Development of a novel scale based on qPCR for rapid and accurate prediction of the number of Aphelenchoides besseyi in paddy rice

Abstract

Rice is one of the most important staple foods worldwide. Aphelenchoides besseyi is one of the most economically damaging seed-borne nematodes and spreads rapidly in rice-growing areas through infested seeds. Although rapid and accurate identification and quantification of A. besseyi are necessary to prevent its spread, infested seeds do not show evident symptoms. Therefore, several molecular methods have been used for detection, but quantification has, as yet, been inaccurate. In this study, a real-time PCR assay was developed to detect and quantify the number of A. besseyi in rice paddy and glume samples from rice-growing areas of Thrace, Turkey. A standard curve (R2 = 0.97, E = 98%, y = −3.35x + 28.16) was generated using Ct values of samples containing known numbers of nematodes. Subsequently, this standard curve was used to estimate the number of nematodes and the scale of infection (low, medium or high) from Ct values of rice field samples. Using this method, the infestation levels of 188 paddy and glume samples from Turkey and 4 samples from Russia were analysed. Of the samples, 96 were found to be infested and 43, seven and four glume samples had low, medium and high levels of infection, respectively. In contrast, the infestation level of many paddy samples was low. For infection level, significant correlation was determined between Ct values and counting under the microscope (r = −0.75, P < 0.0001). The scale developed in this study could be used for quantification of A. besseyi in rice and help to prevent the distribution of infested seeds.