柑橘黃龍病(立枯病)之核酸探針與應用

Abstract

柑橘黃龍病的擬病原綑菌在寄主體內的濃度低且分布不平均，以往須依賴生物檢定法或電顯切片觀察，相當費時且效果不彰。曾嘗試開發單元抗體做為病害之診斷探針，但因偵測結果不夠穩定而無法有效應用。為了研製效果穩定且特異性與敏感度高的病原偵測探針，於是應用現代分子生物學技術，進行病原核酸之選殖，製備出核酸探針以供病害診斷及病原檢疫之應用。從台灣全省各地的柑橘園中，採得各類疑似有黃龍病的柑橘植株，所有疑似有黃龍病的植株皆與核酸探針呈正反應，所有的健康的無毒苗則都沒有反應，由此可看出核酸探針Probe l是一普遍有效的病原探針，對台灣全省各地不同種類的黃龍病病株皆能做有效之偵測。應用黃龍病核酸探針之檢測可以清楚地將假性黃龍病與真正的黃龍病做確切的分辨。此黃龍病病原核酸探針的點漬雜配法[Dot hybridization (DH)]偵測效果穩定且其特異性與敏感度高，只要0.25克以上的材料即可偵測到病原，已廣泛應用於病原之偵測及生態之研究。為求建立一套簡便且有效的病原偵測法，就採樣方式、材料處理、核酸抽取等加以研究訂出標準化技術。純化部位比較，在相同病原核酸純化方法下，中肋部分的偵測效果最好；新鮮的材料經風乾處理使之乾燥，與未乾燥處理的同一新鮮材料相互比較其偵測效果及探針敏感度，結果發現經乾燥處理的材料比未乾燥處理者可提高16倍偵測敏感度。在病原檢疫方面，此核酸探針之特異性除了可檢測台灣各柑橘品種中之黃龍病病原外，亞洲地區國家柑橘似黃龍病綠化病病原亦可與此探針雜配反應，但與南非的熱敏感型綠化病菌系統（Heot-sensitive stroins）則不反應。顯示此探針是對亞洲的耐熱型黃龍病菌系統具特異性的核酸探針，亦證明所測試之亞洲各黃龍病菌系統均屬耐熱型系統（Heot-tolerant stroins)。 The fastidious bacteria causing citrus Huanlungpin, called Likubin (greening) inhabit in sieve tubes of host plants unevenly in low population. In order to prepare highly sensitive and specific diagnostic probes, an attempt was made to prepare nucleic acid probes for disease diagnosis and pathogen detection. Through cloning genomic DNA of the pathogen into plasmid pBS as vector, one stable transformant was selected to prepare biotinelabled DNA probes. Dot hybridization(DH) with the selected DNA probe 1 showed specific and sensitive reaction constantly with DNA extract from greening-affected citrus plants. DH with DNA probe 1 has been commonly applied to detection and ecological study of the pathogen. Detection of pathogen in symptomless citrus plants could be accomplished by the techniques with DNA probes so far developed, which were applied the indexing citrus foundation stocks and seedlings, and epidemiological study. As for the specificity in pathogen identification, the DNA probe could be used for identifying Huanlunpin pathogen in the all citrus cultivars of Taiwan, and also showed strong specific reaction in DH test with GO in citrus samples collected from the Asian countries. However, this DNA probe did not reacted with the heat-sensitive strain of African greening from South Africa. It implied that the DNA probe was just specific to Asian heat-tolerant strain of greening pathogen, and the all test GO isolates from the Asian countries were belonging to the heat-tolerant strain.