Front. Plant Sci. Frontiers in Plant Science Front. Plant Sci. 1664-462X Frontiers Media S.A. 10.3389/fpls.2025.1535877 Plant Science Original Research Herbicide resistance in Leptochloa chinensis (L.) Nees populations from different regions of Jiangsu Province, China: sensitivity differences and underlying mechanisms Xu Peng 1 Wang Ke 2 Ju Yawen 1 Fu Yousheng 1 Zhu Axiu 3 Cao Kaige 1 Wang Hongchun 2 * 1 Huaiyin Institute of Agriculture Science of Xuhuai Area in Jiangsu, Huai’an, China 2 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China 3 Plant Protection and Quarantine Station of Jiangsu Province, Nanjing, China

Edited by: Ye Liu, Nanjing Agricultural University, China

Reviewed by: Minghao Jiang, Anhui Agricultural University, China

Marta Stankiewicz-Kosyl, Warsaw University of Life Sciences, Poland

*Correspondence: Hongchun Wang, whc23@jaas.ac.cn

04 02 2025 2025 16 1535877 28 11 2024 20 01 2025 Copyright © 2025 Xu, Wang, Ju, Fu, Zhu, Cao and Wang 2025 Xu, Wang, Ju, Fu, Zhu, Cao and Wang

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Leptochloa chinensis (L.) Nees, a noxious weed species commonly found in rice fields, has become a significant challenge in Jiangsu Province, China, as it has developed resistance to multiple herbicides due to extensive and continuous herbicide use in recent years. Therefore, this study was conducted to elucidate sensitivity differences and the mechanisms underlying the resistance of L. chinensis (L.) Nees populations to commonly used herbicides across different regions of Jiangsu Province, China. A whole-plant bioassay was used to assess the sensitivity of 46 L. chinensis populations collected from various areas within Jiangsu to several herbicides frequently applied in paddy fields, including: cyhalofop-butyl, fenoxaprop-P-ethyl, pyraclonil, benzobicyclon, anilofos, and oxaziclomefone. After treatment with cyhalofop-butyl, 38 out of 46 populations showed relative resistance-index values that were over four times that of the controls, indicating significant resistance to cyhalofop-butyl. All 41 cyhalofop-butyl-resistant populations showed cross-resistance to fenoxaprop-P-ethyl but remained susceptible to pyraclonil, benzobicyclon, anilofos, and oxaziclomefone. The proportion of populations resistant to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides increased progressively from the south to the north of Jiangsu. Cross-resistance was evident between cyhalofop-butyl and fenoxaprop-P-ethyl; however, all resistant populations were susceptible to pyraclonil, benzobicyclon, anilofos, and oxaziclomefone. Furthermore, mutations in the ACCase gene were identified as a crucial mechanism for cyhalofop-butyl resistance. Specifically, we found ACCase mutations I1781L, W1999C, W2027C/L/S, I2041N, and D2078G in cyhalofop-butyl-resistant L. chinensis populations, among which, W1999C and W2027C accounted for a relatively high proportion, while I1781L, W2027L/S, I2041N, and D2078G were found in one population each. ACCase gene mutations are seemingly a key mechanism for the development of resistance to cyhalofop-butyl, thus, our study provides useful information for developing effective weed-management strategies for controlling this noxious weed species, while ensuring sustainable agricultural practices.

 Leptochloa chinensis (L.) Nees geographical populations herbicide cyhalofop-butyl sensitivity target mutation section-in-acceptance Sustainable and Intelligent Phytoprotection Introduction

Leptochloa chinensis (L.) Nees is a tetraploid (2n=4x=40) belonging to the Chloridoideae subfamily of the Poaceae(grass) family, with high drought and waterlogging resistance, moisture preference, and strong reproductive and tillering abilities. Furthermore, this grass species is globally recognized as a noxious agricultural weed (Chauhan and Johnson, 2008; Wen et al., 2017; Zhu et al., 2018). The spread and harmful effect of L. chinensis in the middle and lower reaches of the Yangtze River Basin in China have shown a clear increasing trend over time, which has been exacerbated by the widespread adoption of direct-seeded rice cultivation practices and prolonged, exclusive use of herbicides, posing a severe threat to rice production security (Wu et al., 2015; Yuan et al., 2022).

