马铃薯晚疫病菌侵染的Solophenyl Flavine荧光染色方法研究

刘甜甜; 周倩; 吴秋云; 熊兴耀

doi:10.11913/PSJ.2095-0837.2016.20316

马铃薯晚疫病菌侵染的Solophenyl Flavine荧光染色方法研究

刘甜甜¹,
周倩^1, ,,
吴秋云²,
熊兴耀^3,4

1. 植物病虫害生物学与防控湖南省重点实验室, 湖南农业大学植物保护学院, 长沙 410128;
2. 湖南省马铃薯工程技术中心, 湖南农业大学园艺园林学院, 长沙 410128;
3. 中国农业科学院蔬菜花卉研究所, 北京 100081;
4. 湖南农业大学作物种质创新与资源利用国家重点实验室培育基地, 长沙 410128

基金项目:

国家自然科学基金项目(31201502)

高校博士学科点专项科研基金项目(20124320120015)

详细信息

作者简介:
刘甜甜(1991-),女,硕士研究生,研究方向为植物病理学(E-mail:872254877@qq.com)。

通讯作者:
周倩.E-mail:zhouqian2617@hunau.edu.cn

中图分类号: Q945.8
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- 文章访问数: 1559
- HTML全文浏览量: 5
- PDF下载量: 1474
出版历程
- 收稿日期: 2015-12-02
- 修回日期: 2015-12-24
- 网络出版日期: 2022-10-31
- 发布日期: 2016-04-27

Fluorescent Staining with Solophenyl Flavine for Infection Observation in Phytophthora infestans

LIU Tian-Tian¹,

ZHOU Qian^1, ,,

WU Qiu-Yun²,

XIONG Xing-Yao^3,4

1. Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Insect Pests, College of Plant Protection, Hunan Agricultural University, Changsha 410128, China;

2. Hunan Provincial Engineering Research Center of Potatoes, Horticulture and Landscape College, Hunan Agricultural University, Changsha 410128, China;

3. Institute of Vegetable and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China;

4. Pre-State Key Laboratory for Germplasm Innovation and Resource Utilization of Crops, Hunan Agricultural University, Changsha 410128, China

Funds:
This work was supported by grants from the National Natural Science Foundation of China(31201502) and the Special Research Fund for Doctoral Program of Higher Education of China(20124320120015).

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摘要: 在植物病害研究中,观察寄主植物被病原菌入侵的过程非常重要。Solophenyl Flavine 7GFE染料可附着于菌丝,在波长为330~380 nm的激发光下被激发出蓝色荧光。为了更好的观察寄主植物中病原真菌的侵染情况,本实验以Solophenyl Flavine 7GFE为染剂,对寄主植物中病原真菌的侵染情况进行了观察研究。结果显示,用0.002%(M/V)Solophenyl Flavine 7GFE溶于0.1 mol/L Tris-HCl (pH 8.5)配制的染色液染色5 min的效果最佳;使用95%乙醇溶液替代0.15%三氯乙酸(W/V)酒精溶液:氯仿(V/V)(4:1)对寄主植物叶片脱色的方法操作简便、毒害较低;染色时省略了番红预染步骤。将改良的染色方法用于晚疫病菌入侵的马铃薯叶片观察取得了良好的效果。该技术是一种改良的、快速有效、安全无毒的观察真菌菌丝入侵植物的荧光染色方法,也适用于观察其他真菌入侵寄主植物组织的过程。

关键词:
Solophenyl Flavine 7GFE /

马铃薯 /

晚疫病菌 /

荧光染色 /

入侵过程

Abstract: Research on host plants invaded by pathogenic fungi have great significance in plant disease. Solophenyl flavine 7GFE can attach to hyphae, which give fluorescence under 330-380 nm light. This paper modified a fluorescence staining method using solophenyl flavine 7GFE to observe fungal hyphae invading plants. Results showed that the most suitable leaf staining condition was 0.002% (M/V) solophenyl flavine 7GFE dissolved in 0.1 mol/L Tris-HCl (pH 8.5) for 5 min. Using 95% ethanol instead of 0.15% trichloroacetic acid (W/V) alcohol solution:chloroform (V/V) 4:1 for decolorization and omission of safranine staining simplified operation and reduced toxicity. The modified staining method was used to observe potato leaves invaded by Phytophthora infestans and achieved good effects. The modified staining method was fast, effective and non-toxic, and was suitable for observation of the invasion process of fungal pathogens and hosts.

