Development of a real-time PCR method for the potato-cyst nematodeGlobodera rostochiensisand the root-knot nematodeMeloidogyne incognita

Abstract

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.

Quantitative detection of Pratylenchus penetrans from soil by using soil compaction and real-time PCR

“…Recently, DNA analysis has been used to identify and quantify soil nematodes (Madani et al, 2005;MacMillan et al, 2006;Sato et al, 2007;Toyota et al, 2008) and to assess nematode community structure (Sato et al, 2006;Okada and Oba, 2008;Donn et al, 2008). For these studies, nematodes must first be extracted from soil by the Baermann methods using funnels and trays, which are time-consuming and yield a recovery rate of around 50% (Ingham, 1994).…”

Development of a direct quantitative detection method for Meloidogyne incognita and M. hapla in andosol and analysis of relationship between the initial population of Meloidogyne spp. and yield of eggplant in an andosol

“…In addition, the density of M. incognita might be overestimated. The primer set RKN amplifies DNA even from M. hapla at an efficiency of 15%, as this defect was already reported in a previous paper (Toyota et al, 2008), indicating that 100 individuals of M. hapla are detected as 43 2 2013 12 Fig. 7.…”

“…, ] reported by Toyota et al (2008) ( Table 1). Primer specificity was evaluated using M. hapla and M. incognita with real-time PCR.…”

“…Nematode densities at transplanting were assessed using a composite soil sample collected from six sites of each plot. The M. incognita primers were reported by Toyota et al (2008). 1 Numbers in parentheses after the species names indicates the accession number.…”

“…The rapid development of DNA technology has been a breakthrough for overcoming the weak points of traditional identification methods (Atkins et al, 2005;Berry et al, 2007). Since Madani et al (2005) reported real-time PCR specific primers for quantitative purposes of the potato cyst nematode Globodera pallida and the sugarbeet cyst nematode Heterodera schachtii, many specific primers have been designed for different plant-parasitic nematodes such as Bursaphelenchus xylophilus (Leal et al, 2007), Pratylenchus penetrans (Sato et al, 2007), M. incognita and G. rostochiensis (Toyota et al, 2008), M. javanica, P. zeae and Xiphinema elongatum (Berry et al, 2008), H. glycines (Goto et al, 2009) and P. thornei (Yan et al, 2012). However, no specific primers have been developed for M. hapla until now.…”