Cyhalofop-butyl is an acetyl-CoA carboxylase (ACCase, EC.6.4.12) inhibitor herbicide traditionally used in paddy fields for the management of Poaceae weeds, including L. chinensis and Echinochloa crus-galli (Ruiz-Santaella et al., 2006). However, the efficacy of cyhalofop-butyl against L. chinensis has been declining owing to its long-term, exclusive use. This has led to the development of resistance among weeds, as indicated by an increasing number of reports from farmers stating that recommended doses are ineffective in controlling the weed. Exclusive use of ACCase inhibitor herbicides over extended periods can induce resistance in weeds. To date, 49 species of weeds have reportedly developed resistance to this class of herbicides globally (Heap, 2024; Heap and Knight, 1986; Maneechote et al., 2005). Thus, a population of L. chinensis resistant to ACCase inhibitors, including cyhalofop-butyl, was identified in Bangkok, Thailand in 2002 (Heap, 2024). Similarly, L. chinensis populations resistant to cyhalofop-butyl were detected in the paddy fields of Hubei Province, China in 2011; L. chinensis populations showing cross-resistance to cyhalofop-butyl and fenoxaprop-P-ethyl were reported from South Korea in 2012 (Sahid et al., 2011; Wei et al., 2015), and L. chinensis populations resistant to cyhalofop-butyl were identified in Malaysia in 2014 (Rahman et al., 2014). Currently, L. chinensis populations resistant to cyhalofop-butyl have been identified in Jiangsu (Deng et al., 2019), Shanghai (Yuan et al., 2022), Anhui (Jiang et al., 2022), and Hunan (Peng et al., 2020). Several mutations, such as Ile-1781-Leu, Leu-1818-Phe, Trp-1999-Cys, Trp-2027-Cys, Trp-2027-Gly, Trp-2027-Leu, Trp-2027-Ser, Ile-2041-Asn, Cys-2088-Arg, and Gly-2096-Ala, in ACCase have been identified as the primary cause of L. chinensis resistance (Kurniadie et al., 2022; Zhang et al., 2022; Zhao et al., 2022; Liao et al., 2024; Deng et al., 2023; Jiang et al., 2024).

In turn, Deng et al. (2019) detected cyhalofop-butyl-resistant populations of L. chinensis in the paddy fields of Jiangsu Province in 2019. However, variation in sensitivity to cyhalofop-butyl and other commonly used herbicides among different geographic populations of L. chinensis in Jiangsu, along with their distribution and the underlying mechanisms responsible for resistance, remain unclear. Thus, in this study, we systematically collected seed samples of L. chinensis from paddy fields across the southern, central, and northern regions of Jiangsu Province to test their sensitivity to the commonly used herbicides, the aryloxyphenoxypropionate(APP) family of ACCase inhibitor cyhalofop-butyl(HRAC 1, inhibition of Acetyl CoA Carboxulase) and fenoxaprop-P-ethyl(HRAC 1, inhibition of Acetyl CoA Carboxulase), the protoporphyrinogen oxidase(PPO) inhibitor pyraclonil(HRAC 14, inhibition of Protoporphyrinogen Oxidase), the 4-hydroxyphenylpyr­uvate dioxygenase inhibitor benzobicyclon(HRAC 27, inhibition of Hydroxyphenyl Pyruvate Dioxygenose), the organophosphorus herbicide anilofos(HRAC 15, inhibition of Very Long-Chain Fatty Acid Synthesis), and the organoheterocyclic herbicide oxaziclomefone(HRAC 30, inhibition of Fatty Acid Thioesterase), with the aim to analyze the distribution of resistant populations and the key molecular mechanisms responsible for resistance. Our study aimed to provide technical guidance for the effective management and control of L. chinensis in paddy fields.

 Materials and methods Seeds

Seeds were randomly collected from various locations across southern and northern Jiangsu Province during seed maturation period, from September to November 2020. The LENJ-001 population is a known susceptible population. Specific collection sites are listed in Table 1 .

Leptochloa chinensis (L.) Nees seed collection sites.

