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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2022, Vol. 48 Issue (6): 830-842    DOI: 10.3785/j.issn.1008-9209.2022.06.151
研究论文     
4种三唑类杀菌剂对香蕉枯萎病菌的抑制效果及其差异性
项丹丹(),杨晓芳,易干军,陶海青,初元琦,李春雨()
广东省农业科学院果树研究所，农业农村部南亚热带果树生物学与遗传资源利用重点实验室/ 广东省热带亚热带果树研究重点实验室，广州 510640
Inhibitory effects of four different kinds of triazole fungicides against Fusarium oxysporum f. sp. cubense and their differences
Dandan XIANG(),Xiaofang YANG,Ganjun YI,Haiqing TAO,Yuanqi CHU,Chunyu LI()
Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization, Ministry of Agriculture and Rural Affairs/Guangdong Provincial Key Laboratory of Tropical and Subtropical Fruit Tree Research, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
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摘要：

香蕉枯萎病由尖孢镰刀菌古巴专化型（Fusarium oxysporum f. sp. cubense, Foc）侵染引起，正严重威胁着我国香蕉产业，目前尚无有效的化学防治措施。本研究通过测定菌丝生长速率比较了香蕉枯萎病菌热带4号生理小种（Foc tropical race 4, Foc TR4）对4种常用三唑类杀菌剂的敏感性，发现丙硫菌唑、戊唑醇、丙环唑、腈菌唑对Foc TR4的抑制作用依次减小。通过菌丝形态观察、相对电导率测定和丙二醛含量测定发现，与对照组相比，4种杀菌剂均可引起Foc TR4菌丝分枝增多、不规则扭曲，表面干瘪、凹陷和扁平化等畸形现象，使得菌丝细胞膜通透性和丙二醛含量显著性增加。三唑类杀菌剂可增大细胞色素P450酶活性并显著性上调细胞色素P450甾醇14α-脱甲基酶（cytochrome P450 sterol 14α-demethylase, CYP51）基因CYP51-1、CYP51-3的表达。本研究还通过分子对接和表面等离子共振试验明确了4种三唑类杀菌剂与Foc TR4中CYP51的互作模式差异，发现虽然丙硫菌唑对Foc TR4的生物活性优于其他3种杀菌剂，但是其与CYP51的亲和力最弱，说明丙硫菌唑与CYP51的作用方式存在特异性。本研究可为防治香蕉枯萎病菌Foc的新型杀菌剂的筛选和合理设计提供一定的理论参考。
关键词： 三唑类杀菌剂;  香蕉枯萎病;  尖孢镰刀菌古巴专化型热带4号生理小种;  化学防治    
Abstract:

The Chinese banana industry is under grave threat by Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc). However, it is still obscure how to control this devastating disease effectively. In this study, the fungicidal activities of four triazole fungicides were tested by hyphae growth rates of Foc tropical race 4 (Foc TR4). The results showed that the fungicidal activities of these four fungicides were prothioconazole＞tebuconazole＞propiconazole＞myclobutanil. The morphology changes and membrane integrity of Foc TR4 in response to these four fungicides were evaluated by morphological observation of hyphae, and detection of relative conductivity and malondialdehyde (MDA) concentrations. Compared with the control group, all these four fungicides can cause abnormalities including increased hyphae branching, irregular distorted, shrivelled, sunken and flattened in hypha cells of Foc TR4. The MDA concentrations and membrane permeability of hypha cells were significantly elevated in all treatment groups. The activities of the cytochrome P450 enzymes were increased significantly so were the expression levels of cytochrome P450 sterol 14α-demethylase (CYP51) genes CYP51-1 and CYP51-3 upon treatment with these fungicides. Molecular docking and surface plasmon resonance (SPR) assays were employed to demonstrate interaction modes of four fungicides with CYP51 of Foc TR4. Even though prothioconazole showed less binding affinity with CYP51 than other three fungicides, it showed the strongest fungicidal activity against Foc TR4, which indicated the specificity of its mode of interaction with CYP51. This study can provide some theoretical references for the screening and rational design of novel fungicides for controlling Fusarium wilt of banana pathogen of Foc.
Key words: triazole fungicides    Fusarium wilt of banana    Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4)    chemical control
收稿日期: 2022-06-15 出版日期: 2022-12-27
CLC:  S 432.1  
基金资助: 广东省基础与应用基础研究基金（区域联合基金-重点项目）(2019B1515120088);广东省现代农业产业技术体系创新团队项目(2022KJ109)
通讯作者: 李春雨     E-mail: xiangdandan@gdaas.cn;lichunyu881@163.com
作者简介: 项丹丹（https://orcid.org/0000-0002-1544-8241），E-mail：xiangdandan@gdaas.cn
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项丹丹
杨晓芳
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陶海青
初元琦
李春雨