Keywords:
Solophenyl Flavine 7GFE /

Phytophthora infestans /

Potato /

Fluorescent staining /

Pathogens infection

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参考文献(22)

[1] 余仲东,李琰,李登武.两类专性寄生真菌侵染植物的组织学染色技术初步研究[J]. 武汉植物学研究, 2005, 23(6):588-591.

Yu ZD, Li Y, Li DW. Histological stain technology study of two plant obligate parasitic fungi with their hosts[J].Journal of Wuhan Botanical Research, 2005, 23(6):588-591.

[2] 殷萍萍,李珊珊,王玉玲,曾会明. 立枯丝核菌(Rhizoctonia solani)对日本结缕草(Zoysia japonica Steud.)的侵染过程研究[J]. 微生物学通报,2015,42(7):1253-1262.

Yin PP, Li SS, Wang YL, Zeng HM. The infection process of Rhizoctonia solani in Zoysia japonica[J]. Microbiology China, 2015, 42(7):1253-1262.

[3] 王庭杰,张亮,韩琼,郑风霞,王天琪,冯娜娜,王太霞. 玉米茎秆细胞壁和组织构建对抗压强度的影响[J]. 植物科学学报,2015,33(1):109-115.

Wang TJ, Zhang L, Han Q, Zheng FX, Wang TQ, Feng NN, Wang TX. Effects of stalk cell wall and tissue on the compressive strength of maize[J]. Plant Science Journal, 2015, 33(1):109-115.

[4]
Chen XR, Cheng BP, Wang XL, Dong SM, Wang YL, Zheng XB, Wang YC.Green fluorescent protein (GFP) as a vital marker for studying the interaction of Phytophthora sojae and soybean[J]. Chinese Science Bulletin, 2009,54(16):2822-2829.

[5]
Sebastien JS, Testa A, Kamoun S, Laurence VM. Use of a green fluorescent protein marker for studying splash dispersal of sporangia of Phytophthora infestans[J]. Eur J Plant Pathol, 2005,112(4):391-394.

[6]
Jin DX, Liu GZ, Zhu HG, Yang XY, Zhang XL. Transformation of upland cotton (Gossypium hirsutum L.) with gfp gene as a visual marker[J]. J Integrative Agriculture, 2012, 11(6):910-919.

[7] 吴娟,邹路阳,曾群安,徐虹.钝顶螺旋藻FtsZ在大肠杆菌中的表达和定位[J].植物科学学报,2013, 31(2):171-177.

Wu J, Zou LA, Zeng QA, Xu H. Expression and localization of FtsZ from Spirulina platensis in Escherichia coli[J]. Plant Science Joumal, 2013, 31(2):171-177.

[8] 徐莹, 刘太国, 何月秋, 陈万权. 绿色荧光蛋白及其在丝状真菌研究中的应用[J]. 植物保护, 2008, 34(6):1-6.

Xu Y, Liu TG, He YQ, Chen WQ. Application of fluorescent protein to the studies on filamentous fungi[J]. Plant Protection, 2008, 34(6):1-6.

[9]
Kilaru S, Schuster M, Studholme D, Soanes D, Lin C, Talbot NJ, Steinberg G. A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici[J]. Fungal Genet Biol, 2015,79(6):125-131.

[10]
Knight NL, Sutherland MW. A rapid differential staining technique for Fusarium pseudograminearum in cereal tissues during crown rot infections[J]. Plant Pathol, 2011, 60(6):1140-1143.

[11] 苏跃, 冯泽蔚, 胡虎.马铃薯试管苗快繁培养基研究[J]. 种子, 2009, 28(3):71-73.