Order Population code Site of collection Geographical location
1 LESZ-001 Tongli Town, Wujiang District, Suzhou City 31°6′N, 120°43′E
2 LESZ-003 Guli Town, Changshu County, Suzhou City 31°40′N, 120°49′E
3 LESZ-005 Daxin Town, Zhangjiagang City, Suzhou City 31°57′N, 120°31′E
4 LEJY-001 Songjiawei Village, Jiangyin City, Wuxi City 31°52′N, 120°7′E
5 LEHS-003 Yuqi Street, Huishan District, Wuxi City 31°42′N, 120°10′E
6 LEYX-003 Dingshu Town, Yixing City, Wuxi City 31°16′N, 119°50′E
7 LELY-003 Nandu Town, Liyang City, Changzhou City 31°26′N, 119°19′E
8 LEJT-001 Zhiqian Town, Jintan District, Changzhou City 31°39′N, 119°28′E
9 LELS-001 Baima Town, Lishui District, Nanjing City 31°34′N, 119°10′E
10 LEJR-001 Shishi Town, Jurong City, Zhenjiang City 31°56′N, 119°9′E
11 LEJR-002 Guozhuang Town, Jurong City, Zhenjiang City 31°48′N, 119°1′E
12 LELH-003 Chengqiao Town, Liuhe District, Nanjing City 32°22′N, 118°43′E
13 LEYZ-004 Qianjin Village, Yizheng City, Yangzhou City 32°18′N, 119°4′E
14 LEYL-001 Yiling Town, Jiangdu District, Yangzhou City 32°28′N, 119°40′E
15 LEJD-001 Daqiao Town, Jiangdu District, Yangzhou City 32°21′N, 119°42′E
16 LEJD-003 Dinggou Town, Jiangdu District, Yangzhou City 32°33′N, 119°39′E
17 LEGY-001 Ganduo Town, Gaoyou City, Yangzhou City 32°49′N, 119°46′E
18 LEGY-002 Ganduo Town, Gaoyou City, Yangzhou City 32°50′N, 119°44′E
19 LERG-001 Changjiang Town, Rugao City, Nantong City 32°6′N, 120°36′E
20 LERG-002 Wuyao Town, Rugao City, Nantong City 32°12′N, 120°32′E
21 LERG-003 Wuyao Town, Rugao City, Nantong City 32°12′N, 120°32′E
22 LEJJ-001 Dongxing Town, Jingjiang City, Taizhou City 31°58′N, 120°9′E
23 LETX-002 Huangqiao Town, Taixing City, Taizhou City 32°14′N, 120°14′E
24 LEXH-002 Xingdong Town, Xinghua City, Taizhou City 32°58′N, 119°54′E
25 LEXH-003 Diaoyu Town, Xinghua City, Taizhou City 33°4′N, 119°58′E
26 LEXH-006 Diduo Town, Xinghua City, Taizhou City 32°50′N, 120°5′E
27 LEHQ-001 Hongqi Farm, Jiangyan District, Taizhou City 32°33′N, 120°1′E
28 LEDF-007 Fangqiang Farm, Dafeng District, Yancheng City 33°29′N, 120°28′E
29 LESY-005 Haitong Town, Sheyang County, Yancheng City 33°48′N, 120°20′E
30 LEFN-001 Chenliang Town, Funing County, Yancheng City 33°41′N, 119°56′E
31 LEHX-001 Huanghai Farm, Xiangshui County, Yancheng City 34°17′N, 120°1′E
32 LEHX-002 Huanghai Farm, Xiangshui County, Yancheng City 34°17′N, 120°1′E
33 LEBH-001 Binhuai Farm, Binhai County, Yancheng City 34°15′N, 120°6′E
34 LEHH-001 Huaihai Farm, Sheyang County, Yancheng City 34°9′N, 120°14′E
35 LESQ-001 Wangji Town, Siyang County, Suqian City 33°52′N, 118°44′E
36 LESQ-002 Chuancheng Town, Siyang County, Suqian City 33°36′N, 118°37′E
37 LESQ-003 Lailong Town, Suyu District, Suqian City 34°2′N, 118°29′E
38 LESN-001 Daodao, Weiji Town, Suining County, Xuzhou City 34°1′N, 117°57′E
39 LESN-002 Weiji Town, Suining County, Xuzhou City 34°1′N, 117°57′E
40 LELSH-001 Gaozhuang, Lianshui County, Huai’an City 33°58′N, 119°5′E
41 LEBYH-001 Huai’an Baoying Lake Farm 33°36′N, 119°1′E
42 LEGY-001 Songzhuang Town, Ganyu District, Lianyungang City 34°46′N, 119°8′E
43 LEGYD-001 Dongxin Farm, Guanyun County, Lianyungang City 34°35′N, 119°22′E
44 LEYT-002 Yuntai Farm, Haizhou District, Lianyungang City 34°39′N, 119°23′E
45 LEDX-003 Dongxin Farm, Lianyun District, Lianyungang City 34°32′N, 119°22′E
46 LENJ-001 Xuanwu District, Nanjing City 30°2’N, 118°52’E
Herbicide treatments

Test chemicals and the manufacturers are shown in Table 2 . The technical materials of cyhalofop -butyl, fenoxaprop-P-ethyl, pyraclonil, benzobicyclon, anilofos, and oxaziclomefone were individually dissolved in 1mL acetone and then diluted to a series of solutions with water containing 0.1% (v/v) Tween-80 aqueous solution.

Herbicides and manufacturers.

Herbicide Test doses(g a.i. ha-1) Manufacturer
98% Cyhalofop-butyl TC 105 Jiangsu Agrochem Laboratory Co., Ltd., Jiangsu, China
96% Fenoxaprop-P-ethyl TC 37.5 Jiangsu Agrochem Laboratory Co., Ltd., Jiangsu, China
97% Pyraclonil TC 210 Hubei Xianghe Precision Chemistry Co., Ltd., Hubei, China
98% Benzobicyclon TC 225 SDS Biotech K.K., Tokyo, Japan
95% Anilofos TC 315 Shandong Binnong Technology Co., Ltd., Shandong, China
97% Oxaziclomefone TC 45 Jiangsu Agrochem Laboratory Co., Ltd., Jiangsu, China

TC, Technical Material.

 Test methods Cultivation of the experimental materials

Whole-plant bioassay was used in this experiment. White plastic pots (7 cm in length and width and 9 cm in height), with holes in the bottom, were filled to approximately three-quarters of their capacity with air-dried fine previously sieved soil and placed inside a plastic turnover box. Water was added to the bottom to ensure the soil was saturated with moisture. The soil used for the tests was a loam (pH 6.7) with 1.6% organic matter content. The seeds were sown separately, covered with a thin layer of soil of approximately 0.2–0.3-cm thick and cultivated in a solar greenhouse (temperature: daytime, 25 ± 5°C, nighttime, 25 ± 5°C; light regime: 12-:12-h light/dark). Once the weeds had emerged uniformly, seedlings were thinned to 12 per pot.

