Conversion of sequence-characterized amplified region (SCAR) bands into high-throughput DNA markers based on RAPD technique for detection of the stem nematode Ditylenchus dipsaci in crucial plant hosts

Abstract

Ditylenchus dipsaci, the stem nematode, is a migratory endoparasite of over 500 species of angiosperms. The main method of D. dipsaci control is crop rotation, but the presence of morphologically indistinguishable host races with different host preferences makes rotation generally ineffective. Therefore, a sensitive, rapid, reliable, as well as cost effective technique is needed for identification of D, dipsaci in biological samples. This study describes the development of species-specific pairs of PCR oligonucleotides for detection and identification of the D. dipsaci stem nematode in various plant hosts. Designed DIT-2 primer pair specifically amplified a fragment of 325 bp, while DIT-5 primer pair always produced a fragment of 245 bp in all D. dipsaci isolates. Two developed SCAR primer pairs were further tested using template DNA extracted from a collection of twelve healthy plant hosts; no amplification was however observed. The developed PCR protocol has proved to be quite sensitive and able to specifically detect D. dipsaci in artificially infested plant tissues.