Preliminary Investigation on Resistance of Beckmannia syzigachne to Clodinafop-Propargyl and Mesosulfuron-Methyl from China

Abstract

Beckmannia syzigachne is one of the most competitive weeds in winter wheat fields in China. In this study, 120 suspected resistant populations of Beckmannia syzigachne were collected from the Anhui, Hubei, Jiangsu, and Shandong Provinces from 2017 to 2019. In total, 110 populations exhibited different levels of resistance to clodinafop-propargyl, 114 populations expressed different levels of resistance to mesosulfuron-methyl, and 105 populations were resistant to both herbicides at different levels. The resistant weeds were mainly distributed in Anhui and Jiangsu Provinces. The detection results of acetyl coA carboxylase (ACCase) and acetolactate synthase (ALS) genes in the resistant populations indicated that ACCase gene mutations occurred in 97 out of 110 populations resistant to clodinafop-propargyl and ALS gene mutations occurred in 25 out of 114 populations resistant to mesosulfuron-methyl. There were several mutation types, including Ile-1781-Leu, Trp-2027-Cys, Ile-2041-Asn, Ile-2041-Val, Asp-2078-Gly, and Gly-2096-Ala in the ACCase sequence and Pro-197-Ser, Pro-197-Thr, Pro-197-His, Pro-197-Leu, Asp-376-Glu, and Trp-574-Leu in the ALS sequence. Among these mutation types, Pro-197-His, Asp-376-Glu, and Trp-574-Leu in the ALS sequence were the first identified in Beckmannia syzigachne.

1. Introduction

American sloughgrass (Beckmannia syzigachne Steud.), an annual weed, is widely distributed in China and is predominant in the wetlands or ditch edges of the Yangtze River Basin and southwestern China [1]. In recent decades, it has been one of the most malignant weeds in winter wheat and oilseed rape fields rotated with rice. The weed infestations cause yield losses of wheat [2,3].

Herbicides are the most popular chemicals used to control weeds in crop fields [4]. In recent decades, acetyl coA carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors have been the primary herbicides used to control B. syzigachne, and these herbicides are of great value to wheat fields. ALS inhibitors such as chlorsulfuron, tribenuron-methyl, bensulfuron methyl, florasulam, and mesosulfuron-methyl and ACCase inhibitors such as diclofop-methyl, fenoxaprop-P-ethyl, clodinafop-propargyl, and pinoxaden have been registered individually in wheat fields in China since the late 1980s. However, because of the single-action site of the two above-mentioned herbicides, weeds easily develop herbicide resistance. Ultimately, an increasing number of resistant populations survived after herbicide treatment, such that the weed was beyond chemical control [5]. Moreover, many scholars have been attracted to the research on resistant weeds, and they found that target-site resistance (TSR) and non-target-site resistance (NTSR) were important reasons for the different levels of weed resistance [6,7,8]. TSRs mainly contain target-site mutations or the overexpression of target genes, and NTSRs work primarily by reducing the absorption or translocation or by increasing the metabolism and sequestration [9,10,11,12].

To date, the resistance of B. syzigachne to herbicides has been described in many papers in China. Initially, the resistance of some weed plants in Jiangsu Province to chlorsulfuron was reported [13]. Then, some researchers found that the mutation in the ACCase gene of the weed led to resistance to fenoxaprop-p-ethyl [14] and discussed the mutant types (Pro-197-Ser/Asp-376-Glu in the ALS gene) as the reasons for resistance to mesosulfuron-methyl [15,16]. For example, B. syzigachne populations have developed high resistance to clodinafop-propargyl in Yutai city, Jining, Shandong Province, in 2016 [17]. The high resistance of the weed caused by different ACCase mutations (Ile-1781-Leu, Trp-2027-Cys, Ile-2041-Asn, Asp-2078-Gly, and Gly-2096-Ala) from Anhui and Jiangsu [18] and ALS with Pro-197-Ser substitution were reported in 2015 and 2018, respectively [19]. Moreover, the pattern of cross-resistance between the mutant homozygote and wild-type plants of B. syzigachne from Jiangsu was compared and clarified [14].

