甘蔗根系内生真菌染色方法探讨  

Method to Stain Endophytic Fungi in Sugarcane Roots

作　　者：廖楠[1,2] 张金莲[2] 李冬萍[2] 汪茜[2] 龙艳艳[2] 谭裕模[3] 李松[4] 陆祖军[1] 陈廷速[2] 

机构地区：[1]广西师范大学生命科学院,广西桂林541004 [2]广西农业科学院微生物研究所,广西南宁530007 [3]广西农业科学院农业资源与环境研究所,广西南宁530007 [4]广西农业科学院甘蔗研究所,广西南宁530007

出　　处：《西南农业学报》2016年第4期814-819,共6页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31360356);国家甘蔗产业技术体系项目(CARS-20-3-5);广西农业科学院重点项目和学科团队项目(2014YP06;2015YT81;2015YZ10)

摘　　要：为了建立一种甘蔗根系内生真菌最佳的染色方法,便于更好观察甘蔗根际土壤丛枝菌根（Arbuscular mycorrhiza,AM）真菌和深色有隔内生真菌（Dark septate endophytes,DSE）对甘蔗根系的侵染情况。将甘蔗根样置于20%的KOH溶液中90℃水浴透明90~120 min;接着加入碱性H2O2脱色60 min;5%的乙酸酸化5 min后,用5%的墨水醋染液（Quink牌纯黑墨水、北京牌蓝黑墨水）、酸性品红、苏丹红Ⅳ、台酚蓝、苯胺蓝为染液,在66℃水浴染色30 min,后用清水浸泡（12 h）脱色后即可镜检。经过碱性H2O2脱色60 min后Quink牌纯黑墨水染色的根系,AM真菌的菌丝和泡囊以及DSE微菌核能够清晰可见。此实验建立的墨水醋染法安全性高而且操作简便,成本低廉。相对于其他染色方法毒性低,对环境的污染小,而且染色效果稳定,更适于观察和镜检拍照。An experiment was conducted to establish a method for staining endophytic fungi in sugarcane roots to observe arbuscular mycorrhizal（AM） fungi and dark septate endophyte（DSE） colonizations. Sugarcane roots were treated with 20 % KOH solution for 90 to 120 min in water bath at 90 ℃,and then the roots were destained by immersing in alkaline H2O2 for 60 min. After acidified in 5 % acetic acid for5 min,the roots were dyed with five stains i. e. vinegar with 5 % ink dye（Black ink of Quink brand,and Dark blue of Beijing brand）,acid fuchsin,Sudan Red Ⅳ,trypan blue,and aniline blue in water bath for 30 min at 66 ℃. Sugarcane roots were cleared by immersing in tap water for 12 h. After clearing with alkaline H2O2 solution for 60 min,the structure of both of hyphae and vesicle of AM fungi and microsclerotia of DSE stained with ink dye（Black ink,Quink brand） could be clearly visible. The ink-vinegar solution staining provided a simple,low toxic and inexpensive technique for staining endophytic fungi in sugarcane roots with excellent staining results,and it is beneficial for observation and photograph,with little pollution to the environment.

关 键 词：甘蔗根系 内生真菌 丛枝菌根真菌 深色有隔内生真菌 染色 

分 类 号：S154.38[农业科学—土壤学]