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研究生:萬宥伶
研究生(外文):Yu-Ling Wan
論文名稱:西瓜蔓割病菌變異菌株之毒力相關基因表現的探討
論文名稱(外文):Expression of virulence-related genes in variants of Fusarium oxysporum f. sp. niveum
指導教授:張碧芳張碧芳引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:50
中文關鍵詞:西瓜蔓割病變異毒力相關基因
外文關鍵詞:watermelon wilt pathogenFusarium oxysporum f. sp. niveumcultural variationvirulence-related gene
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西瓜為台灣地區重要的蔬菜作物之ㄧ,而西瓜蔓割病為其中一個影響西瓜產量甚鉅的土壤傳播性病害。西瓜蔓割病菌 (Fusarium oxysporum f. sp. niveum) 在人工培養一段時間後,菌落型態常發生變異,由原本的野生孢子堆型轉變成菌絲型或黏膜型。根據前人研究顯示,菌落型態變異後,若利用帶菌土接種方式,則該病原菌之變異型感染西瓜之毒力也會因此而降低。本研究目的為探討西瓜蔓割病菌 Fon-H0103 之各培養型態的菌株,在特定培養條件下的生長速率、分生孢子產量、厚膜孢子形成量,及其對寄主西瓜「藍寶」品種毒力之影響。試驗結果顯示,變異型菌株 (黏膜型與菌絲型) 的單一分生孢子在培養基上的菌落直徑發展情況與野生型菌株 (孢子堆型) 間存在差異;在產孢量分析上,黏膜型菌株之分生孢子明顯多於孢子堆型與菌絲型菌株;而在形成厚膜孢子的測試方面,發現黏膜型菌株形成厚膜孢子的能力明顯低於孢子堆型與菌絲型菌株。另外,以浸根方式接種各培養型態的病原菌 (濃度為105 spores/ mL) 於西瓜藍寶品種的幼苗後得知,變異型菌株仍具有毒力,甚至有略高於孢子堆型菌株的趨勢。在分子生物層次的分析方面,以隨機增幅多型性核酸 (RAPD) 技術分析各培養型態之菌株,發現其條帶分佈具一致性,顯示在基因組核酸組成上可能沒有發生變異;進一步利用反轉錄-聚合酶連鎖反應 (RT-PCR) 分析各菌株中與毒力相關基因之表現,結果顯示多數所分析的毒力相關基因在變異型菌株中之表現量較孢子堆型菌株為高,與接種結果吻合,因此推測菌落型態的變異對病原菌造成的影響為降低其殘存於土壤中的能力與纏據植物根部的能力。
Watermelon is one of the most important vegetable crops in Taiwan. Fusarium wilt of watermelon is a soil-borne disease which affects watermelon production severely. Watermelon wilt fungal pathogen, Fusarium oxysporum f. sp. niveum, tends to develop cultural variability from sporodochial type (abundant in aerial mycelium and sporodochia formation ) to mycelial type (only abundant in aerial mycelium) and pionnotal type (lack in aerial mycelium) during sequential culture transfers. According to the previous studies, reduced virulence of the pathogen accompanied with cultural variation when inoculated with infested soil. The aim of this research is to study the effects of cultural variability on the mycelia growth, conidiation, chlamydospore formation, and virulence (by root-dipping inoculation) toward host plant Grand Baby watermelon. The results showed that there were differences in the growth rate between the cultures of sporodochial and the mycelial or pionnotal variants. The pionnotal variant culture, however, produced the most abundant conidia. Nevertheless, during nutrition deficiency in the soil extract, the pionnotal variant produced the least amount of chlamydospores, which are the survival structure of the pathogen. In the results of root-dipping inoculation (105 spores / mL) of Grand Baby watermelon seedlings, the pionnotal variant cultures remained highly virulent, even slightly higher than the sporodochial variant culture. On the molecular biology respect, the RAPD patterns of different cultural variants of Fon-H0103 were identical, showing that the compositions of genomic DNA of these cultural variants were not different. According to the expression results of several virulence-related genes, the pionnotal and mycelial variant cultures showed higher level of expression of some genes in the RT-PCR analysis, which is correlated to the results of root-dipping inoculation. The effects of cultural variability on the pathogen might be the reduced survival ability and colonization to the host roots in the soil.