引用本文:

项丹丹,杨晓芳,易干军,陶海青,初元琦,李春雨. 4种三唑类杀菌剂对香蕉枯萎病菌的抑制效果及其差异性[J]. 浙江大学学报（农业与生命科学版）, 2022, 48(6): 830-842.

Dandan XIANG,Xiaofang YANG,Ganjun YI,Haiqing TAO,Yuanqi CHU,Chunyu LI. Inhibitory effects of four different kinds of triazole fungicides against Fusarium oxysporum f. sp. cubense and their differences. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(6): 830-842.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2022.06.151    或    https://www.zjujournals.com/agr/CN/Y2022/V48/I6/830

目的基因

Target gene

引物序列（5′→3′）

Primer sequence (5′→3′)

CYP51-1F: CACGGCTTCGTTTTCGTGTT
R: CGGTTCGCAACAGTAGAGGT
CYP51-2F: AAGGGTAGTGGGGAGACAGTT
R: GACCAGGCTTCTCAATGTGGA
CYP51-3F: CTGATTTGCCTCCCCTGACTT
R: GAAGTAGGCCGAGTCAGTAGC
G6PDHF: ATATTCCCCGAAACGAGCTT
R: ATGCTGAGACCAGGCAACTT
表1  实时荧光定量PCR引物序列
图1  不同三唑类杀菌剂对 Foc TR4的抑制效果及菌丝形态A.菌落直径；B.菌丝生长抑制率；C.光学显微镜观察菌丝形态；D.扫描电镜观察菌丝形态。
  
图2  不同三唑类杀菌剂对 Foc TR4菌丝细胞相对电导率的影响A.丙硫菌唑；B.戊唑醇；C.丙环唑；D.腈菌唑。
图3  不同三唑类杀菌剂对 Foc TR4菌丝细胞内丙二醛含量的影响短栅上不同小写字母表示在P＜0.05水平差异有统计学意义，n=3。下同。
图4  不同三唑类杀菌剂对细胞色素P450酶活性和靶标基因 CYP51 相对表达量的影响
图5  Foc TR4中CYP51蛋白质的同源建模A. Foc TR4中CYP51蛋白质和各模板间的序列比对；B. CYP51蛋白质的三维结构及对接盒子。
图6  分子对接预测4种三唑类杀菌剂与CYP51的结合模式A.三维互作示意图；B.二维互作示意图。

配体

Ligand

结合自由能

Binding free energy/(kJ/mol)

结合速率常数

Ka/(L/(mol·s))

解离速率常数

Kd/s－1

亲和动力常数

KD/(mol/L)

丙硫菌唑

Prothioconazole

－33.795.19×1021.49×10－42.87×10－7

戊唑醇

Tebuconazole

－34.921.09×1062.63×10－32.42×10－9

丙环唑

Propiconazole

－36.592.20×1051.73×10－27.86×10－8

腈菌唑

Myclobutanil

－34.081.07×1022.30×10－42.15×10－6
表2  不同三唑类杀菌剂与CYP51的分子对接和亲和动力学参数
图7  不同三唑类杀菌剂与CYP51的结合动力学曲线A.丙硫菌唑；B.戊唑醇；C.丙环唑；D.腈菌唑。
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