芋疫霉菌的巢式PCR检测
|
摘要
【目的】建立芋疫霉菌快速、准确的PCR检测技术,为芋疫病流行规律监测和综合防控提供科学依据。【方法】根据芋疫霉菌与其他疫霉菌种类Ypt1基因序列差异,设计了1对芋疫霉菌PCR检测特异引物PCOF/PCOR,并对该引物的特异性、灵敏性和应用性进行了验证。【结果】在优化的反应体系与扩增条件下,PCOF/PCOR引物能特异性地从芋疫霉菌基因组DNA中扩增出1条172 bp的条带,而其他供试病原菌均无扩增条带。在25μL PCR反应体系中,PCOF/PCOR引物对芋疫霉菌基因组DNA的检测灵敏度为100 pg,而以疫霉菌Ypt1基因通用引物ph1F/Yph2R为第一轮引物,PCOF/PCOR为第二轮引物,进行巢式PCR扩增,能检测到10 fg芋疫霉菌基因组DNA,检测灵敏度提高了10 000倍。采用巢式PCR,可从芋疫病发病的叶片和未显症叶片组织中检测到芋疫霉菌,检出率分别为100%和57.5%。【结论】所建立的巢式PCR可应用于芋疫霉菌的快速、特异和高灵敏度检测。
【Objective】To develop a PCR assay for rapid and accurate detection, epidemiology information, and integrated disease management on Phytophthora colocasiae, the pathogen of taro phytophthora blight. 【Method】A pair of species-specific primers, PCOF/PCOR, for P. colocasiae was designed based on the differences in Ras-related protein(Ypt1) gene sequence between P. colocasiae and other species in the same genus. The specificity, sensitivity and applicability of the primers were evaluated. 【Result】 With the optimized reaction conditions and amplification,PCOF/PCOR amplified only a single band of 172 bp with genomic DNA extracted from all P.colocasiae strains, while the other tested pathogens had no corresponding band. The sensitivity of conventional PCR method using PCOF/PCOR as primers was 100 pg of genomic DNA in a 25 μL reaction solution. Whereas, the newly developed nested-PCR performed using Ypt1 gene universal primers ph1 F/Yph2 R for the first-round and PCOF/PCOR for the second-round increased 10 000-fold on the sensitivity to 10 fg. The nested-PCR methodology could positively detected P. colocasiae 100% in diseased leaves or 57.5% in symptom-free infected tissues. 【Conclusion】 The newly established nested-PCR assay could be used for rapid, specific and sensitive detection of P. colocasiae.
【Objective】To develop a PCR assay for rapid and accurate detection, epidemiology information, and integrated disease management on Phytophthora colocasiae, the pathogen of taro phytophthora blight. 【Method】A pair of species-specific primers, PCOF/PCOR, for P. colocasiae was designed based on the differences in Ras-related protein(Ypt1) gene sequence between P. colocasiae and other species in the same genus. The specificity, sensitivity and applicability of the primers were evaluated. 【Result】 With the optimized reaction conditions and amplification,PCOF/PCOR amplified only a single band of 172 bp with genomic DNA extracted from all P.colocasiae strains, while the other tested pathogens had no corresponding band. The sensitivity of conventional PCR method using PCOF/PCOR as primers was 100 pg of genomic DNA in a 25 μL reaction solution. Whereas, the newly developed nested-PCR performed using Ypt1 gene universal primers ph1 F/Yph2 R for the first-round and PCOF/PCOR for the second-round increased 10 000-fold on the sensitivity to 10 fg. The nested-PCR methodology could positively detected P. colocasiae 100% in diseased leaves or 57.5% in symptom-free infected tissues. 【Conclusion】 The newly established nested-PCR assay could be used for rapid, specific and sensitive detection of P. colocasiae.
引文
[1]SHRESTHA S, HU J, FRYXELLl R T, et al.SNP marker identify widely distributed clonal lineages of Phytophthora colocasiae in Vietnam, Hawaii and Hainan island, China [J].Mycologia, 2014, 106(4): 676-685.
[2]王汉荣, 方丽, 茹水江, 等.槟榔芋疫病的识别与防治[J].中国蔬菜, 2009(21): 21-22. WANG H R, SU L, RU S J, et al.Identification and control of taro phytophthora blight [J].China Vegetables, 2009(21): 21-22.(in Chinese)
[3]王向社, 李锐, 胡茂松, 等.海南岛芋疫霉菌生物学特性、致病力、对甲霜灵的敏感性研究[J].热带作物学报, 2001, 22(1): 83-90. WANG X S, LI R, HU M S, et al.Study on the biology, virulence of Phytophthora colocasiae Racib and its sensitivity to metalaxyl [J].Chinese Journal of Tropical Crops, 2001, 22(1): 83-90.(in Chinese)
[4]QUITUGUA R J, TRUJILLO E E.Survival of Phytophthora colocasiae in field soil at various temperatures and water matric potentials [J].Plant Disease, 1998, 82(2): 203-207.