Some B. syzigachne populations have evolved high levels of resistance in winter wheat fields of China, and the resistance levels, resistance distributions, and target gene mutation sites must be further clarified for scientific management. Therefore, a total of 120 B. syzigachne populations were collected in winter wheat fields with long-term applications of herbicides or high survival rates after herbicide treatments from Anhui, Hubei, Jiangsu, and Shandong during 2017–2019 as plant materials to conduct the experiments as follows: (1) assay the resistance to clodinafop-propargyl and mesosulfuron-methyl to screen the resistant populations and clarify the geographical distributions of the resistant populations; and (2) detect the target gene mutation sites in ACCase and ALS to confirm the cause of resistance to both herbicides.

2. Materials and Methods

2.1. Plant Materials

From Anhui, Hubei, Jiangsu, and Shandong Provinces during 2017–2019, 120 populations of Beckmannia syzigachne were collected from winter wheat fields that had a long-term herbicide use history or could survive well after herbicide treatment. Matrix nutrient soil without herbicide application history was purchased and mixed with organic fertilizer and vermiculite in a 4:1:1 ratio (organic matter content ≥ 15%, total nitrogen, phosphorus and potassium contents > 0.83% and pH = 7.0–7.5), and filled into 12 cm diameter pots. Approximately 35–40 seeds of different populations were soaked in gibberellin solution at a concentration of 3.2 g L^–1 for approximately 20 h to break dormancy, washed with clean water, and sown in pots [19]. The susceptible population was selected from Jiangsu Province that had no history of herbicide use and had been identified to be susceptible in a laboratory setting. And it was used as a control to make a sensitivity baseline. The number of plants in each pot was 15 after thinning and before herbicide treatment.

2.2. Detection of Resistance Level to Clodinafop-Propargyl and Mesosulfuron-Methyl

The plants were treated with clodinafop-propargyl and mesosulfuron-methyl to determine the resistance levels at the 3-leaf stage using a moving TeeJet^® XR8002 flat fan nozzle cabinet sprayer at a pressure of 0.275 MPa (Compressed Air Cabinet Sprayer ASS-4, Beijing Research Center for Information Technology in Agriculture, Beijing, China). In the whole plant dose–response assays, the clodinafop-propargyl and mesosulfuron-methyl dose settings for suspected resistant (SR) and susceptible population (S) are shown in

Table 1. Dose settings for clodinafop-propargyl and mesosulfuron-methyl.

Herbicides	Doses (g a.i. ha−1)
clodinafop-propargyl (15%, WP, Syngenta)	S: 0, 8.44 (1/8X), 16.88 (1/4X), 33.75 (1/2X), 67.5 (1X), 135 (2X) SR: 0, 33.75 (1/2X), 67.5 (1X), 135 (2X), 270 (4X), 540 (8X), 1080 (16X)
mesosulfuron-methyl (30 g/L, OD, Bayer)	S: 0, 1.69 (1/8X), 3.38 (1/4X), 6.75 (1/2X), 13.5 (1X), 27 (2X) SR: 0, 6.75 (1/2X), 13.5 (1X), 27 (2X), 54 (4X), 108 (8X), 216 (16X)

WP = wettable powder; OD = oil dispersion; S = susceptible; SR = suspected resistant.

. The recommended doses of the two herbicides were 67.5 g a.i. ha⁻¹ and 13.5 g a.i. ha⁻¹, respectively.

