拟轮枝镰孢菌染色方法与荧光菌株构建  

Staining Method and Fluorescent Strain Construction of Fusarium verticillioides

作　　者：孔铭 朱金洁 祁显涛 谢传晓[1] 刘昌林[1] KONG Ming;ZHU Jinjie;QI Xiantao;XIE Chuanxiao;LIU Changlin(Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081)

机构地区：[1]中国农业科学院作物科学研究所,北京100081

出　　处：《华北农学报》2024年第S1期252-258,共7页Acta Agriculturae Boreali-Sinica

基　　金：科技创新2030—重大项目(2022ZD040200102)。

摘　　要：拟轮枝镰孢菌显色方法的建立可以明确其在玉米植株中的侵染途径,进一步确定其致病作用机制。以拟轮枝镰孢菌为研究对象,探讨FDA、台盼蓝、甲苯胺蓝、酸性品红、碱性品红、FM4-64、转红色荧光载体等显色方法对拟轮枝镰孢菌真菌孢子和接种拟轮枝镰孢菌的玉米幼苗根系的染色效果,建立一种拟轮枝镰孢菌显色示踪法。结果发现,5%碱性品红和1%甲苯胺蓝对拟轮枝镰孢菌孢子染色效果较好,颜色明显、结构清晰,5.00 g/L FDA、0.4%台盼蓝、0.50 g/L酸性品红染色剂进行染色后,拟轮枝镰孢菌孢子能染上极浅的颜色,很难观察到孢子形态;对玉米幼苗根系染色剂染色对于根腐的观察,发现0.4%台盼蓝、1%甲苯胺蓝染色后玉米的根系会产生蓝色和棕色,可以明显看出棕色为玉米被拟轮枝镰孢菌侵染后发生根腐的部位,而0.50 g/L酸性品红和5%碱性品红染色过的则不能明显看出发生根腐的部位;对拟轮枝镰孢菌孢子的红色荧光显色观察发现,FM4-64颜色较深、结构不清晰、对观察时间有限制,而转红色荧光载体后荧光颜色明显、结构清晰、对观察时间无要求,后期可用于拟轮枝镰孢菌引起玉米、小麦等穗(粒)腐病的致病机理研究。综合比较之后,转红色荧光载体的拟轮枝镰孢菌比其他染色方法在致病机理研究等方面更具优势。The establishment of a coloration method for Fusarium verticillioides can clarify its infection pathway in maize plants and further determine its pathogenic mechanism.This study took Fusarium verticillioides as the re-search object to explore the staining effects of FDA,trypan blue,toluidine blue,acid fuchsin,basic fuchsin,FM4-64,and red fluorescence vector on fungal spores of F.verticillioides and the root system of maize seedlings inoculated with F.verticillioides.A coloration method for F.verticillioides was established.The results showed that 5%basic fuchsin and 1%toluidine blue had good staining effects on F.verticillioides spores,with clear color and clear struc-ture.After staining with 5.00 g/L FDA,0.4%trypan blue,and 0.50 g/L acid fuchsin,the F.verticillioides spores could be stained with a very light color,making it difficult to observe the spore morphology.Staining the root system of maize seedlings with stains showed that 0.4%trypan blue and 1%toluidine blue stained the root system of maize with blue and brown colors,allowing clear observation of the brown area as the site of root rot caused by F.verticil-lioides infection in maize.However,staining with 0.50 g/L acid fuchsin and 5%basic fuchsin did not allow clear observation of the site of root rot.Observation of the red fluorescence coloration of F.verticillioides spores showed that FM4-64 had a deeper color and unclear structure,limiting observation time.However,after being transferred to a red fluorescence vector,the fluorescence color was obviousr and the structure was clear,without any time limit for observation.This method can be used in future studies on the pathogenic mechanism of F.verticillioides causing ker-nel rot in maize and wheat.After comprehensive comparison,the F.verticillioides transferred with a red fluorescence vecror had more advantages than other staining methods in terms of research on pathogenic mechanisms.

关 键 词：玉米穗腐病 拟轮枝镰孢菌 荧光载体 接种 染色 

分 类 号：S432.4*4[农业科学—植物病理学] S513[农业科学—农业昆虫与害虫防治]