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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）  2019, Vol. 45 Issue (6): 699-706    DOI: 10.3785/j.issn.1008-9209.2018.12.202
植物保护     
柑橘黑斑病菌与柚黑斑病菌对苯并咪唑类杀菌剂的抗性及其分子机制
曾一冰1(),蒋立强1,李国华2,刘蕊2,李红叶1()
1.浙江大学农业与生物技术学院生物技术研究所，杭州 310058
2.广东省梅州市农业科学院果树研究所，广东 梅州 514071
Resistance and its molecular mechanism of Phyllosticta citricarpa and Phyllosticta citriasiana to benzimidazole fungicide
Yibing ZENG1(),Liqiang JIANG1,Guohua LI2,Rui LIU2,Hongye LI1()
1.Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
2.Fruit Research Institute, Meizhou Academy of Agricultural Sciences, Meizhou 514071, Guangdong, China
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摘要：

为了对柑橘黑斑病和柚黑斑病提出更科学的化学防治方法，该文研究了柑橘黑斑病菌和柚黑斑病菌对杀菌剂多菌灵的抗性频率、抗性水平及其抗性分子机制。结果表明：在江西114个柑橘黑斑病菌株中发现了2个多菌灵抗性菌株，抗性频率为1.75%，抗性系数分别为5 333.6和379.2；在四川32个柑橘黑斑病菌株中发现了1个多菌灵抗性菌株，抗性频率为3.13%，抗性系数为303.3；在浙江23个菌株和重庆3个菌株中没有出现抗性菌株。在广东收集的54个柚黑斑病菌株中发现了1个抗性菌株，抗性频率为1.85%，抗性系数为13 719.5；在广西和福建分别收集的34个柚黑斑病菌株中没有发现抗性菌株。柑橘黑斑病菌和柚黑斑病菌对甲基硫菌灵和多菌灵存在正交互抗性，对嘧菌酯和多菌灵无交互抗性。因此，建议多菌灵可继续在柑橘黑斑病和柚黑斑病防治中使用，鉴于高抗菌株已出现，使用时应与其他作用机制的杀菌剂轮换使用。本实验还发现，柑橘黑斑病菌存在2种抗苯并咪唑类的分子机制，即β-微管蛋白基因上第198位氨基酸位点由谷氨酸突变为赖氨酸（抗性系数为5 333.6的菌株）和第200位氨基酸位点由苯丙氨酸突变为酪氨酸（抗性系数为379.2的菌株）；而柚黑斑病菌抗苯并咪唑类的分子机制则与β-微管蛋白基因第198位氨基酸由谷氨酸突变为丙氨酸有关。
关键词： 柑橘黑斑病菌;  柚黑斑病菌;  苯并咪唑类杀菌剂;  抗药性;  分子机制    
Abstract:

