Assessment of Florpyrauxifen-Benzyl Sensitivity in Echinochloa crus-galli and E. crus-galli var. mitis: A Case Study with 228 Populations in Eastern China

Abstract

Echinochloa crus-galli and E. crus-galli var. mitis are two of the most troublesome rice weeds. Florpyrauxifen-benzyl is one of the most important post-emergence rice herbicides that has been pervasively applied in many countries since 2018. We collected 70 E. crus-galli and 158 E. crus-galli var. mitis populations from rice fields in eastern China in 2022 and tested their sensitivities to florpyrauxifen-benzyl through whole-plant bioassays. A total of 21 days after treatment with florpyrauxifen-benzyl label dose (36 g ai ha⁻¹), 71.4% of E. crus-galli and 70.9% of E. crus-galli var. mitis populations were completely controlled. The GR₅₀ doses (doses causing 50% fresh weight reductions in aboveground parts) of florpyrauxifen-benzyl applied to E. crus-galli populations ranged from 1.4 to 36.9 g ai ha⁻¹, with a baseline sensitivity dose of 4.9 g ai ha⁻¹; those for E. crus-galli var. mitis populations ranged from 1.3 to 97.6 g ai ha⁻¹, with a baseline sensitivity dose of 5.0 g ai ha⁻¹. No significant differences between E. crus-galli and E. crus-galli var. mitis were found in GR₅₀ values. Among 70 E. crus-galli populations, 61.4%, 35.7%, and 2.9% showed no, low, and moderate resistance to florpyrauxifen-benzyl, while among 158 E. crus-galli var. mitis populations, 54.4%, 36.1%, 1.9%, and 1.9% showed no, low, moderate, and high resistance to florpyrauxifen-benzyl, respectively. Moreover, the frequency of florpyrauxifen-benzyl-resistant populations of E. crus-galli var. mitis tended to be higher in southwestern areas.

1. Introduction

Rice (Oryza sativa L.), a staple crop feeding half of the global population, faces critical yield constraints from Echinochloa crus-galli (L.) P. Beauv. and E. crus-galli var. mitis (Pursh) Petermann in eastern China [1,2,3]. These co-occurring species exhibit high morphological and biological similarity, leading farmers to manage them collectively. Spikelets of Echinochloa crus-galli (3–4 mm in length) feature lower lemma extending awns in varying lengths, whereas spikelets of E. crus-galli var. mitis (approximately 3 mm) are awnless or bear <5 mm awns, and their racemes are usually branched [4,5]. Moreover, in our previous study, we surveyed 250 rice fields in eastern China and suggested that E. crus-galli var. mitis exhibits significantly higher dominance over E. crus-galli [6].

Herbicides are the most effective tools for controlling rice weeds. The evolving resistance in Echinochloa weeds poses a pressing threat to sustainable weed management on rice fields [7,8,9,10,11]. Given that the development of a new herbicide typically requires over 10 years of research and substantial financial investment [12], extending the longevity of herbicides is critical for sustainable chemical control [13,14]. Monitoring the resistance of Echinochloa weeds to important herbicides on a regional scale could be fundamental for raising systematical and effective control strategies and for blocking the dissemination of herbicide-resistant populations. Such monitoring requires multi-population studies due to the great inter/intra-specific biological or ecological variations documented for different Echinochloa weed species [6,15,16].

Florpyrauxifen-benzyl (benzyl 4-amino-3-chlor-6-[4-chlor-2-fluor-3-methoxyphenyl]-5-fluor-2-pyridincarboxylate) is a globally used rice herbicide pervasively commercialized in 2018, which delivers effective post-emergence control of diverse weeds, including different Echinochloa weed species. Florpyrauxifen-benzyl-resistant E. crus-galli populations have emerged in major rice-producing regions, including the United States and China [9,10,17]. The establishment of baseline sensitivity is essential for quantifying herbicide resistance evolution and conducting relative comparisons on different spatiotemporal scales [18,19]. The baseline sensitivity of 70 E. crus-galli and 71 E. oryzicola populations to florpyrauxifen-benzyl was determined in Korea [20], suggesting intra- and inter-specific variations. However, no study has reported the baseline sensitivity of E. crus-galli and E. crus-galli var. mitis to this herbicide in China.

