Summary: | <i>Monochoria vaginalis</i> (Burm. f.) C. Presl, belonging to the family Pontederiaceae, is an aquatic herbaceous plant, native to temperate and tropical Asia. The species often occurs in paddy fields as a noxious weed in East Asia, and in the USA, and causes a significant reduction in rice production. The objective of the present research was the evaluation of the resistance levels of <i>M. vaginalis</i> against three chemical groups of acetolactate synthase (ALS)-inhibitor herbicides and other two different groups of herbicides, and the investigation of the mutations in the <i>ALS</i> gene of the resistant biotype of <i>M. vaginalis</i>. Herbicide dose–response experiments showed that the resistant biotype of <i>M. vaginalis</i> was highly resistant to bensulfuron-methyl, moderately resistant to bispyribac-sodium, had low resistance to penoxsulam and 2,4-D dimethyl ammonium, and was susceptible to sulfentrazone. The nucleotide sequences of the <i>ALS gene</i> of resistant and susceptible biotypes showed 14 base substitutions, which caused two amino acid substitutions: Val-143-Ile and Val-148-Ile. It is the first report of the substitutions of amino acids Val-143-Ile and Val-148-Ile in ALS protein. Those mutations may give different resistance spectra against three ALS-inhibitor herbicides: bensulfuron-methyl, bispyribac-sodium, and penoxsulam. Further research is needed to elucidate the molecular basis of target-site resistance mechanisms such as the transformation of the <i>ALS</i> gene of <i>M. vaginalis</i>. It is also necessary to evaluate herbicide mixtures and/or the rotation of herbicide sites of action to control the resistant biotype of <i>M. vaginalis.</i>
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