After 21 days of treatment, the fresh weight of the plants was investigated under each dose treatment to calculate the inhibition rate of fresh weight. The GR₅₀ (the dose that results in a 50% growth reduction in the aboveground biomass) was calculated using a nonlinear log-logistic regression model using Sigma Plot (v. 12.5) according to Equation (1) [20]:

$$y=C+\left\{\left(D-C\right)/\left[1+{(x/{\mathrm{GR}}_{50})}^b\right]\right\}$$

(1)

where C and D, respectively, denote the lower limit and the upper limit, b denotes the slope of the curve, and y is the corresponding percentage of control at herbicide dose x. The resistance index (RI) was calculated by dividing the GR₅₀ value of the resistant population by that of the susceptible population to estimate the resistance level of different populations.

Then, the populations were divided into three resistance levels depending on the RI value: susceptible populations: RI = 1, low resistance level: 1.0 < RI ≤ 3.0, moderate resistance level: 3.0 < RI ≤ 10.0, and high resistance level: RI > 10.0 [21,22].

2.3. Identification of ACCase and ALS Gene Mutations in Resistant Populations

Three surviving plants were randomly selected from each resistant population to each herbicide, and the fresh tissues were cut off and stored in a refrigerator at −80 °C. Genomic DNA was extracted using a DNA extraction kit (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China) according to the manufacturer’s instructions.

In this study, the amplification reaction of the ACCase sequence required one pair of primers: the forward primer was WCACC (5′-AAACTCTGGTGCTCGGATTG-3′), including one conserved site (I1781), and the reverse primer was WCACC-R (5′-TAGGCTTCCATTTGCTCCC-3′), which could obtain six conserved sites (T1999, T2027, I2041, A2078, C2088, and C2096). The 25 μL polymerase chain reaction (PCR) system was composed of 2 μL genomic DNA (75 ng mL⁻¹), 12.5 μL 2 × Taq PCR Master Mix, 0.5 μL of each primer (10 μM), and 9.5 μL ddH₂O (TaKaRa Biotechnology, Dalian, China).

The PCR system of the ALS sequence amplification was the same as those of the amplification of the ACCase sequence, and this reaction required two pairs of primers. The first pair of primers were WCALS-1F (5′-CGCCTTACCCAAACCTACT-3′) and WCALS-1R (5′-ATGCGGCTGCTTGTTCTT-3′), including five conserved sites (A122, P197, A205, F206, D376, and R377), and the second pair of primers were WCALS-2F (5′- ATCCCACCACAATATGCTATCC-3′) and WCALS-2R (5′-TCACAGTTGACCACACTTC-3′), covering three conserved sites (W574, S653, and G654).

3. Results

3.1. Resistance Levels and Distribution of B. syzigachne to Clodinafop-Propargyl

The detection results demonstrate that out of all (120) populations collected, 10 populations were susceptible to clodinafop-propargyl, 26 populations had a low resistance level (21.7%), 10 populations had a moderate resistance level (8.3%), and the others had a high resistance level (61.7%). Comparing the resistance levels among provinces, the populations in Anhui and Jiangsu had severe resistance. Among the 46 populations in Anhui Province, high-, moderate-, and low-resistance-level populations accounted for 58.7% (27), 8.7% (4), and 26.1% (12) of the total, respectively. In 59 populations from Jiangsu Province, 44 (74.6%), 5 (8.5%), and 10 (16.9%) populations had high, moderate, and low resistance levels, respectively, and these populations had no susceptibility to clodinafop-propargyl. Among the thirteen populations collected from Hubei, the population number of high-, moderate-, and low-resistance-level and susceptible populations was one, one, four, and seven, respectively. Both populations from Shandong exhibited high levels of resistance (Table 1 and Figure 1).

The geographical distribution of populations with different resistance levels to clodinafop-propargyl is described in Figure 1. The resistance of B. syzigachne to clodinafop-propargyl was mainly distributed in central and western Jiangsu and southeast and northwest Anhui. Few populations were collected in Hubei and Shandong, and the resistant populations mainly occurred in central Hubei and northwest and southwest Shandong (Figure 1).