Additionally, the baseline sensitivity of Echinochloa spp. to other herbicides has been documented. For example, the baseline sensitivity dose of Echinochloa spp. to tembotrione is 18.0 g ai ha⁻¹, and that of E. crus-galli to flusulfinam is 6.5 g ai ha⁻¹ [11,21]. Although E. crus-galli var. mitis was reported to exhibit herbicide resistance to penoxsulam (30 g ai ha⁻¹), metamifop (120 g ai ha⁻¹), and florpyrauxifen-benzyl (36 g ai ha⁻¹) [22], systematic assessments of its herbicide sensitivities remain lacking.

Jiangsu Province, China, is one of the world’s largest rice planting areas, with double-crop systems (mostly rice–wheat rotation) being used yearly. In Jiangsu, rice is usually transplanted or directly seeded in June and harvested in October. In 2023, Jiangsu Province produced a total of 20,032,490 tons rice from a total planting area of 2,221,020 ha, with a yield of 9019.5 kg ha⁻¹ [23]. Meanwhile, herbicide resistance in Jiangsu is also troublesome [16,24]. To date, no prior studies reported distributions of florpyrauxifen-benzyl-resistant E. crus-galli and E. crus-galli var. mitis in Jiangsu or in other areas in China. In 2022, we collected 70 E. crus-galli and 158 E. crus-galli var. mitis populations across Jiangsu. We conducted a series of experiments with the aims of (1) assessing the sensitivity of both E. crus-galli and E. crus-galli var. mitis to florpyrauxifen-benzyl in rice fields on a regional scale and (2) establishing their baseline sensitivity for future resistance monitoring.

2. Materials and Methods

2.1. Collection of Plant Materials

Seeds of 70 E. crus-galli and 158 E. crus-galli var. mitis populations were collected from rice fields (direct-seeded/transplanted) in 13 cities across Jiangsu Province, China, in October 2022 (Figure 1). Sampling was carried out following a systematic road survey protocol: we drove extensively across rice production regions and randomly surveyed rice fields, with adjacent fields separated by >5 km. Each surveyed site covered an area of approximately 0.1–0.2 ha. From each site, seeds were collected from naturally occurring populations, air-dried, and stored at room temperature before use.

2.2. Whole-Plant Dose–Response Bioassay

Seeds of each population were germinated on two layers of moistened filter paper in 9 cm diameter Petri dishes. Incubation conditions were maintained at 30 °C (day)/25 °C (night) under a 12 h photoperiod. Twelve germinated E. crus-galli and E. crus-galli var. mitis seedlings per population were transplanted into a plastic pot (7 × 7 × 8 cm) with a 2:1 (v:v) mixture of natural soil (collected from deep soil about 30–60 cm below ground of a fallow land) and nutritional soil (pH 5.6 with organic matter content >40%; Nanjing Duole Horticulture Co., Ltd., Nanjing, China) and grown in a greenhouse at Yangzhou University. Seedlings were thinned to nine uniformly sized plants per pot one week after transplantation.

At the 3–4 leaf stage, seedlings were treated with florpyrauxifen-benzyl (label dose: 36 g ai ha⁻¹, Corteva Agriscience, Shanghai, China) using a laboratory sprayer (TBSHIELD, Yangzhou, China) equipped with a 1.1 mm diameter flat fan nozzle (15° jet angle), delivering 450 L ha⁻¹ at 230 kPa. A preliminary dose–response test (0, 18, and 36 g ai ha⁻¹) was conducted to estimate the sensitivity range of each population tested. Based on the injury and survival observed 21 days after treatment, the final dose–response experiment was established using five out of nine florpyrauxifen-benzyl doses (0, 2.25, 4.5, 9, 18, 36, 72, 144, and 288 g ai ha⁻¹). Aboveground fresh weight was recorded 21 days after treatment. Each treatment had four replicates, and the experiment was conducted as two independent runs between May and October 2023.

