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以半巢式PCR偵測樹枝與預採果實中的檬果蒂腐菌

Detection of Quiescent Infection of Mango Stem End Rot Pathogen Lasiodiplodia theobromae in Shoot and pre-plucked Mango Fruit by Seminested PCR

Abstracts


本研究由檬果罹病果實上分得55株菌株,經鑑定爲Botryosphaeria dothidea, B. ribis, Pestalotiopsis sp., Phomopsis sp., Colletotrichum acutatum, C. gloeosporioides,其中6株以形態與核酸序列鑑定爲檬果蒂腐菌Lasiodiplodia theobromae Griffon & Maubl.。由L. theohromae核醣體DNA的ITS區域設計種專一性引子Lth1,與廣效性引子ITS4配對,以最適煉合溫度58℃增幅得到一約420bp的產物,上述其他菌種不會形成產物。爲提升偵測靈敏度,建立半巢式PCR:第一段以ITS5/ITS4廣效性引子對增幅,第二段續以Lth1/ITS4增幅,可使偵測靈敏度達125 fg L. theobromae全量DNA。2004至2005年間,將半巢式PCR偵測技術應用於田間愛文檬果枝條帶菌調查與預採果買檢測。結果發現,三月至五月,病原菌由舊枝侵入當年度新枝,進入果授,舊枝中棲息的蒂腐菌爲重要的果實採收後蒂腐病的感染源。當年度新枝基部偵測率可達45.5%,新舊校交接處(63.3%)與老枝頂端(72.7%)的偵測率最高,老枝15公分處仍能測得蒂腐菌。由屏東、台南和嘉義等檬果主要產區的七個果園,於採收前1-1.5個月收集預採果實,萃取果蒂及其周圍果皮組織DNA,再將預採果實浸潤生長素益收(ethylene)以催熟,調查罹病率。2004年,樣果果實蒂腐病罹病率在0%~82%,半巢式PCR偵測率則在6.3%~76%之間;2005年果質罹病率約爲20%~92.9%,半巢式PCR偵測率則約29.2%~81%。雖然各果園半巢式PCR偵測率與預採果實罹病率不需相符,但得病率超過10%的樣本,半巢式PCR偵測率皆超過10%。依據政府『貯藏期果實病害預測方法』,訂定凡經預測之果園,蒂腐病罹病果實率在10%以下,得選爲日本外銷果園。半巢式PCR偵測以其靈敏與快速的優點,適宜開發成監測與偵測的技術。

Parallel abstracts


A semi nested PCR-based method was developed for specific detection of mango (Mangijera indica L.) stem end rot pathogen Lasiodiplodia theobromae Griffon & Maubl. (=Botryodiplodia theobromae Pat.) in plant tissues. A specific primer Lth 1 was designed from rDNA internal transcribed spacers (ITS) region of L. theobromae. Under stringent PCR conditions, a 420-bp amplicon formed from L. theobromae DNA but not from other stem end rot pathogens such as Botryosphaeria dothidea, B. ribis, Pesralotiopsis sp., Phomopsis sp., Colletotrichum acutatum, and C. gloeosporioides. Seminested PCR using primer pairs of ITS5/ITS4 and Lth 1/ITS4 was sensitive enough to detect 125fg of genomic DNA. The technique was used to detect L. theobromae and study the colonization of the pathogen in shoots and pre-plucked fruits. The pathogen in asymptomatic old branches was detected by seminested PCR. It survived endophytically. During March to May, it grew into new branch and extended to inflorescence. The endophytic pathogen seems to be an important inoculum source of the post-harvest disease. In infected orchards, the pathogen was detected in 45.5% of the base of new shoots, in 63.2% tissues between new shoot and old branch, and in 72.7% old branch top. Pre-plucked mango samples were collected from 7 orchards in Pingtung, Tainan, and Chiayi. The pathogen in fruit stalk and the skin near stalk was detected by seminested PCR. The preplucked samples were treated with ethylene and stem end rot symptoms appeared in 9-12 days, significantly after 12 days. In 2004, 0%-82% fruit samples developed stem end rot and the detection rate of L. theobromae was between 6.3%-76%. In 2005, the disease rate of the fruit was 20%-92.9% and the detection rate was about 29.2%-81%. The detection rate of pre-plucked samples was not always consistent with the disease rate. However, when the detection rates in these samples were higher than 10% by seminested PCR, their disease rates were higher than 10%. Based on the standards of mango export orchards, the mango stem end rot disease rates need to be lower than 10%. The seminested PCR assay showed promise as a monitor tool for the mango stem end rot pathogen.

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