小麦矮腥黑粉菌侵染小麦子房的激光共聚焦显微观察方法
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摘要
本试验利用碘化丙啶(propidium iodide)和Alexa Flour 488(AF 488)标记的麦胚凝集素(WGA)分别对小麦子房细胞和小麦矮腥黑粉菌进行染色,结合激光共聚焦显微镜成像系统获取小麦子房的三维立体图像。该技术可获得清晰的小麦子房细胞图像,并观察小麦矮腥黑粉菌在小麦子房中的侵染状况。该方法将为研究病原菌在寄主体内的分布提供可参考依据。
In this study, we used WGA AF-488 to label the mycelia of Tilletia controversa Kühn(TCK), used propidium iodide to stain the wheat ovary, and then used confocal laser scanning microscopy(CLSM) to observe the infection of TCK throughout the growth of wheat ovary. The images successfully revealed the cells of wheat ovary and the mycelia of TCK in the ovary cells, which contribute a lot to the research of mycelial distribution in the host cells.
In this study, we used WGA AF-488 to label the mycelia of Tilletia controversa Kühn(TCK), used propidium iodide to stain the wheat ovary, and then used confocal laser scanning microscopy(CLSM) to observe the infection of TCK throughout the growth of wheat ovary. The images successfully revealed the cells of wheat ovary and the mycelia of TCK in the ovary cells, which contribute a lot to the research of mycelial distribution in the host cells.
引文
[1] HOFFMAN J A. Bunt of wheat [J]. Plant Disease, 1982: 979-986.
[2] TRIONE E J, HESS W M, STOCKWELL V O. Growth and sporulation of the dikaryons of the dwarf bunt fungus in wheat plants and in culture [J]. Canadian Journal of Botany, 1989, 67(6): 1671-1680.
[3] 郭予元. 中国农作物病虫害[M]. 北京: 中国农业出版社, 2014: 615-618.
[4] 魏淑秋, 章正, 郑耀水. 应用生物气候相似距对小麦矮化腥黑穗病在我国定殖可能性的研究[J]. 北京农业大学学报, 1995, 21(2): 127-132.
[5] 陈克, 章正. 小麦矮腥黑穗病在中国定殖风险分析及区划研究[J]. 植物病理学报, 2002, 32(4): 312-318.
[6] 周益林,段霞瑜,贾文明,等.小麦矮腥黑穗病(TCK)传入中国及其定殖的风险分析研究进展[J].植物保护,2007,33(2):6-10.
[7] KOCHANOVá M, ZOUHAR M, PROKINOVá E, et al. Detection of Tilletia controversa and Tilletia caries in wheat by PCR method [J]. Plant Soil and Environment,2004,50(2):75-77.
[8] MCDONALD J G, WONG E, KLASSEN G R. Differentiation of Tilletia species by rep-PCR genomic fingerprinting[J]. Plant Disease, 2000, 84: 1121-1125.
[9] YUAN Qing, NIAN Siji, YIN Youping, et al. Development of a PCR-based diagnostic tool specific to wheat dwarf bunt, caused by Tilletia controversa[J]. European Journal of Plant Pathology, 2009, 124(4): 585-594.
[10] GAO Li, CHEN Wanquan, LIU Taiguo. Development of a SCAR marker by inter-simple sequence repeat for diagnosis of dwarf bunt of wheat and detection of Tilletia controversa Kühn[J]. Folia Microbiologica, 2010, 55(3): 258-264.
[11] GAO Li, CHEN Wanquan, LIU Taiguo. An ISSR-based approach for the molecular detection and diagnosis of dwarf bunt of wheat, caused by Tilletia controversa Kühn [J]. Journal of Phytopathology, 2011, 159: 155-158.
[12] KNIGHT N L, SUTHERLAND M W. A rapid differential staining technique for Fusarium pseudograminearum in cereal tissues during crown rot infections [J]. Plant Pathology, 2011, 60(6): 1140-1143.
[13] CHIU Wanling, NIWA Y, ZENG Weike, et al. Engineered GFP as a vital reporter in plants [J]. Current Biology, 1996, 6(3): 325-330.
[14] LORANG J M, TUORI R P, MARTINEZ J P, et al. Green fluorescent protein is lighting up fungal biology[J]. Applied and Environmental Microbiology, 2001, 67(5): 1987-1994.
