Development of PrimeTime-Real-Time PCR for Species Identi?cation of Soybean Cyst Nematode (Heterodera glycines Ichinohe, 1952) in North Carolina

摘要

Soybean cyst nematode (SCN) is an obligate, sedentary parasite that is a major pathogen of soybean and accounts for an estimated 1 billion dollars in production losses annually in the United States of America.This paper describes the development of a real-time PCR method for rapid, sensitive, species-speci?c and accurate identi?cation of SCN alone or on mixed populations with other nematodes in North Carolina.The 83-bp DNA fragment of PrimeTime-real-time PCR was designed based on a 477-bpSCN-SCAR marker previously proved to be SCN-speci?c.A total of 44 populations including cyst forming nematodes (Heterodera glycines, H.?ci, H.schachtii, H.trifolii, Cactodera weissi, Globodera tabacum, Meloidodera ?oridensis and other unidenti?ed cyst nematodes) and non-cyst forming nematodes (Ditylenchus dipsaci, Meloidogyne incognita and Xiphinema chambersi) were tested in this study, all SCN populations are tested positive and non-SCN populations negative.This assay for the detection and identi?cation has been successfully applied for testing a single SCN cyst, a 2nd-stage-SCN juvenile, a single SCN egg, up to ten SCN cysts, a 10-fold dilution of a single 2nd-stage-SCN juvenile and 20-fold dilution of one SCN cyst.The assay is not SCN-race speci?c.It gave an accurate positive result when SCN is mixed with other cyst species.Also, nematode universal primers/probes for real-time PCR ampli?cation as a nematode endogenous control to detect the presence of 18S ribosomal RNA (rRNA) gene were employed in this assay, so that a SCN-negativesamplecanbe testedto excludefalsenegative.Thismethodwillbeveryusefulforabroadrangeof researchprograms as well as the regulatory response and management of SCN in North Carolina and other region of the southeastern U.S.A.