 Determination of the sensitivity of <italic>L. chinensis</italic> populations to cyhalofop-butyl

When plants reached the 2–3-leaf stage, stem and leaf spray treatments were applied. Spraying was performed with a 3WPSH-500D bioassay spray tower developed by the Nanjing Institute of Agricultural Mechanization of the Ministry of Agriculture, with a disk diameter of 50 cm, a spindle rotation speed of 6 rpm, nozzle aperture of 0.3 mm, spray pressure of 0.3 MPa, droplet diameter of 100 μm, and a nozzle flow rate of 90 mL/min. The volume of water used was 450 L ha-1. There were six experimental doses: 1/4, 1/2, 1, 2, 4, and 8 times the upper limit of the recommended field application rate of cyhalofop-butyl, which is 105 g a.i.ha-1, resulting in doses of 26.25, 52.5, 105, 210, 420, and 840 g a.i. ha-1, respectively. The herbicide doses were 6.56, 13.12, 26.24, 52.48, 104.96, and 209.92 g a.i. ha-1 for the LENJ-001 population. For each L. chinensis population, an untreated blank control was set up, and four replications per treatment were included. At 20 d after treatment, the fresh weight of the aerial parts of the L. chinensis plants was measured. Each treatment had four replicates, and the experiment was repeated twice. Excel Software was used to analyze the inhibition of biomass accumulation in the areal plant body parts. The statistical analysis software SigmaPlot v.14.0 (Systat Software, San Jose, CA, USA) was used to perform a four parameter double logistic nonlinear regression analysis on the data using the formula y = C+(D-C)/[1+(x/GR50)b] and calculate the herbicide dosage required to inhibit 50% plant growth (GR50). In the formula, x is the specific herbicide dosage, y is the relative fresh weight of weeds under x treatment, C is the lower response limit; D is the upper response limit, and b is the slope of the curve at GR50. Relative resistance index (RI) was calculated as the GR50 of the resistant population divided by the GR50 of the susceptible population. RI ≤ 2 indicates a susceptible population (S), 2 < RI ≤ 5 indicates low resistance (LR), 5 < RI ≤ 10 indicates moderate resistance (MR), and RI > 10 indicates high resistance (HR).

 Analysis of <italic>ACCase</italic> mutations in <italic>L. chinensis</italic> populations

Based on the results of the cyhalofop-butyl-resistance test above, all populations were selected, with at least 10 plants per population. Fresh leaf tissue was sampled for genomic DNA extraction. Primers were designed according to the method described by Jiang et al. (2022) (F: 5′-GTGGTGAGGTTATTTGGATTATTGAC-3′; R: 5′-CACCTTGGAGTTTTGCTTTCAG-3′), covering seven amino acid variation sites known to contribute to ACCase inhibitors resistance (I1781, W1999, W2027, I2041, D2078, C2088, and G2096). The target fragment with 1128 bp was amplified using TaKaRa LA Taq polymerase for PCR under the following conditions: initial denaturation at 98°C for 3 min; 35 cycles at 98°C for 30 s, 56°C for 30 s, and 72°C for 30 s; followed by a final extension at 72°C for 10 min. Subsequently, the polymerase chain reaction (PCR) products were separated using electrophoresis, recovered and purified using agar gel with TIAN gelMidi Purification Kit (Tiangen, Beijing, China). The purified products were cloned into the pMD20-T vector (TaKaRa Biotechnology). The recombinant plasmids containing the gene were extracted and sequenced. In order to distinguish the genes encoding plasticity ACCase in L. chinensis, at least 20 clones were randomly selected from each biological replicate for sequencing. The resulting sequences were aligned and compared using DNAMAN v 6.0 software (Lynnon, Quebec, Canada).

 Determination of the level of sensitivity of <italic>L. chinensis</italic> to other herbicides

When plants reached the 2–3-leaf stage, the stems and leaves were sprayed using a 3WPSH-500D bioassay spray tower described above. Information on the herbicides used in the whole-plant doseresponse experiments are showed in Table 2 . An untreated blank control was prepared for each L. chinensis population; each treatment included four replicates and the experiment was conducted in duplicate. At 20 d after treatment, fresh weight of the aerial parts of L. chinensis was measured, and the rate of inhibition of biomass accumulation was analyzed using Microsoft Excel.

 Results Differences in the sensitivity of <italic>L. chinensis</italic> populations to cyhalofop-butyl

A whole-plant bioassay was used to determine the sensitivity of 46 L. chinensis populations collected from various areas of Jiangsu Province to cyhalofop-butyl, a commonly used aryloxyphenoxypropionate herbicide in paddy fields. The results ( Table 3 ) indicated that 41 out of the 46 L. chinensis populations exhibited 2.07- to 14.58 times resistance to cyhalofop-butyl. Although the GR50 values of LESE-001, LESZ-005, LEJY-001, LELY-001 populations were lower than 50% of the field dose, they were still classified as LR. Several researchers think that if for 50% growth inhibition of the population less than ½ of the recommended herbicide dose is sufficient, then it is not a resistant population. Based this rule, the LESE-001, LESZ-005, LEJY-001, LELY-001 populations should be classified as S. The area south of the Yangtze River in Jiangsu Province was defined as the southern Jiangsu region, the area from the Yangtze River to the northern Jiangsu main irrigation canal was defined as the Central Jiangsu region, and the area north of the northern Jiangsu main irrigation canal was defined as the northern Jiangsu region ( Figure 1 ). Analysis of the distribution of herbicide resistance across different geographic populations in Jiangsu Province revealed that 7 out of 11 populations in the southern Jiangsu region had 2.07- to 4.96 times resistance, with 63.64% of the populations showing resistance. Meanwhile, in the Central Jiangsu region, all 19 populations showed 4.96- to 11.09 times resistance to cyhalofop-butyl, with 100% of the populations being resistant. In turn, in the Northern Jiangsu region, all 15 populations showed 5.57- to 14.58 times resistance, i.e., again, 100% of the populations were resistant.