3.2. Resistance Levels and Distribution of B. syzigachne to Mesosulfuron-Methyl

The results show the evolution of the different resistance levels of 120 populations to mesosulfuron-methyl (Table 1). There were 75 low-resistance populations (62.5%), 10 high-resistance populations (8.3%), 29 moderate-resistance populations (24.2%), and 6 susceptible populations (5.0%) to this inhibitor. Among 46 populations from Anhui, 35 populations (76.1%) exhibited a low resistance level to mesosulfuron-methyl, 8 populations (17.4%) showed a moderate resistance level, and only 1 population (2.2%) had a high resistance level. In 59 populations from Jiangsu, the population number of low, moderate and high resistance levels to mesosulfuron-methyl were 30 (50.8%), 20 (33.9%), and 8 (13.6%), respectively. There were thirteen populations from Hubei Province, including nine low-resistance-level populations and one moderate-resistance-level population. In the two populations from Shandong, one exhibited low resistance to mesosulfuron-methyl, and the other had a high resistance level.

The geographical distribution of populations with different resistance levels to mesosulfuron-methyl is described in Figure 2. The higher-resistance-level populations were mainly distributed in central Jiangsu, southeast Anhui, central Hubei, and the northern and southern parts of Shandong.

3.3. Analysis of the Multiple Resistance to Clodinafop-Propargyl and Mesosulfuron-Methyl in B. syzigachne

The results of multiple resistance to clodinafop-propargyl and mesosulfuron-methyl in one hundred and twenty B. syzigachne populations showed that one population (0.8%) was susceptible to both inhibitors, nine populations were susceptible to clodinafop-propargyl but not to mesosulfuron-methyl (7.5%), and five populations were susceptible to mesosulfuron-methyl but not to clodinafop-propargyl (4.2%). A total of 105 populations (87.5%) were resistant to both herbicides at different levels. In 120 populations, 26 populations (21.7%) were at low resistance to clodinafop-propargyl, among which, 15, 7, and 2 populations expressed low, moderate, and high resistance levels to mesosulfuron-methyl, respectively. Among the ten populations (8.3%) with moderate resistance to clodinafop-propargyl, the populations with low resistance, moderate resistance and high resistance to mesosulfuron-methyl were five, three, and one, respectively. There were 74 populations (61.7%) with high resistance to clodinafop-propargyl, among which the populations with low resistance, moderate resistance, and high resistance to mesosulfuron-methyl were 46, 19, and 7, respectively (Table 1).

In summary, most populations were highly resistant to clodinafop-propargyl and expressed lower resistance (low and moderate resistance levels) to mesosulfuron-methyl (

Table 2. Multiple resistance to clodinafop-propargyl and mesosulfuron-methyl of Beckmannia syzigachne populations from different provinces.

Collection Province	Total Number	The Population Number of Each Resistance Level to Both Herbicides
		RIC = 1	RIC = 1	RIC = 1	RIC = 1	1 < RIC ≤ 3	1 < RIC ≤ 3	1 < RIC ≤ 3	1 < RIC ≤ 3	3 < RIC  ≤ 10	3 < RIC  ≤ 10	3 < RIC  ≤ 10	3 < RIC  ≤ 10	RIC ≥ 10	RIC ≥ 10	RIC ≥ 10	RIC ≥ 10
		RIM = 1	1 < RIM ≤ 3	3 < RIM  ≤ 1	RIM ≥ 10	RIM = 1	1 < RIM ≤ 3	3 < RIM ≤ 10	RIM ≥ 10	RIM = 1	1 < RIM ≤ 3	3 < RIM ≤ 10	RIM ≥ 10	RIM = 1	1 < RIM ≤ 3	3 < RIM ≤ 10	RIM ≥ 10
Anhui	46	0	3	0	0	1	9	2	0	0	1	2	1	1	22	4	0
Hubei	13	1	6	0	0	1	2	1	0	1	0	0	0	0	1	0	0
Jiangsu	59	0	0	0	0	0	4	4	2	0	4	1	0	1	22	15	6
Shandong	2	0	0	0	0	0	0	0	0	0	0	0	0	0	1	0	1
Total	120	1	9	0	0	2	15	7	2	1	5	3	1	2	46	19	7

RI_C = resistance index (RI) of B. syzigachne to clodinafop-propargyl. RI_M = resistance index (RI) of B. syzigachne to mesosulfuron-methyl.

