Sourgrass [Digitaria insularis (L.) Mez ex Ekman] is considered the most troublesome weed in agronomic crops in South America. Overreliance on glyphosate has selected for resistant populations, although the resistance mechanisms remain unknown. Recently, populations were identified that exhibited multiple resistance to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetyl-CoA carboxylase (ACCase) inhibitors, posing a significant challenge due to the lack of alternative control options. This project aimed to identify the resistance patterns and levels to glyphosate and ACCase inhibitors of three suspected resistant populations (P1, P2, and P3), and elucidate the resistance mechanisms. We performed dose–response experiments with clethodim, fluazifop-P-butyl, glyphosate, and pinoxaden to identify the possibility of cross- and multiple resistance and to quantify the resistance levels. We sequenced the ACCase and EPSPS genes to test the hypothesis that target-site mutations were involved in the resistance mechanisms, given the resistance patterns observed. Our results indicated that two of the tested populations, P1 and P2, were multiple resistant to glyphosate and all ACCase-inhibitor classes, while P3 was resistant to glyphosate only. Resistance levels varied by herbicide, with resistance indices ranging from 2.7- to nearly 2,000-fold. We identified an amino acid substitution in ACCase at position 2078 (Asp-2078-Gly), homozygous for both P1 and P2, corroborating the resistance patterns observed. Interestingly, EPSPS sequencing identified multiple heterozygous DNA polymorphisms that resulted in amino acid substitutions at positions 106 (P1 and P2) or at both 102 and 106 (P3), indicating multiple evolutionary origins of glyphosate-resistance evolution. We show for the first time the genetic mechanisms of multiple resistance to glyphosate and ACCase in D. insularis, and provide a thorough discussion of the evolutionary and management implications of our work.