Su Y, Feng ZW, Hu H. Study on culture mediuns for rapid propagation free-virus potato tube seedlings[J]. Seed, 2009, 28(3):71-73.

[12] 李惠霞, 刘永刚, 王蒂, 杨富银. 马铃薯晚疫病菌培养条件的研究[J]. 中国马铃薯,2007, 21(4):203-205.

Li HX, Liu YG, Wang D, Yang FY. Studies on suitable cultural condition of phytophthora infestans[J]. Chinese Potato Journal, 2007, 21(4):203-205.

[13] 李惠霞, 刘永刚, 张俊莲, 王蒂.马铃薯对晚疫病水平抗性的组织细胞学观察[J]. 西北植物学报, 2015, 35(8):1554-1559.

Li HX, Liu YG, Zhang JL, Wang D. Histocytology of interaction between Phytophthora infestans and potato cultivar with horizontal resistance[J]. Acta Botanica Boreali-Occidentalia Sinica, 2015, 35(8):1554-1559.

[14]
Duckett JG, Read DJ. The use of the fluorescent dye,3,3'-dihexyloxacarbocyanine iodide, for selective staining of ascomycete fungi associated with liverwort rhizoids and ericoid mycorrhizal roots[J]. New Phytol, 1991,118(2):259-272.

[15] 陶明福, 车海彦, 罗大全. 海南长春花黄化病植原体的DAPI荧光显微检测[J]. 热带农业工程,2010, 34(2):17-20.

Tao MF, Che HY, Luo DQ. Detection of phytoplasma of Hainan perwinkle yellows disease with DAPI fluorescence microscopy[J]. Tropical Agricultural Engineering,2010, 34(2):17-20.

[16]
Hoch HC, Galvani CD, Szarowski DH, Turner JN. Two new fluorescent dyes applicable for visualization of fungal cell walls[J]. Mycologia, 2005, 97(3):580-588.

[17]
Anderson CT, Carroll A, Akhmetova L, Somerville C. Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots[J]. Plant Physiol, 2010,152(2):787-796.

[18]
Oliver JP, Castro A, Gaggero C, Cascón T, Schmelz EA, Castresana SC, León IP. Pythium infection activates conserved plant defense responses in mosses[J]. Planta, 2009,230(3):569-579.

[19]
Takahara H, Dolf A, Endl E, O'Connell R. Flow cytometric purification of Colletotrichum higginsianum biotrophic hyphae from Arabidopsis leaves for stage-specific transcriptome analysis[J]. Plant J, 2009,59(4):672-683.

[20]
Knight NL, Sutherland MW.Histopathological assessment of wheat seedling tissues infected by Fusarium pseudograminearum[J]. Plant Pathol, 2013,62(3):679-687.

[21]
Leon RF, Raymond EK. Fluorescent microscopy of viable Batrachochytrium dendrobatidis[J]. J Parasitol, 2012, 98(3):509-512.

[22]
Latijnhouwers M, de Wit PJ, Govers F. Oomycetes and fungi:similar weaponry to attack plants[J]. Trends Microbiol, 2003, 11(10):462-469.

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2. 陶凯锋，朱永，王乐骋，张颖铎，李璐. 两种玉凤花属植物的花结构和合蕊柱超微特征及其分类学意义. 广西植物. 2024(01): 89-101 . 百度学术

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文章访问数: 1559

HTML全文浏览量: 5

PDF下载量: 1474

被引次数: 8

出版历程

收稿日期: 2015-12-02

修回日期: 2015-12-24

网络出版日期: 2022-10-31

发布日期: 2016-04-27

目录

摘要
HTML全文
参考文献(22)
相关文章
施引文献(8)
资源附件(0)

