Multiplex endpoint PCR techniques are among the essential diagnostic tools used for identifying the potato cyst nematodes (PCNs: Globodera rostochiensis and G. pallida). Multiplex endpoint PCR assays for PCNs published to date, based on three-step PCR, are not integrated with primers to verify successful PCR in testing non-target species. Besides, carry-over contamination is a serious problem in diagnostic PCR assays. To improve the run time and reliability of the endpoint PCR test, we developed a two-step multiplex PCR identification method for PCNs using the dUTP/UNG carry-over prevention system. For this purpose, primers with high melting temperatures were newly designed to amplify mitochondrial DNA fragments specific to the respective PCN species and the nuclear ribosomal RNA gene fragments as PCR positive controls across cyst nematodes. In addition, the DNA preparation method from juveniles and cysts was simplified using sodium dodecyl sulfate and disposable homogenizers. This multiplex amplification generated amplicons of 150 bp, 287 bp and ca. 450 bp for G. rostochiensis, G. pallida and the non-target cyst nematode species, respectively. No cross-reactions were observed among the tested nematodes including 11 species and 22 populations. In examining mixed juveniles and cysts of PCNs, our method successfully detected both species even in the ratio of one to ten. These PCR runs took ca. 1 h, faster than the other three-step PCR protocols. The new PCR diagnostic method described here is reliable, fast and thus a good alternative to the other assays based on conventional PCR for diagnosis of PCNs.

In 1986, an undescribed Heterodera species found in continuously cultivated soybean fields was reported in Japan. Only dead nematodes of the population are currently available for taxonomic description of this species. In 2016 a new soybean parasitic cyst nematode was described as H. sojae in Korea. These two nematodes showed morphological similarity and thus were expected to be conspecific. In this study, the nucleotide sequences of the nuclear ribosomal RNA gene region were examined to confirm whether the unknown cyst nematode was H. sojae. BLAST results showed that the nucleotide sequences of the large subunit rRNA D2–D3 expansion segments (740 bp) and the internal transcribed spacer (ITS) fragment covering 3’-end of the ITS1, the entire 5.8S rRNA and the partial ITS2 (368 bp) both were by far the most similar to the sequences of H. sojae. Together with morphological similarity including the ambifenestrate vulval cone with a particularly short vulval slit, the unknown cyst nematode was identified as H. sojae. In addition, the duplex PCR assay targeting the ITS2 region to discriminate H. sojae was developed to amplify a 201 bp product specific to H. sojae as well as ca. 450–480 bp products across cyst nematodes. No further detections in Japan since the first report in 1986 and detections limited to a few countries including Korea, China and Japan to date imply H. sojae has occurred for a long time as a minor pest of soybean in East Asia.

A survey for plant-parasitic nematodes (PPN) in 33 sugarcane fields was conducted to elucidate the relationship between the yield loss of sugarcanes (Saccharum spp.) and damage caused by PPNs in Tanegashima Island, Japan. Ring, root knot (dominant species; Meloidogyne incognita), lesion (dominant species; Pratylenchus zeae), spiral, stunt, reniform, stubby root, and dagger nematodes were detected in soil samples obtained from these sugarcane fields near harvest time by both the Baermann and the sugar-flotation extraction methods. Furthermore, model selection for sugarcane yield (t/10 a), with parameters including PPNs detected in this survey, was carried out based on AICc. Nevertheless, no distinct correlations were observed between the total yield and the numbers of each PPN.

Effect of plant-parasitic nematodes on initial growth of sugarcane in Tanegashima Island was examined by means of a pot experiment. Two sugarcane cultivars, NiF8 and Ni22, were grown in pots filled with two soils from sugarcane fields in Tanegashima treated with 0 g, 0.75 g per pot (corresponding to 30 g/m²), or 3.75 g per pot (corresponding to 150 g/m²) of 1.5% fosthiazate granules. Both fosthiazate treatment and cultivar difference influenced sugarcane culm height, shoot dry weight, and root dry weight significantly by ANOVA. In one soil with a high initial density of lesion nematodes, there was a significant difference in growth of Ni22 among fosthiazate treatments, whereas the difference was small and not significant for NiF8. These results suggested that plant parasitic nematodes affect the initial growth of sugarcane in Tanegashima Island soil, and the effect was different between two cultivars.

Parasitic nematodes were found in the two bark beetle species Ips typographus and I. cembrae in Japan. Based on their ecological and morphological characteristics, these nematodes are identified as members of the genus Contortylenchus. They show some differences in not only parasitic manners but also molecular profiles. Ribosomal DNA sequences for small subunit (SSU), and D2-D3 expansion segments of the large subunit (D2-D3 LSU) of the nematodes in the two different beetle species are almost identical, but they show one base-pair substitution in the D2-D3 LSU. These results suggest that the nematodes from I. typographus and I. cembrae are closely related but separate species. Interestingly, the D2-D3 LSU of the nematode in I. typographus was identical to those of both Contortylenchus sp. from the Moscow region, the Russian Federation, and C. diplogaster in I. cembrae from the Czech Republic. This result indicates that the nematode in I. typographus is closely related to or could be conspecific to the European Contortylenchus species, including especially C. diplogaster or a related species, C. pseudodiplogaster.