Development of real-time PCR primers specific to the garlic-damaging potato rot nematode Ditylenchus destructor to quantify its density in soil and outer skin of garlic

The potato rot nematode Ditylenchus destructor is a major threat to garlic production in Aomori Prefecture, Japan. The objectives of this study were i) to design real-time PCR primers specific to D. destructor, and ii) to make calibration curves to evaluate the relationships between the number of D. destructor inoculated to soil or outer skin of garlic and the cycle threshold (Ct) values. Ditylenchus destructor strains were collected from 11 major growing regions in Aomori Prefecture and their ITS regions were sequenced. Strains from different regions showed the same sequence and, thus, real-time PCR primers specific to D. destructor were designed. The specific primers (Ddf and Ddr) matched 100% with 10 out of 11 D. destructor sequences from different countries as well as with the Aomori strains, while there are 7 and 16 bp mismatches in the closest species D. africanus and D. askenasyi, respectively. There were highly significant correlations (soil: y =－1.1221x + 35.225, R2 = 0.9973; outer skin of garlic: y =－1.145x + 35.295, R2 = 0.9883) between the log-transformed numbers of nematodes inoculated (x) and the respective Ct values (y). Based on this calibration curve, the densities of D. destructor in soils were estimated to be 43/10 g of soil in an infested field and 0/10 g of soil in a field without a history of garlic cultivation. These results demonstrated that the presently designed primers are useful to quantify the density of D. destructor in both soil and garlic.