construction of dose–response curves. A resistance factor (RF) of 6.79 was observed for 50% control (C<sub>50</sub>) and 3.92 for 50% growth reduction (GR<sub>50</sub>) for the application of paraquat. An RF of 12.32 was found for C<sub>50</sub> and 4.15 for GR<sub>50</sub> for the application of glyphosate. For the application of chlorimuron, an RF of 11.32 was found for C<sub>50</sub> and 10.96 for GR<sub>50</sub>. This confirms the multiple resistance of the <i>C. sumatrensis</i> biotype to paraquat, glyphosate, and chlorimuron. Population monitoring indicated the presence of <i>C. sumatrensis</i> with multiple resistance in departments of Canindeyú and Alto Paraná, Paraguay.
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