Development of specific primers to Hirschmanniella spp. causing damage to lotus and their economic threshold level in Tokushima prefecture in Japan

Abstract

Nematode diseases caused by Hirschmanniella spp. are becoming a major threat to lotus production in Tokushima Prefecture, a primary production area in Japan. The objectives of this study were: i) to design specific primers to Hirschmanniella spp. causing damage to lotus; ii) to evaluate the relationship between pre-plant density of Hirschmanniella spp. in soil and damage index of lotus; and iii) to establish an economic threshold level. Since two species, Hirschmanniella imamuri and H. diversa, are known as nematode pests on lotus, three real-time PCR primers were designed based on the ITS regions specific to H. imamuri (designated as imaF and imaR), H. diversa (divF and divR), and both species (HdiF and HdiR). The primers imaF and imaR did not react to DNA of nematodes extracted from lotus fields in Tokushima but the primers divF and divR did, suggesting that the damage seen in Tokushima is mainly caused by H. diversa. A calibration curve was made to evaluate the relationship between the number of Hirschmanniella spp. inoculated to soil and the threshold cycle values. The quantification of Hirschmanniella spp. in soil was conducted by real-time PCR method with the primers HdiF and HdiR as well as the Baermann method. The economic threshold level was estimated as ten individuals of Hirschmanniella spp. (100 g fresh soil)−1 with the Baermann method and 50 individuals (100 g oven-dried soil)−1 with real-time PCR.