Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (9): 3511-3528.DOI: 10.1016/j.jia.2024.01.004

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杀菌剂三氟吡啶胺对藤仓镰孢菌抑菌活性及病菌抗药性分子机制研究

  

  • 收稿日期:2023-07-31 修回日期:2024-01-05 接受日期:2023-11-20 出版日期:2025-09-20 发布日期:2025-08-11

Activity of fungicide cyclobutrifluram against Fusarium fujikuroi and mechanism of the pathogen resistance associated with point mutations in FfSdhB, FfSdhC2 and FfSdhD

Yang Sun1, 3, 4, 5*, Yu Liu1, 3, 4, 5*, Li Zhou1, 3, 4, 5, Xinyan Liu1, 3, 4, 5, Kun Wang1, 3, 4, 5, Xing Chen1, 3, 4, 5, Chuanqing Zhang2, Yu Chen1, 3, 4, 5#   

  1. 1 School of Plant Protection, Anhui Agricultural University, Hefei 230036, China

    2 Department of Plant Pathology, Zhejiang Agriculture and Forest University, Hangzhou 311300, China

    3 Key Laboratory of Integrated Crop Pest Management of Anhui Province, School of Plant Protection, Anhui Agricultural University, Hefei 230036, China

    4 Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes, School of Plant Protection, Anhui Agricultural University, Hefei 230036, China

    5 Anhui Province Engineering Laboratory for Green Pesticide Development and Application, School of Plant Protection, Anhui Agricultural University, Hefei 230036, China

  • Received:2023-07-31 Revised:2024-01-05 Accepted:2023-11-20 Online:2025-09-20 Published:2025-08-11
  • About author:#Correspondence Yu Chen, E-mail: chenyu66891@sina.com * These authors contributed equally to this study.
  • Supported by:
    This study was supported by i) the National Natural Science Fundation of China (32302392), ii) the Key Project of the Natural Science Foundation of Anhui Provincial Department of Education, China (2022AH050877 and 2023AH040129), iii) the Programs for the Scientific Research Activities of Academic and Technical Leaders of Anhui Province, China (2020D251), iv) the Development Fund for Talent Personnel of Anhui Agricultural University, China (rc342006 and rc342008), v) the University Synergy Innovation Program of Anhui Province, China (GXXT-2021-059), vi) the Grants from Anhui Agricultural University, China (2020zd27) and vii) the Anhui Province Agricultural Eco-Environmental Protection and Quality Safety Industry Technology System, China.

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摘要：

水稻恶苗病是一种严重危害水稻生产的种传病害，其优势病原菌为藤仓镰孢菌（Fusarium fujikuroi）。为明确新型琥珀酸脱氢酶抑制剂类杀菌剂三氟吡啶胺用于防治水稻恶苗病的应用潜力，评估抗性风险并解析病原抗药性分子机制，本研究采用菌丝生长速率法和孢子萌发抑制法测定了72株藤仓镰孢菌对三氟吡啶胺的敏感性，并建立敏感性基线，继而探究药剂对病原菌形态学和相关生理生化指标的影响，并测定在活体盆栽条件下药剂防效，评估其用于恶苗病防治潜力。进一步采用药剂驯化获得抗性突变体并评估抗性风险及与常用药剂交互抗性，比对分析抗性菌株与亲本菌株Sdh基因序列，鉴定抗性突变位点，最终采用同源替换与分子对接技术揭示抗药性分子机制。研究发现三氟吡啶胺对藤仓镰孢菌具有显著生物活性，生长速率法和孢子萌发法测定EC50分别为0.0410 ± 0.0470 μg mL-1和0.0038 ± 0.0015 μg mL-1，且敏感性频率呈正态分布。三氟吡啶胺可破坏病原菌菌丝和孢子形态，提升细胞膜透性，降低病菌的毒素合成，并抑制琥珀酸脱氢酶活性。此外，活体盆栽条件下三氟吡啶胺对水稻恶苗病的保护作用显著优于对照药剂氰烯菌酯和嘧菌酯。以上结果表明三氟吡啶胺可作为防治水稻恶苗病的理想候选药剂。通过药剂驯化共获得6株抗性突变体菌株，突变频率为3.75 × 10-4，抗性菌株生物适合度均显著低于其亲本菌株，且对SDHI类杀菌剂氟唑菌酰羟胺和氟唑菌苯胺存在交互抗性，而与非同类药剂氰烯菌酯、咯菌腈、咪鲜胺和嘧菌酯等无交互抗性，表明藤仓镰孢菌对三氟吡啶胺存在中等抗性风险。抗性机制研究发现SdhB亚基H248D突变可赋予病原菌中等水平抗药性（20< RF <100）；SdhC2亚基A83V突变可赋予高水平抗药性（RF >100）；SdhD亚基S106F和E166K突变赋予菌株低水平抗性（RF <10）。研究结果可为三氟吡啶胺在水稻恶苗病防治上的科学使用及抗药性治理提供理论依据。

Abstract:

Rice bakanae disease (RBD) is a devastating plant disease caused by Fusarium fujikuroi. This study aimed to evaluate the potential of cyclobutrifluram, a novel succinate dehydrogenase inhibitor (SDHI), to control RBD, and determine the risk and mechanism of resistance to cyclobutrifluram in F. fujikuroi. In vitro experiments showed that cyclobutrifluram significantly inhibited mycelial growth and spore germination, and altered the morphology of mycelia and conidia. Treatment with cyclobutrifluram significantly decreased mycotoxin production and increased cell membrane permeability in F. fujikuroi. The baseline sensitivity of 72 F. fujikuroi isolates to cyclobutrifluram was determined using mycelial growth and spore germination inhibition assays, which revealed EC50 values of 0.0114 – 0.1304 μg mL-1 and 0.0012 – 0.016 μg mL-1, with mean EC50 values of 0.0410 ± 0.0470 μg mL-1 and 0.0038 ± 0.0015 μg mL-1, respectively. Pot experiments demonstrated that the protective effect of cyclobutrifluram against F. fujikuroi was more significant than that of phenamacril and azoxystrobin, indicating that cyclobutrifluram is a promising antifungal agent for the control of RBD. Six cyclobutrifluram-resistant mutants of F. fujikuroi were obtained via fungicide adaptation. Moreover, these mutants exhibited weaker fitness than their parental isolate and positive cross-resistance with other SDHI fungicides, including pydiflumetofen and penflufen; however, no cross-resistance was detected with other classes of fungicides, including phenamacril, fludioxonil, prochloraz, or azoxystrobin. These results indicated that the resistance risk of F. fujikuroi to cyclobutrifluram might be moderate. Sequencing analysis revealed that mutations, including H248D in FfSdhB, A83V in FfSdhC2, and S106F and E166K in FfSdhD, contributed to resistance, which was confirmed by molecular docking and homologous replacement experiments. The results suggest a high potential for cyclobutrifluram to control RBD and a moderate resistance risk of F. fujikuroi to cyclobutrifluram, which are meaningful findings for the scientific application of cyclobutrifluram.

Key words: Fusarium fujikuroi ,  rice bakanae disease ,  cyclobutrifluram ,  succinate dehydrogenase inhibitors ,  resistance risk and mechanisms