利用卡氏白和尼罗红染色观察稻瘟病菌有性世代的结构  

Observation of Sexual Structure of Magnaporthe oryzae via Calcofluor White and Nile Red Staining

作　　者：顾卓侃 李玲[2,3] 王教瑜[2] 柴荣耀[2] 王艳丽[2] 张震[2] 毛雪琴[2] 邱海萍[2] 孙国昌[2] 

机构地区：[1]杭州师范大学生命与环境科学学院,杭州310036 [2]浙江省农业科学院植物保护与微生物研究所,杭州310021 [3]浙江农林大学农业与食品科学学院,浙江临安311300

出　　处：《中国水稻科学》2016年第6期668-672,共5页Chinese Journal of Rice Science

基　　金：国家自然科学基金资助项目(31170136;31470249);浙江省公益技术研究农业项目(2013C32019)

摘　　要：稻瘟病菌(Magnaporthe oryzae)是异宗配合的子囊菌,但至目前,关于其有性世代产生过程和结构的研究相对较少。本研究利用两个稻瘟病菌菌株Guy-11与2539在多种培养基上进行杂交试验,观察有性世代产生情况。结果表明,两菌株在所有参试培养基上杂交后均能产生子囊壳,但子囊壳的数量、产生速度和成熟度各不相同,以燕麦培养基为最佳。为了进一步观察有性世代的结构,我们采用卡氏白和尼罗红对子囊和子囊孢子进行染色和荧光观察。荧光显微镜下,子囊和子囊孢子的细胞壁均能被卡氏白染成清晰的亮蓝色,细胞结构清晰可辨。成熟的子囊壳内可产生大量的子囊,子囊中含有8个子囊孢子,子囊孢子通常含有4个细胞。同时,子囊孢子能够被尼罗红染成橘红色,表明子囊孢子中储藏大量的脂肪类物质。本研究提供了一种有效的观察稻瘟病菌有性世代结构的荧光染色方法,为后续的研究奠定了基础。Magnaporthe oryzae is an economically important heterothallic ascomycete which causes rice blast,the most destructive rice disease worldwide.To the present,sexual generation of the fungus has been relatively less investigated.In the present work,Guy-11 and 2539,two M.oryzae strains in opposite mating types,were cross cultured on six deferent substrates to induce the sexual generation.The strains produced ascocarps on all of the substrates tested,however,the number,forming time and ripeness degree of the ascocarps varied largely with the producing substrates,among which oatmeal agar（OMA）gave the optimum value.In order to better detect the structures of sexual generation,the asci and ascospores were stained with Fluorescent Brightener and Nile red,and examined using a fluorescence microscopy.Under the fluorescence microscope,the cell walls were stained bright blue by Fluorescent Brightener,which greatly improved the visibility of asci and ascospores.Numbers of asci were found in mature ascocarps,with eight ascospores in each ascus,and an ascospore usually has four cells.Meanwhile,the ascospores can be stained bright orangey-red by Nile red,indicating they are rich in lipids.These findings showed that the fluorescence staining is an effective method to observe the structure of sexual generation in M.oryzae.

关 键 词：稻瘟病菌 有性生殖 卡氏白 尼罗红 

分 类 号：S435.111.41[农业科学—农业昆虫与害虫防治]