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The primers PCN280f and NEPCN398r were designed for the quantitative detection of the potato-cyst nematode Globodera rostochiensis using real-time polymerase chain reaction (PCR). One, five, 50, 125 and 250 individuals of the second-stage juveniles (J2) of G. rostochiensis were mixed with various stages of vermiform Caenorhabditis elegans to make a total of 500 individuals and DNA was extracted from the nematode mixture. There was a significant correlation (r 2 = 0.9355, P < 0.001) between the threshold cycle values and the number of G. rostochiensis added. When nematodes were extracted from soils artificially infested with G. rostochiensis to various degrees and real-time PCR was conducted using DNA templates from the nematodes extracted, there was a highly significant correlation in the numbers of G. rostochiensis J2 from the real-time PCR method and morphological identification. Real-time PCR sensitively detected a single G. rostochiensis J2 out of 1,000 individuals of free-living nematodes. Similarly, real-time PCR primers RKNf and RKNr were designed for the detection of the root-knot nematode Meloidogyne incognita. This study demonstrated that the real-time PCR assay for the potato-cyst nematode and the root-knot nematode provides a sensitive and reliable means for the rapid quantification of these vermiform pests.

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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.

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