Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30780
標題: 利用PCR偵測百合萎凋病菌
Detection of Fusarium oxysporum f.sp. lilii by PCR
作者: 施儼育
Shih, Yan-yu
關鍵字: Fusarium oxysporum;百合萎凋病;Detection;PCR;Primer;DNA probe;鐮孢菌;病原菌偵測;核酸探針;聚合連鎖反應
出版社: 植物病理學系
摘要: 
本省百合栽培過程中所遭遇最嚴重問題為土壤傳播性病害,其中百合萎凋病由Fusarium oxysporum Schlecht f.sp. lilii Imle所引起,Fusarium在分類上有多種系統,傳統分類多以菌落形態、孢子大小、厚膜孢子有無、孢子堆產生與否等特性加以歸類,而在分化種的鑑定方面,病原性測試耗日費時,且可能因測試環境差異及種苗帶菌等問題影響測試結果,故希望應用分子生物學技術開發一快速且準確的病原菌偵測方式,以供檢防疫及學術研究上之應用。本試驗以菌株F016、F025、G016為標準病原性菌株,利用隨機核酸增幅多形性聚合連鎖反應(Random amplified polymorphic DNA PCR; RAPD)篩選100個長度為10 nucleotides之核酸引子,藉此篩選出可區別病原性與非病原性菌株之引子OPAT-04(5''-TTGCCTCGCC-3''),經回收其增幅之鑑別性去氧核醣核酸片段,選殖至大腸桿菌(E. coli)質體DNA,以LB培養基增殖後解其核酸序列,藉由所得到的核酸序列設計專一性核酸引子Fusa1-1:5’-CCTAACA ACAGCCCATCCCA-3’與Fusa1-4:5’-GCCAAGGTTCAAGCAAGAGA-3’,以此核酸引子增幅病原性菌株之總量去氧核醣核酸可得長度為474 bp及578 bp的專一片段,經測試其靈敏度可偵測到50pg之DNA,以少量之菌絲經粗略純化步驟後進行聚合連鎖反應即可產生預期之去氧核醣核酸片段,經進一步試驗結果發現無論植株地上部之病徵表現與否,所設計之專一性核酸引子皆可偵測出病原菌的感染纏據,故所設計之專一性核酸引子可實際應用於偵測種球帶菌,但直接應用在土壤帶菌偵測所得到的靈敏度卻不盡理想,可偵測範圍約為每克土壤中有104個以上的繁殖體,且測試結果不甚穩定,經改變方式後以測試土壤在PCNB培養液中經24~36小時增量培養,再加以偵測,發現靈敏度可由原來的每克土壤104繁殖體提高到每克土壤50個繁殖體,故所開發之核酸引子可成功應用在菌絲、植株(種球)、土壤帶菌之偵測。在試驗中所發現之專一性去氧核醣核酸片段只在對百合具有病原性的鐮孢菌總量去氧核醣核酸增幅時產生,故大膽推測此片段可能跟病原性或是病原與寄主之間的辨識有所關連,亦或是演化上產生的專有片段,將此片段之核酸序列與網路上之基因庫比對發現目前所發表的已知片段與本去氧核醣核酸片段相似性皆不高,且所列出之序列多為病毒上的核醣核酸片段,故此專一性去氧核醣核酸片段是否有其特殊功能則有待進一步研究。

Basal rot, root rot and wilt of lilies caused by Fusarium oxysporum f.sp. lilii are the most destructive disease during its growing period in Taiwan. Fusarium oxysporum Schlecht f.sp. lilii Imle is difficult to identified be-cause of its various morphology. However, molecular techniques provide a fast and precise method in identification of pathogen. RAPD was used to screen 100 random primers with total DNA of pathogenic and non-pathogenic Fusarium oxysporum isolates .The primer OPAT-4 (5''-TTGCCTCGCC-3'') was selected, and the specific DNA segment was amplified when total DNA of pathogenic isolates were present. The spe-cific DNA segment was amplified, recovered , cloned ,and sequenced. And the specific primer set Fusa1-1/Fusa1-4 was designed and assembled according to the nucleotide sequence of the specific DNA segment. The sensitivity test of specific primer set showed the minimal quantity of DNA can be detected is about 50pg. And the detection of pathogen from mycelium and infected plant tissue can be conducted using Fusa1-1/Fusa1-4 . As we expected , 474 bps and 578 bps specific DNA segment were produced. However, the detection of pathogen from soil was insensitive. It may be due to the clamydospore form of pathogen ex-isting in soil . As it is difficult to extract DNA from clamydospore, there might not have targeted DNA for PCR. A new procedure was developed. By incubating the soil sample in PCNB broth for 36 hours before PCR detection. This resulted the increased sensitivity of detection from soil to about 50 spores/g soil. The results show the specific primer set we de-veloped can successfully detecting Fusarium oxysporum f.sp. lilii from pure culture, plant tissue and infested soil.
URI: http://hdl.handle.net/11455/30780
Appears in Collections:植物病理學系

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