利用rDNA-RFLP技術鑑識台灣常見劍線蟲屬植物病原線蟲

Abstract

本研究分別利用Xiphinema spp.及Belonolaimus spp.核糖體DNA 18S和5.8S基因序列，設計用以增幅核糖體DNA非轉錄區間ITS1之PCR通用性引子對，分析台灣地區常見之劍線蟲屬(Xiphinema spp.)植物病原線蟲核糖體DNA之差異，結果發現本省常見劍線蟲中形態特徵屬於X. americanum群中之四個種[X. diffusum (Xdi)、X. oxycaudatum (Xoxy)、X. brevicollum (Xbre)、X. incognitum (Xin)]增幅所得之ITS1大小約在850-900bp之間；而X. brasiliense、X. insigne與X. elongatum之ITS1片段長度則皆大於1kb。進一步利用限制酵素片段長度多型性(RFLP)技術分析X. americanum群核糖體DNA之ITS1片段，顯示經MspI、CfoI、DdeI或是HinfI四種限制酵素酵解後，不僅Xdi、Xoxy之限制酵素切割片段差異明顯外，其與Xbre和Xin之限制酵素圖譜亦有相當大的差異性，而Xbre與Xin則利用上述酵素作用後其圖譜均完全一致，無法鑑別，利用AccI限制酵素作用後，則可將Xin酵解為160bp及770bp兩片段，而Xbre則未具有AccI酵素切位；因此利用AccI可以區別Xbre及Xin兩個種之劍線蟲。由本研究結果發現，利用CfoI、MspI、DdeI或是HinfI配合AccI等限制酵素之作用，可以成功鑑別本省常見而不易以形態鑑識的X. diffusum、X. incognitum、X brevicollum及X. oxycaudatum等四種劍線蟲，而未來更可進一步將此rDNA-RFLP技術應用在植物病原線蟲之鑑別與檢測。

PCR analysis of the internal transcribed spacer I ITS I) region was performed to distinguish among, Xiphinema americanum group (X. diffusum, X. incognitum, X. oxycaudatum, X. brevicollum), X. insigne, X. elnogatum and X. brasiliense found in Taiwan. The size of amplified internal transcribed spacers I (ITS1) fragments of X. americanum group were ranged from 850 bp to 900 bp, and larger than 1 kb for other Xiphinema species. In this study, X. americanum group were further differentiated by restriction fragment length differences of ITS1. CfoI, DdeI, HinfI, and MspI could distinguish X. diffusum and X. oxycaudatum from X. incognitum or X. brevicollum. The digestion of AccI was able to further differentiate X. incognitum from X. brevicollum.