[5]刘独臣.四川芋疫病发生规律及防治技术[J].长江蔬菜, 2013, 18: 118-119. LIU D C.Epidemiology and control of taro phytophthora blight in Sichuan [J].Journal of Changjiang Vegetables, 2013, 18: 118-119.(in Chinese)
[6]VASGUEZ E A.Yield loss in taro due to Phytophthora leaf blight [J].Journal of Root Crops, 1990, 16: 48-50.
[7]方辉, 张惠琴, 陈孝赏, 等.双炔酰菌胺防治红香芋疫病的效果及应用技术[J].浙江农业科学, 2016, 57(6): 899 -900,911. FANG H, ZHANG H Q, CHEN X S, et al.Effect and application technology of mandipropamid in control taro phytophthora blight [J].Journal of Zhejiang Agricultural Sciences, 2016, 57(6):899-900, 911.(in Chinese)
[8]莫俊杰, 胡汉桥, 梁钾贤, 等.芋疫病抗病性鉴定及不同品系遗传多样性分析[J].广东海洋大学学报, 2012, 32(4): 67-72. MO J J, HU H Q, LIANG J X, et al.Resistance identification of taro to Phytophthora colocasiae and genetic diversity analysis of Colocasia esculenta [J].Journal of Guangdong Ocean University, 2012, 32(4): 67-72.(in Chinese)
[9]NATH V S, SENTHIL M, HEGDE V M, et al.Evaluation of fungicides on Indian isolates of Phytophthora colocasiae causing leaf blight of taro [J].Archives of Phytopathology and Plant Protection, 2013, 46(5): 548-555.
[10]LIN M J,KO W H.Occurrence of isolates of Phytophthora coloscsiae in Taiwan with homothallic behavior and its significance [J].Mycologia, 2008, 100(5): 727-734.
[11]叶泉清, 钟佳铃, 陈媚, 等.槟榔芋疫霉菌生物学特性、致病力测定及田间防治药剂筛选[J].南方农业学报, 2016, 47(4): 588-593. YE Q Q, ZHONG J L, CHEN M, et al.Biological characteristics, virulence of Phytophthora colocasiae Racib.From Colacasia esculenta L.var. cormosus Chang and fungicides screening for field control [J].Journal of Southern Agriculture, 2016, 47(4): 588-593.(in Chinese)
[12]NATH V S, HEGDE V M, JEEVA M L, et al.Rapid and sensitive detection of Phytophthora colocasiae responsible for the taro blight using conventional and real-time PCR assay [J].FEMS Micobiology Letters, 2014, 352: 174-183.
[13]MARTIN F N, ABAD Z G, BALCI Y, et al.Identification and detection of Phytophthora: reviewing our progress, identifying our needs [J].Plant Disease, 2012, 96(8): 1080-1103.
[14]ROLLINS L, COSTS K, ELLIOTT M, et al.Comparison of five detection and quantification methods for Phytophthora ramorum in stream and irrigation water [J]. Plant Disease, 2016, 100(6): 1202-1211.
[15]王晓杰, 康振生, 黄丽丽.PCR技术在植物病害检测中的应用[J].云南农业大学学报, 2005, 20(2): 179-182. WANG X J, KANG Z S,HUANG L L.Application of PCR technology on the detection of plant disease [J]. Journal of Yunnan Agricultural University, 2005, 20(2): 179-182.(in Chinese)
[16]DRENTH A, WAGELS G, SMITH B, et al.Development of a DNA-based method for detection and identification of Phytophthora species [J].Australasian Plant Pathology, 2005, 35: 147-159.
[17]李依韦, 银玲.rDNA-ITS序列分析在植物病原真菌分类鉴定中的应用[J].内蒙古民族大学学报(自然科学版), 2012, 27(1): 66-67. LI Y W,YIN L.Application of rDNA-ITS sequences in plant disease fungi classification and identification [J].Journal of Inner Mongolia University for Nationalities (Natural Sciences), 2012, 27(1): 66-67.(in Chinese)
[18]傅华英, 葛丹凤, 李晓燕, 等.甘蔗赤条病菌巢式PCR检测[J].植物保护学报, 2017, 44(2): 276-282. FU H Y, GE D F, LI X Y, et al.Nested-PCR detection of Acidovorax avenae subsp.avenae, the pathogen of red stripe on sugarcane [J].Journal of Plant Protection, 2017, 44(2): 276-282.(in Chinese)
[19]KONIG S, SCHWENKBIER L, POLLOK S, et al.Potential of Ypt1 and ITS gene regions for the detection of Phytophthora species in a lab-on-a-chip DNA hybridization array [J].Plant Pathology, 2015, 64: 1176-1189.
