Discrimination of Bursaphelenchus xylophilus and Bursaphelencus mucronatus by PCR-RFLP technique

Abstract

A polymerase chain reaction restriction fragment length polymorphism analysis was used to discriminate isolates of Bursaphelenchus xylophilus and B. mucronatus. The amplifications of B. xylophilus isolates yielded one fragment of approximately 890 bp and that of B. mucronatus was about 930 bp. Digestion of amplified products of each nematode isolate with five restriction endonucleases revealed the following results: 1) Dra I digestion of the internal transcribed spacer (ITS) products of B. xylophilus populations yielded two fragments of 510 and 380 bp. Dra I could not digest the ITS products of B. mucronatus populations; 2) Sal I could not digest the ITS products of all B. xylophilus populations, but it could digest those of B. mucronatus populations into two fragments, which were 720 and 220 bp; 3) digested products of four B. xylophilus populations by Msp I yielded two fragments of 530 and 360 bp, except GZ02, which could not be digested. B. mucronatus populations yielded three fragments: 340, 290, and 180 bp; 4) all populations of B. xylophilus and B. mucronatus could not be digested by Apa I; 5) digestion of the ITS products of B. xylophilus and B. mucronatus yielded two fragments of 520 and 370 bp, and 530 and 400 bp respectively. The restriction endonucleases Dra I and Sal I could be used to identify B. xylophilus and B. mucronatus. Because the results of digestion of B. xylophilus and B. mucronatus were markedly different, they were very easy to be identified and applied; Msp I and Xho I were not suitable for identification of B. xylophilus and B. mucronatus and Apa I could not identify and distinguish between B. xylophilus and B. mucronatus.