Pathogenicity and Nutritional Requirement Tests of Xylella fastidiosa Pear and Grape Strains Isolated in Taiwan

Xylella fastidiosa Wells (Xf) 為一植物導管侷限細菌，可感染多種重要經濟作物並造成病害，如葡萄皮爾斯病 (Pierce''s disease, PD)，自然界中，此菌可藉由昆蟲傳播。而在台灣，Xf則為梨葉緣焦枯病 (Pear leaf scorch disease, PLS)及葡萄皮爾斯病 (Pierce''s disease, PD)的病原菌。本實驗利用由台灣梨樹分離之Xylella fastidiosa菌株 PLS 235、PLS 244及由葡萄分離之PD 5-1-2、GM菌株，分別對梨樹 (Pyrus pyrifolia)、蟛蜞菊 (Wedelia trilobata)、菸草 (Nicotiana tabacum) 及日日春 (Catharanthus roseus) 進行接種測試，以了解台灣分離之梨葉緣焦枯病菌與葡萄皮爾斯病菌是否能感染不同植物，以及病原菌可否在接種植物導管中移行。梨樹接種PLS及PD菌株之接種測試只於初次分離實驗中出現一次PCR正反應，並且無法從葉柄中分離出病原菌；蟛蜞菊之葉柄病原菌分離與PCR檢測結果皆為無檢出，顯示其並非Xylella fastidiosa之寄主；另菸草及日日春接種試驗中，四菌株皆分別有PCR 檢測正反應之樣本，且可從葉柄中成功分離病原菌，顯示台灣分離之 X. fastidiosa 可在菸草及日日春中生長並在導管中進行系統性移行。在實驗的過程中，發現上述四種接種的植物並未出現病徵，與接種水的對照組外觀上無明顯差異，可能與接種植物之生長環境與營養供給有關，又或許 Xf 菌株確實不會在這些測試植物中顯現病徵，而這些假設均需更進一步實驗才能證明。另外，為測試此四種菌株的培養營養需求，以Color changing units (CCU)方式分別測試PD2培養基中的Succinate、Trisodium citrate、MgSO4、Hemin Cl及Bovine serum albumin (BSA) 是否為其生長必需成分。結果顯示PLS 235、PLS 244、PD 5-1-2及GM等 Xf 菌株可在無添加succinate的培養基生長，但移除 PD2 中的 MgSO4後，PLS 235、PLS 244 及GM等菌株的生長狀況明顯較差，顯示在試管中培養 Xf 菌株時需添加適量 MgSO4。PD及PLS菌株於去除trisodium citrate之PD2培養基中生長狀況良好，顯示此成分可能並非生長必需物質。而在去除succinate及citrate的培養基中，細菌完全無法生長，但可以在添加malate或α-ketoglutarate後回復。在移除Bovine serum albumin (BSA) 的 PD2 培養基，PLS 及 PD 菌株的生長明顯降低。測試不同濃度 BSA 對 Xf 菌株的生長時，發現降低 BSA 濃度至0.4 g/L 可促進細菌的生長。由營養需求測試結果可知 PLS 與 PD 菌株中存在菌株間生理及生化特性的差異，經由適當的調整培養基中的成分，可達到促進不同植物來源的Xf菌株在培養基上生長的目的。Xylella fastidiosa is a xylem-limited bacterium (XLB); it causes many diseases on a wide host range of plant, including Pierce’s disease of grapevine. In nature, the bacterium is transmitted by insect vectors. In Taiwan, Xylella fastidiosa is the causal bacterium of pear leaf scorch disease (PLS) and Pierce’s disease (PD). To understand if the bacterium infects other plants, i.e. it can migrate within the xylem vessels or cause Xf-elicited disease symptoms, X. fastidiosa pear strains PLS 235, PLS 244 and grape strain PD 5-1-2 and GM were used to inoculate pear (Pyrus pyrifolia), periwinkle (Catharanthus roseus), tobacco (Nicotiana tabacum) and wedelia (Wedelia trilobata) by stem-stabbing method to find out the possible alternative hosts of X. fastidiosa in Taiwan. The inoculation of pear with PLS and PD strains showed only one PCR positive result in the first recovery of pear petioles, and no bacteria can be re-isolated from the inoculated pears. In wedelia, PLS bacteria cannot be detected by PCR in or recovered from petiole elutents, indicating that wedelia is not a host of PLSB. The results of PCR detection and bacterial isolation showed that X. fastidiosa strain PLS 235, PLS 244, PD 5-1-2 and GM can infected and migrate within the xylem vessels of tobacco and periwinkle plants, indicating that tobacco and periwinkle might be alternative host plants of Taiwan-isolated X. fastidiosa. However, Xf strains did not elicit apparent disease symptom on tobacco or periwinkle, which might be due to the inappropriate environmental conditions where the inoculated plants were kept. For testing in-vitro nutrient requirements of PD and PLS bacteria, a color changing unit (CCU) method was applied to test the ingredients of disodium succinate, trisodium citrate, MgSO4, hemin Cl and bovine serum albumin (BSA) in PD2 medium. The results showed that all tested Xf strains can grow in PD2 medium without succinate, but the growth of PLS235, PLS244 and GM strains of Xf were greatly compromised without the addition of MgSO4 , indicating that MgSO4 is essential for culturing Xf. In the modified PD2 medium without succinate and citrate, bacterial growth was completely halted, and the growth can be restored by replacing succinate and citrate with malate or α-ketoglutarate. Removal of bovine serum albumin (BSA) from PD2 medium compromised the Xf growth. Moreover, the concentration of BSA in PD2 medium can be adjusted to 0.4 g/L to promote the growth of PLS bacteria. Taken together, the nutrient requirement tests reveal strain-specific physiological and biochemical properties among Xf PLS and PD bacteria. Adjustment of the nutrients will optimize the formulation of a culture medium to promote the growth of Xf bacteria from different plant hosts.