稻田稗草对五氟磺草胺的抗性机制及其防治药剂筛选

doi:10.3864/j.issn.0578-1752.2023.14.007

中国农业科学 ›› 2023, Vol. 56 ›› Issue (14): 2713-2723.doi: 10.3864/j.issn.0578-1752.2023.14.007

稻田稗草对五氟磺草胺的抗性机制及其防治药剂筛选

张丽雅¹(), 李奇¹(), 史珊珊¹, 马雨梦¹, 刘亚琪¹, 赵超伟¹, 王鹤如², 操海群¹, 廖敏¹, 赵宁¹()

¹ 安徽农业大学植物保护学院/作物有害生物综合治理安徽省重点实验室，合肥 230036
² 天长市农业科技中心植保站，安徽天长 239300

收稿日期:2023-04-11 接受日期:2023-05-14 出版日期:2023-07-16 发布日期:2023-07-21
通信作者:
赵宁，E-mail：zhaon@ahau.edu.cn
联系方式: 张丽雅，E-mail：3012596087@qq.com。李奇，E-mail：qigi12345@163.com。张丽雅和李奇为同等贡献作者。
基金资助:
安徽省科技重大专项(201903a06020033); 安徽农业大学人才引进科研启动项目(rc342004); 安徽农业大学大学生创新训练项目(X202210364248)

Resistance Mechanism of Barnyard Grass (Echinochloa crus-galli) to Penoxsulam and Screening Herbicides for Its Control in Rice Fields

ZHANG LiYa¹(), LI Qi¹(), SHI ShanShan¹, MA YuMeng¹, LIU YaQi¹, ZHAO ChaoWei¹, WANG HeRu², CAO HaiQun¹, LIAO Min¹, ZHAO Ning¹()

¹ School of Plant Protection, Anhui Agricultural University/Anhui Province Key Laboratory of Crop Integrated Pest Management, Hefei 230036
² Tianchang Agricultural Science and Technology Center Plant Protection Station, Tianchang 239300, Anhui

Received:2023-04-11 Accepted:2023-05-14 Published:2023-07-16 Online:2023-07-21

摘要/Abstract

摘要：

【目的】稗草（Echinochloa crus-galli）是我国水稻田主要恶性杂草之一，五氟磺草胺等乙酰乳酸合成酶（acetolactate synthase，ALS）抑制剂类除草剂是防治稻田稗草的主要除草剂种类。本研究团队前期在安徽省天长市水稻主产区发现疑似五氟磺草胺抗性稗草种群AHTC-01，明确其对稻田不同种类除草剂的抗性水平及可能的抗性分子机制，为抗性稗草有效防治、延缓其抗药性进一步发展提供理论依据。【方法】采用温室盆栽法在整株水平上测定稗草种群AHTC-01对五氟磺草胺的抗性水平及对不同除草剂的抗性模式，并通过靶标基因测序和实时荧光定量PCR（real-time quantitative PCR，RT-qPCR）分析探索其靶标抗性分子机制。【结果】相比敏感稗草种群AHFY-01，疑似抗性稗草种群AHTC-01已对五氟磺草胺产生高水平抗性，抗性倍数（resistance index，RI）为620。靶标抗性机制分析表明，AHTC-01种群ALS基因拷贝2（ALS2）第574位氨基酸由色氨酸（Trp）突变为亮氨酸（Leu），其种群突变频率为100%；在五氟磺草胺处理后12 h，抗性稗草种群AHTC-01 ALS相对表达量为敏感稗草种群AHFY-01的2.26倍。AHTC-01同时对其他3种ALS抑制剂类除草剂双草醚、嘧啶肟草醚、甲氧咪草烟产生不同水平交互抗性，抗性倍数分别为8.24、13.36、20.36；但是对乙酰辅酶A羧化酶（acetyl-CoA carboxylase，ACCase）抑制剂氰氟草酯、精噁唑禾草灵和烯草酮，4-羟基苯基丙酮酸双氧化酶（4-hydroxyphenylpyruvate dioxygenase，HPPD）抑制剂三唑磺草酮，合成生长素类（synthetic auxin mimic）氯氟吡啶酯等其他作用机制除草剂依旧较为敏感。【结论】稗草种群AHTC-01靶标基因ALS2第574位氨基酸突变和ALS过量表达是其对五氟磺草胺产生抗性的主要原因之一，该抗性机制同时赋予其对不同ALS抑制剂的交互抗性。农田生产实际中，可轮换使用其他作用机制除草剂对其进行有效防治。

