Fluorescent Staining with Solophenyl Flavine for Infection Observation in <i>Phytophthora infestans

LIU Tian-Tian; ZHOU Qian; WU Qiu-Yun; XIONG Xing-Yao

doi:10.11913/PSJ.2095-0837.2016.20316

Plant Science Journal > 2016 > 34(2): 316-324. > DOI: 10.11913/PSJ.2095-0837.2016.20316 CSTR: 32231.14.PSJ.2095-0837.2016.20316

Citation: LIU Tian-Tian, ZHOU Qian, WU Qiu-Yun, XIONG Xing-Yao. Fluorescent Staining with Solophenyl Flavine for Infection Observation in Phytophthora infestans[J]. Plant Science Journal, 2016, 34(2): 316-324. DOI: 10.11913/PSJ.2095-0837.2016.20316

LIU Tian-Tian, ZHOU Qian, WU Qiu-Yun, XIONG Xing-Yao. Fluorescent Staining with Solophenyl Flavine for Infection Observation in Phytophthora infestans[J]. Plant Science Journal, 2016, 34(2): 316-324. DOI: 10.11913/PSJ.2095-0837.2016.20316

Citation: LIU Tian-Tian, ZHOU Qian, WU Qiu-Yun, XIONG Xing-Yao. Fluorescent Staining with Solophenyl Flavine for Infection Observation in Phytophthora infestans[J]. Plant Science Journal, 2016, 34(2): 316-324. DOI: 10.11913/PSJ.2095-0837.2016.20316

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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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Received Date: December 02, 2015

Revised Date: December 24, 2015

Available Online: October 31, 2022

Published Date: April 27, 2016

Graphical Abstract

Abstract

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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References (22)

References

[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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[5] CHANG Jing-Ling, DENG Xiao-Li, ZHANG Jun-Xia, ZHAO Xu-Na, YANG Jian-Lei. GC-MS Analysis of Linolenic Acid and Linoleic Acid in Chinese Trichosanthes kirilowii Oil[J]. Plant Science Journal, 2009, 27(5): 564-568.

[6] LI Miao, LOU Yi-Ceng, YANG Han, HU Jia-Xing, SU Feng-Ping. Study on the Fingerprint Chromatogram of the Compound Giant Knotweed Rhizome by High Performance Liquid Chromatography[J]. Plant Science Journal, 2009, 27(4): 446-450.

[7] HE Ya-Ting, LI Ming, LIU Wen-Zhi, ZHANG Quan-Fa, DANG Gao-Di. Comparison of Gas Exchange Traits of 30 Plant Species in Subalpine Meadow in Foping National Reserve of Qinling Mountains[J]. Plant Science Journal, 2007, 25(5): 451-456.

[8] MA Jin-E, JIN Ze-Xin, LI Jun-Min. Analysis of Flavonoids in Endangered Plant Sinocalycanthus chinensis Using Thin Layer Chromatography[J]. Plant Science Journal, 2007, 25(4): 366-370.

[9] LEI An-Ping, HU Zhang-Li, WONG Yuk-Shan, TAM Fung-Yee. Bioconcentration and Metabolism of Polycyclic Aromatic Hydrocarbons (PAHs) by Algae[J]. Plant Science Journal, 2005, 23(3): 291-298.

[10] Mei Xinguo. A LOW-PRESSURE CHROMATOGRAPHIC APPARATUS AND ITS USE IN PHYTOCHEMISTRY[J]. Plant Science Journal, 1986, 4(3): 297-302.

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[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]	Kang Hongwei, Yue Kangjie, Liu Huixin, Wang Jiali, Tian Xuping. Effect of sex and leaf shape on gas exchange parameters and chlorophyll fluorescence characteristics in Sabina chinensis (L.) Ant[J]. Plant Science Journal, 2024, 42(6): 791-799. DOI: 10.11913/PSJ.2095-0837.23391
[2]	Han Jia-Xin, Zheng Hao, Zhang Qiong, Zhong Cai-Hong. Research advances in the metabolism and regulation of carbohydrate in fruit trees[J]. Plant Science Journal, 2020, 38(1): 143-149. DOI: 10.11913/PSJ.2095-0837.2020.10143
[3]	Zu Kui-Ling, Dong Shu-Bin, Li Jian-Xia, Xu Shen-Jian, Zhao Liang-Cheng. Differentially expressed genes analysis of terpenoid biosynthesis related to aril development in Celastrus orbiculatus Thunb.[J]. Plant Science Journal, 2017, 35(2): 276-282. DOI: 10.11913/PSJ.2095-0837.2017.20276
[4]	LIANG Jia-Wen, LIU Ai-Jie, MA Bing-Xin, WANG You-Wei. Simultaneous Determination of Six Flavonoid Compounds in Lotus Leaves by High Performance Liquid Chromatography[J]. Plant Science Journal, 2015, 33(6): 861-866. DOI: 10.11913/PSJ.2095-0837.2015.60861
[5]	CHANG Jing-Ling, DENG Xiao-Li, ZHANG Jun-Xia, ZHAO Xu-Na, YANG Jian-Lei. GC-MS Analysis of Linolenic Acid and Linoleic Acid in Chinese Trichosanthes kirilowii Oil[J]. Plant Science Journal, 2009, 27(5): 564-568.
[6]	LI Miao, LOU Yi-Ceng, YANG Han, HU Jia-Xing, SU Feng-Ping. Study on the Fingerprint Chromatogram of the Compound Giant Knotweed Rhizome by High Performance Liquid Chromatography[J]. Plant Science Journal, 2009, 27(4): 446-450.
[7]	HE Ya-Ting, LI Ming, LIU Wen-Zhi, ZHANG Quan-Fa, DANG Gao-Di. Comparison of Gas Exchange Traits of 30 Plant Species in Subalpine Meadow in Foping National Reserve of Qinling Mountains[J]. Plant Science Journal, 2007, 25(5): 451-456.
[8]	MA Jin-E, JIN Ze-Xin, LI Jun-Min. Analysis of Flavonoids in Endangered Plant Sinocalycanthus chinensis Using Thin Layer Chromatography[J]. Plant Science Journal, 2007, 25(4): 366-370.
[9]	LEI An-Ping, HU Zhang-Li, WONG Yuk-Shan, TAM Fung-Yee. Bioconcentration and Metabolism of Polycyclic Aromatic Hydrocarbons (PAHs) by Algae[J]. Plant Science Journal, 2005, 23(3): 291-298.
[10]	Mei Xinguo. A LOW-PRESSURE CHROMATOGRAPHIC APPARATUS AND ITS USE IN PHYTOCHEMISTRY[J]. Plant Science Journal, 1986, 4(3): 297-302.

Fluorescent Staining with Solophenyl Flavine for Infection Observation in Phytophthora infestans

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