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

基于土壤宏基因组的线虫病诊断新方法的开发

基本信息

批准号：
17380048
负责人：
TOYODA Koki
金额：
$ 9.04万
依托单位：
Tokyo University of Agriculture and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17380048/
关键词：
Pratylenchus penetrans Meloidogyne incognita Globodera rostochiensis Heterodera glycines denaturing gradient gel electrophoresis Baermann soil compaction nematode diagnosis キタネグサレセンチュウ自活性線虫 PCR-DGGE リアルタイムPCR 定量PCR 群集構造微生物

项目摘要

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.

开发了实时荧光定量PCR特异性引物，用于定量检测根部病斑线虫、根结线虫、根结线虫、马铃薯囊线虫、大豆囊线虫。然后，将这种使用实时 PCR 的检测方法与从土壤中直接提取 DNA 相结合。我们发现，通过在严重压实后提取DNA（火山灰土壤为1.4g cm^-3>），它们的检测得到显着增强。一般方法需要3天以上的时间才能统计出植物寄生线虫的数量。然而，我们采用土壤压实和实时PCR相结合的方法，可以在4小时内检测到它们。我们建立了PCR-DGGE方法来评估使用常规方法从土壤中提取的线虫的群落结构，发现线虫群落结构（主要由自由生活的线虫组成）可能参与估计根部病斑线虫的损害。在4年的现场调查中，研究了根部病斑线虫和线虫的初始密度。在含有穿甲虫的田地中，在密度小于 2.5 个/20g^1 土壤的情况下，未观察到萝卜受到损害。相比之下，在密度超过 30 株 20g^1 土壤的田地中，总是观察到严重的损害。在含有 P. penetrans 的田地中，密度为 3.4 至 12 个个体 20g^1 土壤中，线虫造成的损害差异很大，但可以通过线虫群落结构的差异来估计。本研究表明，对四种植物寄生线虫的土壤压实和实时 PCR 检测为快速定量这些日本主要害虫提供了灵敏而可靠的方法。