利用卡氏白和尼罗红染色观察稻瘟病菌有性世代的结构

doi:10.16819/j.1001-7216.2016.6035

中国水稻科学 ›› 2016, Vol. 30 ›› Issue (6): 668-672.DOI: 10.16819/j.1001-7216.2016.6035

• • 上一篇

利用卡氏白和尼罗红染色观察稻瘟病菌有性世代的结构

顾卓侃^1,², 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,^*()

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

收稿日期:2016-03-03 修回日期:2016-07-27 出版日期:2016-11-10 发布日期:2016-11-10
通讯作者: 王教瑜,孙国昌
基金资助:
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)

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

Zhuo-kan GU^1,², Ling LI^2,³, Jiao-yu WANG^2,^*(), Rong-yao CHAI², Yan-li WANG², Zhen ZHANG², Xue-qin MAO², Hai-ping QIU², Guo-chang SUN^2,^*()

¹ School of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036, China
² Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
³ College of Agriculture and Food Science, Zhejiang A&F University, Ling'an 311300, China

Received:2016-03-03 Revised:2016-07-27 Online:2016-11-10 Published:2016-11-10
Contact: Jiao-yu WANG, Guo-chang SUN

摘要/Abstract

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。

关键词: 稻瘟病菌, 有性生殖, 卡氏白, 尼罗红

Abstract:

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.

Key words: Magnaporthe oryzae, sexual generation, Calcofluor White, Nile Red

中图分类号:

S435.111.4⁺1

顾卓侃, 李玲, 王教瑜, 柴荣耀, 王艳丽, 张震, 毛雪琴, 邱海萍, 孙国昌. 利用卡氏白和尼罗红染色观察稻瘟病菌有性世代的结构[J]. 中国水稻科学, 2016, 30(6): 668-672.

Zhuo-kan GU, Ling LI, Jiao-yu WANG, Rong-yao CHAI, Yan-li WANG, Zhen ZHANG, Xue-qin MAO, Hai-ping QIU, Guo-chang SUN. Observation of Sexual Structure of Magnaporthe oryzae via Calcofluor White and Nile Red Staining[J]. Chinese Journal OF Rice Science, 2016, 30(6): 668-672.

图/表 3

图1 各培养基上子囊壳的形成情况 A-燕麦培养基(OMA)、马铃薯葡萄糖培养基(PDA)、淀粉酵母培养基(Starch)、基本培养基(MM)、V8培养基(V8)子囊壳形成情况;M,成熟的子囊壳;U,不成熟的子囊壳;标尺=1000 μm。B-显微镜下成熟子囊壳的形态;标尺=100 μm。C-各培养基上子囊壳的形成数量统计。

Fig. 1. Perithecia formed on different media. A, Perithecia formed on oatmeal agar (OMA), potato dextrose agar (PDA), starch yeast medium (Starch), minimal medium (MM) and V8 medium (V8). M, Mature perithecia; U, Unmatured perithecia. Bars=1000 μm. B, Microscopical analysis of morphology of perithecia. Bars=100 μm. C, Number of total and mature perithecia formed on different media.

图2 子囊与子囊孢子的卡氏白染色观察 A~D-大量游离的子囊,标尺=100 μm; E-一个完整的子囊,标尺=10 μm; H-放大的子囊孢子,标尺=10 μm。

Fig. 2. Structure of perithecia stained with Calcofluor white (Fluorescent Brightener) under a fluorescent microscopy. A-D, Asci migrated from a perithecium, Bars=100 μm; E and F, An intact ascus, bar=10 μm; G and H, Magnified image of an ascospore, bar = 10 μm.

图3 子囊与子囊孢子的尼罗红染色 A~B-标尺=100 μm; C~D-标尺=10 μm。

Fig. 3. Perithecia and ascospores stained with Nile red. A and B, Bar =100 μm; C and D, bar =10 μm.

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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

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