梨葉緣焦枯病

Abstract

梨葉緣焦枯病(pear leaf scorch)為台灣特有之病害，普遍發生於横山梨(Pyrus pyrifolia)栽植區，梨葉緣焦枯病菌之培養性狀及具波浪狀細胞壁(ripple cell wall) 的特性與侷限導管細菌(xylem-limited bacteria) Xylella fastidiosa 相同，但梨葉緣焦枯病菌與其他寄主植物來源之X. fastidiosa 菌株 間無血清相關性，且以X. fastidiosa 廣效性引子對272-1-int/272-2-int 及 RST31/RST33 對梨葉緣焦枯病菌菌株進行polymerase chain reaction (PCR) 反應時，亦無任何基因產物。利用隨機引子(OPA11)進行random amplified polymorphic DNA (RAPD)分析，成功獲得對梨葉緣焦枯病菌獨有核酸片段大小為1412 bp，從核酸序列中設計得到一組專一性引子對PLS-F/PLS-R 可增幅出416 bp 大小之專一性基因產物。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。利用RAPD-PCR、enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR)及BOX-PCR 三種DNA 指紋圖譜技術分析所得之親緣樹狀圖皆可將8 種寄主植物之 X. fastidiosa 菌株區分為4 個菌群，分別為：(1)梨菌群、(2)夾竹桃及葡萄菌群、(3)桑椹菌群及(4)胡桃樹、李樹、桃樹及無花果菌群。此等指紋圖譜技術均可有效鑑別梨葉緣焦枯病菌，並顯示其為 X. fastidiosa 菌群中唯一之獨立菌系。進一步利用鄰聚法（Neighbor Joining）進行10 種不同寄主植物之 X. fastidiosa 菌株系統親緣分析（phylogentic analysis），由16S rRNA 序列及16S-23S rRNA 區間序列所構建的親緣演化樹（phylogentic tree）皆可明顯區分為2 個菌群，分別為梨葉緣焦枯病菌菌群及其他9 種寄主植物X. fastidiosa 菌群，而其他寄主植物X. fastidiosa 菌群皆可再細分4 個次菌群，分別為(1)夾竹桃菌群、(2)葡萄及桑椹菌群、(3)咖啡及柑橘菌群及(4)胡桃樹、李樹、桃樹及無花果菌群。由以上結果顯示，梨葉緣焦枯病菌為Xylella 屬內僅侷限於台灣獨立的菌群與其他寄主植物X. fastidiosa 菌株間為較低同源性，應可支持梨葉緣焦枯病菌為Xylella 屬內亞種分類地位，甚至可能為新種。本文研發有關侷限導管細菌PCR 鑑定與偵測技術，未來有潛力應用於梨葉緣焦枯病菌之蟲媒及中間寄主植物快速偵測。合理的管理栽培制度應可減輕梨葉緣焦枯病之嚴重度，剷除罹病株，可減少其感染源，無性繁殖時應避免剪取罹病株之枝條扦插或芽供接穗之用，化學治療方面，注射四環黴素於每年5 月及10 月各注射一次，有相當程度之療效，配合增施有機肥可維持病樹正常生產力。 Pear leaf scorch (PLS) disease, the only Xylella fastidiosa-induced disease reported from Taiwan, is commonly found in areas where the variety Hengshan (Pyrus pyrifolia) is grown. Strains of PLS bacterium shares similarities to strains of X. fastidiosa from other host origins in the requirement of complex medium and the rippled cell wall in cell structure. 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 general primer sets of RST31/RST33 and 272-int/272-int-2 used to detect strains of X. fastidiosa from other hosts. A fragment of 1412 bp in size amplified for PLS strains with a random primer OPA11 by random amplified polymorphic DNA(RAPD) technique was identified as specific for PLS strains. A set of specific primer PLS-F/PLS-R amplified a 416 bp fragment was designed from the sequence data for PLS strains. The PLS bacterium could be detected by using the specific primer set in PLS diseased leaf tissues collected from fields.The DNA fingerprintings generated by RAPD-PCR, enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR) and BOX-PCR of all strains of X. fastidiosa from eight hosts. The constructed dendrograms showed that all strains of PLS bacterium were clusted together and separate from other X. fastidiosa strains, and the latter wereclustered into three groups: one consisted of mulberry strains, another consisted of grape and oleanderstrains, and the third comprised pecan, plum, peach and sycamore strains. Our results indicate that the DNA fingerprintings amplified by arbitrary primers are useful to differentiate pear leaf scorchbacteria from other X. fastidiosa strains. Further compared the phylogenetic relationship based on 16S rRNA sequence and 16S-23S rRNA spacer region of various strains from ten hosts using neighbor joining method. All strains of X. fastidiosa from different host were distinguished two groups, pear group and other host group. The other host group were subdivided into four subgroup: (1)oleander, (2)grape and mulberry, (3)citrus and coffee, and (4) pecan, peach, plum and sycamore. According to the above 16S rRNA sequence and 16S-23S rRNA spacer region data, pear leaf scorch strains were much low homology in comparison with strains of X. fastidiosa from other host and induced disease which only reported in Taiwan. We postulated pear leaf scorch strains could be a new subspecies or species within the genera of Xylella. The developed PCR identified techniques of X. fastidiosa could be a useful tool for rapid detection of PLS bacterium in insect vectors and alternative hosts in Taiwan in the future. The rational cultural pratices, such as roguing diseased plant, avoiding bud or cutting from infected trees for vegetable reproduction and increasing amendments of organic matters, could alleviate disease symptoms. Injection of oxytetracycline solution in May and October partially suppressed disease development.