[1]	余仲东,李琰,李登武.两类专性寄生真菌侵染植物的组织学染色技术初步研究[J]. 武汉植物学研究, 2005, 23(6):588-591. Yu ZD, Li Y, Li DW. Histological stain technology study of two plant obligate parasitic fungi with their hosts[J].Journal of Wuhan Botanical Research, 2005, 23(6):588-591.
[2]	殷萍萍,李珊珊,王玉玲,曾会明. 立枯丝核菌(Rhizoctonia solani)对日本结缕草(Zoysia japonica Steud.)的侵染过程研究[J]. 微生物学通报,2015,42(7):1253-1262. Yin PP, Li SS, Wang YL, Zeng HM. The infection process of Rhizoctonia solani in Zoysia japonica[J]. Microbiology China, 2015, 42(7):1253-1262.
[3]	王庭杰,张亮,韩琼,郑风霞,王天琪,冯娜娜,王太霞. 玉米茎秆细胞壁和组织构建对抗压强度的影响[J]. 植物科学学报,2015,33(1):109-115. Wang TJ, Zhang L, Han Q, Zheng FX, Wang TQ, Feng NN, Wang TX. Effects of stalk cell wall and tissue on the compressive strength of maize[J]. Plant Science Journal, 2015, 33(1):109-115.
[4]	Chen XR, Cheng BP, Wang XL, Dong SM, Wang YL, Zheng XB, Wang YC.Green fluorescent protein (GFP) as a vital marker for studying the interaction of Phytophthora sojae and soybean[J]. Chinese Science Bulletin, 2009,54(16):2822-2829.
[5]	Sebastien JS, Testa A, Kamoun S, Laurence VM. Use of a green fluorescent protein marker for studying splash dispersal of sporangia of Phytophthora infestans[J]. Eur J Plant Pathol, 2005,112(4):391-394.
[6]	Jin DX, Liu GZ, Zhu HG, Yang XY, Zhang XL. Transformation of upland cotton (Gossypium hirsutum L.) with gfp gene as a visual marker[J]. J Integrative Agriculture, 2012, 11(6):910-919.
[7]	吴娟,邹路阳,曾群安,徐虹.钝顶螺旋藻FtsZ在大肠杆菌中的表达和定位[J].植物科学学报,2013, 31(2):171-177. Wu J, Zou LA, Zeng QA, Xu H. Expression and localization of FtsZ from Spirulina platensis in Escherichia coli[J]. Plant Science Joumal, 2013, 31(2):171-177.
[8]	徐莹, 刘太国, 何月秋, 陈万权. 绿色荧光蛋白及其在丝状真菌研究中的应用[J]. 植物保护, 2008, 34(6):1-6. Xu Y, Liu TG, He YQ, Chen WQ. Application of fluorescent protein to the studies on filamentous fungi[J]. Plant Protection, 2008, 34(6):1-6.
[9]	Kilaru S, Schuster M, Studholme D, Soanes D, Lin C, Talbot NJ, Steinberg G. A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici[J]. Fungal Genet Biol, 2015,79(6):125-131.
[10]	Knight NL, Sutherland MW. A rapid differential staining technique for Fusarium pseudograminearum in cereal tissues during crown rot infections[J]. Plant Pathol, 2011, 60(6):1140-1143.
[11]	苏跃, 冯泽蔚, 胡虎.马铃薯试管苗快繁培养基研究[J]. 种子, 2009, 28(3):71-73. Su Y, Feng ZW, Hu H. Study on culture mediuns for rapid propagation free-virus potato tube seedlings[J]. Seed, 2009, 28(3):71-73.
[12]	李惠霞, 刘永刚, 王蒂, 杨富银. 马铃薯晚疫病菌培养条件的研究[J]. 中国马铃薯,2007, 21(4):203-205. Li HX, Liu YG, Wang D, Yang FY. Studies on suitable cultural condition of phytophthora infestans[J]. Chinese Potato Journal, 2007, 21(4):203-205.
[13]	李惠霞, 刘永刚, 张俊莲, 王蒂.马铃薯对晚疫病水平抗性的组织细胞学观察[J]. 西北植物学报, 2015, 35(8):1554-1559. Li HX, Liu YG, Zhang JL, Wang D. Histocytology of interaction between Phytophthora infestans and potato cultivar with horizontal resistance[J]. Acta Botanica Boreali-Occidentalia Sinica, 2015, 35(8):1554-1559.
[14]	Duckett JG, Read DJ. The use of the fluorescent dye,3,3'-dihexyloxacarbocyanine iodide, for selective staining of ascomycete fungi associated with liverwort rhizoids and ericoid mycorrhizal roots[J]. New Phytol, 1991,118(2):259-272.
[15]	陶明福, 车海彦, 罗大全. 海南长春花黄化病植原体的DAPI荧光显微检测[J]. 热带农业工程,2010, 34(2):17-20. Tao MF, Che HY, Luo DQ. Detection of phytoplasma of Hainan perwinkle yellows disease with DAPI fluorescence microscopy[J]. Tropical Agricultural Engineering,2010, 34(2):17-20.
[16]	Hoch HC, Galvani CD, Szarowski DH, Turner JN. Two new fluorescent dyes applicable for visualization of fungal cell walls[J]. Mycologia, 2005, 97(3):580-588.
[17]	Anderson CT, Carroll A, Akhmetova L, Somerville C. Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots[J]. Plant Physiol, 2010,152(2):787-796.
[18]	Oliver JP, Castro A, Gaggero C, Cascón T, Schmelz EA, Castresana SC, León IP. Pythium infection activates conserved plant defense responses in mosses[J]. Planta, 2009,230(3):569-579.
[19]	Takahara H, Dolf A, Endl E, O'Connell R. Flow cytometric purification of Colletotrichum higginsianum biotrophic hyphae from Arabidopsis leaves for stage-specific transcriptome analysis[J]. Plant J, 2009,59(4):672-683.
[20]	Knight NL, Sutherland MW.Histopathological assessment of wheat seedling tissues infected by Fusarium pseudograminearum[J]. Plant Pathol, 2013,62(3):679-687.
[21]	Leon RF, Raymond EK. Fluorescent microscopy of viable Batrachochytrium dendrobatidis[J]. J Parasitol, 2012, 98(3):509-512.
[22]	Latijnhouwers M, de Wit PJ, Govers F. Oomycetes and fungi:similar weaponry to attack plants[J]. Trends Microbiol, 2003, 11(10):462-469.

