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浙江大学学报(农业与生命科学版)  2017, Vol. 43 Issue (5): 589-598    DOI: 10.3785/j.issn.1008-9209.2017.02.201
植物保护     
以防治油菜菌核病为目标的核盘菌乙酰乳酸合酶抑制剂的筛选
温雪玮,陈泱泱,武斌,胡东维,梁五生*
浙江大学农业与生物技术学院生物技术研究所/农业部作物病虫分子生物学重点实验室,杭州 310058
Screening of acetolactate synthase inhibitors against Sclerotinia sclerotiorum for the purpose of controlling rapeseed Sclerotinia disease
WEN Xuewei, CHEN Yangyang, WU Bin, HU Dongwei, LIANG Wusheng*
(Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects/Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China)
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摘要:

为开发新的油菜菌核病防治药剂,选取7种乙酰乳酸合酶(acetolactate synthase, ALS)潜在抑制剂(氯嘧磺隆、苄嘧磺隆、氯磺隆、甲嘧磺隆、咪唑乙烟酸、灭草喹和磺胺噻唑)作为3类ALS酶抑制剂(磺酰脲类、咪唑啉酮类、磺胺噻唑类)的代表,对其防治油菜菌核病的性能进行测试。结果显示,1.0 mg/L灭草喹、磺胺噻唑、氯嘧磺隆、苄嘧磺隆和氯磺隆对核盘菌ALS酶活性有较强的抑制作用,而相同剂量的甲嘧磺隆的抑制作用微弱,咪唑乙烟酸无抑制作用。相应地,半最大效应浓度(half-maximal effective concentration,EC50)值显示,核盘菌的菌丝生长对灭草喹、磺胺噻唑、氯嘧磺隆、苄嘧磺隆和氯磺隆的抑制作用较敏感,而对甲嘧磺隆和咪唑乙烟酸的敏感性较弱。氯嘧磺隆、苄嘧磺隆、氯磺隆、灭草喹和甲嘧磺隆对油菜叶片ALS酶活性有强烈的抑制作用,咪唑乙烟酸和磺胺噻唑的抑制作用明显比上述5种抑制剂弱。氯嘧磺隆、苄嘧磺隆、氯磺隆、灭草喹、甲嘧磺隆和咪唑乙烟酸处理对油菜幼苗生长发育有明显的负面影响,主要表现为生长速率下降,植株明显比对照矮小,叶片变小,叶色变黄。磺胺噻唑处理对油菜幼苗生长发育的负面影响明显比上述6种抑制剂弱。选择对核盘菌ALS酶活性和菌落生长的抑制作用都较强、而对油菜ALS酶活性的抑制作用和对油菜幼苗生长的负面影响最弱的磺胺噻唑进行抗核盘菌侵染试验。结果显示,磺胺噻唑处理显著地降低了核盘菌对油菜叶片的侵染程度,对油菜菌核病产生了较明显的防治效果。上述研究结果表明,磺胺噻唑可作为分子结构基础来进一步开发以核盘菌ALS酶为作用靶标的油菜菌核病防治剂。

Abstract:

Rapeseed is an oil crop with quite large growing areas in China, and also one of the main oil crops in the world. As one of the most serious diseases in rapeseed, Sclerotinia disease, caused by the fungal pathogen Sclerotinia sclerotiorum, can result in significant yield loss. At present, no rape cultivars are available that are resistant to S. sclerotiorum. Using fungicides is the main measure to control rapeseed Sclerotinia disease in China. Carbendazim, as a common fungicide, has been frequently applied to control rapeseed Sclerotinia disease for long. Increasing evidence of fungicide resistance in
populations of S. sclerotiorum has been found. Therefore, new fungicides are constantly being requested to control rapeseed Sclerotinia disease. Acetolactate synthase (ALS) is the enzyme that catalyzes the first step in the branched-chain amino acid biosynthesis pathway in plants and microbes, and is the target of some herbicides. Recently, some studies have reported the development of new antibiotics to control bacterial pathogens or new fungicides to control fungal pathogens of human beings with ALS as the action target. However, there are few studies on developing new fungicides to control fungal
pathogens of plants with ALS as the action target.
In the present study, seven potential ALS inhibitors (chlorimuron-ethyl, bensulfuron-methyl, chlorsulfuron, sulfometuronmethyl, imazethapyr, imazaquin and sulfathiazole) were used to determine the effectiveness of controlling rapeseed Sclerotinia disease. Chlorimuron-ethyl, bensulfuron-methyl, chlorsulfuron and sulfometuron-methyl were chosen as the representatives of sulfonylurea-type ALS inhibitors. Imazethapyr and imazaquin were the representatives of imidazolinone-type ALS inhibitors, and sulfathiazole was the representative of sulfathiazole-type ALS inhibitors.
The results indicated that the activity of ALS in S. sclerotiorum could not been inhibited by 1.0 mg/L imazethapyr. In contrast, the treatment with 1.0 mg/L chlorimuron-ethyl, bensulfuron-methyl, chlorsulfuron, sulfometuron-methyl, imazaquin and sulfathiazole showed more or less inhibitory effects on the ALS activity of S. sclerotiorum. Among them, imazaquin had the strongest inhibitory effect on ALS, but sulfometuron-methyl showed the weakest inhibitory effect. Imazaquin and sulfathiazole could effectively suppress the growth of S. sclerotiorum colony on potato dextrose agar plates at the concentration of 0.1 mg/L.
Chlorimuron-ethyl, bensulfuron-methyl and chlorsulfuron had inhibitory effect at the concentration of 1.0 mg/L. However, sulfometuron-methyl and imazethapyr showed weak inhibitory effect on the growth of S. sclerotiorum colony even at the concentration of 50 mg/L. The half-maximal effective concentrations (EC50) of imazaquin, sulfathiazole, chlorimuron-ethyl, bensulfuron-methyl and chlorsulfuron were determined to be less than 10 mg/L, but EC50 values of sulfometuron-methyl and imazethapyr were determined to be more than 50 mg/L. All the seven potential ALS inhibitors showed inhibitory effects on the activity of ALS in rapeseed leaves at the concentration of 1.0 mg/L. The inhibitory effects of chlorimuron-ethyl, bensulfuronmethyl, chlorsulfuron, imazaquin and sulfometuron-methyl were much stronger than those of imazethapyr and sulfathiazole. The inhibitory effect of sulfathiazole was the weakest among the seven inhibitors. Significant damaging influences were observed on rapeseed seedlings by using chlorimuron-ethyl, bensulfuron-methyl, chlorsulfuron, imazaquin, sulfometuron-methyl and imazethapyr at the concentration of 1.0 mg/L, such as decreasing growth rate, dwarfing, reducing leaf size and leaf yellowing, but no significant influences were observed by treating rapeseed seedlings with sulfathiazole. The use of sulfathiazole could exert remarkable inhibitory effects against ALS activity and colony growth of S. sclerotiorum without giving rise to harmful influence on rapeseed seedlings. Thus, the effect of sulfathiazole on S. sclerotiorum infection to rapeseed was tested.  The result indicated that the treatment with sulfathiazole significantly reduced the infection scale of S. sclerotiorum in rapeseed leaves.
In conclusion, the present research indicated that S. sclerotiorum was sensitive to imazaquin, sulfathiazole, chlorimuron-ethyl, bensulfuron-methyl and chlorsulfuron other than sulfometuron-methyl and imazethapyr. On the other hand, rapeseed was sensitive to all the seven ALS inhibitors but imazethapyr and sulfathiazole. It is suggested that sulfathiazole treatment is an effective strategy to control rapeseed Sclerotinia disease caused by S. sclerotiorum. Therefore, this study concludes that sulfathiazole is a potential molecular structure basis for controlling rapeseed Sclerotinia disease with ALS of S. sclerotiorum as an action target.

收稿日期: 2017-02-20 出版日期: 2017-04-28
CLC:  S 432.44  
基金资助: 国家自然科学基金(31370279);浙江省自然科学基金(LY13C140001);浙江省教育厅科研计划项目(Y201327327)
通讯作者: 梁五生(http://orcid.org/0000-0001-9360-1187)     E-mail: liangws@zju.edu.cn
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引用本文:

温雪玮,陈泱泱,武斌,胡东维,梁五生. 以防治油菜菌核病为目标的核盘菌乙酰乳酸合酶抑制剂的筛选[J]. 浙江大学学报(农业与生命科学版), 2017, 43(5): 589-598.

WEN Xuewei, CHEN Yangyang, WU Bin, HU Dongwei, LIANG Wusheng. Screening of acetolactate synthase inhibitors against Sclerotinia sclerotiorum for the purpose of controlling rapeseed Sclerotinia disease. Journal of Zhejiang University (Agriculture and Life Sciences), 2017, 43(5): 589-598.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2017.02.201        http://www.zjujournals.com/agr/CN/Y2017/V43/I5/589

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