关键词: 稗草, 五氟磺草胺, 乙酰乳酸合成酶, 基因突变, 基因过量表达

Abstract:

【Objective】Barnyard grass (Echinochloa crus-galli) is one of the main malignant weeds in rice fields in China. Acetolactate synthase (ALS) inhibitors, such as penoxsulam, are the main herbicides for controlling E. crus-galli in rice fields. Previously, our research team identified a suspected penoxsulam-resistant E. crus-galli population, AHTC-01, in the main rice production area of Tianchang City, Anhui Province, China. The objective of this study is to clarify its resistance levels to major herbicides, investigate the possible resistance molecular mechanisms, and to provide a theoretical basis for the effective control of resistant E. crus-galli and the delay of further development of herbicide resistance.【Method】Using the greenhouse potting method, the resistance levels of AHTC-01 to penoxsulam and its resistance patterns to different herbicides were determined at the whole-plant level. The target resistance molecular mechanism was explored through target gene sequencing and real-time quantitative PCR (RT-qPCR) analysis.【Result】Compared with the susceptible E. crus-galli population AHFY-01, the suspected resistant population AHTC-01 had developed high-level resistance to penoxsulam, with a resistance index (RI) of 620. Analysis of the target-site based resistance mechanisms showed that the ALS2 gene copy in the AHTC-01 population had a mutation from tryptophan (Trp) to leucine (Leu) at codon position 574, with a population mutation frequency of 100%. At 12 h after penoxsulam treatment, the relative expression level of ALS in the resistant E. crus-galli population AHTC-01 was 2.26 times of that in the susceptible E. crus-galli population AHFY-01. AHTC-01 also exhibited varying levels of cross-resistance to three other ALS inhibitors, bispyribac sodium, pyribenzoxim, and imazamox, with RIs of 8.24, 13.36, and 20.36, respectively. However, it remained susceptible to other herbicides with different modes of action (MOAs), including the acetyl-CoA carboxylase (ACCase) inhibitors cyhalofop-butyl, fenoxaprop-P-ethyl, and clethodim, the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor tripyrasulfone, and the synthetic auxin mimic florpyrauxifen-benzyl.【Conclusion】Mutation of the ALS2 at amino acid position 574 and ALS overexpression are one of the main reasons for the resistance of E. crus-galli population AHTC-01 to penoxsulam, which also confers cross-resistance to different ALS inhibitors. In actual agricultural production, effective control of this type of resistant E. crus-galli can still be achieved by rotating the use of other herbicides with different MOAs.

Key words: Echinochloa crus-galli, penoxsulam, acetolactate synthase (ALS), gene mutation, gene overexpression

张丽雅, 李奇, 史珊珊, 马雨梦, 刘亚琪, 赵超伟, 王鹤如, 操海群, 廖敏, 赵宁. 稻田稗草对五氟磺草胺的抗性机制及其防治药剂筛选[J]. 中国农业科学, 2023, 56(14): 2713-2723.

ZHANG LiYa, LI Qi, SHI ShanShan, MA YuMeng, LIU YaQi, ZHAO ChaoWei, WANG HeRu, CAO HaiQun, LIAO Min, ZHAO Ning. Resistance Mechanism of Barnyard Grass (Echinochloa crus-galli) to Penoxsulam and Screening Herbicides for Its Control in Rice Fields[J]. Scientia Agricultura Sinica, 2023, 56(14): 2713-2723.

