J. Phytopathology 152, 229–234 (2004) Ó 2004 Blackwell Verlag, Berlin ISSN 0931-1785 Crop Protection Department, Agricultural Research Centre, Merelbeke, Belgium Molecular Characterization of Cyst Nematode Species (Heterodera spp.) from the Mediterranean Basin using RFLPs and Sequences of ITS-rDNA M.Madani 1, N.Vovlas 2, P.Castillo 3, S. A.Subbotin 4 and M.Moens 1,51,5 AuthorsÕ addresses: 1Crop Protection Department, Agricultural Research Centre, Burg, Van Gansberghelaan 96, 9820 Merelbeke, Belgium; 2Istituto per la Protezione delle Piante, Sezione di Bari: Nematologia Agraria, Consiglio Nazionale delle Ricerche (C.N.R.), Via G. Amendola 165/A, 70126 Bari, Italy; 3Instituto de Agricultura Sostenible, Consejo Superior de Investigaciones Cientificas (C.S.I.C.), Apdo. 4084, 14080 Cordoba, Spain; 4Institute of Parasitology of the Russian Academy of Sciences, Leninskii Prospect 33, Moscow 119071, Russia; 5University of Gent, Laboratory for Agrozoology, Coupure 555, 9000 Gent, Belgium (correspondence to S. A. Subbotin. E-mail: [email protected]) With 2 figures Received August 6, 2003; accepted February 11, 2004 Keywords: Heterodera carotae, Heterodera ciceri, Heterodera fici, Heterodera filipjevi, Heterodera goettingiana, Heterodera hordecalis, Heterodera humuli, Heterodera mediterranea, Heterodera ripae, Heterodera schachtii, internal transcribed spacer region, phylogenetic relationships, molecular identification Abstract plant growth and yield may be suppressed (Baldwin Fifteen populations of cyst-forming nematodes belong- and Mundo Ocampo, 1991). ing to 11 known and one unidentified species collected Currently, the genus Heterodera contains more than in countries bordering the Mediterranean Sea were 60 species (Wouts and Baldwin, 1998). In the Mediterra- studied using polymerase chain reaction restriction nean Basin several cyst nematode species have been fragment length polymorphism (PCR–RFLP) and reported on herbaceous and woody plants (Greco and internal transcribed spacer (ITS)-rDNA sequences. Brandonisio, 1981; Di Vito and Inserra, 1982; Vovlas RFLP profiles generated by the restriction enzymes et al., 1985; Ferris and Greco, 1992; Castillo et al., AluI, AvaI, Bsh1236I, HaeIII, Hin6I, MvaI, PstI and 1999). These species belong to several taxonomic groups RsaI are presented for Heterodera carotae, H. ciceri, within the genus Heterodera: Avenae group (H. filipjevi, H. fici, H. filipjevi, H. goettingiana, H. hordecalis, H. hordecalis, H. latipons); Goettingiana group (H. caro- H. humuli, H. mediterranea, H. ripae and H. schachtii. tae, H. goettingiana, H. mediterranea); Humuli group Molecular data support the first detection of H. filipjevi (H. humuli, H. ripae); and Schachtii group (H. ciceri, H. from wheat in Italy and H. ripae from nettle in fici, H. schachtii). Some of these species have been dem- Greece. A relative high level of sequence divergence onstrated to be highly pathogenic to legumes (Greco between populations of H. hordecalis was observed. et al., 1988, 1991), wheat (Greco and Brandonisio, This suggests that two species might presently be 1987), sugar beet (Greco et al., 1982), carrot (Greco grouped under this taxon. The phylogenetic relation- et al., 1993), fig seedlings (Di Vito and Inserra, 1982) ship between the Mediterranean cyst-forming nema- and olive planting stocks (Castillo and Vovlas, 2002). tode species is analysed based on the ITS-rDNA Within each Heterodera group, only minor morpho- sequences. logical and morphometrical differences can distinguish all of these species from each other. Identification Introduction based on morphology and morphometrics of cysts and Cyst-forming nematodes are highly specialized and second-stage juveniles is time-consuming and requires economically important soil-borne parasites attacking a lot of skill. However, accurate identification and numerous agricultural crops worldwide. Injury to pathogenic characterization of Heterodera spp. attack- crops by these nematodes is probably second in ing crops in the Mediterranean Basin are needed as an importance to damage caused by root-knot nematodes. initial step in designing effective control measures. This Parasitism of cyst nematodes is characterized by the is especially important when searching for possible establishment of permanent feeding sites (syncitia) in sources of host-plant resistance against Heterodera spe- the cortex, endodermis, pericycle and vascular paren- cies (Cook and Noel, 2002). In addition, rapid and chyma of the host tissues, acting as a metabolic sink accurate identification is highly significant for quaran- to which photosynthesis are mobilized. Consequently tine purposes of important crop plants. U. S. Copyright Clearance Centre Code Statement: 0931–1785/2004/1524–0229 $ 15.00/0 www.blackwell-synergy.com 