[15] OREN L, EZRATI S, COHEN D, et al. Early events in the Fusarium verticillioides-maize interaction characterized by using a green fluorescent protein-expressing transgenic isolate[J]. Applied and Environmental Microbiology, 2003, 69(3): 1695-1701.
[16] RAJASEKARAN K, CARY J W, COTTY P J, et al. Development of a GFP-expressing Aspergillus flavus strain to study fungal invasion, colonization, and resistance in cottonseed [J]. Mycopathologia, 2008, 165(2): 89-97.
[17] 徐莹, 刘太国, 何月秋, 等. 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.
[18] ANDARGIE M, LI Luoye, FENG Aiqing, et al. Development of a GFP-expressing Ustilaginoidea virens strain to study fungal invasion and colonization in rice spikelets [J]. South African Journal of Botany, 2015, 97: 16-24.
[19] 刘甜甜,周倩,吴秋云,等.马铃薯晚疫病菌侵染的Solophenyl Flavine荧光染色方法研究[J].植物科学学报,2016,34(2):316-324.
[20] HOCH H C, GALVANI C D, SZAROWSKI D H, et al. Two new fluorescent dyes applicable for visualization of fungal cell walls [J]. Mycologia, 2005, 97(3): 580-588.
[21] 李惠霞, 刘永刚, 张俊莲, 等. 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.
[22] FERNANDEZ J A, DURAN R. Hypodermic inoculation, a rapid technique for producing dwarf bunt in wheat [J]. Plant Disease Reporter,1978, 62(4): 336-337.
[23] 姚卓, 陈万权, 高利, 等. 小麦矮腥黑粉菌冬孢子萌发和室内人工接种方法的探索[J]. 植物保护, 2014, 40(3): 122-126.
[24] MIDOUX P, GRIVET J P, DELMOTTE F, et al. The binding of monosaccharides to wheat germ agglutinin: Fluorescence and NMR investigations [J]. Biochemical and Biophysical Research Communications, 1984, 119(2): 603-611.
[25] LAMPRECHT A, SCH?FER U F, LEHR C. Characterization of microcapsules by confocal laser scanning microscopy: structure, capsule wall composition and encapsulation rate [J]. European Journal of Pharmaceutics and Biopharmaceutics, 2000, 49(1): 1-9.
[26] HONG Ren, LIU Yong, SUN Jingsan. Observation of rice embryo sac development with confocal laser scanning microscopy [J]. Acta Botanica Sinica, 1998, 40(9): 786-789.
[27] KELLIHER T, WALBOT V. Emergence and patterning of the five cell types of the Zea mays anther locule [J]. Developmental Biology, 2011, 350(1): 32-49.
[28] GAO Li, KELLIHER T, NGUYEN L, et al. Ustilago maydis reprograms cell proliferation in maize anthers [J].Plant Journal for Cell and Molecular Biology,2013,75(6):903-914.
[29] 蔚慧欣,高利,沈慧敏,等.小麦矮腥黑粉菌在小麦体内侵染过程的显微观察[J].中国科学:生命科学,2016,46(5):637-645.
[2] TRIONE E J, HESS W M, STOCKWELL V O. Growth and sporulation of the dikaryons of the dwarf bunt fungus in wheat plants and in culture [J]. Canadian Journal of Botany, 1989, 67(6): 1671-1680.
[3] 郭予元. 中国农作物病虫害[M]. 北京: 中国农业出版社, 2014: 615-618.
[4] 魏淑秋, 章正, 郑耀水. 应用生物气候相似距对小麦矮化腥黑穗病在我国定殖可能性的研究[J]. 北京农业大学学报, 1995, 21(2): 127-132.
[5] 陈克, 章正. 小麦矮腥黑穗病在中国定殖风险分析及区划研究[J]. 植物病理学报, 2002, 32(4): 312-318.
[6] 周益林,段霞瑜,贾文明,等.小麦矮腥黑穗病(TCK)传入中国及其定殖的风险分析研究进展[J].植物保护,2007,33(2):6-10.
[7] KOCHANOVá M, ZOUHAR M, PROKINOVá E, et al. Detection of Tilletia controversa and Tilletia caries in wheat by PCR method [J]. Plant Soil and Environment,2004,50(2):75-77.
[8] MCDONALD J G, WONG E, KLASSEN G R. Differentiation of Tilletia species by rep-PCR genomic fingerprinting[J]. Plant Disease, 2000, 84: 1121-1125.
