Galium aparine L. var. echinospermum (Wallr.) Cuf., an annual or winter annual broadleaf weed in the Rubiaceae family, has become troublesome due to its herbicide resistance in the wheat fields of the Huang-Huai-Hai region in China. Four cleavers populations (JS-15, SD-10, JS-22, and AH-20) were collected from the wheat fields of Jiangsu, Shandong, and Anhui Province, where florasulam control was absent. This study identified the herbicide resistance patterns and investigated the mechanism underlying the florasulam resistance. The whole-plant dose-response experiments revealed a notable variation in the degree of resistance exhibited by three specific populations towards florasulam, in comparison to the most sensitive populations (S and AH-9), with the highest resistance index reaching 841.4. Acetolactate Synthase gene-sequencing assay found that R plants from JS-15, JS-22, and AH-20 had a Trp-574-Leu mutation, while no known ALS resistance mutations were discovered in SD-10 plants. In vitro ALS enzyme activity assays also indicated that the extractable ALS from the resistant plants of JS-15, JS-22, and AH-20 was greatly resistant to florasulam relative to that of the susceptible. Additionally, according to the resistance rating system, all resistant populations were susceptible to Carfentrazone-ethyl + MCPA-Na and Bipyrazone + Fluroxypyr-methyl. AH-20, JS-15, and JS-22 exhibited resistance to selected ALS, 4-hydroxyphenylpyruvate dioxygenase, and photosystem Ⅱ complex inhibitors, demonstrating RR and RRR resistance profiles, whereas AH-9 displayed S-sensitivity to virtually all tested agents. SD-10, on the other hand, showed RR and RRR resistance to HPPD and PSII inhibitors, but exhibited S-sensitivity specifically to tribenuron-methyl. These findings indicate that a target site-based mechanism drives the resistance to the ALS inhibitor florasulam in cleavers populations, but NTSR resistance may have also contributed (was not investigated). Other herbicides with different sites of action were tested and were active against cleavers.