2.3. Data Analysis

Data from the two repeated trials were compared through ANOVA using SPSS software (v. 26.0). The data were pooled due to the nonsignificant interaction between the two repeated trials.

A four-parameter log-logistic dose–response curve was fitted to test responses of fresh weight to florpyrauxifen-benzyl treatments [25], using the “drc” add-on package in R 4.4.2 [26] as follows:

Y = d + (a − d)/[1 + (x/GR₅₀)^b]

(1)

where Y represents the fresh weight reduction response at dose x of florpyrauxifen-benzyl, d is the lower limit, a is the upper limit, GR₅₀ is the dose of florpyrauxifen-benzyl causing a 50% reduction in fresh weight, and b is the slope. Reduction was generated using the fresh weights and then ranked within the different responses according to the method used in our previous work [17,27].

The frequency distribution of GR₅₀ of florpyrauxifen-benzyl to E. crus-galli or E. crus-galli var. mitis populations was estimated using the Shapiro–Wilk test with Origin 2021 software. The baseline sensitivity to florpyrauxifen-benzyl of E. crus-galli and E. crus-galli var. mitis was fitted by the Gaussian function test [16]:

$$y=y_0+{\displaystyle \frac{A}{\left(\omega \sqrt{\frac{\pi }{2}}\right)}}{e}^{-2{\left({\displaystyle \frac{x-x_c}{\omega }}\right)}^2}$$

(2)

where $y_0$ is the offset; A is the area; ω is the width; and $x_{\mathrm{c}}$ is the center. Resistance factors (RFs) were calculated by dividing the GR₅₀ value of each population by baseline sensitivity dose to the herbicide. Using criteria reported by Beckie and Tardif [28], populations were described as having no resistance (RF < 2), low resistance (RF 2–5), moderate resistance (RF6–10), or high resistance (RF > 10).

To compare significant differences in the average GR₅₀ value of 13 cities of seed collection sites, least significant differences were used when variances were homogeneous, and Dunnett’s T3 test was used if variances were not homogeneous. Correlation between the longitudes and latitudes of seed collection sites and GR₅₀ values of E. crus-galli and E. crus-galli var. mitis were determined with SPSS using correlate analysis. To compare the above GR₅₀ values between E. crus-galli and E. crus-galli var. mitis, an independent sample t-test (for overall populations) and a paired sample t-test (for E. crus-galli and E. crus-galli var. mitis populations co-occurring on the same field) were used. To analyze influences of planting method (transplanting or direct-seeded) and rice subspecies (subsp. japonica or indica) on the sensitivity of E. crus-galli and E. crus-galli var. mitis, general linear models were applied, where the GR₅₀ value was the dependent variable and the planting method and rice species were independent variables. The data are presented as the mean ± SE.

3. Results

3.1. Sensitivity to Label or Half Label Dose of Florpyrauxifen-Benzyl

The fresh weight reduction in E. crus-galli at a 0.5 × label dose of florpyrauxifen-benzyl (18 g ai ha⁻¹) ranged from 25.0% to 100% (84.7% on average) (Figure 2), and 61.4% of E. crus-galli populations exhibited >85% fresh weight reduction. When the dose of florpyrauxifen-benzyl increased to the label dose (36 g ai ha⁻¹), the fresh weight reduction in E. crus-galli ranged from 49.6% to 100% (97.0% on average), and seedlings of 71.4% of E. crus-galli populations were completely killed.