[20]LAN C Z, LIU P Q, LI B J, et al.Development of a specific PCR assay for the rapid and sensitive detection of Phytophthora capsici [J].Australasian Plant Pathology, 2013, 42: 379-384.
[21]杨万风, 刘艳, 刘翔, 等.巢式PCR检测菜豆细菌性萎蔫病菌[J].浙江农业学报, 2015, 27(7): 1202-1207. YANG W F, LIU Y, LIU X, et al.Detection of Curtobacterium flaccumfaciens pv.flaccumfaciens using nested PCR [J].Acta Agriculturae Zhejiangensis, 2015, 27(7): 1202-1207.(in Chinese)
[22]ZHANG Z G, LI Y Q, FAN H, et al.Molecular detection of Phytophthora capsici in infected plant tissues, soil and water [J].Plant Pathology, 2006, 55(6): 770-775.
[23]赵杰.ITS序列分析及其在植物真菌病害分子检测中的应用[J].陕西农业科学, 2004(4): 35-37. ZHAO J.ITS sequence analysis and its application in molecular detection of plant fungal diseases [J].Shanxi Journal of Agricultural Sciences, 2004(4): 35-37.(in Chinese)
[24]SCHENA L, COOKE D E L.Assessing the potential of regions of the nuclear and mitochondrial genome to develop a “molecular tool box” for the detection and characterization of Phytophthora species [J].Journal of Microbiological Methods, 2006, 67: 70-85.
[25]VOLOSSIOUK T, ROBB E J, NAZAR R N.Direct DNA extraction for PCR-mediated assays of soil organisms [J].Applied and Enviromental Microbiology, 1995,61(11): 3972-3976.
[2]王汉荣, 方丽, 茹水江, 等.槟榔芋疫病的识别与防治[J].中国蔬菜, 2009(21): 21-22. WANG H R, SU L, RU S J, et al.Identification and control of taro phytophthora blight [J].China Vegetables, 2009(21): 21-22.(in Chinese)
[3]王向社, 李锐, 胡茂松, 等.海南岛芋疫霉菌生物学特性、致病力、对甲霜灵的敏感性研究[J].热带作物学报, 2001, 22(1): 83-90. WANG X S, LI R, HU M S, et al.Study on the biology, virulence of Phytophthora colocasiae Racib and its sensitivity to metalaxyl [J].Chinese Journal of Tropical Crops, 2001, 22(1): 83-90.(in Chinese)
[4]QUITUGUA R J, TRUJILLO E E.Survival of Phytophthora colocasiae in field soil at various temperatures and water matric potentials [J].Plant Disease, 1998, 82(2): 203-207.
[5]刘独臣.四川芋疫病发生规律及防治技术[J].长江蔬菜, 2013, 18: 118-119. LIU D C.Epidemiology and control of taro phytophthora blight in Sichuan [J].Journal of Changjiang Vegetables, 2013, 18: 118-119.(in Chinese)
[6]VASGUEZ E A.Yield loss in taro due to Phytophthora leaf blight [J].Journal of Root Crops, 1990, 16: 48-50.
[7]方辉, 张惠琴, 陈孝赏, 等.双炔酰菌胺防治红香芋疫病的效果及应用技术[J].浙江农业科学, 2016, 57(6): 899 -900,911. FANG H, ZHANG H Q, CHEN X S, et al.Effect and application technology of mandipropamid in control taro phytophthora blight [J].Journal of Zhejiang Agricultural Sciences, 2016, 57(6):899-900, 911.(in Chinese)
[8]莫俊杰, 胡汉桥, 梁钾贤, 等.芋疫病抗病性鉴定及不同品系遗传多样性分析[J].广东海洋大学学报, 2012, 32(4): 67-72. MO J J, HU H Q, LIANG J X, et al.Resistance identification of taro to Phytophthora colocasiae and genetic diversity analysis of Colocasia esculenta [J].Journal of Guangdong Ocean University, 2012, 32(4): 67-72.(in Chinese)
[9]NATH V S, SENTHIL M, HEGDE V M, et al.Evaluation of fungicides on Indian isolates of Phytophthora colocasiae causing leaf blight of taro [J].Archives of Phytopathology and Plant Protection, 2013, 46(5): 548-555.
