An Evaluation of the Diversity of Fluorescent Pseudomonads in Maize Rhizosphere Using 16S-23S rDNA Intergenic Spacer Region Restriction Fragment Length Polymorphisms and the Biolog GN Plate Method

利用16S-23S rDNA基因區問限制酵素片段多型性及Biolog GN plate評估玉米根圈螢光假單胞菌之多樣性

張臻穎(Chen-Ying Chang) ； 趙震慶(Chen-Ching Chao) ； 趙維良(Wei-Liang Chao)

《臺灣農業化學與食品科學》 45卷2期 (2007/06) Pp. 67-75

https://doi.org/10.6578/TJACFS.2007.009

摘要

根圈螢光假單胞菌的多樣性極高，欲分辨該菌種的不同菌株極不容易。本研究建立16S-23S rDNA基因區間限制酵素片段多型性分析法(16S-23S IGS RFLP)來分析玉米根圈螢光假單胞菌之多樣性，同時使用生理鑑定法(Biolog GN plate)來辨識篩選到的分離株，並且將基因分析法與生理鑑定法的結果相互比較。16S-23S IGS RFLP分析法能在相同菌株間產生一致的限制酵素切割圖譜，而分離株的限制酵素切割圖譜經非加權配對群叢分析法(unweighted-Pair-group method with arithmetic averag，UPGMA)分析後，則能依照其菌株之不同分為三個群叢，分離株的分佈型態又與16S rDNA序列分析推算出的親緣關係相符。而Biolog GN plate分析法則將已經16S rDNA序列分析法鑑定為Pseudomonas Putida的分離株鑑定為Pseudomonas fragi或Pseudomonas syringae，根據各分離株之生理代謝特性進行的群叢分析結果，僅能將全部分離株依照其菌種的不同分為二個群叢。因此，本研究的結果顯示16S-23S IGS RFLP分析法不僅解析度高於Biolog GN plate，且適合用於分析玉米根圈土壤中螢光假單胞菌之多樣性。

關鍵字

螢光假單胞菌 ； 根圈 ； 16S-23S IGS RFLP ； Biolog GN plate

並列摘要

The strains of rhizosphere fluorescent Pseudomonas are diverse and their identification is difficult. To assess this diversity in maize rhizosphere, we developed a new method that used 16S-23S rDNA intergenic spacer region restriction fragment length polymorphisms (16S-23S IGS RFLP). At the same time, we also used a phenotypical method (Biolog GN plate) to delineate their phenotypes. An unweighted-pair-group method with arithmetic average (UPGMA) cluster analysis of 16S-23S IGS RFLP band patterns divided all isolates into three strain-specific clusters, which corresponded consistently to the phylogenetic tree based on 16S rDNA sequencing data. Isolates identified as Pseudomonas putida by 16S rDNA sequence analysis were identified as Pseudomonas fragi or Pseudomonas syringae by the Biolog GN plate method. The UPGMA cluster analysis of Biolog GN substrate utilization patterns divided all isolates into two species-specific clusters. Therefore, we concluded that the 16S-23 S IGS RFLP method was more sensitive than the Biolog GN method to differentiate between the strain-specific level making it the more suitable of the two methods for analyzing the diversity of fluorescent Pseudomonas in maize rhizosphere soil.

並列關鍵字

Fluorescent Pseudomonas ； Rhizosphere ； 16S-23S IGS RFLP ； Biolog GN plate