Development of a quantification method with real-time PCR for three Pratylenchus species causing damage to chrysanthemum in Japan

Three Pratylenchus species, P. penetrans , P. pseudocoffeae and P. kumamotoensis , are major threats to chrysanthemum production in Japan. To develop a quantification method for these three Pratylenchus species in soil using real-time PCR, we designed two new specific primer sets for P. pseudocoffeae and P. kumamotoensis and applied the primer set NEG, developed previously, for P. penetrans . Relationships between the threshold cycle (Ct: y ) values and number of nematodes inoculated (log 2 (no. (20 g soil) −1 ): x ) were for P. penetrans y = − 0 . 9502 x + 35 . 99 , P. pseudocoffeae y = − 1 . 238 x + 35 . 39 and P. kumamotoensis y = − 0 . 9613 x + 36 . 50 . The quantification of these Pratylenchus spp. was conducted using 16 soil samples. Pratylenchus penetrans and P. kumamotoensis were detected from seven and one soil samples, respectively, while no P. pseudocoffeae was detected. These results demonstrated that the presently designed primers are useful to quantify the densities of three Pratylenchus spp. in chrysanthemum fields in Japan.