目次
中文摘要-------------------------------------------------------------------------------------------i
英文摘要------------------------------------------------------------------------------------------ii
前言------------------------------------------------------------------------------------------------1
材料與方法---------------------------------------------------------------------------------------6
一、供試菌株來源與培養------------------------------------------------------------------6
二、變異菌株之取得------------------------------------------------------------------------6
三、菌株之生理特性分析------------------------------------------------------------------6
(一)生長測試-------------------------------------------------------------------------------6
(二)分生孢子產量測試-------------------------------------------------------------------7
(三)厚膜孢子形成能力測試-------------------------------------------------------------7
四、核酸之萃取與分析---------------------------------------------------------------------7
(一)去氧核醣核酸之萃取----------------------------------------------------------------7
(二)核糖核酸之萃取----------------------------------------------------------------------8
(三)RNA變性膠體電泳之分析---------------------------------------------------------8
(四)隨機增幅核酸多型性分析----------------------------------------------------------9
(五)反轉錄-聚合酶連鎖反應分---------------------------------------------------------9 (六)RT-PCR結果之量化分析-----------------------------------------------------------9
五、基因之選殖----------------------------------------------------------------------------- 10
六、接種試驗-浸根接種------------------------------------------------------------------- 10
七、數據統計分析-------------------------------------------------------------------------- 11
結果---------------------------------------------------------------------------------------------- 12
一、變異菌株之取得----------------------------------------------------------------------- 12
二、生長測試-------------------------------------------------------------------------------- 12
三、分生孢子產量測試-------------------------------------------------------------------- 12
四、厚膜孢子形成能力測試-------------------------------------------------------------- 13
五、浸根接種試驗-------------------------------------------------------------------------- 13
六、基因之選殖----------------------------------------------------------------------------- 13
七、隨機增幅核酸多型性分析----------------------------------------------------------- 13
八、以反轉錄-聚合酶連鎖反應分析各毒力相關基因的表現---------------------- 14
討論---------------------------------------------------------------------------------------------- 15
參考文獻---------------------------------------------------------------------------------------- 20
圖表---------------------------------------------------------------------------------------------- 27



















表目次
表一、Fon-H0103 各培養型態菌株之生長特性---------------------------------------27
表二、本研究中使用的引子名稱及其序列---------------------------------------------28
表三、各基因片段選殖定序後與其他同源性基因經核酸比對後之相同性------29
表四、各選殖基因之蛋白質功能及其在各培養型態菌株中之表現---------------30





















圖目次
圖一、本研究中西瓜蔓割病各發病等級之病徵呈現---------------------------------31
圖二、Fon-H0103 各型態菌株在減糖 PDA 培養基上生長二週之情形---------33
圖三、西瓜蔓割病菌 Fon-H0103 各培養型態之菌株在減糖 PDA 上菌落直徑
發展的情況-------------------------------------------------------------------------------34
圖四、西瓜蔓割病菌 Fon-H0103 各培養型態之菌株的分生孢子產量----------35
圖五、西瓜蔓割病菌 Fon-H0103 各培養型態之菌株在土壤萃取液中厚膜孢子
產生的情況-------------------------------------------------------------------------------36
圖六、以浸根接種方式觀察 Fon-H0103各培養型態之菌株在藍寶西瓜幼苗造
成發病的情況----------------------------------------------------------------------------37
圖七、以隨機引子OPA-02、OPA-11、OPM-12 及OPJ-20 對 Fon-H0103不同
培養型態之菌株進行 RAPD 增幅之結果-----------------------------------------39
圖八、以反轉錄-聚合酶連鎖反應分析訊息傳導相關基因 fmk1 與 fgb1 在西瓜
蔓割病菌 Fon-H0103 各培養型態之菌株中表現的情形-----------------------40
圖九、以反轉錄-聚合酶連鎖反應分析 pH 訊息傳導相關基因 pacC 在西瓜蔓
割病菌 Fon-H0103 各培養型態之菌株中表現的情形--------------------------42
圖十、以反轉錄-聚合酶連鎖反應植物細胞壁分解酵素相關基因 pl1 與 xlnR
在西瓜蔓割病菌 Fon-H0103 各培養型態之菌株中表現的情形--------------43
圖十一、以反轉錄-聚合酶連鎖反應分析真菌細胞壁生合成相關基因 rho1 與 gas1 在西瓜蔓割病菌 Fon-H0103 各培養型態之菌株中表現的情形-------45
圖十二、以反轉錄-聚合酶連鎖反應分析產孢相關基因 foSTUA 在西瓜蔓割病
菌 Fon-H0103 各培養型態之菌株中表現的情形------------------------------- 47
圖十三、以反轉錄-聚合酶連鎖反應分析逆境相關基因 sod 與 wc1 在西瓜蔓
割病菌 Fon-H0103 各培養型態之菌株中表現的情形---------------------------48
圖十四、以反轉錄-聚合酶連鎖反應分析粒線體運輸蛋白基因 fow1在西瓜蔓割
病菌 Fon-H0103 各培養型態之菌株中表現的情形------------------------------50
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