Developing a real-time PCR diagnostic method for a potential threat to chrysanthemum, Paratylenchus dianthus

Chrysanthemum is a very popular flower in Japan and is known to be infected by many soil-borne plant pathogens including nematodes. A nematode survey in six chrysanthemum fields in Okinawa, Japan, found Pratylenchus, Meloidogyne, and Paratylenchus (P. dianthus). The first two genera are known as plant pathogens against chrysanthemum, however, Paratylenchus dianthus has not been reported previously. Chrysanthemum seedlings were grown in pots containing soil infected only with P. dianthus for two months in 2017 and 2018. The nematicide imicyafos was applied in triplicates to half of the pots (treated) while the other half were left without the nematicide (non-treated). Plant height and dry plant weight of the imicyafos treated plants exceeded those of the control plants. Also, single-photon avalanche diode value of chrysanthemum leaves was higher in imicyafos treated plants than in the non-treated plants at two-month after planting. The results suggest that P. dianthus may suppress the growth of chrysanthemum. For high-throughput nematode diagnosis, a real-time PCR primer set specific to P. dianthus was developed and its sensitivity to quantify P. dianthus was confirmed with a proper calibration curve. The calibration curve was developed in a simplified approach by using serially diluted DNA extracted from individual nematodes.