桉树焦枯病菌巢式聚合酶链反应快速检测方法的建立与应用  

Development and application of nested polymerase chain reaction for rapid detection of Cylindrocladium scoparium on Eucalyptus

作　　者：谯天敏[1] 张静[1] 麻文建 朱天辉[1] 郑磊[1] 

机构地区：[1]四川农业大学林学院,四川雅安625014

出　　处：《浙江大学学报（农业与生命科学版）》2015年第5期497-504,共8页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家科技基础条件平台基金(2005DK21207-13)

摘　　要：桉树焦枯病是威胁桉树生长的首要病害,建立准确、有效的桉树焦枯病巢式聚合酶链反应(polymerase chain reaction,PCR)快速检测技术是桉树焦枯病前期诊断的必要手段。本研究针对柱枝双孢霉(Cylindrocladium scoparium)菌株的β-微管蛋白(beta-tubulin)基因上保守序列极强的靶基因区域序列进行特异性引物BT-S-1/BTA-1的设计和通用引物BT-T1-S/BT-CYLTUBIR-A的合成,分别利用常规PCR和巢式PCR技术对引物特异性和灵敏度进行检测和比较,同时本研究也对所建立的巢式PCR用于桉树焦枯病原菌快速检测的田间时效性进行验证。试验结果表明:利用beta-tubulin基因序列通用引物BT-T1-S/BT-CYLTUBIR-A对全部供试菌株进行扩增,所有参试菌株均可扩增出1条约500bp的条带;而单独使用特异性引物BT-S-1/BT-A-1进行常规PCR扩增时仅病原菌能够扩增出1条148bp的明亮条带;当利用通用引物作为第1轮引物,以稀释10倍后的第1轮PCR产物作为第2轮PCR模板,利用特异性引物进行巢式PCR扩增时,也可扩增出上述148bp大小的明亮条带,且巢式PCR的扩增效果较常规PCR具有明显的视觉优越性;灵敏度检测试验表明,巢式PCR的灵敏度可检测到5fg/μL,较常规PCR可以扩增出的极限(DNA质量浓度为5pg/μL)至少提高了103倍;田间时效检测试验也说明巢式PCR较常规PCR具有更高的准确度和灵敏性,可达到田间检测的要求。本研究建立的巢式PCR检测技术可有效应用于桉树焦枯病的早期诊断。Eucalyptus,as one of the most important components of the forestry resources,was originated in Australia,and then was cultivated worldwide including China.Prior to the early twentieth century,with the increasing introduction of this tree species,Eucalyptus dieback,caused by Cylindrocladium scoparium（teleomorph：Calonectria morganii）,was frequently affected in almost regions of China.Also it has historically been an important disease of Eucalyptus,which was defined as a key national plant quarantine disease in China,on January 5,1996.In Southern China,this disease affected Eucalyptus throughout its range of cultivation and caused severe yield loss and tree mortality.Cylindrocladium scoparium,especially in late infection,can cause a large damage to the growth of Eucalyptus,as well as dieback,even all branches withered severely.Even in early infection,it can easily lead to reddish-brown spots,wilting leafs,and droping branches.However,thus far,it is still a great constraint to the cultivation and production expansion of Eucalyptus in many provinces of China,such as Sichuan,Fujian,and Guangxi.Therefore,to establish a rapid and effective detection technology has an important significance for control and prevention of Eucalyptus dieback.A total of 19 isolates of fungus,including C.scoparium were obtained from diseased Eucalyptus in Sichuan Province,and three isolates（C.scoparium and Cylindrocladium ilicicola）were purchased from Chinese academy of Forestry.Based on its conserved domain beta-tubulin gene,two pairs of specific primers BT-S-1/BT-A-1and universal primers BT-T1-S/BT-CYLTUBIR-A were severally designed by the aid of the software Premier 5.0,and then were synthesized respectively,and the specificities and sensitivities of these two pairs were tested with the method of universal polymerase chain reaction（PCR）and nested-PCR.Meanwhile,the field tests were conducted.The results showed that a band of 500 bp with the universal primers BT-T1-S/BT-CYLTUBIR-A was amplified among these isolates,while only a

关 键 词：巢式聚合酶链反应 桉树焦枯病 快速检测 丽赤壳属 柱枝双孢霉 

分 类 号：Q939.5[生物学—微生物学] S432.4[农业科学—植物病理学]