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除草剂 Herbicide	系列剂量 Series of doses (g a.i.·hm^-2)
除草剂 Herbicide	抗性种群AHTC-01 Resistant population AHTC-01	敏感种群AHFY-01 Susceptible population AHFY-01
五氟磺草胺Penoxsulam	0, 60, 120, 240, 480, 960, 1920	0, 0.04, 0.12, 0.37, 1.11, 3.33, 10
双草醚Bispyribac-sodium	0, 45, 90, 180, 360, 720, 1440	0, 5.60, 11.30, 22.50, 45, 90, 180
嘧啶肟草醚Pyribenzoxim	0, 37.50, 75, 150, 300, 600, 1200	0, 4.70, 9.40, 18.80, 37.50, 75, 150
甲氧咪草烟Imazamox	0, 24, 48, 96, 192, 384, 768	0, 3, 6, 12, 24, 48, 96
氰氟草酯Cyhalofop-butyl	0, 0.49, 1.48, 4.44, 13.33, 40, 120	0, 0.49, 1.48, 4.44, 13.33, 40, 120
精噁唑禾草灵Fenoxaprop-P-ethyl	0, 0.10, 0.50, 2.50, 12.40, 62.10, 310.50	0, 0.02, 0.10, 0.50, 2.50, 12.40, 62.10
烯草酮Clethodim	0, 0.03, 0.17, 0.86, 4.32, 21.60, 108	0, 0.03, 0.17, 0.86, 4.32, 21.60, 108
三唑磺草酮Tripyrasulfone	0, 0.04, 0.22, 1.08, 5.40, 27, 135	0, 0.04, 0.22, 1.08, 5.40, 27, 135
氯氟吡啶酯Florpyrauxifen-benzyl	0, 0.15, 0.44, 1.33, 4, 12, 36	0, 0.15, 0.44, 1.33, 4, 12, 36

种群 Population	整株剂量响应曲线参数 Parameters for whole-plant dose-response curves				GR₅₀ (g a.i.·hm^-2)	抗性倍数 Resistance index
种群 Population	C	D	b	R²	GR₅₀ (g a.i.·hm^-2)	抗性倍数 Resistance index
AHTC-01	26.90 (9.20)	93.25 (16.53)	5.72 (6.23)	0.91	155.00 (53.80)	620
AHFY-01	32.89 (3.82)	102.17 (7.65)	1.89 (0.67)	0.99	0.25 (0.06)	-

物种/种群 Species/Population	各氨基酸的位置和碱基及相应氨基酸序列 The amino acid position, relative sequence of nucleotide and derived amino acid
物种/种群 Species/Population	122	197	205	376	377	574	653	654
拟南芥 A. thaliana	GCA	CCT	GCT	GAT	CGT	TGG	AGT	GGT
拟南芥 A. thaliana	Ala	Pro	Ala	Asp	Arg	Trp	Ser	Gly
AHFY-01	GCC	CCC	GCC	GAT	CGT	TGG	AGC	GGT
AHFY-01	Ala	Pro	Ala	Asp	Arg	Trp	Ser	Gly
AHTC-01	GCC	CCC	GCC	GAT	CGT	TTG	AGC	GGT
AHTC-01	Ala	Pro	Ala	Asp	Arg	Leu	Ser	Gly

除草剂 Herbicide	GR₅₀(g a.i.·hm^-2)		抗性倍数 Resistance index
除草剂 Herbicide	AHTC-01	AHFY-01	抗性倍数 Resistance index
双草醚Bispyribac-sodium	190.09 (8.30)	23.07 (2.56)	8.24
嘧啶肟草醚Pyribenzoxim	207.05 (45.02)	15.50 (6.68)	13.36
甲氧咪草烟Imazamox	197.32 (55.85)	9.69 (2.18)	20.36
氰氟草酯Cyhalofop-butyl	7.79 (0.45)	5.74 (0.35)	1.37
精噁唑禾草灵Fenoxaprop-P-ethyl	3.44 (0.27)	2.06 (0.30)	1.67
烯草酮Clethodim	4.75 (2.20)	3.27 (1.55)	1.45
三唑磺草酮Tripyrasulfone	5.27 (1.80)	5.04 (2.58)	1.05
氯氟吡啶酯Florpyrauxifen-benzyl	1.04 (0.12)	1.31 (0.49)	0.80

稻田稗草对五氟磺草胺的抗性机制及其防治药剂筛选

Resistance Mechanism of Barnyard Grass (Echinochloa crus-galli) to Penoxsulam and Screening Herbicides for Its Control in Rice Fields

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