Accurate detection of chestnut ink disease causing Phytophthora katsurae by nested PCR

Abstract

We designed two specific primer pairs, KatI 3F/KatI 5R and KatI 4F/KatI 5R to detect P. katsurae by PCR and each primer produced specific amplicons of 747 bp and 721 bp, respectively. To assess the specificity and sensitivity of primer, 10-fold serial dilutions of P. katsurae genomic DNA were made ranging in concentration from 1 mgml⁻¹ to 1 pgml⁻¹. Using nested PCR, the limits of detection for P. katsurae were 100 ngml⁻¹ and 10 ngml⁻¹ of DNA, respectively. To identify the limits of detection for P. katsurae zoospores, zoospore suspensions were serially diluted 10-fold from 10 × 10⁵ to 10 × 10¹ before being extracted. The limit of detection for both primer sets was 10 × 10¹. We applied to test tissue and soil samples from chestnut stands that had been infested by P. katsurae in Gongju, Chungnam Province, and Hadong, Gyeongsangnam Province, and could detect specific P. katsurae amplicons from trunk tissue collected from sites containing infected trees in nested PCR. These results show that nested PCR may be a useful tool for the accurate and sensitive detection of P. katsurae infection.