利用聚合酵素連鎖反應鑑定檬果黑斑病菌

Identification of Xanthomonas campestris pv. mangiferaeindicae Using the Polymerase Chain Reaction

黃秀珍(H. C. Huang) ； 張治安(J. A. Chang) ； 林元春(Y. C. Lin) ； 朱木貴(M. K. Chu) ； 林信成(H. C. Lin) ； 徐世典(S. T. Hsu)

《植物病理學會刊》 6卷1期 (1997/03) Pp. 1-9

https://doi.org/10.6649/PPB.199703_6(1).0001

摘要

檬果黑斑病菌(Xanthomonas campestris pv. mangiferaeindicae，簡稱XCM)H15菌株之全量DNA (total DNA)經限制酵素BamHI處理後，與載體pBluescript SK接合，再轉型到E. coli DH10B菌株，構築一基因庫，由其中逢機選取之3個選殖株(clones)，分別命名爲 pXcM58、pXCM24及pXCM21，其插入片段大小分別爲5.8，2.4，及2.1 kb。這些選殖株以random priming的方式製作以非放射性物質Digoxigenin-11-dUTP(DIG)標識做爲核酸探針(probe)，並以南方轉漬法(Southern blotting)及點漬法(dot blotting)分析分離自臺灣不同地區或不同品種檬果的各供試黑斑病菌株，眞菌炭疽病菌及其他植物病原細菌染色體DNA之雜合關係。試驗結果顯示此三種探針皆可專一性地與黑斑病菌之染色體發生雜合現象，但pxCM24 也可與X. c. pv. vesicatoria發生微弱雜合訊號。在聚合酵素連鎖反應(polymerase chain reaction，簡稱PCR)的硏究上，由pXCM21、pXCM24、pXCM24之次選殖株pXCM24-1及pXCM58之次選殖株pXCM58-P1的部份DNA序列中分別設計了4對引子P21-3/P21-7、P24-3/P24-7、P24-3/P24-1-7及P58-P1-3/P58-P1-7。這4對引子以XCM菌株染色體爲模版時，可專一性的增幅到預期的片段大小分別爲1.9，2.2，0.9及1 kb ,其偵測率分別爲82.9%、100%、94.3%及100%。針對偵測性最高的兩對引子P24-3/P24-7及P58-P13/P58-P1-7測試其敏感性，能偵測純化的DNA約爲10-100g，菌量約爲100-500個細胞。由以上結果顯示，這2對引子在未來於田間檬果黑斑病之診斷上應具有應用潛力。

關鍵字

檬果黑斑病菌 ； 核酸探針 ； 聚合酵素連鎖反應

並列摘要

A genomic library of Xanthomonas campestris pv. mangiferaeindicae (XCM) strain H15 was constructed in the pBluescript SK. and was transformed into E. coli DH10B. Three recombinant clones, designated as pXCM58, pXCM24, and pXCM21 respectively, were randomly selected and labelled by the nonradioactive system with Digoxigenin-11-dUTP (DIG) using the random priming method. In Southern-blot hybridization and dot-blot hybridization analyses these three probes specifically hybridized to XCM strains but not to other microorganisms including Erwinia carotovora, E. chrysamthermi, Pseudomonas solanacerum, X. c. begoniae, X. c. campestris, X. c citri, X. c. diffenbachiae X. c. pruni, X. c. vesicatoria, and Colletotrichum gloeosporioides chromosomes. Four sets of primers. P21-3/P21-7, P24-3/P24-7, P24-3/P24-1-7, and P58-P1-3/P58-PI-7, were generated from the nucleotide sequences of insert DNAs in pXCM21, pXCM24. pXCM24-1 (subcloned from pXCM24) and pXCM58-P1 (subcloned from pXCM58), respectively. These four primer sets specifically amplify 1.9, 2.2, 0.9 and 1 kb DNA fragments respectively, using chromosomal DNAs of XCM strains as templates in PCR (polymerase chain reaction) assay; the rates of detection for the four primer sets using 35 strains of XCM were 82.9%. 100%. 94.3% and 100%, respectively. In sensitivity test. P24-3/P24-7 and P58-P1-3/P58-P1-7 detected the minimal DNA amount of 10-100 fg, and minimal number of 100-500 cells. The results indicate that two primer sets, P24-3/P24-7 and P58-P1-3/P58-P1-7, can be potentially developed to diagnose naturally bacterial black spot infected fruits using PCR technique.

並列關鍵字

Xanthomonas campestris pv. Mangiferaeindicae ； DNA probes ； polymerase chain reaction