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PCR-RFLP analyses, reproduction tests on sweet potato and taro, as well as inter-RFLP-phenotypic hybridization were performed using 20 isolates of Pratylenchus coffeae and two isolates of P. penetrans to identify relationships between these nematodes. F194 and F195 primers yielded rDNA ITS region amplification products of ca. 1,080 bp (Japanese and Indonesian isolates), and ca.1,020 bp (Guatemalan isolate) that were digested using Hinf I, Alu I, Dde I, and Hha I restriction endonucleases. There were four distinct DNA fragment patterns for isolates of P. coffeae that were designated to be RFLP phenotypes A, B, C and D. All the Japanese isolates corresponded to RFLP-phenotypes A, B or C. The Indonesian isolate was phenotype A and the Guatemalan isolate was phenotype D. The Pf/Pi ratios 55 days or 90 days after inoculation to sweet potato in a greenhouse experiment differed between phenotypes. Phenotype A isolates had the greatest Pf/Pi of 1.1 -17.3. Phenotype B and C had values of 0.05 -0.40 and 0.12 -0.36 respectively. Reproduction on taro in a greenhouse experiment differed among phenotypes. Phenotypes A and B had greater reproduction (Pf/Pi>1) than did phenotypes C (Pf/Pi = 0). Laboratory hybridization tests between phenotypes A X B, A X C, and B X C generated abundant Fi hybrids, though reciprocal breedings of A X D and A X P. penetrans failed to reproduce. Inbreedings of Fis obtained from A x C were rarely successful, those from A X B generated a few F2s in 1/3 of replications, and those from B X C generated F2s in 2/3 of replications, suggesting incomplete reproductive isolation among the three rDNA RFLP phenotypes of P. coffeae. The allopatric distribution accompanied by distinct host adaptation, thermal adaptation and incomplete reproductive isolation exemplify microevolution in the P. coffeae species complex. Jpn. J. Nematol. 33 (2), 57-76 (2003). Key words: host compatibility, hybridization, host preference, thermal adaptation, PCR-RFLP, sweet potato, taro.The coffee root-lesion nematode, Pratylenchus coffeae (Zimmerman, 1898) Filipjev and Schuurmans Stekoven, 1941 is an important pest for a variety of crops primarily in tropical and subtropical zones of the world, but is also reported from temperate zones such as northern Japan (Gotoh, 1974;Loof, 1960; Sher 1 This research was conducted during 1996-1997 at the Kyushu National Agricultural Experiment Station; most parts of this report were presented at the SON 36th annual meeting, Tucson, Arizona, 19-23, 1997. and Allen, 1953). Isolates of P. coffeae in Japan are a species complex with extensive inter-specific variation (Mizukubo, 1992a). Pratylenchus pseudocoffeae was removed from this complex (Mizukubo, 1992a;Mizukubo, 1992b). The remaining members of this complex exhibit extensive differences in host compatibility and ribosomal DNA (rDNA) endonuclease-digestion-patterns (Orui, 1996;Mizukubo and Sano, 1997). The geographic isolates that form the species complex in Japan have differing host compatibility to sweet potato, radish, kidney bean, tomato, egg...

Yukio ORUI1In order to use random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) for the identification of ten Meloidogyne species in Japan, experiments were conducted on the screening of primers and on the extraction of template DNA. Of 120 random sequence 10-mer primers (OPA, OPB, OPC, OPD, OPF, and OPG kits; javanica, by using the template DNA extracted from many adult females with the extraction method of highly pure DNA and by using that from a single J2 with the lysis buffer.The RAPD patterns were presumed to be considerably affected by the magnesium concentration in template DNA. Jpn. J. Nematol. 29 (2): 7-15.