1.	胡梦露，李宗艳，任书娴，杨建伟，伍倩，冯尧，叶松菩. 云南26种石斛种质资源的形态分类与亲缘关系. 江苏农业科学. 2025(01): 191-200 . 百度学术
2.	陶凯锋，朱永，王乐骋，张颖铎，李璐. 两种玉凤花属植物的花结构和合蕊柱超微特征及其分类学意义. 广西植物. 2024(01): 89-101 . 百度学术
3.	贺漫媚，代色平，陈秀萍，吴俭峰，刘国锋，阮琳，王伟. 17种石斛属植物表型性状多样性分析. 植物资源与环境学报. 2024(02): 71-79+90 . 百度学术
4.	涂国章，张显强. DNA条形码技术在石斛分类鉴定中的应用进展. 食品安全质量检测学报. 2023(02): 154-160 . 百度学术
5.	尚明越，王嘉乐，周莹，张满常，刘颖琳，段宝忠. 濒危紫皮石斛叶绿体基因组结构及系统发育分析. 中草药. 2023(19): 6424-6433 . 百度学术

马铃薯晚疫病菌侵染的Solophenyl Flavine荧光染色方法研究

作者简介: 刘甜甜(1991-),女,硕士研究生,研究方向为植物病理学(E-mail:872254877@qq.com)。

通讯作者: 周倩.E-mail:zhouqian2617@hunau.edu.cn

计量

出版历程

Fluorescent Staining with Solophenyl Flavine for Infection Observation in Phytophthora infestans

期刊类型引用(5)

其他类型引用(3)

计量

出版历程

目录

作者简介:
刘甜甜(1991-),女,硕士研究生,研究方向为植物病理学(E-mail:872254877@qq.com)。

通讯作者:
周倩.E-mail:zhouqian2617@hunau.edu.cn