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

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.