For E. crus-galli var. mitis populations, fresh weight reduction ranged from 9.0% to 100% at a florpyrauxifen-benzyl dose of 18 g ai ha⁻¹ (83.5% on average) and from 16.2% to 100% (94.1% on average) at a florpyrauxifen-benzyl 36 g ai ha⁻¹ dose (Figure 3). With the 18 g ai ha⁻¹ treatment, 65.8% of E. crus-galli var. mitis populations (104/158) showed >85% fresh weight reduction, which increased to 86.1% (136/158) at the label dose. Complete mortality was observed in 19.6% and 70.9% of E. crus-galli var. mitis populations with 18 and 36 g ai ha⁻¹ treatments, respectively. The proportion of E. crus-galli var. mitis populations with <60% fresh weight reduction decreased by 12.6% when the florpyrauxifen-benzyl dose was increased from 18 to 36 g ai ha⁻¹.

3.2. Florpyrauxifen-Benzyl Resistance

The GR₅₀ values of florpyrauxifen-benzyl ranged from 1.4 to 36.9 g ai ha⁻¹ through 70 E. crus-galli populations (8.1 on average; 5.7 on median) (Figure 4 and Figure S1). For 158 E. crus-galli var. mitis populations, the GR₅₀ values of florpyrauxifen-benzyl ranged from 1.3 to 97.6 g ai ha⁻¹ (9.3 on average; 6.1 on median). The GR₅₀ values of florpyrauxifen-benzyl in E. crus-galli and E. crus-galli var. mitis were all concentrated around 5 g ai ha⁻¹ ($x_{\mathrm{c}}$ values were 4.9 in E. crus-galli and 5.0 in E. crus-galli var. mitis). Relative to the average GR₅₀ value, 46/70 (65.7%) E. crus-galli populations and 115/158 (72.8%) E. crus-galli var. mitis populations exhibited GR₅₀ values below this dose.

Based on the GR₅₀$x_{\mathrm{c}}$ (4.9 g ai ha⁻¹) value of E. crus-galli, 43, 25, and 2 E. crus-galli populations showed no resistance (61.4%), low resistance (35.7%), and moderate resistance (2.9%), respectively. For E. crus-galli var. mitis (GR₅₀$x_{\mathrm{c}}$ = 5.0 g ai ha⁻¹), 86, 57, 3, and 3 E. crus-galli var. mitis populations showed no resistance (54.4%), low resistance (36.1%), moderate resistance (1.9%), and high resistance (1.9%), respectively. The GR₅₀$x_{\mathrm{c}}$ values could serve as the baseline sensitivity doses of E. crus-galli and E. crus-galli var. mitis.

3.3. Inter- and Intra-Specific Differences in Florpyrauxifen-Benzyl Resistance

The control efficacy of florpyrauxifen-benzyl on E. crus-galli at different doses was analogous to that on E. crus-galli var. mitis, with control efficacy on E. crus-galli being somewhat better. Meanwhile, the GR₅₀ values of E. crus-galli and E. crus-galli var. mitis maintained similar proportions across different ranges defined by the label dose of florpyrauxifen-benzyl. No significant differences (p > 0.05) were observed in GR₅₀ values between E. crus-galli and E. crus-galli var. mitis through an independent sample t-test; additionally, no significant difference was found in the GR₅₀ values between direct-seeded and transplanted rice fields (Tables S1 and S2). Moreover, in 38 rice fields collecting both E. crus-galli and E. crus-galli var. mitis seeds, no significant difference was found in the GR₅₀ values between E. crus-galli and E. crus-galli var. mitis populations when using a paired sample t-test (Table S3). However, the SI₅₀ (the ratio between the GR₅₀ values of the least and most sensitive populations) value showed greater variability in florpyrauxifen-benzyl responses among E. crus-galli var. mitis populations (75.1-fold) than among E. crus-galli populations (26.4-fold).

The average GR₅₀ value of E. crus-galli in 13 different cities showed no significant difference (

Table 1. Number of populations and average GR₅₀ values of florpyrauxifen-benzyl to the two Echinochloa species collected from 13 cities in Jiangsu Province, China.

