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

利用实时荧光定量PCR技术，分别对马铃薯根腐线虫Pratylenchus lanthans、南方根结线虫Meloidogyne incognita、马铃薯胞囊线虫Globodera rostochiensis和大豆胞囊线虫Heterodera glycines进行了检测。然后，将实时荧光PCR检测方法与直接提取土壤DNA方法相结合。我们发现，通过在重度压实（在火山灰土壤的情况下为1.4g cm^）后提取DNA，它们的检测显著增强<-3>。一般方法计算植物寄生线虫数需要30天以上。本研究建立了PCR-DGGE方法，对常规方法提取的土壤线虫群落结构进行了研究，发现线虫群落结构以自由生活线虫为主，这可能与线虫对土壤的危害程度有关。研究了根腐线虫的危害与线虫初始密度的关系。在每平方克土壤中含有2.5株以下的黑腐霉的田地中，没有观察到萝卜受到损害。相比之下，在密度超过30个个体/每克土壤的田地中，总是观察到严重的损害。在每20克土壤中含有3.4至12个个体的密度下，线虫造成的损害是相当可变的，但可以通过线虫群落结构的差异来估计。这项研究表明，土壤压实和实时荧光PCR检测对四种植物-寄生线虫提供了一种灵敏和可靠的手段，快速量化这些主要害虫在日本。

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.