Comparison of promidium monoazide and Reagent D for discrimination of viable Aphelenchoides besseyi using real-time PCR

Aphelenchoides besseyi Christie, 1942 is a seed-borne nematode and causes economic yield losses in rice. Information on the viability of A. besseyi in stored rice seeds is important to manage this nematode. In this study, propidium monoazide (PMA) and Reagent D, combined with qPCR, were used for discrimination of viable A. besseyi individuals. A. besseyi individuals that were live, stored for 1 year or heat-treated (40 or 60 °C, 15 min) were exposed to PMA and Reagent-D dyes. In addition, A. besseyi individuals not treated with any chemicals were used as a control. DNA was then isolated from all individuals and real-time PCR was carried out. Ct values for samples treated with Reagent-D and PMA were different from untreated samples. Results showed that live individuals of the rice white tipe nematode could be distinguished from non-viable individuals using Reagent-D or PMA combined with qPCR.