City	E. crus-galli		E. crus-galli var. mitis
	Number of Populations	Average GR50 Value (g ai ha−1)	Number of Populations	Average GR50 Value (g ai ha−1)
Changzhou	6	5.7	15	7.7 ab
Huaian	7	4.1	13	5.8 b
Lianyungang	4	11.7	11	7.6 ab
Nanjing	/	/	17	12.7 ab
Nantong	10	11.0	12	7.4 ab
Suqian	5	9.9	10	5.7 b
Suzhou	7	7.1	11	13.0 ab
Taizhou	4	8.1	11	8.4 ab
Wuxi	7	6.9	10	8.3 ab
Xuhzou	1	24.8	11	5.4 b
Yancheng	7	5.8	13	8.5 ab
Yangzhou	4	8.8	15	17.4 a
Zhenjiang	8	8.1	9	8.5 ab

Note: Different letters indicate significant differences among 13 cities.

). For E. crus-galli var. mitis, the average GR₅₀ value in Yangzhou city (17.4 g ai ha⁻¹) was significantly higher than that in the other 12 cities, and the average GR₅₀ values in Huaian (5.8 g ai ha⁻¹), Suqian (5.7 g ai ha⁻¹), and Xuzhou (5.4 g ai ha⁻¹) cities were significantly lower.

Moderate and high resistance to florpyrauxifen-benzyl remains sporadic in E. crus-galli and E. crus-galli var. mitis in Jiangsu Province. Notably, GR₅₀ values of E. crus-galli var. mitis exhibit a significant positive correlation with latitude and longitude (Figure 5), indicating higher resistance frequencies in southwestern populations.

4. Discussion

Florpyrauxifen-benzyl, one of the most important herbicides used in rice production throughout the world, was reported to be highly effective in controlling Echinochloa weed species [9,16]. In Jiangsu Province, China, florpyrauxifen-benzyl was used for six years before the seeds were collected for this study. Our results unequivocally demonstrate the high intrinsic efficacy of florpyrauxifen-benzyl against a majority of tested populations of both Echinochloa species tested, confirming its potential as a valuable tool for managing these pernicious rice weeds. The average GR₅₀ value (8.1 g ai ha⁻¹) of E. crus-galli for florpyrauxifen-benzyl in this study was lower than that reported in Korea (11.33 g ai ha⁻¹). The highest resistance factor of E. crus-galli (26.4-fold) in this study was higher than that of E. crus-galli in Korea [20]. Despite showing greater intraspecific variability, E. crus-galli populations in this study were more sensitive to florpyrauxifen-benzyl than those collected from Korea. Furthermore, together, E. crus-galli var. mitis populations showed higher variability in florpyrauxifen-benzyl resistance than E. crus-galli. Our previous study with these populations also found higher variability of E. crus-galli var. mitis in seed germination indices than E. crus-galli [6].

The baseline sensitivity of a weed to herbicides is critical for monitoring the development of herbicide resistance, which is the cornerstone for future resistance management decisions [16,29]. Without this initial reference, discerning the early emergence of resistant biotypes from natural population variation or confounding environmental influences becomes significantly more challenging. In our study, the GR₅₀$x_{\mathrm{c}}$ value served as the baseline sensitivity dose. Florpyrauxifen-benzyl-resistant E. crus-galli populations were reported in different rice planting areas [10,30,31]. In 2022, a study with 93 mixed samples of Echinochloa weeds suggested florpyrauxifen-benzyl resistance in Jiangsu Province [32]. In many studies on herbicide resistance, the control was the most sensitive population and was more important than the baseline sensitivity dose. In this study, 97% of populations (E. crus-galli and E. crus-galli var. mitis) tested showed different levels of florpyrauxifen-benzyl resistance, which does not coincide with the fact that most populations (71.4% of E. crus-galli and 70.9% of E. crus-galli var. mitis populations) tested were totally killed by the florpyrauxifen-benzyl label dose. The GR₅₀ value of the E. crus-galli populations most sensitive to florpyrauxifen-benzyl ranges from 0.4 to 6.2 in studies on florpyrauxifen-benzyl-resistant [7,22,33,34]. When the GR₅₀$x_{\mathrm{c}}$ values for these species (instead of the GR₅₀ value of the most sensitive population) are used to calculate the sensitivity index among populations, proportions of populations at different florpyrauxifen-benzyl resistance levels could be more reasonable. To date, there are only two studies reporting florpyrauxifen-benzyl-resistant E. crus-galli var. mitis [35,36], and neither pinpoint GR₅₀ values. In our study, proportions of florpyrauxifen-benzyl-resistant populations were similar among the two species, as well as the GR₅₀$x_{\mathrm{c}}$ value (5.0 g ai ha⁻¹ for E. crus-galli var. mitis and 4.9 g ai ha⁻¹ for E. crus-galli). Therefore, the GR₅₀$x_{\mathrm{c}}$ value could be the baseline sensitivity dose used for calculating the resistance factors of Echinochloa spp. In addition, the GR₅₀$x_{\mathrm{c}}$ value also served as the baseline sensitivity dose of E. glabrescens to florpyrauxifen-benzyl [16].

