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Table 1 Root-knot nematode (Meloidogyne spp.) isolates used in this study Species Isolate are1 chi1 ex1 ex2 ex3 ex5 ex6 ex7 ex8 fa1 ha1 inc2 inc3 inc5 jav1 ma1 par5 Geographic origin Pato Branco, RS, Brazil Valks, the Netherlands Lavras, MG, Brazil Rondonopolis, MS, Brazil Lavras, MG, Brazil Carazo, Nicaragua Cajon, Costa Rica Cartago, Costa Rica Cruz Grande, Honduras Baexem, the Netherlands Caxias, RS, Brazil Londrina, PR, Brazil Londrina, PR, Brazil Tierras Morenas, Costa Rica Pelotas, RS, Brazil Hojancha, Costa Rica Apucarana, PR, Brazil
M. arenaria M. chitwoodi M. exigua
M. fallax M. hapla M. incognita
M. javanica M. mayaguensis M. paranaenesis
Bgl II products (≈ 270 bp) was electroeluted, ligated into the plasmid vector pBS and used to transform competent Escherichia coli DH12S cells according to standard procedures (Sambrook et al., 1989). The transformants were selected on ampicillin (100µg/ mL) agar plates containing X-gal (80µg/mL) and IPTG (120µg/ mL), and screened by colony hybridization using the≈ 270 bp DNA fragments isolated from the gel as a probe. The nucleotide sequ
ence of inserts from 20 recombinant clones selected at random and corresponding to putative monomers was determined by Eurogentec (Herstal, Belgium). Searches for restriction sites and direct repeats were performed using MACMOLLY, version 1.0. Multiple sequence alignments were done using CLUSTAL W (Thompson et al., 1994). DNA sequence data were compared with the EMBL and GENBANK databases by using the National Center for Biotechnology Information B LAST version 2.0 server (Altschul et al., 1997).Squash-blot procedure For each nematode isolate tested, second-stage juveniles, females, egg-masses and galls were hand-picked from infested tomato roots and placed on a nylon membrane. The nematodes or the plant fragments were then ruptured by gentle pressure with a yellow, at-tipped micropipet tip. Squashed materials were lysed by layering, successively, the lter on Whatman 3MM paper soaked with 10% SDS (2 min), 0.5 M NaOH/2.5 M NaCl (two times, 5 min each), and 3 M sodium acetate, pH 5 (three times, 2 min each). The lter was dried at room temperature (30 min) and then baked at 80°C for 1 h. Prehybridization and hybridization of the lter were carried out as described above with a probe consisting of a 32P-labelled cloned satellite DNA monomer.
E X P E R I M E N T A L P RO C E D U RE SNematode isolates Seventeen RKN isolates belonging to nine different species (Table 1), were maintained on tomatoes ( Lycopersicon esculentum L. cv. Saint Pierre) grown at 20°C in a greenhouse. They were speci cally identi ed morphologically and according to their isoesterase electrophoretic pattern (Carneiro et al., 2000). Eggs were collected from infested roots, concentrated by centrifugation at 2000 g for 2 min in a 30% sucrose solution, washed in distilled water, pelleted in a microcentrifuge and stored at 80°C until use. DNA puri cation and analysis For each nematode isolate, total genomic DNA was puri ed from 100 to 200µL of eggs, as described previously (Piotte et al., 1994). Standard procedures were used for restriction endonuclease digestion, electrophoresis, transfer to nylon membranes, radioactive labelling and hybridization (Sambrook et al., 1989). Hybridizations were performed overnight at 65°C. Conditions for washing consisted of 65°C in a 1× SSC, 0.1% SDS nal solution. Cloning and sequence analysis Genomic DNA of M. exigua isolate ex1 was digested with a set of restriction endonucleases (listed in Fig. 1), separated on a 1% agarose gel, and stained with ethidium bromide. To ensure complete digestion, incubations were performed for 4 h at 37°C with 10 units of enzyme/µg of DNA. The most prominent band in
ACKNOWLEDGEMENTSOnivaldo Randig is the recipient of a PhD scholarship from the Conselho Nacional de Desenvolvimento Cientí co e Tecnológico (CNPq) of Brazil, which is gratefully acknowledged. This work was nancially supported by the‘Fond Commun INRA-CIRAD’ programme (AIP P0221).
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tschul, S.F., Madden, T.L., Shaffer, A.A., Zhang, Z., Zhang, J., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search program. Nucl. Acids Res. 25, 3389–3402. Bachmann, L., Schibel, J.M., Raab, M. and Sperlich, D. (1993) Satellite DNA as a taxonomic marker. Biochem. Syst. Ecol. 21, 3–11. Beridze, T.G. (1986) Satellite DNA. Berlin: Springer-Verlag. Campos, V.P., Sivapalan, P. and Gnanapragasam, N.C. (1990) Nematode parasites of coffee, cocoa and tea. In: Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. (Luc, M., Sikora, R.A. and Bridge, J., eds), pp. 387– 430. London: CAB International.
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