大丽轮枝菌致病性相关突变体快速筛选体系的建立

doi:10.3864/j.issn.0578-1752.2015.14.006

摘要/Abstract

摘要： 【目的】建立适合于大丽轮枝菌（Verticillium dahliae）致病性相关突变体的快速筛选体系，为突变库中与致病性相关突变体的系统筛选和鉴定提供技术支撑。【方法】棉花幼苗接种于5.0×10⁴、5.0×10⁵、5.0×10⁶、5.0×10⁷和5.0×10⁸孢子/mL的孢子悬浮液30 min，通过病情指数调查明确引起棉花黄萎病发生的病原菌浓度范围；对培养于培养瓶的大丽轮枝菌分别加入15、25、35和45 mL的灭菌水，利用血球计数板检测洗脱的孢子浓度，明确不同体积灭菌水对洗脱孢子悬浮液浓度的影响；以保存于96孔板的突变体为单位，在培养皿上进行单孢分离并挑取单个孢子于培养瓶中扩繁培养，培养后于培养瓶中直接加入适宜体积灭菌水洗脱孢子，并将棉花幼苗直接置于含有孢子悬浮液的培养瓶中处理30 min，接种后继续培养14 d并调查结果；致病性相关突变体的可靠性检验采用定量菌液蘸根接种法，每个突变体接种30株棉花幼苗，3个重复，孢子浓度为5.0×10⁶孢子/mL，每株棉花幼苗按接种5 mL菌液计算，处理30 min，分别在第5、8、11和14天调查病情指数。【结果】明确了适合于大丽轮枝菌致病力快速鉴定的接种孢子浓度为＞5.0×10⁵孢子/mL；建立了大丽轮枝菌培养方法，单孢纯化培养5 d，培养瓶中扩大培养9 d，确定了快速定量制备孢子悬浮液的洗脱体积为25 mL，测试的20个突变体的洗脱孢子浓度范围在（2.55±0.58）×10⁶—（1.72±0.25）×10⁷孢子/mL；优化了单孢分离、扩大培养、孢子悬浮液制备、接种、继续培养和结果统计等环节，建立了大丽轮枝菌致病性快速鉴定流程，进一步统筹设计构建了大丽轮枝菌致病性相关突变体快速筛选体系；测试表明该体系1人1个循环共7个流程可完成1 344个突变体筛选，周期54 d，工作量为21人日；采用定量菌液蘸根法重复验证结果，突变体致病力同样显著下降，与快速筛选体系的鉴定结果一致，表明该体系适用于大丽轮枝菌致病性相关突变体的快速筛选。【结论】通过棉花幼苗种植、突变体单孢分离、扩繁培养、孢子悬浮液制备、接种、结果统计等环节的优化和标准化，构建了适合于大丽轮枝菌致病性相关突变体的快速筛选体系，为后续致病相关基因的分离提供了技术支撑。

关键词: 大丽轮枝菌, 致病性相关突变体, 致病性鉴定, 快速筛选体系

Abstract: 【Objective】 The objective of this study is to construct a rapid screening system of pathogenicity-related mutants in Verticillium dahliae, which will provide a technical support to identification of T-DNA mutant library.【Method】The cotton roots were inoculated with V. dahliae conidial suspension by root-dipping method for 30 min, including five concentrationsof 5.0×10⁴, 5.0×10⁵, 5.0×10⁶, 5.0×10⁷and 5.0×10⁸conidia/mL, to determine the suitable concentration range of causing the obvious Verticillium wilt. The culture flasks were used for the V. dahliae culture, and 15, 25, 35 and 45 mL of sterilized water were added into the flasks to elute the conidial. The concentration of conidial was calculated by the hemocytometer to detect the effect of sterilized water volume on the conidial concentration. Based on the unit of mutants in 96-well plates, the single spore of mutants was isolated on the petri dish and expanding cultured in the flasks, then the sterilized water at suitable volume was directly added into the flasks to elute the conidial and the cotton seedling roots were dipped into the conidial suspension in the flasks for 30 min, subsequently the infected cotton seedlings were cultured for 14 d and the disease index was investigated. A rapid screening system of pathogenicity-related mutantsin V. dahliae was constructed after optimization of cotton planting, mutants single spore isolation, expanding culture, inoculation, and results investigation. The quantitative inoculation method was used for validation of the reliability results of pathogenicity-related mutants, 30 cotton seedlings with three replicates for each mutant, the concentration of suspension was 5.0×10⁶conidia/mL, 5 mL suspension for each seedling, and inoculated for 30 min, finally the disease indexes of pathogenicity-related mutants were investigated on 5, 8, 11 and 14 d after inoculation.【Result】The conidial concentration up to 5.0×10⁵conidia/mL could obviously infect the cotton and cause the Verticillium wilt symptom. A standard culture method of V. dahliae was constructed which includes single spore isolation for 5 days and expanding culture in standard culture flasks for 9 days. The appropriate volume of sterilized water used for eluting the conidial suspension was 25 mL. The test results showed that the concentration for eluting the conidial suspension of 20 mutants were（2.55±0.58)×10⁶-(1.72±0.25)×10⁷conidia/mL. Subsequently, the inoculation steps were optimized for construction of the rapid process of V. dahliae pathogenicity identification, which includes single spore isolation, expanding culture, conidial suspension preparation, inoculation, and results investigation. Moreover, a rapid screening system of pathogenicity-related mutants in V. dahliae was constructed after coordinative design. The test results showed that one people could finish 1 334 mutants within 54 days by running one cycle which includes seven processes, and the work load is only 21 man-days. The validation of target mutants by quantitative inoculation method showed that the pathogenicity of these mutants was significantly reduced, consistent with the results of the rapid screening system, which indicated that the rapid screening system is appropriate for the screening of pathogenicity-related mutants in V. dahliae.【Conclusion】A rapid screening system ofpathogenicity-related mutants in V. dahliae was constructed after optimization and standardization of cotton planting, mutants single spore isolation, expanding culture, preparation of the conidial suspension, inoculation, and results investigation, which will provide a technical support for the pathogenicity-related gene research in future.

Key words: Verticillium dahliae, pathogenicity-related mutant, pathogenic identification, rapid screening system

王新艳，张丹丹，桂月晶，李楠洋，徐明，陈捷胤，戴小枫. 大丽轮枝菌致病性相关突变体快速筛选体系的建立[J]. 中国农业科学, 2015, 48(14): 2747-2756.

WANG Xin-yan, ZHANG Dan-dan, GUI Yue-jing, LI Nan-yang, XU Ming, CHEN Jie-yin, DAI Xiao-feng. Construction of a Rapid Screening System of Pathogenicity-Related Mutants in Verticillium dahliae[J]. Scientia Agricultura Sinica, 2015, 48(14): 2747-2756.

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