一种快速、简易、可靠的培养皿内青枯菌接种方法

doi:10.1016/j.jia.2023.05.020

Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (12): 3709-3719.DOI: 10.1016/j.jia.2023.05.020

一种快速、简易、可靠的培养皿内青枯菌接种方法

收稿日期:2023-02-01 接受日期:2023-04-10 出版日期:2023-12-20 发布日期:2023-12-08

Seedling Petri-dish inoculation method: A robust, easy-to-use and reliable assay for studying plant–Ralstonia solanacearum interactions

CAO Peng^1*, CHEN Jia-lan^2*, LI Ning-ning^2*, ZHANG Shuang-xi¹, WANG Rong-bo³, LI Ben-jin³, LIU Pei-qing³, AN Yu-yan^1#, ZHANG Mei-xiang^1#

¹ National Engineering Laboratory for Endangered Medicinal Resource Development in Northwest China/Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry of Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi’an 710119, P.R.China
² Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, P.R.China
³ Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Fuzhou 350013, P.R.China

Received:2023-02-01 Accepted:2023-04-10 Online:2023-12-20 Published:2023-12-08
About author:CAO Peng, E-mail: chaopenn@snnu.edu.cn; CHEN Jia-lan, E-mail: chenjialan05@foxmail.com; LI Ning-ning, E-mail: lnn1047541675@163.com; #Correspondence AN Yu-yan, E-mail: anyuyan@snnu.edu.cn; ZHANG Mei-xiang, E-mail: meixiangzhang@snnu.edu.cn * These authors contributed equally to this study.
Supported by:
This work was supported by the National Natural Science Foundation of China (32072399 and 32272641), the Fundamental Research Funds for the Central Universities (GK202201017 and GK202207024), and the Program of Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, China (MIMCP-202203).

摘要/Abstract

摘要：

茄科劳尔氏菌，简称青枯菌(Ralstonia solanacearum)，是一种分布广泛的土传细菌病原菌，该病原菌从根部侵染植物，最终引发青枯病(Bacterial wilt disease)。青枯菌寄主范围广泛，可在200多种植物包括马铃薯、番茄、辣椒、烟草、花生等重要作物上引起毁灭性的植物病害，被列为世界上第二大细菌性植物病原菌，给全世界的农作物生产带来巨大威胁。研究并解析青枯菌与植物互作的分子机制可为青枯病防控策略的建立奠定基础。然而，目前科研中主要采用的青枯菌土壤灌根法接种方法存在实验周期长、所需植物培养空间大、且条件不容易控制等问题，限制了青枯菌与植物互作研究的开展。因此，亟需一种快速、操作简易、且结果稳定的青枯菌接种方法。为此，本文建立了一种培养皿内青枯菌接种方法(Petri-dish inoculation method)，该方法具备接种后发病稳定、操作简易和实验周期短等特点，可广泛用于青枯菌与植物互作研究。为了证明该方法在青枯菌与植物互作研究中的有效性，本文利用引发植物防御作用的分子模式Pep1，拟南芥感病突变体npr1，以及具有不同毒力水平的青枯菌突变体ΔhrcV和ΔRipAC，研究了青枯菌皿内接种方法在评估植物抗病性和青枯菌致病性中的适用性和高效性。同时，我们利用皿内接种方法再现了前人对4个烟草品种的抗性评估结果，表明该方法可用于不同植物种质材料的抗性评估和筛选。鉴于此方法具有快速、操作简单、节省空间，人力和财力，以及所需接种量少的优点，该方法可用于快速大规模青枯菌突变体致病性分析以及植物对青枯菌抗性种质材料的筛选和鉴定。青枯菌皿内接种方法也可为其他土传病原菌的接种提供参考。

Abstract:

Ralstonia solanacearum causes a lethal bacterial wilt disease in many crops, leading to huge losses in crop production every year. Understanding of plant–R. solanacearum interactions will aid to develop efficient strategies to control the disease. As a soilborne pathogen, R. solanacearum naturally infects plants via roots. A huge limitation in studying plant–R. solanacearum interactions is the large variation of R. solanacearum infection assay due to the variable soil conditions and uneven inoculum exposure. Here, we developed a robust and reliable Petri-dish inoculation method which allows consistent and stable infection in young plant seedlings. This method is easy to use, takes about only 10 days from seed germination to the completion of inoculation assay, and requires less inoculum of bacteria as well as growth chamber space. We proved the efficacy of the seedling Petri-dish inoculation method by analyzing plant defense primed by molecular patterns, resistance of defense-related plant mutants, and virulence of R. solanacearum mutants. Furthermore, we demonstrated that the seedling Petri-dish inoculation method can be applied to other host plants such as tobacco and has great potential for high-throughput screening of resistant plant germplasms to bacterial wilt in the future.

Key words: bacterial wilt , Ralstonia solanacearum , Petri-dish inoculation method , virulence , resistance , defense priming

CAO Peng, CHEN Jia-lan, LI Ning-ning, ZHANG Shuang-xi, WANG Rong-bo, LI Ben-jin, LIU Pei-qing, AN Yu-yan, ZHANG Mei-xiang. 一种快速、简易、可靠的培养皿内青枯菌接种方法[J]. Journal of Integrative Agriculture, 2023, 22(12): 3709-3719.

CAO Peng, CHEN Jia-lan, LI Ning-ning, ZHANG Shuang-xi, WANG Rong-bo, LI Ben-jin, LIU Pei-qing, AN Yu-yan, ZHANG Mei-xiang. Seedling Petri-dish inoculation method: A robust, easy-to-use and reliable assay for studying plant–Ralstonia solanacearum interactions[J]. Journal of Integrative Agriculture, 2023, 22(12): 3709-3719.

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一种快速、简易、可靠的培养皿内青枯菌接种方法

Seedling Petri-dish inoculation method: A robust, easy-to-use and reliable assay for studying plant–Ralstonia solanacearum interactions

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