梨葉緣焦枯病病菌與其他寄主侷限導管細菌之親緣相關性及研發PCR鑑定技術

Phylogenetic Relationships between Pear Leaf Scorch Bacteria and Strains of Xylella fastidiosa from Other Host, then Development of PCR Identified Technique

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

《臺中區農業改良場特刊》 75號 (2005/12) Pp. 255-290

https://doi.org/10.29563/ZHWHGX.200512.0008

摘要

梨葉緣焦枯病（pear leaf scorch）為台灣特有之病害，亦為限制梨產業之重要病害，其病原菌為侷限導管細菌（xylem-limited bacteria; Xylella fastidiosa Wells），為具有波浪狀細胞壁且歸類為不易培養之原核生物（fastidious prokaryrote），過去研究顯示梨葉緣焦枯病菌與其他寄主之X.fastidiosa菌株間為低血清相關性（serology relation），同時X. fastidiosa專一性引子對272-int-1/272-int-2及RST31/RST33對國內各地梨葉緣焦枯病病菌菌株進行PCR反應，則無任何條帶產生，因此梨葉緣焦枯病病菌菌株與其他寄主之X. fastidiosa 菌株間之親緣關係（phylogentic relationship）值得進一步釐清；利用ERIC-PCR、RAPD-PCR及BOX-PCR等技術分析國內各地之梨葉緣焦枯病病菌菌株及其他寄主之X. fastidiosa菌株之DNA，其他寄主植物包括夾竹桃（oleander）、胡桃（pecan）、李（plum）、桃（peach）、桑（mulberry）、葡萄（grapevine）及無花果（sycamore），分析結果皆可將試驗菌株區分為兩大群，分別為梨菌株群與其他寄主之X. fastidiosa菌株群，其中其他寄主之X. fastidiosa菌群可以再分為三個菌群：（I）夾竹桃菌群、（II）胡桃、李、桃及無花果菌群及（III）桑及葡萄菌群；進一步進行16S rDNA及16-23S rDNA區間序列之分析，分別選殖得到特定核酸片段大小介於1537～1540bp及512～540bp，其中基因序列顯示梨葉緣焦枯病病菌菌株與其它寄主之X. fastidiosa菌株相似值分別介於0.982～0.987及0.874～0.876；利用由鄰聚法（neighbor joining）進行親緣相關性分析，由16S rDNA序列及16-23S rDNA區間序列所得親緣樹（phylogentic tree）皆可將菌株分為兩大群，分別為梨菌株群及其它寄主之X. fastidiosa菌株群。由指紋圖譜分析及特定核酸序列分析之結果，顯示梨葉緣焦枯病病菌菌株應為Xylella屬內獨立的菌株群，由此結果應可支持梨葉緣焦枯病菌菌株應為Xylella亞種以上分類地位，甚至可能為新種。為快速鑑別梨葉緣焦枯病菌菌株，利用OPA11隨機引子進行RAPD分析，成功篩選出梨葉緣焦枯病病菌獨有核酸片段大小為1412bp，進一步進行轉殖及序列解序，並設計得一組專一性引子對為PLS-F/PLS-R（5'-TGGACGTTGTGGTATCGGTG-3'/ 5'- TTGAAGTTGACGTGTGGCTG-3'），此一引子可對梨葉緣焦枯病病菌增幅出416bp專一性產物片段，但其他寄主之X. fastidiosa 菌株及其他對照一般病原細菌菌株之基因體核酸皆無法增幅產物片段，其靈敏度最高可以到10pg基因體核酸，並可直接進行田間罹病梨樹組織之檢測，此引子有潛力應用於偵測田間中間寄主植物及蟲媒之病原菌。

關鍵字

梨葉緣焦枯病 ； 侷限導管細菌 ； 親緣相關性 ； PCR鑑定技術

並列摘要

Pear leaf scorch (PLS) disease was unique and limited in the cultivation of pear in Taiwan. It was caused by strains of Xylella fastidiosa, xylem-limited bacteria, belonged to fastidious prokaryote that had a ripple cell wall structure. The early study showed PLS strains were low serology relation in comparison of strains of X. fastidiosa from other host. Then, for polymerase chain reaction (PCR) test, none DNA fragment was amplified from PLS strains with two specific sets of RST31/RST33 and 272-int/272-int-2 those to detect and identify of X. fastidiosa. Therefore, to determine the taxonomic relatedness between PLS bacterium and strains of X. fastidiosa from other host, we conducted DNA fingerprinting -enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR), random amplified polymorphic DNA-PCR (RAPD-PCR), and BOX-PCR assays and sequenced the 16S rDNA and 16-23S rDNA intergenic spaces region using PLS bacterium and strains of X. fastidiosa from other host that included oleander, pecan, plum, peach, mulberry, grapevine and sycamore. Under dendrogram depict the genetic relationships using RAPD, ERIC-PCR and BOX-PCR assays, all strains were distinguished two major groups- pear group and other host group, and strains of X. fastidiosa from other host could be grouped into three subgroups -(I) oleander group; (II) pecan, plum, peach, and sycamore group; (III) mulberry and grapevine group. The specific DNA fragment size cloned from 16S rDNA sequence and 16-23S rDNA intergenic spacesr region sequence from PLS bacterium and strains of X. fastidiosa from other host was 1537∼1540bp and 512∼540bp, while the similarity of those specific fragment DNA sequence from PLS bacterium in comparison with strains of X. fastidiosa was 0.982 ∼ 0.987 and 0.874 ∼ 0.876. Under neighbour-joining method showed phylogenetic relationships, all strains were distinguished two groups -pear group and other host group. The PLS bacterium was low closely related in comparison of strains of X. fastidiosa from other host. The data of DNA fingerprinting and intergenic spacer DNA sequence showed PLS bacterium did not belong to the X. fastidiosa genomic species. We postulated the PLS bacterium could be a new species within the genera of Xylella , but further elucidate it combined other characters. For development of a rapid PCR technique to identify and detect PLS bacterium, a specific random amplified polymorphic DNA product (PLS-OPA11) was isolated from a PLS strain. The specific fragment of DNA was cloned, sequenced, and found to be 1412bp in length. A set of specific primer PLS-F/PLS-R (5'-TGGACGTTGTGGTATCGGTG-3' /5'- TTGAAGTTGACGTGTGGCTG -3') was obtained to utilize these sequence data for PLS strains. The primer set amplified a 416 bp DNA fragment from PLS strains, but not from strains of X. fastidiosa from other host and strains of other general phytobacteria. The minimum amount of DNA from X. fastidiosa that could be amplified by PCR was 10pg. The PLS bacterium could be directly detect by PCR technique using pear diseased tissue. A developed PCR primer technique would be a potential to exploit for detection of PLS bacterium within vector and alternative host plants in future.

並列關鍵字

pear leaf scorch ； Xylella fastidiosa ； phylogenetic relationships ； PCR identified technique