落叶松八齿小蠹伴生致病菌的特异性快速检测

Specific and rapid detection of the pathogenic fungi associated with Ips subelongatus

落叶松八齿小蠹Ips subelongatus严重危害落叶松Larix spp.林,造成了巨大的经济和生态损失,被列为中国林业危险性有害生物。长喙壳类真菌(ophiostomatoid fungi)是小蠹虫伴生菌中的最主要类群,在小蠹虫危害寄主甚至导致寄主死亡的过程中起到了重要作用。一些长喙壳类真菌是重要的植物病原物,具有强致病力。实现病原菌的特异性和快速检测是明确病害分布和评估其危害的前提条件。为克服传统组织分离受多种因素制约的缺陷,对2种病原菌进行快速特异性聚合酶链式反应(PCR)检测,以内源转录间隔区(ITS)为目标序列,对比Endoconidiophora属和Ophiostoma属的14个近缘种,分别筛选出可以从目标真菌类群以及寄主组织中扩增出特异性片段的种特异性(SS-)PCR引物对OK1/OK2和CFU1/CFU2。引物对OK1/OK2可以从O.olgensis中特异性扩增出1条248 bp的清晰明亮条带,引物对CFU1/CFU2可以从E.fujiensis中特异性扩增出1条251 bp的清晰明亮条带。使用特异性引物能够直接从感病韧皮部组织中检测出O.olgensis和E.fujiensis。

Ips subelongatus, a native bark beetle categorized as a dangerous forest pest in China having caused huge losses to larch forests, and ophiostomatoid fungi, important plant pathogens with strong pathogenicity which numerous studies have shown to be the main group of fungi associated with bark beetles, have played a key role in the process of beetle damage, sometimes killing tree hosts. This study on specific and rapid detection of pathogens, a prerequisite for clarifying the distribution of diseases and assessing their hazards, was designed to overcome the shortcomings of traditional tissue isolation, which normally influenced by a variety of anthropological factors. Two pathogens Ophiostoma olgensis and Endoconidiophora fujiensis were specifically detected by polymerase chain reaction（PCR）. Internal transcribed spacer（ITS） sequences were used as a target to design species specific（SS-） PCR primers through comparison of 14 sibling species from the genera Endoconidiophora and Ophiostoma. Results showed that two pairs of primers（OK1/OK2 and CFU1/CFU2）, which could amplify specific sequences from the fungal flora and host tissues, were screened out. Primer pairs OK1/OK2 and CFU1/CFU2 specifically amplified a clear and bright band of 248 bp from O. olgensis and 251 bp from E. fujiensis. Thus, O. olgensis and E. fujiensis were detected by the SS-PCR from phloem tissues in which the two fungi had been previously isolated.