Two kinds of applied molecular skills to detect Bursaphelenchus xylophilus

In order to determine the reliability of two methods, SCAR marker and real-time PCR, developed for detecting pine wood nematode, seven pine wood isolates and three identified nematode strains (Bursaphelenchus xylophilus, B. mucronatus and Aphelenchoides macronucleatus) were assayed with the two methods. The traditional method of morphological identification was also used to determine the seven isolates samples. Results showed that 1) with the method of SCAR marker, isolates 2, 3, 4 and 7 showed a common specific electrophoretic band of 860 bp, as did by pine wood nematode B. xylophilus, whereas isolates 1, 5 and 6, and B. mucronatus and Aphelenchoides macronucleatus did not produce the specific band. That suggests that isolates 2, 3, 4 and 7 contained B. xylophilus, while isolates 1, 5 and 6 did not. 2) With the method of real-time PCR identification, fluorescent signal was detected from samples prepared from isolate 2, 3, 4, 7 and strain B. xylophilus, on the contrary, the rest isolates showed no signals and cycle threshold (Ct values). 3) With morphological identification, it was revealed that isolate 2, 4 and 7 contained B. xylophilus while isolate 3 contained B. xylophilus as well as other nematodes. However, isolate 1, 5 and 6 did not contain B. xylophilus. Therefore, the two molecular methods showed a consistent result with morphological identification. Moreover, the two methods are highly specific and time-saving. SCAR marker method costs two hours to perform, while real-time PCR method takes only one hour. Both techniques make it possible to determine rapidly existence of juvenile pine wood nematodes with a simple and easily readable result.