山东省多主棒孢对三种常用杀菌剂的敏感性监测及对氟吡菌酰胺的抗性

doi:10.3864/j.issn.0578-1752.2022.07.008

摘要/Abstract

摘要：

【目的】由多主棒孢（Corynespora cassiicola）引起的黄瓜靶斑病是世界公认的黄瓜三大病害之一,严重影响着黄瓜的产量和品质。随着防治药剂的连续使用,其抗药性问题日益突出。本研究旨在明确山东省多主棒孢对常用杀菌剂的抗性情况,为黄瓜靶斑病的药剂防治提供理论依据,同时筛选高效混配药剂为多主棒孢的抗药性治理提供数据支持。【方法】从山东省不同地区采集、分离纯化获得140株多主棒孢,采用室内菌丝生长速率法测定多主棒孢对3种常用杀菌剂（苯醚甲环唑、咪鲜胺和氟吡菌酰胺）的敏感性,并利用Modeller v9.19程序同源建模及AutoDock4.2.6分子对接软件包分析多主棒孢对氟吡菌酰胺的抗性。结合室内菌丝生长抑制和离体叶片防治效果试验筛选氟吡菌酰胺和咪鲜胺的复配最佳配比。【结果】经过异常值分析并剔除,敏感菌株频率分布均呈单峰,分别确定苯醚甲环唑对多主棒孢121株敏感菌株的EC₅₀范围为0.29—6.80 μg·mL^-1,平均EC₅₀值为2.44 μg·mL^-1;咪鲜胺对129株敏感菌株的EC₅₀范围为0.01—0.57 μg·mL^-1,平均EC₅₀值为0.16 μg·mL^-1;而氟吡菌酰胺对136株多主棒孢的EC₅₀范围为0.56—47.54 μg·mL^-1,平均EC₅₀值为6.94 μg·mL^-1。经测序分析发现31.62%多主棒孢菌株SdhC亚基发生S73P点突变。分子对接结果表明,SdhC亚基上发生S73P突变后,氨基酸的刚性增强,并且空间位阻变大,使氟吡菌酰胺与Ser73之间的氢键作用消失,从而亲和力降低,导致抗药性的发生。交互抗性分析结果表明氟吡菌酰胺与咪鲜胺不存在交互抗性,两者以7﹕3混用增效系数1.84,表现为增效作用,且相同剂量处理下,混剂的防治效果高于两个单剂处理的防治效果。【结论】山东省不同地区多主棒孢群体对不同杀菌剂的敏感性存在差异,对氟吡菌酰胺已产生不同程度的抗性,其中SdhC上点突变S73P的发生频率相对较高,导致病原菌琥珀酸脱氢酶与氟吡菌酰胺的结合能力下降。氟吡菌酰胺与咪鲜胺以7﹕3混用能够提高防治黄瓜靶斑病的效果,建议生产上可将氟吡菌酰胺与咪鲜胺交替或混合使用,用于治理目前多主棒孢对氟吡菌酰胺等SDHI类杀菌剂的抗性问题。

关键词: 多主棒孢, 黄瓜靶斑病, 敏感性, S73P突变, 分子对接, 增效作用

Abstract:

