An Improved Technique for Isolation and Characterization of Single-Spore Isolates of Plasmodiophora brassicae
An Improved Technique for Isolation and Characterization of Single-Spore Isolates of Plasmodiophora brassicae
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DOI：
10.1094/pdis-03-21-0480-re
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发表时间：
2021-12-01
期刊：
影响因子：
4.5
通讯作者：
Feng, Hui
中科院分区：
文献类型：
--
作者：
Lv, Mingcan;Liu, Yifan;Feng, Hui
根瘤病是由油菜恶性疟原虫引起的一种土传病害，在世界范围内广泛发生在十字花科作物上。甘蓝型油菜疫霉通常以多种致病类型混合存在，阻碍了十字花科作物对油菜油菜型油菜疫霉抗性机制的研究。本研究以沈阳中国田间采集的根瘤菌为病原源，通过对接种苗龄、寄主接种方法和植物培养方法的优化，建立了一套高效的甘蓝型油菜根腐病菌单孢子分离体系。对单孢子分离法的操作步骤进行了优化：用2日龄的幼苗接种，用冷冻箱(100×2.0ml)代替培养皿，加入营养液培养，并对单孢子进行显微镜观察。该系统大大提高了侵染成功率，获得了4个致病类型(4、8、9、11)的单孢子分离物。随后，利用优化后的体系对中国地区5个田间采集的甘蓝型油菜单孢菌株进行了分离鉴定。从每个区域分离到大约4到9个致病型。其中，病原菌4型最为常见。这项研究提供了有价值的信息来源，最终可用于宿主P。甘蓝型油菜相互作用。
Clubroot, caused by Plasmodiophora brassicae, is a soilborne disease that occurs in cruciferous crops worldwide. P. brassicae usually exists as a mixture of several pathotypes, which has hampered the research on resistance mechanisms of cruciferous crops against P. brassicae. In this study, clubroot galls were collected from a field in Shenyang, China, as a pathogen source to develop an efficient protocol for a single-spore isolation system of P. brassicae by optimizing the seedling age for inoculation, host inoculation method, and plant culture method. The operational steps of the single-spore isolation method were optimized as follows: the use of 2-day-old seedlings for inoculation, substituting a cryobox (100 x 2.0-ml vials) for culture dishes, the addition of nutrient solution culture, and microscopic observations of single spores. The rate of infection success was substantially improved, and single-spore isolates of four pathotypes (4, 8, 9, and 11) were acquired in this system. Subsequently, the optimized system was used to isolate and characterize the pathotypes of single-spore isolates of P. brassicae collected from five fields in regions in China. Approximately four to nine pathotypes were isolated from each region. Among these, pathotype 4 was the most prevalent. This study provides a source of valuable information that can eventually be used for the genetic analysis of host-P. brassicae interaction.