230 Madani et al. The internal transcribed spacer region (ITS) of the Baldwin, 1998). Heterodera latipons from Rostov ribosomal DNA repeat unit is a good candidate for region, Russia (Ferris et al., 1999) was re-sequenced molecular taxonomic and phylogenetic studies. The ri- and also included in this study. bosomal genes flanking this region are highly con- served allowing the construction of primers that enable DNA extraction, PCR, RFLP and sequencing polymerase chain reaction (PCR) amplification of the The total DNA from one or several cysts was extrac- highly variable ITS region between them. Sequence ted using proteinase K in the worm lysis buffer as des- variation in this region yields restriction fragment cribed by Subbotin et al. (2000). The ITS region of length polymorphisms (RFLPs), which can be used for rDNA was amplified by PCR with the forward TW81 taxonomic goals. Developed during recent years, primer (3¢-GTTTCCGTAGGTGAACCTGC-5¢) and PCR-ITS-rDNA diagnostics are a reliable tool for a reverse AB28 primer (3¢-ATATGCTTAAGTTCAGC- precise and quick identification of cyst nematodes. GGGT-5¢). Two to eight microlitres of the PCR prod- Comparisons of RFLP profiles and sequences of the uct was digested by one of the following restriction ITS-rDNA of unknown nematodes with those pub- enzymes: AluI, AvaI, Bsh1236I, HaeIII, CfoI, MvaI, lished or deposited in GenBank (Ferris et al., 1994; RsaI and PstI in the buffer stipulated by the manufac- Thie´ ry and Mugnie´ ry, 1996; Orui, 1997; Szalanski turer. The digested DNA was run on a 1.5% TAE et al., 1997; Subbotin et al., 1999, 2000, 2001; Sabo buffered agarose gel, stained with ethidium bromide et al., 2001; Tanha Maafi et al., 2003) facilitate quick and photographed and analysed using Kodak Scientific identification of most species of cyst nematodes. Imaging System (Rochester, NY, USA) under UV The objectives of this study were: (i) to molecularly light. PCR products were sequenced using the TW81, characterize cyst-forming nematode populations collec- AB28 primer and internal reverse 5.8SM5 primer ted in countries bordering the Mediterranean Sea based (Zheng et al., 2000) with the BigDye Terminator Cycle on RFLPs and sequence of the ITS region, (ii) to study Sequencing Ready Reaction Kit (PE Applied Biosys- the phylogenetic relationships of some species based on tems, Benelux, the Netherlands). The resulting prod- maximum parsimony and minimum evolution analyses ucts were purified and sequences were run on a 377 of the alignment of the ITS sequences. DNA Sequencer (PE Applied Biosystems, Warrington, UK). The sequences obtained in this study were Materials and Methods deposited in GenBank under accessions numbers Nematode populations AY347917–AY347927). The exact lengths of restriction The study comprised a total of 16 isolates of cyst- fragments from the PCR products were obtained by forming nematodes belonging to 10 known and one virtual digestion of the sequences using WebCutter 2.0. unidentified species (Table 1). Cysts were either obtained from the nematode collections maintained in Sequence and phylogenetic analysis the Istituto per la Protezione delle Piante, Sezione di Only sequences of ITS1-5.8S-ITS2 were used for analy- Bari, Italy, and the Instituto de Agricultura Sostenible, ses. Newly obtained sequences and the sequences Cordoba, Spain, or were directly collected in orchards obtained from publications and the GenBank (Ferris and agricultural fields. After their extraction from soil et al., 1999; Sabo et al., 2001; Subbotin et al., 2001, cysts were kept in dry condition at room temperature. 2003; Tanha Maafi et al., 2003) were aligned with Clu- Identification was carried out on the basis of morpho- stalX1.64 with default options. Meloidodera alni and metrics and morphological characteristics of cyst vul- Cryphodera brinkmani were used as outgroup taxa. val cones and second-stage juveniles (Wouts and Sequence alignment was analysed with an equally Table 1 Species Host-plant Location Study Species and populations of Heterodera used in the present Heterodera carotae Daucus carota Zapponeta, Italy RFLP, sequencing study Heterodera ciceri Cicer arietinum Aleppo, Syria RFLP Heterodera fici Ficus carica Acores, Portugal RFLP Heterodera fici Ficus carica Epirus, Greece RFLP Heterodera fici Ficus carica Potenza, Italy RFLP Heterodera filipjevi Triticum sp. Foggia, Italy RFLP, sequencing Heterodera goettingiana Pisum sativum Ostuni, Italy RFLP Heterodera hordecalis Triticum sp. Zapponeta, Italy RFLP, sequencing Heterodera hordecalis Triticum sp. Israel sequencing Heterodera humuli Humulus lupulus Braganc¸ a, Portugal RFLP, sequencing Heterodera latipons Elymus repens Rostov, Russia sequencing Heterodera mediterranea Pistacia lentiscus Torre Canne, Italy RFLP, sequencing Heterodera