A novel nematode diagnostic method using the direct quantification of major plant-parasitic nematodes in soil by real-time PCR

Abstract

We have developed a direct quantification method using real-time PCR for various plant-parasitic nematodes. Firstly, specific primers were designed for the root-knot nematode Meloidogyne incognita , the root-lesion nematode Pratylenchus penetrans , the potato cyst nematode Globodera rostochiensis and the soybean cyst nematode Heterodera glycines . A DNA extraction method was then developed beginning with 20 g of soil, a relatively large amount of soil but a necessary amount in the consideration of heterogeneous distribution of nematodes in soil. To estimate the density of the target nematode in soil, calibration curves for each plant-parasitic nematode were obtained by inoculating different numbers of the target nematode and then extracting DNA from the soils. The detection limit was 4-5 nematodes (20 g soil) −1 . This method was applied to nematode diagnostics. Soil sampling was done when transplanting of radish and sweet potato in fields was taking place, and the density of plant-parasitic nematodes was measured using both the Baermann funnel extraction method and real-time PCR methods. In some soils, P. penetrans and M. incognita were not found with the Baermann method but were detected with the real-time PCR method. At harvest, damage to crops was evaluated and its relationship with initial densities was investigated. The real-time PCR method more precisely predicted damage to radish and sweet potato by nematodes and was considered to be a powerful tool in the diagnosis of nematode diseases.