).

3.4. The Detection Results of ACCase and ALS Gene Mutations in B. syzigachne Populations Resistant to Clodinafop-Propargyl and Mesosulfuron-Methyl

Of the 120 populations collected during 2017~2019, 97 of the populations that developed resistance to clodinafop-propargyl had mutations in the ACCase sequence. The number of mutant populations in the ACCase sequence from the Anhui, Hubei, Jiangsu, and Shandong Provinces was 36, 5, 54, and 2, respectively. In this study, some populations mutated at a single point and some were double-site mutations in the ACCase sequence. The single-point mutations were identified at the sites of Ile1781 (32.7%), Trp2027 (10.9%), Ile2041 (38.2%), Asp2078 (37.3%), and Gly2096 (5.5%) (Figure 3). The population number of Ile-1781-Leu mutations in detected populations from the Anhui, Hubei, Jiangsu, and Shandong Provinces were 16, 1, 17, and 2, respectively. The proportion of the Ile-2041-Asn mutation was 27.9%, 33.3%, and 45.8% in the detected populations from Anhui, Hubei, and Jiangsu, respectively. The Ile-2041-Val mutation had only one population in all of the mutant populations in Anhui. The Asp-2078-Gly mutation was 34.9%, 16.7%, and 42.4% of the detected populations from Anhui, Hubei, and Jiangsu, respectively. For the double-site mutations in the ACCase sequence, the Ile-1781-Leu mutation co-occurred with Trp-2027-Cys, Ile-2041-Asn, or Asp-2078-Gly, Trp-2027-Cys occurred simultaneously with Ile-2041-Asn or Asp-2078-Gly, and Ile-2041-Asn co-existed with Asp-2078-Gly mutations (

Table 3. Mutation analysis of the ACCase gene in resistant Beckmannia syzigachne populations.

Mutations	The Population Number a of Different Provinces				Total
	Anhui	Hubei	Jiangsu	Shandong
Number of detection populations	43	6	59	2	110
Number of mutation populations	36	5	54	2	97
Single-point mutation
Ile-1781-Leu	16	1	17	2	36
Trp-2027-Cys	4	1	7	0	12
Ile-2041-Asn	12	2	27	0	41
Ile-2041-Val	1	0	0	0	1
Asp-2078-Gly	15	1	25	0	41
Gly-2096-Ala	1	0	5	0	6
Double-site mutation
Ile-1781-Leu and Trp-2027-Cys	1	1	0	0	2
Ile-1781-Leu and Ile-2041-Asn	3	0	2	0	5
Ile-1781-Leu and Asp-2078-Gly	3	0	2	0	5
Trp-2027-Cys and Ile-2041-Asn	0	0	1	0	1
Trp-2027-Cys and Asp-2078-Gly	0	0	1	0	1
Ile-2041-Asn and Asp-2078-Gly	0	0	2	0	2

^a: The number of each mutation type in B. syzigachne population with the ACCase gene mutation detected in each province.

).

As described in

Table 4. Mutation analysis of the ALS gene in resistant Beckmannia syzigachne populations.

Mutations	The Population Number a of Different Provinces				Total
	Anhui	Hubei	Jiangsu	Shandong
Number of detection populations	44	10	58	2	114
Number of mutation populations	3	1	20	1	25
Pro-197-Ser	1	0	6	0	7
Pro-197-Thr	1	0	8	0	9
Pro-197-His	1	0	1	0	2
Pro-197-Leu	0	0	2	0	2
Asp-376-Glu	0	0	1	0	1
Trp-574-Leu	0	1	2	1	4

^a: The number of each mutation type in B. syzigachne population in the ALS gene mutation detected in each province.