Sensitivity of different geographical populations of Leptochloa chinensis (L.) Nees, to cyhalofop-butyl.

Entry Population Logistic equation Correlation coefficient GR50 g a.i. ha-1 Susceptibility Mutation Frequency of plants with mutation(s) % RI*
1 LESZ-001 y=-9.1964 + 109.4264/(1+(x/37.0971) -2.2368) 1.0000 37.0971 LR WT – 2.53
2 LESZ-003 y=-53.6186 + 154.2167/(1+(x/23.7359) -1.7999) 0.9999 23.7359 S – – 1.62
3 LESZ-005 y=-17.2467 + 117.5376/(1+(x/30.3213)-1.9665) 0.9999 30.3213 LR WT – 2.07
4 LEJY-001 y=6.8889 + 93.1705/(1+(x/39.2875) -2.6445) 1.0000 39.2875 LR WT – 2.68
5 LEHS-003 y=25.2840 + 74.8558/(1+(x/63.3494)-4.3050) 1.0000 63.3494 LR WT – 4.32
6 LEYX-003 y=-28.2482 + 128.5793/(1+(x/26.9161)-1.7531) 0.9993 26.9161 S – – 1.83
7 LELY-003 y=-10.4796 + 110.8085/(1+(x/31.4017)-2.0106) 1.0000 31.4017 LR WT – 2.14
8 LEJT-001 y=-94.2302 + 194.3745/(1+(x/18.5684)-1.7307) 0.9990 18.5648 S – – 1.27
9 LELS-001 y=16.7391 + 78.8823/(1+(x/79.5397)-3.4533) 0.9902 79.5397 MR W2027C 90 5.42
10 LEJR-001 y=11.9962 + 82.8761/(1+(x/72.7335)-2.5779) 0.9833 72.7335 LR WT – 4.96
11 LEJR-002 y=-129.5258 + 229.5656/(1+(x/16.1023)-1.6053) 0.9965 16.1023 S – – 1.10
12 LELH-003 y=14.6115 + 80.9054/(1+(x/73.0230)-3.4343) 0.9921 73.0230 LR WT – 4.98
13 LEYZ-004 y=18.1072 + 76.2527/(1+(x/87.7839)-5.4614) 0.9861 87.7839 MR W2027C 100 5.98
14 LEYL-001 y=8.4036 + 79.0351/(1+(x/82.1600)-3.5896) 0.9651 82.1600 MR W2027C 100 5.60
15 LEJD-001 y=9.4320 + 88.7853/(1+(x/72.8151)^-2.2299) 0.9966 72.8151 LR WT – 4.96
16 LEJD-003 y=-5.4165 + 101.7864/(1+(x/104.7670)-1.2963) 0.9928 104.7670 MR W2027C 100 7.14
17 LEGY-001 y=7.7252 + 84.6088/(1+(x/75.8842)-2.6572) 0.987 75.8842 MR W2027L 80 5.17
18 LEGY-002 y=8.3153 + 87.4225/(1+(x/77.1450)-2.5675) 0.9898 77.1450 MR I2041N 70 5.26
19 LERG-001 y=9.0839 + 85.7091/(1+(x/85.0290)-2.4508) 0.9788 85.0290 MR W1999C 70 5.79
20 LERG-002 y=6.5286 + 92.8018/(1+(x/82.4145)-1.8677) 0.9875 82.4145 MR WT – 5.62
21 LERG-003 y=-0.7142 + 93.4364/(1+(x/102.3075)-1.6654) 0.9918 102.3075 MR W2027C 100 6.97
22 LEJJ-001 y=-12.4920 + 104.4448/(1+(x/82.3830)-1.3574) 0.9800 82.3830 MR I1781L 90 5.61
23 LETX-002 y=11.9585 + 81.4586/(1+(x/76.1895)-3.6082) 0.9846 76.1895 MR W2027C 90 5.19
24 LEXH-002 y=4.7908 + 84.3971/(1+(x/82.0110)-2.8081) 0.9862 82.0110 MR W1999C 90 5.59
25 LEXH-003 y=6.6055 + 84.8186/(1+(x/102.8400)-2.3092) 0.9964 102.8400 MR W2027C 100 7.01
26 LEXH-006 y=6.1199 + 95.4984/(1+(x/74.3895)-1.6790) 0.9986 74.3895 MR W1999C 90 5.07
27 LEHQ-001 y=7.6368 + 82.5764/(1+(x/83.4090)-2.7359) 0.9876 83.4090 MR W2027C 100 5.68
28 LEDF-007 y=4.5508 + 93.8466/(1+(x/162.8010)-1.7952) 0.9995 162.8010 HR W2027S 100 11.09
29 LESY-005 y=7.2280 + 88.2909/(1+(x/81.0360)-3.1872) 0.9928 81.0360 MR W2027C 90 5.52
30 LEFN-001 y=0.9214 + 93.8226/(1+(x/81.9435)-1.8817) 0.9978 81.9435 MR W2027C 100 5.58
31 LEHX-001 y=3.6921 + 84.0791/(1+(x/167.4600)-1.9868) 0.9995 167.4600 HR W2027C 100 11.41
32 LEHX-002 y=-1.0350 + 105.9160/(1+(x/156.9210)-1.2119) 0.9995 156.9210 HR W1999C 100 10.69
33 LEBH-001 y=7.8959 + 84.2202/(1+(x/207.4485)-2.0099) 0.9979 207.4485 HR W2027C 100 14.14
34 LEHH-001 y=1.9036 + 95.0790/(1+(x/150.8805)-1.6903) 0.9976 150.8805 HR W2027C 90 10.28
35 LESQ-001 y=7.2445 + 87.2385/(1+(x/149.8895)-1.7711) 0.9976 149.8995 HR W2027C 100 10.21
36 LESQ-002 y=-2.6873 + 121.1010/(1+(x/213.9915)-0.9235) 0.907 213.9915 HR W2027C 100 14.58
37 LESQ-003 y=6.3374 + 88.2822/(1+(x/121.3575)-1.6015) 0.9878 121.3575 MR W1999C 100 8.27
38 LESN-001 y=4.5054 + 93.4969/(1+(x/106.6470)-1.5236) 0.998 106.6470 MR WT – 7.27
39 LESN-002 y=-26.3543 + 138.2501/(1+(x/97.4535)-0.8147) 0.9994 97.4535 MR W1999C 100 6.64
40 LELSH-001 y=-16.0025 + 116.9730/(1+(x/81.7770)-1.1393) 0.9951 81.7770 MR WT – 5.57
41 LEBYH-001 y=6.5731 + 85.5943/(1+(x/202.1970)-1.7603) 0.9985 202.1970 HR W1999C 100 13.78
42 LEGY-001 y=1.4817 + 94.8060/(1+(x/147.0945)-1.3893) 0.9973 147.0945 HR W2027C 100 10.02
43 LEGYD-001 y=4.1636 + 95.5794/(1+(x/203.1885)-1.5127) 0.9963 203.1885 HR W1999C 100 13.85
44 LEYT-002 y=-6.0716 + 101.6062/(1+(x/121.9425)-1.3588) 0.9993 121.9425 HR D2078G 100 8.31
45 LEDX-003 y=-7.9602 + 104.9971/(1+(x/126.5910)-1.1405) 0.9998 126.5910 HR W2027 100 8.63
46 LENJ-001 y=-167.1343 + 267.6653/(1+(x/14.6750)-1.7750) 0.9999 14.6750 S

