- 期刊
Diagnosis of Paulownia Witches' Broom Disease Using PCR Amplified Phytoplasma 16S rDNA as a Molecular Marker
利用PCR擴增植物菌質體16S核糖體核酸片段以診斷泡桐簇葉病
摘要
利用聚合酶聯鎖反應及對植物菌質體16S核糖體核酸具有專一性的引子，可用來診斷泡桐簇葉病。由罹病泡桐組織萃取之核酸經聚合酶聯鎖反應後可擴增一條1.2kb大小之核酸條帶，而以健康泡桐核酸為模板則無擴增核酸出現。聚合酶聯鎖反應以進行32個循環結果最佳。反應中加入DMSO會降低產物的量。由葉脈萃取之核酸作為泡桐簇葉病之診斷材料雪敏度最高，當罹病植株之核酸被稀釋至150pg時仍可檢測到被擴增的1.2kb植物菌質體16S核糖體核酸片段。為確認被擴增的1.2kb核酸片段為泡桐簇葉病植物菌質體16S核糖體核酸，共試驗了24種限制酶切割該段1.2kb核酸片段，結果包括ApaI、AvaI、BamHI、BgII、DraI、HindIII、MluI、PstI、SalI、XbaI、及XhoI等11限制酶在1.2kb核酸片段上無切點。面包括AluI、EcoRI、HaeIII、HhaI、HinfI、HpaII、KpnI、MseI、RsaI、Sau3A、ScaI、TaqI及ThaI等13限制酶在1.2kb核酸片段上有1至4切點，其核酸限制圖譜顯示泡桐簇葉病植物菌質體16S核糖體核酸屬於16SrI-D群。利用此技術分析林試所蓮花池分所第一及第二菌圖栽植之由大祟引進耐簇葉病四川及貴州種源泡桐，共分析95株，結果貴州及四川種源泡桐之感病率分別為14%與33%，顯示二種源均易感病，但貴州種源病微較不明顯，生長較佳，具較高之耐病性。
並列摘要
A diagnostic technique based on polymerase chain reaction (PCR) using primer pairs designed specifically against plant phytoplasma 16S rDNA sequence reported previously by Lee et al. (1993a) was optimized for field survey of paulownia witches' broom disease. A 1.2 kb DNA fragment was amplified from witches'-broom-disease-infected paulownia but not from healthy paulownias. PCR at 32 cycles gave the highest amount of amplification products and the addition of dimethyl sufoxide reduced the amount of amplification products. Total DNA extracted from leaf vein gave the highest sensitivity of phytoplasma detection in field-grown paulownias. The diagnostic 1.2 kb phytoplasma rDNA fragment was still detectable when the template DNA was diluted down to about 150pg. A total of 24 commercially available restriction enzymes were used in the restriction analysis of the 1.2kb phytoplasma 16S rDNA fragment. Among them, ApaI, AvaI, BamHI, BgII, DraI, HindIII, MluI, PstI, SalI, XbaI, and XhoI did not digest the 1.2 kb rDNA fragment. The enzymes AluI, EcoRI, HaeIII, HhaI, HinfI, HpaI, KpnI, MseI, RsaI, Sau3A, ScaI, TaqI, and ThaI produced from two to five restriction fragments which ranged from 50 to 1130 bp. The restriction patterns indicated that paulownia witches' broom phytoplasma was in the 16SrI-D group as previous reported. A total of 95 potentially witches'-broom-disease-tolerand P. fortunei trees introduced from Kweichow and Szuchuan Provinces of mainland China were screened for the presence of phytoplasma infection. The infection rate for the Kweichow and Szuchuan provenances were 14% and 33%, respectively, indicating both provenances were highly susceptible. However, P. fortunei trees of the Kweichow provenance showed very few symptoms suggesting some degree of disease tolerance.