, in this study, a total of 114 populations from four provinces were selected to clone the ALS sequence to clarify the molecular mechanisms of resistance, and 25 populations had gene mutations. The number of mutant populations in the detected populations from Anhui, Hubei, Jiangsu, and Shandong were 3, 1, 20, and 1, respectively. In the ALS sequence, there were several mutation types at the 197 site (Pro-197-Ser, Pro-197-Thr, Pro-197-His, Pro-197-Leu), the 376 site (Asp-376-Glu), and the 574 site (Trp-574-Leu) (Figure 4). Mutations at site 197 were mainly found in Anhui and Jiangsu, and the populations with Pro-197-Ser and Pro-197-Thr mutations were more numerous than other mutation types. The Asp-376-Glu mutation was found only in Jiangsu. The Trp-574-Leu mutation was found in all provinces except Anhui (Table 4). It was noticed that the ACCase or ALS genes of three plants from one population might have different mutation types.

In this paper, the three detected samples from a population may have no mutation, or the same or different single-point mutations and double-site mutations.

4. Discussion

Mesosulfuron-methyl and clodinafop-propargyl, which were registered in China in 2003 and 2009, have been widely applied to control B. syzigachne and other weeds in wheat fields around the middle and lower reaches of the Yangtze River because of their high efficacy and low toxicity. Before their registration, ALS inhibitors chlorsulfuron, metsulfuron-methyl, and ACCase inhibitors, including diclofop-methyl, fenoxaprop-P-ethyl, and haloxyfop-R-methyl, had been used in wheat or in oilseed rape fields in these areas for many years to effectively control B. syzigachne since the late 1980s. With the application of new commercialized herbicides in wheat or oilseed fields, the dominant weed species and their resistance to herbicides with different mode of action have developed and changed gradually [23].

According to the results of an investigation into the herbicide application history in experimental materials, it was found that many areas in Jiangsu Province and Anhui Province have applied clodinafop-propargyl for over 10 years and diclofop-methyl or other ACCase herbicides for over 30 years. Other areas have used mesosulfuron-methyl for 8 years and chlorsulfuron for 20 years. However, in collection areas in Hubei Province, the application history of different herbicides was different, including fenoxaprop-P-ethyl for 10 years and clodinafop-propargyl for 5 years, and clodinafop-propargyl was used in combination with mesosulfuron-methyl in recent years. As we know, long-term and continuous use of herbicides with the same or similar mechanisms, especially for over 10 years, can lead weed resistance easily [24]. The resistance of Backmannia syzigachne and Alopecurus japonicum to chlorsulfuron were found in 1996 in China [25]. The resistance caused by gene mutations of B. syzigachne to mesosulfuron-methyl and clodinafop-propargyl were confirmed in 2017 in China, and the resistance levels of populations to herbicides were detected [14,26,27,28,29].

The results indicate that the populations at high resistance levels to clodinafop-propargyl and mesosulfuron-methyl mainly occurred in central and western Jiangsu, southeastern Anhui, and central Hubei in this paper. Some populations were at high resistance levels to clodinafop-propargyl and moderate resistance levels to mesosulfuron-methyl in northwestern Anhui. Regarding multiple resistance, most populations were resistant to both herbicides, particularly in Anhui and Jiangsu and a few fields in Shandong. Populations from Hubei Province were at low or moderate resistance levels to the two herbicides. These populations expressed not only high resistance levels, but also had a great quantity of seeds, which could account for many researchers’ concerns about weed resistance [30]. Due to the differences in herbicide application history, the mode of action and crop rotation system may be the keys to different resistance levels of weed to different herbicides in different regions.

