Enhanced Emission of Monoterpene 3-Carene in Pinus densiflora Infected by Pine Wood Nematode and Characterization of 3-Carene Synthase

Abstract

Pinus densiflora is an important pine species in Northeast Asia due to its historical, cultural, and economic values. Pine wood nematode (PWN) seriously damages P. densiflora, causing the pine wilt disease (PWD). Changes of phyto-compounds in resin and monoterpenes in P. densiflora after infection of PWN were studied. The changes were identified by GC-MS in control and infected P. densiflora. Among the resin phytochemicals (in P. densiflora), 3-carene was distinctly enhanced after PWN inoculation. The emitted plant volatile monoterpenes were analyzed by HS-SPME/GC-MS. It was observed that the amount of 3-carene enhanced conspicuously after infection of PWNs in both P. densiflora and P. koraiensis at 9.7 and 54.7 times, respectively. 3-Carene synthase gene (Pd3-cars) of P. densiflora was isolated and functionally characterized by transgenic tobacco overexpressing Pd3-cars. Integration and expression of transgenic tobacco were confirmed by genomic and RT-PCR analysis. The Pd3-cars gene was expressed in transgenic tobacco plants. Furthermore, the production of 3-carene was identified by HS-SPME/GC-MS analysis as the volatile compounds emitted from leaves of transgenic tobacco. Treatment of 3-carene to PWNs showed a mild nematicidal activity with 45.98% mortality at the concentration of 10 mg/mL. The current findings may apply to the early diagnosis of pine wilt disease infected by PWNs through enhanced emission of 3-carene.