[9] YUAN Qing, NIAN Siji, YIN Youping, et al. Development of a PCR-based diagnostic tool specific to wheat dwarf bunt, caused by Tilletia controversa[J]. European Journal of Plant Pathology, 2009, 124(4): 585-594.
[10] GAO Li, CHEN Wanquan, LIU Taiguo. Development of a SCAR marker by inter-simple sequence repeat for diagnosis of dwarf bunt of wheat and detection of Tilletia controversa Kühn[J]. Folia Microbiologica, 2010, 55(3): 258-264.
[11] GAO Li, CHEN Wanquan, LIU Taiguo. An ISSR-based approach for the molecular detection and diagnosis of dwarf bunt of wheat, caused by Tilletia controversa Kühn [J]. Journal of Phytopathology, 2011, 159: 155-158.
[12] KNIGHT N L, SUTHERLAND M W. A rapid differential staining technique for Fusarium pseudograminearum in cereal tissues during crown rot infections [J]. Plant Pathology, 2011, 60(6): 1140-1143.
[13] CHIU Wanling, NIWA Y, ZENG Weike, et al. Engineered GFP as a vital reporter in plants [J]. Current Biology, 1996, 6(3): 325-330.
[14] LORANG J M, TUORI R P, MARTINEZ J P, et al. Green fluorescent protein is lighting up fungal biology[J]. Applied and Environmental Microbiology, 2001, 67(5): 1987-1994.
[15] OREN L, EZRATI S, COHEN D, et al. Early events in the Fusarium verticillioides-maize interaction characterized by using a green fluorescent protein-expressing transgenic isolate[J]. Applied and Environmental Microbiology, 2003, 69(3): 1695-1701.
[16] RAJASEKARAN K, CARY J W, COTTY P J, et al. Development of a GFP-expressing Aspergillus flavus strain to study fungal invasion, colonization, and resistance in cottonseed [J]. Mycopathologia, 2008, 165(2): 89-97.
[17] 徐莹, 刘太国, 何月秋, 等. 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.
[18] ANDARGIE M, LI Luoye, FENG Aiqing, et al. Development of a GFP-expressing Ustilaginoidea virens strain to study fungal invasion and colonization in rice spikelets [J]. South African Journal of Botany, 2015, 97: 16-24.
[19] 刘甜甜,周倩,吴秋云,等.马铃薯晚疫病菌侵染的Solophenyl Flavine荧光染色方法研究[J].植物科学学报,2016,34(2):316-324.
[20] HOCH H C, GALVANI C D, SZAROWSKI D H, et al. Two new fluorescent dyes applicable for visualization of fungal cell walls [J]. Mycologia, 2005, 97(3): 580-588.
[21] 李惠霞, 刘永刚, 张俊莲, 等. 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.
[22] FERNANDEZ J A, DURAN R. Hypodermic inoculation, a rapid technique for producing dwarf bunt in wheat [J]. Plant Disease Reporter,1978, 62(4): 336-337.
[23] 姚卓, 陈万权, 高利, 等. 小麦矮腥黑粉菌冬孢子萌发和室内人工接种方法的探索[J]. 植物保护, 2014, 40(3): 122-126.
[24] MIDOUX P, GRIVET J P, DELMOTTE F, et al. The binding of monosaccharides to wheat germ agglutinin: Fluorescence and NMR investigations [J]. Biochemical and Biophysical Research Communications, 1984, 119(2): 603-611.
[25] LAMPRECHT A, SCH?FER U F, LEHR C. Characterization of microcapsules by confocal laser scanning microscopy: structure, capsule wall composition and encapsulation rate [J]. European Journal of Pharmaceutics and Biopharmaceutics, 2000, 49(1): 1-9.
[26] HONG Ren, LIU Yong, SUN Jingsan. Observation of rice embryo sac development with confocal laser scanning microscopy [J]. Acta Botanica Sinica, 1998, 40(9): 786-789.
[27] KELLIHER T, WALBOT V. Emergence and patterning of the five cell types of the Zea mays anther locule [J]. Developmental Biology, 2011, 350(1): 32-49.
[28] GAO Li, KELLIHER T, NGUYEN L, et al. Ustilago maydis reprograms cell proliferation in maize anthers [J].Plant Journal for Cell and Molecular Biology,2013,75(6):903-914.
[29] 蔚慧欣,高利,沈慧敏,等.小麦矮腥黑粉菌在小麦体内侵染过程的显微观察[J].中国科学:生命科学,2016,46(5):637-645.
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