Since the resistance occurred, many reports have confirmed resistance mutations and the activity of ALS and ACCase enzymes in some populations [9,16,31] and reported the expression of non-target resistance genes [27,28,29,31,32,33]. Research has reported widespread resistance to fenoxaprop-P-ethyl and mesosulfuron-ethyl in B. syzigachne in wheat fields in Anhui Province due to target-site mutations (TSR) or changes in metabolic enzyme activity (NTSR) [22]. These results deeply explain the resistance situation and resistance mechanism. From 97 resistant populations with mutations in the ACCase sequence of B. syzigachne, 5 resistant mutation types that have been reported were identified in Anhui and Jiangsu, including Ile-1781-Leu, Trp-2027-Cys, Ile-2041-Asn, Asp-2078-Gly, and Gly-2096-Ala [34,35]. In this study, Ile-1781-Leu, Trp-2027-Cys, Ile-2041-Asn, and Asp-2078-Gly mutations in Hubei and the Ile-1781-Leu mutation in Shandong were identified. The Ile-2041-Val mutation type was found in Anhui Province, which had not been reported in B. syzigachne before. Moreover, there were some double mutation types in the ACCase sequence, mainly at 1781 site, the 2027 site, the 2041 site, and the 2078 site (Ile-1781-Leu and Trp-2027-Cys, Ile-1781-Leu and Ile-2041-Asn, Ile-1781-Leu and Asp-2078-Gly, Trp-2027-Cys and Ile-2041-Asn, Trp-2027-Cys and Asp-2078-Gly, and Ile-2041-Asn and Asp-2078-Gly). From 25 B. syzigachne populations with mutations in the ALS sequence, the Pro-197-Ser, Pro-197-Thr, and Pro-197-Leu mutation types were obtained in this research and also were found in other reports [19,36,37]. And Pro-197-His, Asp-376-Glu, and Trp-574-Leu mutations were not reported in B. syzigachne in other papers. Such flexible mutations of the two sequences explained the cause of resistance in these regions forcefully.

In addition, mutations at different gene sites were selective for resistance to different herbicides. According to previous reports about ALS sequence, amino acid mutations at site 197 were resistant to SU herbicides and sensitive to IMI herbicides, while amino acid mutations at site 574 were resistant to both herbicides. Homozygous mutations of Asp-376-Glu showed different resistance levels to the SU herbicides and IMI herbicides [38,39]. Amino acid mutations at seven sites in ACCase maybe express different tolerances to APP herbicides, but this is unclear and needs to deeper research. In this paper, amino acid mutations at site 197 and 574 in the ALS sequence and at site 1781, 2024, 2027, 2078, and 2096 in the ACCase sequence expressed high resistance to the two herbicides in most populations, and which sites caused high resistance needs to be further explored. In short, the detection of resistant weeds’ gene sites is very helpful for us to understand the multi-resistance situation. And the results of resistance monitoring in the field can provide help for farmers to use herbicides scientifically and rationally.

This study is aimed at monitoring the resistance of Beckmannia syzigachne in wheat fields, but it has some limitations. The results of this study have preliminarily defined the resistance and geographical distribution of Beckmannia syzigachne, and the specific resistance level and cross-resistance of the resistance population will be further purified and measured again in a follow-up study.

5. Conclusions

In summary, this survey not only indicated the resistance levels and resistance distribution of B. syzigachne in China, but also confirmed resistance gene mutations of the populations resistant to clodinafop-propargyl and mesosulfuron-methyl, as well as some new discoveries of gene sequence mutations. These data can provide great help in managing the resistance of B. syzigachne. In practice, agricultural fields, the environment, and ecology can be managed and regulated as a whole, and the scale of resistance populations can be controlled by reducing the use of clodinafop-propargyl and smesosulfuron-methyl through an Integrated Pest Management (IPM) system by, for example, the following: (1) rotating different types of herbicide or rotating crops, (2) planting wheat at intervals, and using herbicide at reasonable dosage.