*Resistance index value.

Distribution of different geographical populations of Leptochloa chinensis (L.) Nees in Jiangsu Province, China.

 Analysis of <italic>ACCase</italic> mutations in resistant population of <italic>L. chinesis</italic>

An amplified sequence fragment of 1,128 bp including seven known mutation sites showed >96% homology with the corresponding genes of Alopecurus myosuroides, a species closely related to L. chinensis. A comparison of the translated amino acid sequences with those of A. myosuroides revealed mutations I1781L, W1999C, W2027C/L/S, I2041N, and D2078G in ACCase of cyhalofop-butyl-resistant L. chinensis populations in the paddy fields of Jiangsu Province ( Table 3 ); however, no mutations were detected at the C2088 or G2096 sites. Mutations W1999C and W2027C were relatively prevalent, whereas mutations I1781L, W2027L/S, I2041N, and D2078G were each found in a different population.

No resistance mutations were observed in LR populations with an RI of 4.98 or below. Similarly, no resistance mutations were detected in the LELSH-001 population with an RI of 5.57, or the LESN-001 population, with an RI of 7.27. The LELSH-001 and LESN-001 populations ranked as MR to cyhalofop-butyl without any mutations in the ACCase amino acid sequence, suggesting that resistance may be attributed to non-target site mechanisms or enhanced ACCase expression. The exact causes of this resistance warrant further investigation ( Table 3 ).

 Cross resistance and multiple resistance in <italic>L. chinensis</italic> populations

After treatment with fenoxaprop-P-ethyl, another member of the aryloxyphenoxypropionate family of ACCase inhibitor herbicides, at 37.5 g a.i. ha-1, the inhibition rates of biomass accumulation in the aerial parts of the LEJY-001, LELY-003, and LEJR-002 populations were all >80%, whereas those of the remaining 43 populations were all <80% ( Table 4 ). Among the 30 populations with a biomass inhibition rate of <50% following treatment with fenoxaprop-P-ethyl at 37.5 g a.i. ha-1, 15 were from the northern Jiangsu region and 15 from the Central Jiangsu region, accounting for 100% of the total number of populations in northern and 75% in Central Jiangsu, respectively.

Biomass reduction of different geographical populations of Leptochloa chinensis (L.) Nees by herbicide treatment.

