Use of Loop-Mediated Isothermal Amplification for Detection of Ophiostoma clavatum, the Primary Blue Stain Fungus Associated with Ips acuminatus

ABSTRACTLoop-mediated isothermal amplification (LAMP) is an alternative amplification technology which is highly sensitive and less time-consuming than conventional PCR-based methods. Three LAMP assays were developed, two for detection of species of symbiotic blue stain fungi associated withIps acuminatus, a bark beetle infesting Scots pine (Pinus sylvestris), and an additional assay specific toI. acuminatusitself for use as a control. In common with most bark beetles,I. acuminatusis associated with phytopathogenic blue stain fungi involved in the process of exhausting tree defenses, which is a necessary step for the colonization of the plant by the insect. However, the identity of the main blue stain fungus vectored byI. acuminatuswas still uncertain, as well as its frequency of association withI. acuminatusunder outbreak and non-outbreak conditions. In this study, we employed LAMP technology to survey six populations ofI. acuminatussampled from the Southern Alps.Ophiostoma clavatumwas detected at all sampling sites, whileOphiostoma brunneo-ciliatum, reported in part of the literature as the main blue stain fungus associated withI. acuminatus, was not detected on any of the samples. These results are consistent with the hypothesis thatO. clavatumis the main blue stain fungus associated withI. acuminatusin the Southern Alps. The method developed in the course of this work provides a molecular tool by which it will be easy to screen populations and derive important data regarding the ecology of the species involved.