Sensitivity and resistance risk analysis of Didymella bryoniae populations to fluopyram

doi:10.1016/j.jia.2023.11.044

Journal of Integrative Agriculture

2024, Vol. 23

Issue (7): 2306-2317 DOI: 10.1016/j.jia.2023.11.044

Plant Protection

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Sensitivity and resistance risk analysis of Didymella bryoniae populations to fluopyram

Zhiwen Wu, Xiaowei Cai, Xuewei Mao, Mingguo Zhou, Yiping Hou^#

College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

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摘要

由黑腐球壳菌（Didymella bryoniae）引起的西瓜蔓枯病是西瓜上的重要病害，严重影响西瓜的产量和品质。目前针对西瓜蔓枯病菌的防治依然是以化学防治为主，琥珀酸脱氢酶抑制剂类杀菌剂（SDHI）氟吡菌酰胺已登记用于防治西瓜蔓枯病多年，但田间菌株对氟吡菌酰胺的敏感性是否有变化及其抗性风险尚未知晓。因此本研究在有氟吡菌酰胺用药历史的田间分离获得69株西瓜蔓枯病菌，采用菌丝生长速率法测定了其对氟吡菌酰胺的敏感性，结果表明，氟吡菌酰胺对69株西瓜蔓枯病菌的有效抑制中浓度（EC₅₀）为0.0691 到 0.3503 μg mL^-1，变异系数为5.07，平均EC₅₀为0.1579 ± 0.0669 μg mL^-1，敏感性基线为单峰曲线，田间未发现氟吡菌酰胺的抗性菌株；氟吡菌酰胺对西瓜蔓枯病菌的最低抑制浓度（MIC）为3 μg mL^-1。通过药剂驯化获得了6株氟吡菌酰胺抗性突变体（2株中抗和4株低抗）且抗性均可稳定遗传；与出发菌株相比，所有抗性突变体生长速率显著下降而绝大部分突变体菌丝干重无显著变化，对不同胁迫因子的敏感性显著上升；除了低抗菌株XN51FR-1与出发菌株致病力一致外，其他抗性突变体的致病力均显著下降，以上结果说明抗性突变体的适合度低于出发菌株。氟吡菌酰胺抗性突变体与本研究中的6种SDHI类杀菌剂均存在正交互抗性，而与不同作用机制的杀菌剂氟啶胺和戊唑醇不存在交互抗性。综上，西瓜蔓枯病菌对氟吡菌酰胺的抗性风险为中等。序列比对发现低抗菌株XN30FR-1琥珀酸脱氢酶B亚基（sdhB）存在3个新的氨基酸点突变，分别为K258N、A259P 和 H277N，中抗菌株XN52FR-1 也存在H277N的点突变，其他抗性菌株不存在点突变。本研究明确了田间西瓜蔓枯病菌对氟吡菌酰胺的敏感性，评估了其抗性风险，对SDHI类杀菌剂在蔓枯病防治上的应用、延缓抗性的产生及延长药剂的使用寿命具有重要的指导意义，同时也为蔓枯病菌对SDHI类杀菌剂的抗性机制研究提供理论参考。

Abstract

Fluopyram is an succinate dehydrogenase inhibitors (SDHI) fungicide that has been registered in China to control gummy stem blight (GSB) in watermelons for many years. However, whether the field pathogens of GSB are still sensitive to fluopyram or not is unknown. Therefore, we collected 69 Didymella bryoniae isolates from the fields that usually use fluopyram to control GSB to determine the sensitivity change. The EC₅₀(50% inhibition effect) values of fluopyram against D. bryoniae ranged from 0.0691 to 0.3503 μg mL^–1and the variation factor was 5.07. The mean EC₅₀ value was (0.1579±0.0669) μg mL^–1and the curve of sensitivity was unimodal. No resistant strains were found in the isolates, which means that the pathogens were still sensitive to fluopyram. The minimal inhibition concentration (MIC) of fluopyram against D. bryoniae was 3 μg mL^–1. Four low-resistant mutants and two medium-resistant mutants were obtained using fungicide taming and the resistance of mutants could be inherited stably. The growth rate of mutants decreased significantly compared with that of wild-type strains while the biomass of most mutants was similar to that of wild-type strains. The sensitivity of most resistant mutants to various stresses was increased compared with that of wild-type strains. The virulence of mutants receded except for low-resistant mutant XN51FR-1, which had the same lesion area as XN51 on the watermelon leaves. The results indicated that the fitness of resistant mutants was decreased compared with that of wild-type strains. The cross-resistance assay indicated that fluopyram-resistant mutants were positive cross-resistant to all six SDHI fungicides in this test but were still sensitive to fluazinam and tebuconazole. So the resistance risk of D. bryoniae to fluopyram was moderate. In addition, we found that the SdhB gene of low-resistant mutant XN30FR-1 had three new point mutations at positions K258N, A259P, and H277N. Medium-resistant mutant XN52FR-1 showed a mutation at position H277N and other mutants did not have any point mutation.

Keywords: fluopyram D. bryoniae sensitivity risk assessment

Received: 31 July 2023 Accepted: 17 October 2023

Fund:

This study was sponsored by the National Key R&D Program of China (2022YFD1400900), the National Natural Science Foundation of China (32272585), and the Fundamental Research Funds for the Central Universities, China (KYCXJC2023003).

About author: #Correspondence Yiping Hou, E-mail: houyiping@njau.edu.cn

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Zhiwen Wu, Xiaowei Cai, Xuewei Mao, Mingguo Zhou, Yiping Hou. 2024. Sensitivity and resistance risk analysis of Didymella bryoniae populations to fluopyram. Journal of Integrative Agriculture, 23(7): 2306-2317.

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