Intra-regional and inter-regional variations in herbicide sensitivity are notable [37]. Florpyrauxifen-benzyl-resistant E. crus-galli var. mitis populations were more frequently collected from southwestern regions. This distribution pattern may result from localized governmental promotion of novel herbicides for resistant weed control, coupled with E. crus-galli var. mitis’s higher prevalence—factors potentially accelerating florpyrauxifen-benzyl resistance evolution in agroecosystems. Where weed infestations were severe, the probability of resistance selection was often elevated [38]. Some E. crus-galli populations had already developed resistance before the commercialization of florpyrauxifen-benzyl [31]. Prior and ongoing intense selection pressure from herbicides sharing similar modes of action (e.g., synthetic auxins like quinclorac or 2,4-D, though differing in chemical structure) or potentially multiple resistant mechanisms may have pre-selected individuals with reduced susceptibility to florpyrauxifen-benzyl [28,39]. Over-reliance on and potentially sub-optimal use of florpyrauxifen-benzyl (e.g., repeated applications within a season or across consecutive seasons without rotation) rapidly enriched these resistant individuals. The intensive double cropping system per year, frequent agricultural machinery transportation, and highly developed water systems and transportation networks in Jiangsu create an environment highly conducive to the selection and spread of herbicide-resistant weeds [16,40].

Fortunately, the low baseline sensitivity values observed indicate a potent control effect achievable at the label dose of florpyrauxifen-benzyl. Nevertheless, the concerning emergence of resistance threatens the long-term sustainability of this important herbicide. To safeguard the efficacy of florpyrauxifen-benzyl and ensure sustainable weed management, the implementation of proactive, diversified resistance mitigation strategies is imperative [41,42]. Initially, florpyrauxifen-benzyl must be rotated with herbicides possessing different modes of action (e.g., ACCase inhibitors, ALS inhibitors, and DOXP synthase inhibitors) to delay resistance evolution [22,43,44]. Furthermore, applying florpyrauxifen-benzyl at effective doses and utilizing precise application technologies are also of key importance [45]. Moreover, promoting cultural practices (e.g., crop rotation where feasible, the stale seedbed technique, and use of competitive rice varieties) and mechanical weeding to reduce overall reliance on herbicides should be highlighted [6]. In addition, the baseline established herein could be a reference for monitoring the development of florpyrauxifen-benzyl resistance.

5. Conclusions

While florpyrauxifen-benzyl remains a highly effective herbicide against susceptible Echinochloa populations, the confirmed presence of high-level resistance necessitates immediate and concerted action. Integrated weed management principles to reduce selection pressure and exploit diverse control tactics could be essential to preserve the utility of florpyrauxifen-benzyl and secure rice production at the regional scale. Mechanisms conferring florpyrauxifen-benzyl in Echinochloa species merit many more studies.