Case study on a modified method to quantify the density of some soil-borne plant-parasitic nematodes in a simpler and less expensive way

Abstract

Quantification of plant-parasitic nematodes (PPN) in soil with real-time PCR is a useful diagnosis to estimate damage to crops. However, previously reported methods involve high consumable and labor costs. The objectives of this study were to combine previously reported methods for soil pretreatment, DNA extraction and real-time PCR to quantify the density of soil-borne PPN in a simpler and less expensive way and to confirm the usefulness of a new simple method. Soils infested with either Heterodera glycines (soybean cyst nematode), Ditylenchus destructor (potato rot nematode) or Meloidogyne incognita (root-knot nematode) were ball-milled. DNA was then extracted with phosphate buffer and purified with a commercially available column. Real-time PCR was conducted to quantify the target nematodes. The cycle threshold (Ct) values obtained by the new method showed highly significant correlations with those by the conventional method for all three species (R² > 0.75). Significant correlations (R² > 0.987) were also obtained between the Ct values and the numbers of nematodes inoculated into soils. The DNA extraction from 6 samples by the new simple method required only 1 hr and about $4.8 of consumables, while that by the conventional method required 3 hr and about $12 of consumables. These results demonstrate that the method consisting of ballmilling and simple DNA extraction enables rapid and less expensive quantification of nematodes in soils.