Leite R P, Minsavage G V, Bonas U, Stall R E
Department of Plant Pathology, University of Florida, Gainesville 32611.
Appl Environ Microbiol. 1994 Apr;60(4):1068-77. doi: 10.1128/aem.60.4.1068-1077.1994.
Three pairs of oligonucleotide primers specific for different regions of the hrp gene (hypersensitive reaction and pathogenicity) cluster of Xanthomonas campestris pv. vesicatoria were designed and tested for amplification of DNA isolated from a large number of different bacteria. DNA sequences related to the hrp genes were successfully amplified from X. fragariae and from 28 pathovars of X. campestris. No DNA amplification occurred with genomic DNA from phytopathogenic strains of X. campestris pv. secalis, X. campestris pv. translucens, and X. albilineans or from nonpathogenic opportunistic xanthomonads and phytopathogenic strains of the genera Acidovorax, Agrobacterium, Clavibacter, Erwinia, Pseudomonas, and Xylella. The DNA from those bacteria also failed to hybridize to hrp-specific fragments in Southern blot analysis. DNA fragments amplified with a particular primer pair were of identical size from each of the different phytopathogenic xanthomonads. However, restriction analysis of these fragments by using frequently cutting endonucleases revealed variation in the pattern for these hrp-related fragments amplified from the different Xanthomonas strains. The restriction patterns generated for the different fragments allowed distinction of the strains representing a pathovar or species of phytopathogenic xanthomonads. We believe that DNA amplification with hrp-specific oligonucleotide primers is a highly sensitive and specific method that can be applied for detection and identification of phytopathogenic xanthomonads.
针对野油菜黄单胞菌疮痂致病变种hrp基因（过敏反应和致病性）簇的不同区域设计了三对寡核苷酸引物，并对从大量不同细菌中分离得到的DNA进行扩增测试。与hrp基因相关的DNA序列成功地从草莓黄单胞菌以及野油菜黄单胞菌的28个致病变种中扩增出来。而从野油菜黄单胞菌小麦变种、野油菜黄单胞菌半透明变种、白叶条斑病菌的植物致病性菌株，或从非致病性机会性黄单胞菌以及食酸菌属、土壤杆菌属、棒形杆菌属、欧文氏菌属、假单胞菌属和木质部菌属的植物致病性菌株的基因组DNA中未发生DNA扩增。在Southern印迹分析中，这些细菌的DNA也未能与hrp特异性片段杂交。用特定引物对扩增得到的DNA片段在每个不同的植物致病性黄单胞菌中大小相同。然而，使用常用的内切核酸酶对这些片段进行限制性分析发现，从不同黄单胞菌菌株扩增得到的这些hrp相关片段的图谱存在差异。不同片段产生的限制性图谱可区分代表植物致病性黄单胞菌一个致病变种或物种的菌株。我们认为，用hrp特异性寡核苷酸引物进行DNA扩增是一种高度灵敏且特异的方法，可用于检测和鉴定植物致病性黄单胞菌。
