Molecular techniques are prevailing in nematode identification and quantification, for which DNA extraction from nematodes is essential. However, the published DNA extraction methods are laborious and often require various expensive consumables and high-end equipment. In order to prepare DNA templates for a high-throughput real-time PCR assay, the present study modified a conventional DNA extraction method of Naklha et al. (2010) from nematode suspensions with ordinary lab equipment and achieved results and advantages comparable with two other conventional methods. The results of real-time PCR assays for quantifying Pratylenchus zeae, Tylenchorhynchus leviterminalis and Hoplolaimus sp. using the new protocol were highly correlated with those obtained by morphological counts and comparable to or more sensitive than those obtained by two conventional methods. Although the new protocol took over 100 min for DNA extraction, the manual processing took less than 10 min, i.e., half to one-fourth of the other methods. The running cost was less than half to one-tenth of the other methods.

Quantification of plant-parasitic nematodes (PPN) in soil with real-time PCR is a useful diagnosis to estimate damage to crops. However, previously reported methods involve high consumable and labor costs. The objectives of this study were to combine previously reported methods for soil pretreatment, DNA extraction and real-time PCR to quantify the density of soil-borne PPN in a simpler and less expensive way and to confirm the usefulness of a new simple method. Soils infested with either Heterodera glycines (soybean cyst nematode), Ditylenchus destructor (potato rot nematode) or Meloidogyne incognita (root-knot nematode) were ball-milled. DNA was then extracted with phosphate buffer and purified with a commercially available column. Real-time PCR was conducted to quantify the target nematodes. The cycle threshold (Ct) values obtained by the new method showed highly significant correlations with those by the conventional method for all three species (R² > 0.75). Significant correlations (R² > 0.987) were also obtained between the Ct values and the numbers of nematodes inoculated into soils. The DNA extraction from 6 samples by the new simple method required only 1 hr and about $4.8 of consumables, while that by the conventional method required 3 hr and about $12 of consumables. These results demonstrate that the method consisting of ballmilling and simple DNA extraction enables rapid and less expensive quantification of nematodes in soils.

Thelastomatid nematodes (Nematoda: Oxyurida: Thelastomatoidea) are obligatory parasites that occur naturally in the hindgut of arthropods. Their origin and impact in the host is still unknown. Previous studies showed that the presence of thelastomatid nematodes in the gut of cockroaches (Periplaneta fuliginosa and P. americana) could influence the composition of their hindgut microflora. Through a metagenomic approach (16S rRNA V3-V4 sequencing), we have characterized the hindgut microbiome of P. japonica in the presence of thelastomatid nematodes (L1986, natural parasitic nematode Protrellus sp. present as a natural infection condition; and L1987, non-native parasitic nematode Leidynema appendiculatum present as an artificial infection condition). The hindgut microbiome of P. japonica in both conditions were mainly composed of Bacteroidetes, Firmicutes and Proteobacteria. Moreover, the natural and artificial infection by thelastomatid nematodes lead to shifts in the relative abundance of these main resident flora as seen in P. americana. The OTUs percentage of Bacteroidetes and Proteobacteria were higher in P. japonica infected with Protrellus sp. (L1986) than in P. japonica infected by L. appendiculatum (L1987), while OTUs from Firmicutes phylum was higher in L1987 than in L1986. This study fosters a detailed investigation in the role played by these animal parasites in their host insect.

Tobacco cyst nematode (TCN) Globodera tabacum is able to develop and reproduce on tomato plants and eggplants. As a consequence, plants with a heavy infestation show stunted growth. The most effective and environmentally benign method for managing plant nematodes is through exploitation of host resistance. However, comparatively little information is available on the relative rates of reproduction of TCN on different tomato cultivars and resistance to TCN has not been studied in detail. In the present study, we compared the response of several tomato cultivars to inoculation with TCN in order to identify those with greatest resistance. As a result, five new TCN-resistant tomato cultivars, ‘Doctor-K’, ‘Sugar-Lamp’ ‘Carol-10’, ‘Carol-Queen’, and ‘Chelsea-mini’, were identified.

It is important to clarify the nematode race composition in a resistance test field where many crop varieties are cultivated, because several nematode races may occur. According to parasitism to sweet potatoes, the southern root-knot nematode (Meloidogyne incognita) population in a field in Katori-city showed a race SP4-like response, although the dominant race had been determined SP6. To clarify the reason, we examined the race composition using 16 nematode lines each derived from a single egg-mass. We concluded that average responses based on the dominant SP6 and the minor SP2 might have shown the response of SP4 as a population.