中文摘要------------------------------------------------------------------------------------------i 英文摘要-----------------------------------------------------------------------------------------iii 目錄------------------------------------------------------------------------------------------------v 表目錄-------------------------------------------------------------------------------------------vii 圖目錄------------------------------------------------------------------------------------------viii 前言----------------------------------------------------------------------------------------------01 一、病原菌Xylella fastidiosa之源起---------------------------------------------------------01 二、病原菌Xylella fastidiosa之分類、寄主、分佈及傳播----------------------------------01 三、Xylella fastidiosa之生長及培養特性----------------------------------------------------02 四、台灣梨葉緣焦枯病之沿革----------------------------------------------------------------03 五、台灣葡萄皮爾斯病之沿革----------------------------------------------------------------04 六、實驗目的-------------------------------------------------------------------------------------05 材料與方法-------------------------------------------------------------------------------------07 一、寄主植物接種測試-----------------------------------------------------------------------07 (一)病原菌保存與製備-------------------------------------------------------------------07 (二)測試植物之種類與製備-------------------------------------------------------------07 (三)病原菌之接種-------------------------------------------------------------------------07 1.梨樹接種試驗-----------------------------------------------------------------------------08 2.蟛蜞菊接種試驗--------------------------------------------------------------------------08 3.菸草接種試驗-----------------------------------------------------------------------------08 4.日日春接種試驗--------------------------------------------------------------------------09 (四)植物組織分離及PCR檢測病原菌-----------------------------------------------09 1.由植物組織分離病原菌------------------------------------------------------------09 2.由植物組織中萃取DNA-----------------------------------------------------------09 3.聚合酶連鎖反應(Polymerase chain reaction, PCR)與電泳分析------------10 二、營養需求測試-----------------------------------------------------------------------------10 (一)細菌懸浮液製備-------------------------------------------------------------------10 (二)培養基製備--------------------------------------------------------------------11 (三)營養需求測試--------------------------------------------------------------------11 1.生長必需成分測試 ---------------------------------------------------------------------11 2.必需營養成分最適濃度測試-----------------------------------------------------------11 結果----------------------------------------------------------------------------------------------13 一、寄主植物接種測試----------------------------------------------------------------------13 二、營養需求測試---------------------------------------------------------------------------14 三、BSA濃度調整測試-----------------------------------------------------------------------15 討論----------------------------------------------------------------------------------------------16 參考文獻----------------------------------------------------------------------------------------21 圖表----------------------------------------------------------------------------------------------26 附錄-------------------------------------------------------------------------------------------41 表目錄 表一、蟛蜞菊接種Xylella fastidiosa之PCR檢測及病原菌分離結果-------------26 表二、菸草接種Xylella fastidiosa之PCR檢測及病原菌分離結果----------------27 表三、日日春接種Xylella fastidiosa之PCR檢測及病原菌分離結果-------------29 表四、本實驗使用之引子對----------------------------------------------------------------30 表五、Xylella fastidiosa PD及PLS菌株之營養需求測試結果 ---------------------31 表六、Xylella fastidiosa PD及PLS菌株之營養需求測試結果 ---------------------32 表七、PD2 medium 添加不同濃度牛血清蛋白 (BSA) 對Xylella fastidiosa PLS菌株生長的影響 (CCU/mL) -------------------------------------------------------------------33 圖目錄 圖一、以不含Disodium succinate之PD2培養基測試X. fastidiosa PLS 235、PLS 244、PD 5-1-2與GM四菌株之培養基變色情形-------------------------------------------34 圖二、以不含MgSO4之PD2培養基測試X. fastidiosa PLS 235、PLS 244、PD 5-1-2與GM四菌株之培養基變色情形--------------------------------------------------35 圖三、以不含BSA之PD2培養基測試X. fastidiosa PLS 235、PLS 244、PD 5-1-2及GM菌株之培養基變色情形 ---------------------------------------------------------36 圖四、以不含Trisodium citrate之PD2培養基測試X. fastidiosa PLS 235、PLS 244、PD 5-1-2及GM菌株之培養基變色情形 -------------------------------------------37 圖五、以不含Hemin Cl之PD2培養基測試X. fastidiosa PLS 235、PLS 244、PD 5-1-2及GM菌株之培養基變色情形-------------------------------------------------------38 圖六、以去除succinate及trisodium citrate並加入malate (1 g/L) 之PD2培養基測試X. fastidiosa PLS 235、PLS 244、PD 5-1-2及GM菌株的培養基試管變色情形 ----------------------------------------------------------------------------------------------------39 圖七、以去除succinate及trisodium citrate並加入α-ketoglutarate (1 g/L) 之PD2培養基測試X. fastidiosa PLS 235、PLS 244、PD 5-1-2及GM菌株的培養基試管變色情形-------------------------------------------------------------------------------------------------4