Quantification of lesion nematode (Pratylenchus zeae), stunt nematode (Tylenchorhynchus leviterminalis), spiral nematode (Helicotylenchus dihystera), and lance nematode (Hoplolaimus columbus), parasites of sugarcane in Kitadaito, Okinawa, Japan, using real-time PCR

Abstract

The lesion nematode (Pratylenchus zeae), the stunt nematode (Tylenchorhynchus leviterminalis), the spiral nematode (Helicotylenchus dihystera), and the lance nematode (Hoplolaimus columbus) were major plant-parasitic nematode (PPN) species identified in the sugarcane fields on Kitadaito Island, Okinawa, Japan, for which real-time PCR primer sets were developed. Since the four PPN species ubiquitously inhabited Kitadaito, P. penetrans, which was confirmed to be absent in Kitadaito, was used as a benchmark to establish a soil calibration curve. Kitadaito soil samples were inoculated with juveniles and adults of P. penetrans at densities of 8, 32, 128, and 256/10 g fresh soil, and a soil calibration curve was obtained: y =－0.95x + 34.83 (y = Ct values, x = log2 (the number of P. penetrans ). The soil calibration curve was consistent with a calibration curve derived from serially diluted DNA samples of handpicked single P. penetrans (y =－1.01x + 34.73), after adjusting the DNA dilution rates to the densities in soil. The results suggested that calibration curves developed using serially diluted DNA samples (y =－1.13x + 37.00, y =－0.99x + 30.76, y =－1.06x + 35.52, and y =－1.00x + 28.96 for P. zeae, T. leviterminalis, H. dihystera, and H. columbus, respectively) may be appropriate to quantify target PPN in Kitadaito soil.