Population Inhibition (%) of biomass accumulation Population Inhibition (%) of biomass accumulation
Fenoxaprop-P-ethyl Pyraclonil Benzobicyclon Anilofos Oxaziclomefone Fenoxaprop-P-ethyl Pyraclonil Benzobicyclon Anilofos Oxaziclomefone
LESZ-001 71.25 100 100 100 100 LEXH-002 25.45 100 100 100 100
LESZ-003 68.65 100 100 100 100 LEXH-003 36.45 100 100 100 98.75
LESZ-005 75.62 100 100 100 100 LEXH-006 48.25 100 100 100 100
LEJY-001 80.36 100 100 100 100 LEHQ-001 22.35 100 100 100 97.75
LEHS-003 76.95 100 100 100 100 LEDF-007 19.85 100 100 100 100
LEYX-003 66.35 100 100 100 100 LESY-005 35.68 100 100 100 99.85
LELY-003 80.25 100 100 100 100 LEFN-001 42.35 100 100 100 100
LEJT-001 100 100 100 100 100 LEHX-001 12.35 100 100 100 100
LELS-001 60.55 100 100 100 100 LEHX-002 15.47 100 100 100 92.35
LEJR-001 77.50 100 100 100 100 LEBH-001 24.78 100 100 100 100
LEJR-002 100 100 100 100 100 LEHH-001 25.65 100 100 100 90.05
LELH-003 54.45 100 100 100 95.45 LESQ-001 28.35 100 100 100 100
LEYZ-004 45.35 100 100 100 97.20 LESQ-002 15.65 100 100 100 100
LEYL-001 50.35 100 100 100 92.35 LESQ-003 12.85 100 100 100 94.35
LEJD-001 60.21 100 100 100 98.85 LESN-001 34.62 100 100 100 100
LEJD-003 45.85 100 100 100 100 LESN-002 29.63 100 100 100 100
LEGY-001 52.36 100 100 100 95.35 LELSH-001 21.35 100 100 100 100
LEGY-002 44.38 100 100 100 100 LEBYH-001 18.75 100 100 100 97.55
LERG-001 35.86 100 100 100 95.8 LEGY-001 34.15 100 100 100 100
LERG-002 41.25 100 100 100 100 LEGYD-001 40.35 100 100 100 100
LERG-003 33.68 100 100 100 96.85 LEYT-002 25.68 100 100 100 95.8
LEJJ-001 36.85 100 100 100 90.35 LEDX-003 19.68 100 100 100 100
LETX-002 42.85 100 100 100 100 LENJ-001 100 100 100 100 100

Treatment with protoporphyrinogen oxidase-inhibitor herbicide pyraclonil at 210 g a.i. ha-1, novel bicyclooctane herbicide benzobicyclon at 225 g a.i. ha-1, and organophosphorus herbicide anilofos, at 315 g a.i. ha-1 resulted in 100% inhibition of biomass accumulation across all 46 L. chinensis populations in Jiangsu Province.

Treatment of L. chinensis with 45 g a.i. ha-1 of the organoheterocyclic herbicide oxaziclomefone at the two-leaf stage led to <97% inhibition of biomass accumulation in 10 out of the 46 L. chinensis populations collected from Jiangsu Province, including six populations located in Central and four in northern Jiangsu, whereas the other 36 populations showed 100% inhibition of biomass accumulation.

 Discussion

Cyhalofop-butyl and fenoxaprop-P-ethyl are ACCase inhibitor herbicides (Wei et al., 2015) that have been used continuously for many years in Jiangsu Province as common herbicidal options for controlling Poaceae weeds in paddy fields. In this study, we found that 41 out of the 46 geographical populations of L. chinensis present in Jiangsu Province have developed significant resistance to cyhalofop-butyl, and 43 showed significant cross-resistance to fenoxaprop-P-ethyl, with resistance rates reaching 91%–93%. This explains the failure of conventional recommended dosages of cyhalofop-butyl and fenoxaprop-P-ethyl in effectively controlling L. chinensis infestations in many areas of Jiangsu Province. The results of resistance distribution of different geographical populations in Jiangsu Province showed that 63.64% of the resistance populations were in southern Jiangsu Province. In the middle of Jiangsu Province, the resistant population accounted for 100%. In northern Jiangsu, the resistant population of L. chinensis accounted for 100%. Furthermore, we observed a gradual increase trend in the proportion of L. chinensis populations resistant to ACCase inhibitor herbicides from the southern to the northern region of Jiangsu, possibly owing to the higher frequency of use of these herbicides in the latter (Zhu et al., 2024).

