Field-evolved resistance of goosegrass to glyphosate is due to double or single mutation in EPSPS , or amplification of EPSPS leads to increased transcription and protein levels. Glyphosate has been used widely in the south of China. The high selection pressure from glyphosate use has led to the evolution of resistance to glyphosate in weeds. We investigated the molecular mechanisms of three recently discovered glyphosate-resistant Eleusine indica populations (R1, R2 and R3). The results showed that R1 and R2 had double Thr102Ile and Pro106Ser mutation and a single mutation of Pro106Leu in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, respectively. Escherichia coli containing the mutated EPSPS genes was tolerant to glyphosate. EPSPS activity in R1 and R2 plants was higher than in the sensitive plants. There was no amino acid substitution in EPSPS gene in R3. However, expression of EPSPS in R3 plants was higher than in glyphosate-susceptible (S) population (13.8-fold) after glyphosate treatment. EPSPS enzyme activity in both R3 and S plants was inhibited by glyphosate, while shikimate accumulation in R3 was significantly lower than for the S population. Further analysis revealed that the genome of R3 contained 28.3-fold more copies of the EPSPS gene than that of susceptible population. EPSPS expression was positively correlated with copy number of EPSPS. In conclusion, mutation of the EPSPS gene and increased EPSPS expression are part of the molecular mechanisms of resistance to glyphosate in Eleusine indica.

The dinitroaniline herbicides (particularly trifluralin) have been globally used in many crops for selective grass weed control. Consequently, trifluralin resistance has been documented in several important crop weed species and has recently reached a level of concern in Australian Lolium rigidum populations. Here, we report novel mutations in the L. rigidum α-tubulin gene which confer resistance to trifluralin and other dinitroaniline herbicides. Nucleotide mutations at the highly conserved codon Arg-243 resulted in amino acid substitutions of Met or Lys. Rice calli transformed with the mutant 243-Met or 243-Lys α-tubulin genes were 4- to 8-fold more resistant to trifluralin and other dinitroaniline herbicides (e.g., ethalfluralin and pendimethalin) compared to calli transformed with the wild type α-tubulin gene from L. rigidum. Comprehensive modeling of molecular docking predicts that Arg-243 is close to the trifluralin binding site on the α-tubulin surface and that replacement of Arg-243 by Met/Lys-243 results in a spatial shift of the trifluralin binding domain, reduction of trifluralin-tubulin contacts, and unfavorable interactions. The major effect of these substitutions is a significant rise of free interaction energy between α-tubulin and trifluralin, as well as between trifluralin and its whole molecular environment. These results demonstrate that the Arg-243 residue in α-tubulin is a determinant for trifluralin sensitivity, and the novel Arg-243-Met/Lys mutations may confer trifluralin resistance in L. rigidum.

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BACKGROUND: A Lolium rigidum population collected from Western Australia was previously reported as highly resistant to dinitroaniline herbicides mainly due to a Val-202-Phe substitution in the target site -tubulin protein. To further determine the contribution of the 202 mutation to resistance, two sub-populations, respectively comprising the 202 mutant and wild-type (WT) individuals, were isolated from within the same resistant population and subject to dinitroaniline herbicide doses. A rice transgenic study was conducted to demonstrate whether the amino acid substitution at the 202 residue confers resistance. In addition, as indicated in the phenotyping and genotyping study, non-target enhanced trifluralin metabolism was further examined in the same population. RESULTS: The 202 mutants were more resistant than the wild-type plants. Rice calli transformed with the L. rigidum mutant -tubulin gene (Val-202-Phe) were more resistant to dinitroaniline herbicides relative to calli transformed with the wild-type gene. Also, enhanced trifluralin metabolism was detected in the 202 mutants in comparison to the susceptible seedlings. CONLCUSION: Both target-site Val-202-Phe -tubulin mutation and non-target-site enhanced trifluralin metabolism co-exist in this dinitroaniline-resistant L. rigidum population.

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Resistance to the pre-emergence herbicide trifluralin is increasing in Australian annual ryegrass ( Lolium rigidum) populations. Three L. rigidum populations (R1, R2, and R3) collected from Australian grain fields were identified with trifluralin resistance. Both target-site and nontarget-site resistance mechanisms were investigated. No target-site α-tubulin mutations were detected in populations R1 and R3, while an Arg-243-Lys mutation was found in R2. Compared with the three trifluralin-susceptible populations, enhanced [C]-trifluralin metabolism, quantified by measuring the amount of [C] label partitioning into the polar phase of a hexane:methanol system, was identified in all the three resistant populations. This is the first report of metabolic resistance to trifluralin. Coevolution of target-site and nontarget-site resistance to trifluralin is occurring, and metabolic resistance is not rare in L. rigidum populations in Australia. A method was established for trifluralin metabolic resistance detection, overcoming the difficulties of quantifying this highly volatile herbicide by chromatographic methods.