Nematology, 2001, Vol. 3(4), 365-371 A rapid method for the identication of the soybean cyst nematode Heterodera glycines using duplex PCR Sergei A. SUBBOTIN 1;¤ , Deliang PENG 2 and Maurice MOENS 3 1 Institute of Parasitology of Russian Academy of Sciences, Leninskii prospect 33, Moscow, 117071, Russia 2 Institute of Plant Protection of Chinese Academy of Agricultural Sciences, Beijing 100094, China 3 Agricultural Research Centre, Crop Protection Department, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium Received: 1 July 2000; revised: 1 February 2001 Accepted for publication: 6 February 2001 Summary – A method for rapid identication of juveniles and cysts of the soybean cyst nematode based on PCR with species specic primers is described. The PCR assay was tested on 53 populations originating from China, Russia, USA and Brazil. A single cyst or second stage juvenile of Heterodera glycines alone or in a mixture with other soil inhabiting nematodes was detectable. Keywords – cysts, second stage juveniles, species specic primers. The soybean cyst nematode Heterodera glycines Ichi- nohe, 1952 is a major pest on soybeans in China, the USA and the Russian Far East. The species is also re- ported from Argentina, Brazil, Canada, Colombia, Egypt, Indonesia, Japan and Korea (Noel, 1985; Eroshenko et al., 1990; Baldwin & Mundo-Ocampo, 1991; Liu et al., 1997; Evans & Rowe, 1998). H. glycines belongs to the schachtii group which includes species only differing in minor morphological and morphometrical characters (Graney & Miller, 1982; Sikora & Maas, 1985). Identi- cation of these species requires considerable skill and is time consuming and difcult, even for taxonomists. In practice, cyst forming nematodes are assumed to be H. glycines if found infesting a eld with a soybean history (Riggs & Niblack, 1993). DNA-based diagnostics provide an attractive solution to problems associated with identication. Molecular probes developed for detection of H. glycines (Besal et al., 1988) are not yet widely available (Riggs & Niblack, 1993). Moreover, this approach has several limitations for routine diagnostics because it requires both a large amount of DNA and some technical skill. The develop- ment of the polymerase chain reaction (PCR) technology has opened new opportunities in nematode diagnostics. Restriction fragment length polymorphism (RFLP) analy- ses of the internal transcribed spacers (ITS) of the ribo- * Corresponding author, e-mail: s.subbotin@clo.fgov.be Present address: Agricultural Research Centre, Crop Protection Department, Burg. van Gansborghelaan 96, 9820 Merelbeke, Belgium. somal DNA (rDNA) became a popular tool for identi- cation of cyst forming nematode species (Thiéry & Mug- niéry, 1996; Bekal et al., 1997; Orui, 1997; Subbotin et al., 1997, 1999, 2000; Szalanski et al., 1997). It has been shown that digestion of the PCR product obtained after amplication of the ITS1-5.8S-ITS2 region with differ- ent combinations of restriction enzymes allows clear sep- aration of most cyst forming nematode species from each other. H. glycines can be distinguished from other species (H. ciceri, H. medicaginis, H. schachtii and H. trifolii ) of the schachtii sensu stricto group by the restriction enzyme AvaI (Subbotin et al., 2000; Zheng et al., 2000). PCR with specic primer combinations or multiplex PCR constitutes a major development in DNA diagnos- tics and allow the detection of one or several species in a mixture by a single PCR test, decreasing diagnostic time and costs. This technology has found wide application in medicine for diagnosis of infective and genetic diseases, and in plant pathology for the diagnosis of fungal (Bridge et al., 1998) and bacterial pathogens (Louws et al., 1999). In nematology, this diagnostic tool has been developed for identication of Globodera pallida and G. rostochiensis (Mulholland et al. 1996; Bulman & Marshall, 1997; Ful- laondo et al., 1999), Pratylenchus penetrans and P. scrib- neri (Setterquist et al., 1996), Meloidogyne chitwoodi and M. fallax (Petersen et al., 1997), M. chitwoodi and c ° Koninklijke Brill NV, Leiden, 2001 365