Nematodes often engage with diverse pathogens. Prior research has unveiled nematode-specific microsporidia and fungus-related obligate intracellular pathogens with both terrestrial and marine nematodes across the United States and select European countries. However, our comprehension of microsporidia diversity infecting nematodes in Asia, particularly Japan, remains limited. This study aimed to delve into the diversity of microsporidia-infecting nematodes and unearth novel interactions by investigating wild-isolated bacterivorous and fungivorous nematodes in Japan. Our investigation, successfully isolated 12 strains of microsporidia, among them an unidentified species (MKI37), from bacterivorous nematodes. Notably, MKI37 was found to infect Distolabrellus veechi, a nematode species previously unrecognized as a host for microsporidia. Intriguingly, no infections were detected in fungivorous nematodes. Our findings suggest a more pronounced diversity of microsporidia-infected nematodes among bacterivorous nematodes.

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Occurrence of plant parasitic nematodes (PPN) on blueberry has been reported in North America. However, there is no available report on nematodes in blueberry cultivated in Japan, where its cultivation area is increasing, especially in suburban areas. This study aimed to develop quantitative methods using real-time PCR to enable direct quantification and identification of PPN from blueberry field soils in Japan. Nematodes were isolated from soil samples collected from six commercial blueberry fields located in Kanagawa Prefecture and Tokyo Metropolis, Japan. Pratylenchus penetrans, Helicotylenchus dihystera, Meloidogyne incognita, Paratrichodorus renifer, Tylenchorhynchus claytoni, and Criconema mutabile were the PPN identified by molecular methods. Among them, new primer sets for P. renifer, T. claytoni and C. mutabile and a modified primer set for H. dihystera were designed in the internal transcribed spacer 1 region of ribosomal RNA, and calibration curves for direct quantification of these PPN from soil using realtime PCR were obtained. The quantification limits for these nematodes obtained by real-time PCR were 84.4/20 g soil for P. penetrans, 9.1/20 g soil for M. incognita, 10.4/20 g soil for P. renifer, 19.4/20 g soil for C. mutabile, 1.6/20 g soil for T. claytoni, and 23.2/20 g soil for H. dihystera. Moreover, these primers did not amplify DNA extracts of non-target PPN individuals found in the blueberry soils or amplified with very low efficiency (Ct value ≧ 34.3). This study identified plant-parasitic nematodes in blueberry fields in Japan for the first time and developed their molecular quantification methods.

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Nematodes can contribute to the nutrient flow in above and below-ground forest ecosystems. Although previous studies have shown nematode taxonomic composition in bark near breast height, the vertical distribution of nematodes in bark is unknown. To elucidate the distribution and specific taxa of nematodes on trees, we examined the nematode taxonomic composition and molecular phylogenetic position of dominant nematode taxa in Japanese cedar bark at different heights from 1 m to 10 m. Nematodes were found in all barks regardless of height. Fungivorous Aphelenchoididae nematodes were dominant at all tree heights with a relative abundance of more than 86%. Phylogenetic analysis based on the COI region showed Aphelenchoididae derived from cedar barks were in the same clade regardless of tree height. Phylogenetic analysis based on the SSU region assigned the nematodes to three clades with more than 92% bootstrap values. In the phylogenetic analysis based on the COI region, Aphelenchoididae derived from cedar barks were located in the same clade regardless of tree height and in a separate clade from those living in litter and soil. Thus, nematodes dwell in cedar bark at a height of 10 m above ground, and fungivorous Aphelenchoididae was predominant at all tree heights. Moreover, the same species of Aphelenchoididae were distributed on all cedar barks regardless of tree height. Thus, bryophytes attached to cedar bark were the main habitats for nematodes. Among them, fungivorous nematodes can indirectly be involved in the decomposition process of barks in this study.

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During a nematological survey in a natural forest in Sapporo, Hokkaido, Japan, spherical to lemonshaped cysts thought to be Heterodera, were found in the rhizosphere of Japanese elm (Ulmus davidiana var. japonica). Considering the vegetation of the sampling place, the nematodes seemed to be an unreported cyst nematode species in Japan. A cyst nematode species that parasitizes Japanese elm in the Primorsky territory, the Russian Far East, was described in 2001 as H. vallicola. Therefore, we hypothesized that the cyst nematode population belonged to H. vallicola and examined its morphological features and nucleotide sequences to determine the species identity. The morphometrics of the cysts and second-stage juveniles were generally consistent with those of H. vallicola. In addition, the sequences of the mitochondrial cytochrome c oxidase subunit I gene and the internal transcribed spacer region of the ribosomal RNA gene were the most similar to those of H. vallicola. According to the results, we determined that this nematode belongs to H. vallicola.

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Sugar beet cyst nematode (SBCN), Heterodera schachtii, was first detected in 2017 from a highland vegetable production area of Hara village, Nagano Prefecture, central Japan. We should consider two important characteristics of nematode biology for application of control measures to prevent resurgence after emergency control by fumigation: 1) the local SBCN population can parasitize not only Brassicaceae and Amaranthaceae crops but also tomatoes; 2) its seasonal occurrence pattern could be complex due to single adult females producing eggs with different hatching properties, and to the possibility of two generations per year in the infestation area. We should also consider the local farming system: the size of an individual crop field is small, e.g., mean 1.5 ha for broccoli, a good host of SBCN, and the farmers have a tendency to produce a single crop item exclusively. Among the crop items cultivated in the infestation area, celery, parsley, lettuce, maize, and zucchini were determined nonhosts. However, resistant host crop varieties are not available currently. Fumigation, organophosphate application, 2 month-cultivation of trap crops, 2 month-fallow, and 4 month-cultivation of nonhost crops reduced nematode population densities by minimum est imates of 60, 40, 40, 30 and 10%, respectively. Here, we propose crop rotation systems integrated with these individual control measures to keep SBCN at an undetectably low density after emergency control.

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Sugar beet cyst nematode (SBCN) has occurred in Hara Village, Nagano Prefecture, Japan since 2017. Phytosanitary measures have been taken to eradicate and prevent the spread of this pest. Among commercially available leaf radish green manure varieties, ‘Kobuberi daikon’ has been used as a trap crop against SBCN. In preliminary tests among leaf radish green manure varieties not yet commercially available, ‘KGM1804’ (Kaneko Seeds Co., Ltd.), showed SBCN resistance. In this study seedlings of ‘KGM1804’, ‘Kobuberi daikon’ or cabbage in plastic cups were each inoculated with 1,500 SBCN juveniles to evaluate effectiveness as trap crops. No cysts were observed on ‘KGM1804’ or ‘Kobuberi daikon’ at the end of the test period, whereas an average 243 cysts were produced on cabbage. Root exudates of the two leaf radish varieties stimulated hatching of SBCN eggs. The leaf radish varieties also had comparable growth in fields of Hara Village. These results suggest ‘KGM1804’ and‘ Kobuberi daikon’ are equally effective as SBCN trap crops. ‘KGM1804’ is now commercially available as the variety ‘Shisu kurin’ in Japan.

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