【Objective】Corynespora target spot caused by Corynespora cassiicola is recognized as one of the three most serious worldwide diseases of cucumber, which seriously affects the yield and quality of cucumber. With the continuous use of commercial fungicides, resistance problem of C. cassiicola increased seriously. The objective of this study is to (i) clarify the resistance profile of C. cassiicola in cucumber from Shandong Province to three common fungicides; (ii) provide a theoretical basis for the fungicide control of Corynespora target spot of cucumber; (iii) provide data support for the screening of highly efficient mixed fungicides for the resistance management of C. cassiicola. 【Method】A total of 140 C. cassiicola isolates were isolated from Shandong Province. The sensitivity of C. cassiicola to difenoconazole, prochloraz and fluopyram was investigated using mycelium growth rate method. The relationship of SdhC-S73P and fluopyram-resistance was analyzed using Modeller v9.19 and AutoDock4.2.6 software. The optimal ratio of fluopyram and prochloraz was also screened using in vitro mycelial growth method and detached leaf assay. 【Result】After data outlier analysis and elimination, the EC₅₀ values of the 121 C. cassiicola isolates to difenoconazole ranged from 0.29 to 6.80 μg·mL ^-1, and produced an unimodal distribution with a mean of 2.44 μg·mL ^-1. The EC₅₀ values of the 129 C. cassiicola isolates to prochloraz ranged from 0.01 to 0.57 μg·mL^-1 with a mean of 0.16 μg·mL ^-1, and also produced an unimodal distribution. The EC₅₀ values of the 136 C. cassiicola isolates to fluopyram ranged from 0.56 to 47.54 μg·mL^-1, with a mean of 6.94 μg·mL ^-1. Many fluopyram-resistant isolates with different resistant factors were detected, and the mutation frequency of fluopyram-resistant isolates containing S73P in SdhC was 31.62%. Molecular docking results showed that after the S73P mutation occurred on the SdhC subunit, the rigidity of the amino acid increased, and the steric hindrance became larger, so that the hydrogen bond between fluopyram and Ser73 disappeared, so the affinity was reduced, leading to the occurrence of fluopyram resistance. No cross-resistance was detected between fluopyram and prochloraz. The highest synergistic effect of fluopyram and prochloraz was 1.84 at a ratio of 7﹕3. The control efficacy of the mixture (fluopyram﹕prochloraz=7﹕3) against Corynespora target spot was higher than that of single fungicide (fluopyram or prochloraz) at the same dose. 【Conclusion】The sensitivity of C. cassiicola from different regions to fungicide is different in Shandong Province. Most of C. cassiicola isolates showed fluopyram resistance and many point mutations in SdhB, SdhC and SdhD were detected. Among them, the frequency of S73P mutation in SdhC was the highest. The control efficacy of the mixture of fluopyram and prochloraz in 7﹕3 against Corynespora target spot in cucumber was more excellent compared with that of single fungicide. It is suggested that the fluopyram can be used alternately or in mixture with prochloraz to control the resistance of C. cassiicola to SDHI fungicides such as fluopyram.

Key words: Corynespora cassiicola, Corynespora target spot, fungicide sensitivity, S73P mutation, molecular docking, synergistic effect

李桂香,李秀环,郝新昌,李智文,刘峰,刘西莉. 山东省多主棒孢对三种常用杀菌剂的敏感性监测及对氟吡菌酰胺的抗性[J]. 中国农业科学, 2022, 55(7): 1359-1370.

LI GuiXiang,LI XiuHuan,HAO XinChang,LI ZhiWen,LIU Feng,LIU XiLi. Sensitivity of Corynespora cassiicola to Three Common Fungicides and Its Resistance to Fluopyram from Shandong Province[J]. Scientia Agricultura Sinica, 2022, 55(7): 1359-1370.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.07.008

https://www.chinaagrisci.com/CN/Y2022/V55/I7/1359

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基因 Gene	引物名称 Primer name	引物序列 Primer sequence (5′-3′)	引物长度 Length of primer (bp)	扩增片段大小 Length of product (bp)
SdhB	CcSdhB-F	ATGGCTTGCACACGCGCTTT	20	1106
SdhB	CcSdhB-R	CTACGTGAAAGCCATGCTC	19	1106
SdhC	CcSdhC-F	ATGGCTTCCCAGCGCGTCTT	20	629
SdhC	CcSdhC-R	TAAACAAACAGAGAATAGTA	20	629
SdhD	CcSdhD-F	ATGAAGCGCACCTCG CCAATC	21	938
SdhD	CcSdhD-R	AAAGATCCTAATAATCACATGTATCCAAAC	30	938

点突变类型 Point mutation type	菌株数量 Number of isolates	抑制中浓度 EC₅₀(μg·mL^-1)	比值 Ratio (%)
无突变No mutation	42	0.56-2.42	30.88
SdhB H278Y	9	0.56-1.19	6.62
SdhB I280V	19	1.58-24.63	13.97
SdhC S73P	43	1.88-47.54	31.62
SdhC N75S	5	1.42-11.31	3.68
SdhC H134R	8	0.61-18.84	5.88
SdhD G135V	10	2.66-18.65	7.35