Identification of Pratylenchus penetrans (COBB) by PCR Using ITS-Based Species-Specific Primers

Abstract

For identifying Prutylencus penetrans, species-specific primers (PP1 and PP2) were developed, based on the sequences of the interscribed spacer regions (ITS1 and ITS2) of the rDNA. When the purified DNA from seven Pratylencl7us species was subjected to PCR using these priiners, the 462 bp fragment was generated only for P. penetrans. The same fragment was obtained with DNA from the crude lysate of single juvenile and of adult (male and female) P. penetrai~s. Jpn. J. New~atol. 28 (1/2). 1-7 (1998). Key words: identification, ITS-rDNA, PCR, Pratylencl7f~ps ck7ctrans. Among al1 Pratylencl~us species inhabiting in Japan (191, P. penetrans is one of the most important root lesion nematodes, and damages roots of many plants (5, 7 ) . Namely, it is a major pest of radish, carrot, burdock, lettuce, butterbur, and strawberry. The nematode-induced primary damage is a death of plant root cells around the nematodes, leading to a local lesion formation. Stunting, chlorosis, root pruning, and sparseness are successive symptoms (12). For a successful management of nematode pests, a rapid and reliable identification of the causa1 species is critically important. Although a nematode identification is prin~arilyb ased on morphological characteristics, it is difficult even for taxonomists due to variations in diagnostic features. Isozyme analysis has been used to distinguish some Pratylenchus species ( I l ) , but it requires a large arnount of nematode homogenate. Therefore, it is not suitable for nematode identificati011 at organismic level. Using species-specific DNA probes, dot-blot analysis has been used to identify such nematodes as Globodera (21, Meloidogyne (1, 16) and Bz~rsaphelenchzb~u, t it is timeconsuming and requires radioactive detection facilities. The development of polymerase chain reaction (PCR) technology opened a way to a plant pathogen diagnosis (8). In this paper, we report newly developed P. penetrnizs-specific priiners which based on the interna1 transcribed spacer (ITS) regioris of the rDNA. 'Laboratory of Plant Nematology, Hokkaido National Hgricultural Experiment Station, Hitsuiigaoka-l, Toyohira-ku, Sapporo 062-8555, Japan. ZLaboratory of Plant Nematology, Kyushu National Agricultural Experiment Station, Suya 2412, Nishigoshi, Kikuchi-gun, Kumamoto, 861-1192, Japan. MATERIALS AND METHODS Nemntode Pratylenchus penetrnns, P. coffene, P. v~~lnuPs.. loosi, P. brachyurus, P. zeae, and P. crenatus were used. These nematodes were isolated from different geographical locations and hosts (Table 1). They were monoxenically maintained on alfalfa callus on Krusberg medium (15) and active nematodes were harvested by the Baerniann-funnel method. DNA extractio~z Nematodes were crushed with mortar and pestle and then incubated at 37°C for 3 h in 200 p1 of extraction buffer (10 mM Tris -HCl: pH 8.0, 10 mM EDTA, 100 mM NaCl, 0.5% SDS, and 200 pg/ml proteinase K). The lysate thus obtained was extracted with phenol/chloroform/isoamyl alcohol (25: 24: 1) (17). Nucleic acids were precipitated with ethanol and then dissolved in TE buffer (10 mM Tris-HC1: pH 8.0, 1 mM EDTA). The crude lysates were also prepared by crushing a single juvenile or adult nematode