Quantitative detection of the potato cyst nematode, Globodera pallida, and the beet cyst nematode, Heterodera schachtii, using Real-Time PCR with SYBR green I dye

Abstract

The potato cyst nematode Globodera pallida and the beet cyst nematode Heterodera schachtii are major nematode pests in world agriculture. Precise identification and knowledge about the number of nematodes in field soil are necessary to develop effective integrated pest control. Here we report the results of the Real-Time PCR assay for the rapid detection and quantification of G. pallida and H. schachtii. Using species specific primers and SYBR green I dye, we were able to detect a single second stage juvenile of cyst forming nematodes in samples. The specificity of the reaction was confirmed by the lack of amplification of DNAs from other Heterodera or Globodera species. Validation tests showed a rather high correlation between real numbers of second stage juveniles in a sample and expected numbers detected by Real-Time PCR. Reasons for observed differences in sensitivity and reliability of quantification detection for two species as well as other problems of Real-Time PCR are discussed. The Real-Time PCR assay with SYBR green I dye targeting fragments of the ITS-rDNA provided a sensitive means for the rapid and simultaneous detection and quantification of juveniles of these pests.