Fusarium graminearum sensu stricto和F.asiaticum 3种分子鉴定方法的比较

摘要

Fusarium graminearum sensu stricto和F.asiaticum是中国主要的禾谷镰刀菌复合种(FGSC)成员,为了提高这2个种的鉴定效率,选取48个引起小麦赤霉病和玉米茎腐病的FGSC菌株,比较了系统发育谱系分析、PCR-RFLP(聚合酶链式反应-限制性片段长度多态性)和多重PCR 3种分子鉴定方法.系统发育谱系分析首先扩增菌株的EF-1α和Tri101两个基因的部分序列,测序之后利用MEGA 6.06软件构建系统发育树,结果表明,有11个菌株与F.asiaticum聚在一起,37个菌株与F.graminearum sensu stricto聚为一枝,此法繁琐、复杂、成本高,但鉴定结果准确.PCR-RFLP基于组蛋白H3基因特殊的酶切位点,设计引物H3dStyI/H3R1,通过PCR和酶切反应进行鉴定,电泳检测发现,有11个菌株为F.asiaticum,37个菌株为F.graminearum sensu stricto,该法工作量大且对操作过程要求高.多重PCR基于CYP51A基因设计特异引物FaF/FaR和FgF/FgR进行鉴定,电泳检测结果表明,有11个菌株为F.asiaticum,37个菌株为F.graminearum sensu stricto,此法操作简单、效率高.以上3种分子鉴定方法结果一致,但多重PCR方法具有快速、准确、高效的特点.%Fusarium graminearum sensu stricto and F.asiaticum are major members of Fusarium graminearum species complex(FGSC) in China.In this study,to improve the identification efficiency of these two species,48 members of FGSC collected from plants showing Fusarium head blight (FHB) or maize stalk rot(MSR) were identified using three molecular identification methods.One method was amplification of partial sequences of two genes EF-1α and Tri101,and then sequencing and phylogenetic analyses by MEGA 6.06 software.The results showed that 11 isolates were clustered together with F.asiaticum and 37 isolates were clustered together with F.graminearum sensu stricto.This method was cumbersome,complex and costly,but the identification results were accurate.A second method was polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).Based on the specific restriction sites of histone H3 gene,primers H3dSty Ⅰ and H3R1 were designed.11 isolates were identified as F.asiaticum and 37 isolates were F.graminearum sensu stricto.This method required lots of work and high operation process.The third method was a multiplex PCR.Based on the CYP51A gene,primers FaF/FaR and FgF/FgR were designed.11 isolates were identified as F.asiaticum and 37 isolates were F.graminearum sensu stricto.This method was simple and efficient.These results were similar,while the multiplex PCR method was more rapid,accurate and efficient.