Real-Time PCR as a Tool for Detection and Identification of Bursaphelenchus xylophilus and B. mucronatus Based on Trace Amounts of Their DNA Left in the Vector—The Pine Sawyer Beetle, Monochamus galloprovincialis

The quarantine nematode Bursaphelenchus xylophilus is a causative agent of the pine wilt disease. In Europe, the nematode is vectored by Monochamus galloprovincialis which is also a natural vector of the native, nonpathogenic B. mucronatus. Based on the established regulations, long-term detection surveys of pine wood and vector insects for the presence of B. xylophilus are obligatory in all EU member countries. The main objective of the presented research was to use the real-time PCR technique for the fast and precise detection, and identification of Bursaphelenchus nematodes associated with M. galloprovincialis ssp. pistor occurring in Poland. The research conducted on beetles collected in forest stands in Poland revealed exclusive presence of the native, nonpathogenic nematode B. mucronatus. At the beginning of the beetle flight season (June–July), the use of the real-time PCR allowed detection of B. mucronatus, in the case of its physical presence in the body of the dissected M. galloprovincialis. However, at the end of the flight season (September–October), the DNA of B. mucronatus was also found in some beetles with no physical presence of nematodes in tracheas or under elytra. The univoltine biology of the beetle, gradual decline in dispersal juveniles to zero in/on the beetle body, and positive results of real-time PCR reaction could indicate that in some of the beetles earlier loaded with nematodes, at the end of the season, only their remains were left after the live nematodes' departure during the vector's maturation feeding and/or egg laying. The obtained results confirm extremely high sensitivity of the real-time PCR method used in detection and identification of both nematode species which may simultaneously occur in M. galloprovincialis beetles. It allows for precise detection of the nematodes based on their remains left in/on the vector insects.