[10]LIN M J,KO W H.Occurrence of isolates of Phytophthora coloscsiae in Taiwan with homothallic behavior and its significance [J].Mycologia, 2008, 100(5): 727-734.
[11]叶泉清, 钟佳铃, 陈媚, 等.槟榔芋疫霉菌生物学特性、致病力测定及田间防治药剂筛选[J].南方农业学报, 2016, 47(4): 588-593. YE Q Q, ZHONG J L, CHEN M, et al.Biological characteristics, virulence of Phytophthora colocasiae Racib.From Colacasia esculenta L.var. cormosus Chang and fungicides screening for field control [J].Journal of Southern Agriculture, 2016, 47(4): 588-593.(in Chinese)
[12]NATH V S, HEGDE V M, JEEVA M L, et al.Rapid and sensitive detection of Phytophthora colocasiae responsible for the taro blight using conventional and real-time PCR assay [J].FEMS Micobiology Letters, 2014, 352: 174-183.
[13]MARTIN F N, ABAD Z G, BALCI Y, et al.Identification and detection of Phytophthora: reviewing our progress, identifying our needs [J].Plant Disease, 2012, 96(8): 1080-1103.
[14]ROLLINS L, COSTS K, ELLIOTT M, et al.Comparison of five detection and quantification methods for Phytophthora ramorum in stream and irrigation water [J]. Plant Disease, 2016, 100(6): 1202-1211.
[15]王晓杰, 康振生, 黄丽丽.PCR技术在植物病害检测中的应用[J].云南农业大学学报, 2005, 20(2): 179-182. WANG X J, KANG Z S,HUANG L L.Application of PCR technology on the detection of plant disease [J]. Journal of Yunnan Agricultural University, 2005, 20(2): 179-182.(in Chinese)
[16]DRENTH A, WAGELS G, SMITH B, et al.Development of a DNA-based method for detection and identification of Phytophthora species [J].Australasian Plant Pathology, 2005, 35: 147-159.
[17]李依韦, 银玲.rDNA-ITS序列分析在植物病原真菌分类鉴定中的应用[J].内蒙古民族大学学报(自然科学版), 2012, 27(1): 66-67. LI Y W,YIN L.Application of rDNA-ITS sequences in plant disease fungi classification and identification [J].Journal of Inner Mongolia University for Nationalities (Natural Sciences), 2012, 27(1): 66-67.(in Chinese)
[18]傅华英, 葛丹凤, 李晓燕, 等.甘蔗赤条病菌巢式PCR检测[J].植物保护学报, 2017, 44(2): 276-282. FU H Y, GE D F, LI X Y, et al.Nested-PCR detection of Acidovorax avenae subsp.avenae, the pathogen of red stripe on sugarcane [J].Journal of Plant Protection, 2017, 44(2): 276-282.(in Chinese)
[19]KONIG S, SCHWENKBIER L, POLLOK S, et al.Potential of Ypt1 and ITS gene regions for the detection of Phytophthora species in a lab-on-a-chip DNA hybridization array [J].Plant Pathology, 2015, 64: 1176-1189.
[20]LAN C Z, LIU P Q, LI B J, et al.Development of a specific PCR assay for the rapid and sensitive detection of Phytophthora capsici [J].Australasian Plant Pathology, 2013, 42: 379-384.
[21]杨万风, 刘艳, 刘翔, 等.巢式PCR检测菜豆细菌性萎蔫病菌[J].浙江农业学报, 2015, 27(7): 1202-1207. YANG W F, LIU Y, LIU X, et al.Detection of Curtobacterium flaccumfaciens pv.flaccumfaciens using nested PCR [J].Acta Agriculturae Zhejiangensis, 2015, 27(7): 1202-1207.(in Chinese)
[22]ZHANG Z G, LI Y Q, FAN H, et al.Molecular detection of Phytophthora capsici in infected plant tissues, soil and water [J].Plant Pathology, 2006, 55(6): 770-775.
[23]赵杰.ITS序列分析及其在植物真菌病害分子检测中的应用[J].陕西农业科学, 2004(4): 35-37. ZHAO J.ITS sequence analysis and its application in molecular detection of plant fungal diseases [J].Shanxi Journal of Agricultural Sciences, 2004(4): 35-37.(in Chinese)
[24]SCHENA L, COOKE D E L.Assessing the potential of regions of the nuclear and mitochondrial genome to develop a “molecular tool box” for the detection and characterization of Phytophthora species [J].Journal of Microbiological Methods, 2006, 67: 70-85.
[25]VOLOSSIOUK T, ROBB E J, NAZAR R N.Direct DNA extraction for PCR-mediated assays of soil organisms [J].Applied and Enviromental Microbiology, 1995,61(11): 3972-3976.