【作者】 任海英； 戚行江； 梁森苗； 郑锡良；

【Author】 REN Hai-ying;QI Xing-jiang;LIANG Sen-miao;ZHENG Xi-liang;Institute of Horticulture,Zhejiang Academy of Agricultural Sciences;

【机构】 浙江省农业科学院园艺研究所；

【摘要】 凋萎病是近年来危害杨梅的主要病害。为了快速灵敏的检测杨梅凋萎病菌(Pestalotiopsis versicolor和P.microspora),本研究开发了常规PCR和SYBR Green实时荧光定量PCR技术各一套。利用P.versicolor(JN861773)和P.microspora(JN861776)的ITS1-5.8S rRNAITS2序列的相同部分设计引物对(Pvm1L/Pvm1R)。该引物对利用常规PCR技术能特异性扩增出杨梅凋萎病菌的188 bp目标产物,而对照菌株则呈阴性。该常规PCR体系能够检测人工接种后21天和田间自然发病的有病症杨梅组织中的凋萎病菌,检测下限是0.6×10~5拷贝数。利用Pvm1L/Pvm1R进行SYBR Green实时荧光定量PCR,检测灵敏度是常规PCR的100倍,检测下限是0.6×10~3拷贝数,能够检测出人工接种及田间已经感染但是尚未表现症状的杨梅组织中的凋萎病菌。这两项技术,简单、快速、灵敏,特异性强,可以应用于杨梅凋萎病的诊断和苗木检疫。更多还原

【Abstract】 Twig blight disease is one of the main diseases of bayberry(Myrica rubra Sieb.& Zucc).To develop an effective method to detect and quantify the pathogen,we used conventional PCR and SYBR Green real-time PCR assays for Pestalotiopsis versicolor and P.microspora.A conventional primer set(PvmlL and PvmlR) was designed from a common sequence of the internal transcribed spacer(ITS1-5.8S rRNA-ITS2) region of the ribosomal RNA gene of P.versicolor(JN861773) and P.microspora(JZV861776).The 188 bp segments of DNA were amplified from 30 isolates of P.versicolor and 30 isolates of P.microspora.No product was amplified from isolates of 11 other fungal genera.Conventional PCR was able to detect the pathogens on symptomatic,artificially infected bayberry plants 21 days after inoculation.The limit of detection was 0.6 x 105 copies of Pestalotiopsis DNA.In addition,the Pvm1 L and Pvm1 R primers were successfully adapted to SYBR Green real-time PCR and compared with conventional PCR.Real-time PCR had a limit 100 times lower(0.6 x 10) and was able to detect the pathogens in symptomless,artificially inoculated and naturally infected plants.The conventional and SYBR Green real-time PCR developed in this study were simple,fast,sensitive,and specific.Either can be used to detect Pestalotiopsis spp.from infected bayberry in the field.更多还原