Ludwigia prostrata Roxb. is a problematic weed in rice fields in China, and acetolactate synthase (ALS)-inhibiting herbicides (e.g., bensulfuron-methyl) are widely used for the management of broadleaf weeds. Recently, a L. prostrata biotype (JS-R) that failed to be controlled with ALS-inhibiting herbicides was found in Jiangsu Province, China. This study aims to determine the level and molecular mechanism of resistance to bensulfuron-methyl in this JS-R biotype, and evaluate the cross-resistance spectrums to other ALS-inhibiting herbicides. The dose-response assays indicated that the JS-R L. prostrata biotype had evolved 21.2-fold resistance to bensulfuron-methyl compared to the susceptible biotype (JS-S). ALS gene sequencing revealed that a nucleotide mutation (CCA to TCA) at codon 197, resulting in a Pro-197-Ser mutation, was detected in the resistant plants. Moreover, the JS-R biotype contained the Pro-197-Ser resistance mutation showed cross-resistance to pyrazosulfuron-ethyl (12.0-fold), but was sensitive to penoxsulam, bispyribac-sodium and imazethapyr, which may serve as alternative herbicides to control the resistant L. prostrata biotype. This is the first confirmation of a L. prostrata biotype resistant to bensulfuron-methyl due to a Pro-197-Ser resistance mutation in the ALS gene.

Ammannia multiflora Roxb. is a dominant broadleaf weed which is a serious problem in southern China rice fields, and ALS-inhibiting herbicides have been used for its control for a long time. Excessive reliance of ALS-inhibiting herbicides has led to herbicide resistance in A. multiflora. In this study, 10 A. multiflora populations from Jiangsu province of China were collected, and the resistance levels and target-site resistance mechanisms to ALS-inhibiting herbicides bensulfuron-methyl and penoxsulam were investigated. The dose response assays showed that 8 populations evolved resistance to bensulfuron-methyl (9.1 to 90.9-fold) and penoxsulam (5.0 to 103.1-fold). Amplification of ALS genes indicated that there were three ALS genes (AmALS1, AmALS2, and AmALS3) in A. multiflora. Sequence analysis revealed amino acid mutations at Pro197 in either AmALS1 (Pro-197-Ala, Pro-197-Ser, and Pro-197-His) or AmALS2 (Pro197Ser and Pro197Arg) in resistant populations, and no mutations were found in AmALS3. Moreover, two independent mutations (Pro197Ala in AmALS1 and Pro197Ser in AmALS2, or Pro197Ala in AmALS1 and Pro197Arg in AmALS2) coexisted in two resistant populations, respectively. In addition, the auxin mimic herbicides MCPA and florpyrauxifen-benzyl, PSII inhibitor bentazon and PPO inhibitor carfentrazone-ethyl can effectively control the resistant A. multiflora populations. Our study demonstrated the wide prevalence of ALS-inhibiting herbicides-resistant A. multiflora populations in Jiangsu province, the diversity of Pro197 mutations in ALS genes, and provides alternative herbicide options for controlling resistant A. multiflora populations.

Ammannia auriculata is a troublesome broadleaf weed, widely distributed in the paddy fields of southern China. In this study, 10 biotypes of A. auriculata were sampled from Yangzhou City, China, where the paddy fields were seriously infested with A. auriculata, and their resistance levels to acetolactate synthase (ALS) inhibitor bensulfuron-methyl were determined. The whole-plant response assays showed that nine A. auriculata biotypes were highly resistant (from 16.4- to 183.1-fold) to bensulfuron-methyl in comparison with a susceptible YZ-S biotype, and only one YZ-6 biotype was susceptible. ALS gene sequencing revealed that three ALS gene copies existed in A. auriculata, and four different amino acid substitutions (Pro197-Leu, -Ala, -Ser, and -His) at site 197 in the AaALS1 or 2 genes were found in eight resistant biotypes. In addition, no amino acid mutations in three ALS genes were found in the YZ-3 biotype. These results suggested that target-site mutations or non-target-site resistance mechanisms were involved in tested resistant A. auriculata biotypes. Finally, a cleaved amplified polymorphic sequence (CAPS) marker was identified to rapidly detect the Pro197 mutations in A. auriculata.

Glyphosate is a non-selective herbicide and is widely used for weed control in non-cultivated land in China. One susceptible (S) and five putative glyphosate-resistant (R1, R2, R3, R4, and R5) Eleusine indica biotypes were selected to investigate their resistance levels and the potential resistance mechanisms. Based on the dose–response assays, the R3 and R5 biotypes showed a low-level (2.4 to 3.5-fold) glyphosate resistance, and the R1, R2, and R4 biotypes exhibited a moderate- to high-level (8.6 to 19.2-fold) resistance, compared with the S biotype. The analysis of the target-site resistance (TSR) mechanism revealed that the P106A mutation and the heterozygous double T102I + P106S mutation were found in the R3 and R4 biotypes, respectively. In addition, the similar EPSPS gene overexpression was observed in the R1, R2, and R5 biotypes, suggesting that additional non-target-site resistance (NTSR) mechanisms may contribute to glyphosate resistance in R1 and R2 biotypes. Subsequently, an RNA-Seq analysis was performed to identify candidate genes involved in NTSR. In total, ten differentially expressed contigs between untreated S and R1 or R2 plants, and between glyphosate-treated S and R1 or R2 plants, were identified and further verified with RT-qPCR. One ATP-binding cassette (ABC) transporter gene, one aldo-keto reductases (AKRs) gene and one cytochrome P450 monooxygenase (CytP450) gene were up-regulated in R1 or R2 plants. These results indicated that EPSPS overexpression, single or double mutation was a common TSR mechanisms in E. indica. Additional NTSR mechanisms could play an essential role in glyphosate resistance. Three genes, ABCC4, AKR4C10, and CYP88, could serve as important candidate genes and deserve further functional studies.

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