Research evidence indicates that target-site resistance mechanisms, such as mutations or increased expression of ACCase, along with non-target-site resistance mediated by enhanced metabolism, are the two primary mechanisms underlying weed resistance to ACCase inhibitor herbicides (Han et al., 2016; Wang et al., 2013). Often, target-site mutations within ACCase are the principal cause of herbicide resistance (Beckie and Tardif, 2012). Thus, to date, 14 types of amino acid mutations at seven amino acid sites within the ACCase carboxyltransferase (CT) domain have been identified in weeds that are resistant to ACCase inhibitor herbicides, namely, I1781L, I1781V, I1781T, W1999C/S/L, W2027C, I2041N, I2041N, D2078G, C2088R, G2096A, and G2096S (Beckie and Tardif, 2012; Délye et al., 2005; Guo et al., 2017; Powles and Yu, 2010). In this study, we identified only previously reported resistance mutations within the ACCase CT domain of resistant L. chinensis populations and did not detect any new type of mutation. Previous research has shown that mutations at multiple sites can coexist within the same resistant weed species. For instance, Zhang and Powles (2006) reported six different amino acid mutations in the ACCase CT domain of resistant Italian ryegrass, while Yu et al. (2013) found three distinct ACCase mutations on a single plant of wild oats. Furthermore, Phongphitak et al. (2014) conducted a cross-breeding experiment with fenoxaprop-P-ethyl-resistant L. chinensis in Kedah, Malaysia, and found that resistance in this case might be controlled by a single gene. Similarly, Yu et al. (2017) discovered a mutation from tryptophan to cysteine at site 2027 in L. chinensis populations highly resistant to cyhalofop-butyl, marking the first case of detection of a target-gene mutation in this weed species, with the mutation rate exceeding 50%. Additionally, Wu et al. (2019) identified a replacement of tryptophan with serine at site 1999 in the ACCase CT domain of cyhalofop-butyl-resistant L. chinensis populations in paddy fields in East China, possibly one of the important factors leading to the observed resistance. So far, Ile-1781-Leu, Leu-1818-Phe, Trp-1999-Cys, Trp-2027-Cys, Trp-2027-Gly, Trp-2027-Leu, Trp-2027-Ser, Ile-2041-Asn, Cys-2088-Arg, and Gly-2096-Ala, in ACCase have been identified as the primary cause of L. chinensis resistance to Cyhalofop-butyl (Kurniadie et al., 2022; Zhang et al., 2022; Zhao et al., 2022; Liao et al., 2024; Deng et al., 2023; Jiang et al., 2024). In this study, resistant L. chinensis populations in Jiangsu Province showed mutations I1781L, W1999C, W2027C/L/S, I2041N, and D2078G in ACCase, with no mutations found at the C2088 or G2096 sites. In particular, the W1999C and W2027C mutations were relatively prevalent, whereas I1781L, W2027L/S, I2041N, and D2078G each occurred in a different population. Gene mutations of the target enzyme ACCase are a crucial mechanism underlying the resistance of L. chinensis populations to cyhalofop-butyl and fenoxaprop-P-ethyl in the paddy fields of Jiangsu province. Nonetheless, the LELSH-001 and LEHX-002 populations, which showed no ACCase amino acid mutations, were categorized as MR to cyhalofop-butyl, suggesting that their resistance may result from non-target mechanisms or enhanced ACCase expression. It had been found that the GSTF1, GSTU1, GSTU6, CYP707A5, CYP71C4, and CYP 71C1 genes expressed highly in the R population may be responsible for cyhalofop-butyl resistance in L. chinensis. (Zhang et al., 2022; Cao et al., 2023). The causes of resistance to cyhalofop-butyl in the LELSH-001 and LEHX-002 populations need to further investigation. All 41 cyhalofop-butyl-resistant L. chinensis populations collected in Jiangsu Province were susceptible to oxaziclomefone, with the recommended doses achieving over 90% control efficacy. Similarly, the regular recommended doses of pyraclonil, benzobicyclon, and anilofos showed 100% efficacy in inhibiting biomass accumulation in the 46 geographic populations of L. chinensis in Jiangsu Province studied herein, effectively controlling the weed.

Scientific rotation of herbicide types with different mechanisms of action is an effective strategy for controlling noxious weeds and preventing herbicide-resistance development (Evans et al., 2016; Tranel and Wright, 2002). This study revealed that L. chinensis populations across different regions of Jiangsu province have developed significant resistance to two members of the aryloxyphenoxypropionate family of ACCase inhibiting herbicides, cyhalofop-butyl and fenoxaprop-P-ethyl. However, all populations were susceptible to the PPO-inhibitor herbicide pyraclonil, the novel bicyclooctane herbicide benzobicyclon, the organophosphorus herbicide anilofos, and the organoheterocyclic herbicide oxaziclomefone. These findings are consistent with those reported by Gu et al. (2021) and Tian et al. (2022), who noted that pyraclonil and benzobicyclon could be used for resistance management in L. chinensis. Therefore, using either cyhalofop-butyl or fenoxaprop-P-ethyl alone should be minimized in agricultural practice. Instead, adopting tank mixes and rotation strategies using pyraclonil, benzobicyclon, anilofos, and oxaziclomefone could help delay the development and spread of herbicide resistance in L. chinensis populations.

 Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

 Author contributions

PX: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. KW: Data curation, Writing – review & editing. YJ: Formal Analysis, Writing – review & editing. YF: Writing – review & editing. AZ: Investigation, Writing – review & editing. KC: Investigation, Writing – review & editing. HW: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing.

 Funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Jiangsu Agricultural Science and Technology Innovation Fund (grant number CX(22)5003), and by the National Natural Science Foundation of China (grant number 31801755).

 Acknowledgments

This is a short text to acknowledge the contributions of specific colleagues, institutions, or agencies that aided the efforts of the authors.

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Generative AI statement

The author(s) declare that no Generative AI was used in the creation of this manuscript.

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