Development of a direct quantitative detection method for Meloidogyne incognita in sandy soils and its application to sweet potato cultivated fields in Tokushima prefecture, Japan

Abstract

AbstractA real-time PCR (quantitative PCR: qPCR)-based detection method of the root-knot nematode Meloidogyne incognita was developed for sandy soils, the major soil type in sweet potato cultivated fields in Tokushima prefecture, Japan. Different numbers (5, 20, 80, 200 and 500) of second-stage juveniles (J2) were artificially added into 20 g of an air-dried sandy soil not containing M. incognita. To make homogenous samples, soil was homogenised by two different ways (ground with either a mortar and pestle or ball mill) and then 0.5 g of the soil was used for DNA extraction. There was a strong negative correlation in each homogenisation method between the cycle threshold number (Ct) and inoculated numbers of M. incognita J2. The Ct values were consistently lower and their variations among replicates were smaller in the samples ground with ball mill, suggesting that grinding with ball mill may be suitable for the preparation of soil for DNA extraction. Sandy soils were collected from sweet potato fields in Tokushima prefecture at the transplanting and harvesting times. Damage to sweet potato caused by M. incognita was also evaluated in some of the fields. At the transplanting time, no M. incognita was extracted in all the soils by the Baermann funnel method, while detection in the qPCR method ranged from zero to 4 210 000 J2 equivalent (20 g soil)–1. Heavy damage was observed in fields with more than 500 equivalent M. incognita J2 (20 g soil)–1. By contrast, very few galls were observed in fields with fewer than four individuals (20 g soil)–1. At harvest, zero to >1000 individuals of M. incognita was counted by the Baermann method and there was a significant correlation in estimated numbers of M. incognita between the two methods. However, the estimated numbers were 15 times higher in the qPCR method than in the Baermann method. These results indicate that direct quantification of M. incognita based on the qPCR method might enable a sensitive diagnosis to predict damage by the nematode.