In order to control citrus black spot (CBS, Phyllosticta citricarpa) and pummelo black spot (PBS, Phyllosticta citriasiana) effectively, we evaluated the resistance frequency, resistance level and their molecular mechanisms of these two pathogen populations collected from seven provinces of China to the fungicide carbendazim. The results showed that two carbendazim-resistant (Cab-R) isolates were found from the Jiangxi subpopulation of P. citricarpa out of 114 strains. The resistant frequency was 1.75%, and their coefficient of resistance were 5 333.6 and 379.2, respectively. One Cab-R strain with coefficient of resistance of 303.3 was found in the Sichuan subpopulation of P. citricarpa out of 32 strains, and the resistant frequency was 3.13%. However, none of the Cab-R isolates was detected from the Zhejiang (23 strains) and the Chongqing (3 strains) subpopulations. For P. citriasiana, one Cab-R strain, whose coefficient of resistance was 13 719.5, was obtained in the Guangdong subpopulation (54 strains in total), and the resistant frequency was 1.85%. None of the Cab-R strains was found in the Guangxi (34 strains) and the Fujian (34 strains) subpopulations. In addition, there was a positive cross-resistance between thiophanate-methyl and carbendazimin P. citricarpa and P. citriasiana, while there was no cross-resistance between azoxystrobin and carbendazim. Based on the results obtained, we suggest that the fungicide carbendazim can still be used in controlling CBS and PBS in the fields, but the rotation of the fungicides with different antifungal mechanisms is necessary given the existence of high-resistant strains. Finally, the two molecular mechanisms of carbendazim-resistance of P. citricarpa were found. One was a mutation at the amino acid (aa) 198 of β-tubulin gene, in which the glutamic acid changed to lysine (the strain with a coefficient of resistance of 5 333.6), and the other one was a mutation at the aa 200 of β-tubulin gene, in which the phenylalanine changed tyrosine (the strain with a coefficient of resistance of 379.2). However, the molecular mechanism of the carbendazim-resistance of P. citriasiana was a mutation at the aa 198 in the β-tubulin gene, in which the glutamic acid changed to alanine.
Key words: Phyllosticta citricarpa    Phyllosticta citriasiana    benzimidazole fungicide    fungicide resistance    molecular mechanism
收稿日期: 2018-12-20 出版日期: 2020-01-20
CLC:  S 436.66  
基金资助: 国家重点研发计划“柑橘化肥农药减施技术集成研究与示范”(2017YFD0202000);国家现代农业（柑橘）产业技术体系专项(CARS-26)
通讯作者: 李红叶     E-mail: 923400848@qq.com;hyli@zju.edu.cn
作者简介: 曾一冰（https://orcid.org/0000-0001-8671-0342），E-mail：923400848@qq.com
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引用本文:

曾一冰,蒋立强,李国华,刘蕊,李红叶. 柑橘黑斑病菌与柚黑斑病菌对苯并咪唑类杀菌剂的抗性及其分子机制[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(6): 699-706.

Yibing ZENG,Liqiang JIANG,Guohua LI,Rui LIU,Hongye LI. Resistance and its molecular mechanism of Phyllosticta citricarpa and Phyllosticta citriasiana to benzimidazole fungicide. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(6): 699-706.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.12.202    或    http://www.zjujournals.com/agr/CN/Y2019/V45/I6/699

引物名称 Primer name引物序列 Primer sequence扩增片段长度 Length of amplified product/bp
PA-bT-F5′-CCCGACAACTTCGTCTTTGG-3′711
PA-bT-R5′-CCTCGACCTCCTTCATCGAG-3′
表1  扩增柑橘黑斑病菌β-tubulin基因部分序列的引物信息

来源

Source

敏感菌株1）

Cab-S isolates1)

抗性菌株2）

Cab-R isolates2)

抗性频率

Resistant frequency/%

抗性系数

Coefficient of resistance

总计 Total17231.74高抗
江西 Jiangxi11421.75379.2, 5 333.6
四川 Sichuan3213.13303.3
浙江 Zhejiang2300
重庆 Chongqing300
表2  柑橘黑斑病菌种群对多菌灵抗性水平评价
图1  柑橘和柚黑斑病菌部分β-微管蛋白基因扩增结果M：DNA分子质量标志物；CK：对照（双蒸水）。
图 2  柑橘黑斑病菌β-微管蛋白基因部分氨基酸序列比对
图 3  柑橘黑斑病菌β-微管蛋白基因部分核苷酸序列比对

来源

Source

敏感菌株1）

Cab-S isolates1)

抗性菌株2）

Cab-R isolates2)

抗性频率

Resistance frequency/%

抗性系数

Coefficient of resistance

总计 Total12210.82高抗
广东 Guangdong5411.8513 719.5
广西 Guangxi3400
福建 Fujian3400
表3  柚黑斑病菌种群对多菌灵抗性水平评价
图 4  柚黑斑病菌β-微管蛋白基因部分氨基酸序列比对
图 5  柚黑斑病菌β-微管蛋白基因部分核苷酸序列比对
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