Nested PCR을 통한 참다래 궤양병균 (Pseudomonas syringae pv. actinidiae)의 검출

Abstract

참다래 잎으로부터 궤양병균인 Pseudomnoas syringae pv. actinidiae를 배양하여 PCR을 통해 검출하는 방법을 개발하였다. Nested PCR을 위해 두 set의 primer를 식물 독소인 coronatine의 생합성에 관여하는 유전자 cfl의 염기서열로부터 설계하였다. 이 primer set를 사용하여 665rhk 310-bp의 절편이 증폭되었으며 nested PCR을 통한 궤양병균의 검출한계는 20 CFU/ml이었다. 4 그루의 참다래 나무로부터 노란색 무리로 나타나는 궤양병 초기 증상을 보이는 잎을 채취하여 pepton-sucrose 액체배지에 넣어 <TEX>$16^{\circ}$</TEX>C에서 12시간 배양 한뒤 PCR 을 시행한 결과 한 시료에서 예상했던 밴드가 증폭되었고 이듬해 봄 이 나무가 궤양병에 감염되었음을 확인하였다. A PCR method that combines biological and enzymatic amplification of PCR targets was developed for the detection of Pseudomonas syringae pv. actinidiae on kiwifruit leaves. A nested PCR was performed with primers designes from the coding sequence of the cfl gene, which is involved in production of the phytotoxin coronatine. The first and second primer sets efficiently amplified expected 665 and 310-bp fragments, respectively. With two successive amplifications, as few as 20 CFU/ml of P. syringae pv. actinidiae could be detected on ethidium bromide-stained agarose gel. Leaf samples were collected from 4 kiwifruit trees showing yellow halo spots on leaves and incubated in pepton-sucrose broth for 12 h at <TEX>$16^{\circ}$</TEX>C before PCR amplification. Positive detection was obtained with one sample, which was proved as a diseased plant in the next spring.