應用聚合酵素連鎖反應偵測梨葉緣焦枯病菌

Specific Detection of Xylella fastidiosa Strains Causing Pear Leaf Scorch by Polymerase Chain Reaction

蘇秋竹(C. C. Su) ； 楊文仁(W. J. Yang) ； 徐世典(S. T. Hsu) ； 曾國欽(K. C. Tzeng)

《植物病理學會刊》 17卷3期 (2008/09) Pp. 183-194

https://doi.org/10.6649/PPB.200809_17(3).0001

摘要

梨葉緣焦枯病(Pear leaf scorch)爲台灣橫山梨(Pyrus pyrifolia)栽植區特有之病害，其病原菌爲局限導管細菌(Xylella fastidiosa Wells)，但梨葉緣焦枯病菌與其他寄生植物來源之X. fastidiosa菌株間無血清相關性，且以X. fastidiosa廣效性引子對272-int-1/272-int-2及RST31/RST33對梨葉緣焦枯病菌菌株進行PCR反應時，亦無任何基因産物。爲研發快速鑑定及偵測梨葉緣焦枯病菌之PCR技術。本研究利用隨機引子進行RAPD分析，成功獲得對梨葉緣焦枯病菌獨有之約1,400 bp大小核酸片段，進一步將此核酸片段轉殖及序列解序，確認此核酸片段大小爲1,412 bp（GenBank注冊編碼爲DQ452418），從所得核酸序列中設計得到一組專一性引子PLS-F/PLS-(5'-TGGACGTTGTGGTATCGGTG-3'/5'-TTGAAGTTGACGTGTGG CTG-3')，此一引對測試梨葉緣焦枯病菌30個菌株皆可增幅出416 bp大小之專一性基因産物，但測試來自於夾竹桃(oleander)、胡桃樹(pecan)、李樹(plum)、桃樹(peach)、葡萄(grape)、桑椹(mulberry)及無花果(sycamore)等7種寄生植物之X. fastidiosa 34個菌株及一般植物病原細菌10個菌株之基因體核皆無法增幅出基因産物。此一引子對可直接進行梨葉緣焦枯病田間罹病梨樹組織之PCR檢測，具有潛力可應用於田間寄主植物及蟲媒內病原菌偵測。

關鍵字

梨葉緣焦枯病菌 ； 專一性引子對 ； 聚合酵素連鎖反應 ； 偵測

並列摘要

Pear leaf scorch (PLS) disease is unique its Taiwan and only found its areas where the Hengshen variety (Pyrus pyrifolia) is grown, It is caused by Xylella fastidiosa, a xylem limited bacterium, However, PLS strains are not serologically related to strains of X. fastidiosa from other hosts. Likewise, no DNA fragment was amplified from PLS strains with two universal primer sets of RST31/R5T33 and 272 int/272-int-2 used to detect strains of X. fastidiosa from other hosts. in this study, we developed a polymerase chain reaction (PCR) technique to specifically identify and detect PLS bacterium. The random amplified polymorphic DNA technique was employed and a fragment of 1,412 bp in site was specifically amplified with a random primer OPA11 for PLS strains (GenBank accession no. DQ452418). A set of specific primer PLS-F/PLS-R (5' TGGACGTTGTGGTATCGG1 G-3'/5'-TTGAAGTTGACGTGTGGCTG-3') designed from the 1,412-bp DNA amplified a 416 bp fragment front all 30 PLS strains tested hut not from 34 strains of X. fastidiosa originally isolated from other hosts, including oleander, pecan, plum, peach, grape. mulberry and sycamore, nor from 10 strains of plant pathogenic bacteria other than Xylwella. The primer set can efficiently detect PLS bacterium in PLS diseased leaf tissues collected from fields. We proposed the PER technique developed in this study could he a useful tool for rapid detection of PLS bacterium in potential insect vectors and alternative hosts in the future.

並列關鍵字

Xylella fastidiosa ； pear leaf scorch ； specific primer set ； polymerase chain reaction ； detection