Development of a novel diagnosis method for nematode diseases based on soil metagonome

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Specific primers in real-time PCR were developed for the quantitative detection of the root-lesion nematode Pratylenchus penetrans the root-knot nematode Meloidogyne incognita, the potato-cystnematode Globodera rostochiensis and the soybean-cyst nematode Heterodera glycines. Then, this detection method using real-time PCR was combined with adirect DNA extraction from soil. We found that their detection was significantly enhanced by extracting DNA after heavily compaction (1.4g cm^<-3> in case of volcanic ash soils). In general method, it takes more than 3days to count the number of the plant-parasitic nematodes. However, our method using a combination of soil compaction and real-time PCR enabled their detection within 4hours.We established a PCR-DGGE method to evaluate community structure of nematodes extracted from soil using the conventional method and found that nematode community structure, predominantly consisted of free-living nematodes, might be involved in estimating damages by the root-lesion nematode.In 4years field survey, relationship between damage by the root-lesion nematode and initial density of the nematode was studied. No damage was observed in the radishes in fields containing P. penetrans at a density of less than 2.5 individuals 20g^1 soil. In contrast, severe damage was always observed in fields at a density of more than 30 individuals 20g^1 soil. In fields containing P. penetrans at a density of 3.4 to 12 individuals 20g^1 soil, damage by the nematode was quite variable, but could be estimated by the difference in the nematode community structure.This study demonstrated that the soil compaction and real-time PCR assay for the four kinds of plant-parasitic nematodes provides a sensitive and reliable means for the rapid quantification of these the major pests in Japan.