PCR-RFLP Identification of Three Major Meloidogyne Species in Korea

Partial mitochondrial DNA from single female or second stage juvenile (J2) of root-knot nematodes was amplified by PCR (polymerase chain reaction), and the further analysis by RFLP (restriction fragment length polymorphism) provided discriminatory profiles useful for three major Meloidogyne species, M arenaria, M incognita, and M hapla, in Korea. The nematode DNA was extracted individually in nuclease-free water. The sizes of PCR product (1.7 kb and 500 bp) and restriction patterns obtained from single female nematodes were consistent with the results from single J2 within the same species. M. hapla was easily differentiated from the two other root-knot nematode species by the size of the PCR products. A fragment of 500 bp was generated from M. hapla, while M. arenaria, and M. incognita produced an 1.7 kb fragment in PCR amplification. M arenaria could be distinguished from M. incognita by analysis of restriction enzyme digestion with HinfI or Alu I. Hinf I had no digestion site in mitochondrial DNA of M. arenaria; however, it generated 1.3 kb and 400 bp fragments in M. incognita. Alu I digestion resulted in 1 kb, 460 bp, and 250 bp fragments in M. arenaria, but showed different digestion patterns in M. incognita by generating 800 bp, 460 bp, 250 bp, and 150 bp fragments.