| 摘要 |
Xanthomonas campestris pv. citri is a quarantine organism under United States and International law because of the serious disease of citrus, citrus bacterial canker disease, which is caused by the organism. We have cloned, in vector pUC9, a 4.2-kb BamHI fragment of plasmid DNA from a typical strain of this pathogen and demonstrated that this DNA fragment specifically identifies the pathogen. The procedure involves isolation and cultivation of the bacterium, chemical isolation of its DNA, digestion of the DNA by restriction endonucleases and analysis by Southern or dot blotting using the cloned DNA fragment as biotin-labeled hybridization probe. A subclone has been made from the original 4.2-kb BamHI fragment which has sensitivity and specificity equal or greater than the original clone and which is approximately 572 bp in length. All tested strains of the most virulent form of the pathogen, type A, have a BamHI fragment of 4.2-kb which hybridizes with either probe. Other less pathogenic forms of the bacterium have BamHI fragments greater than 20kb in size. Thus not only are all strains of the pathogen detected with this probe, but sub-pathovar assignment of unknown strains is also facilitated. Strains of X. campestris which cause another non-threatening disease of citrus, citrus bacterial spot disease, are not detected by the probes. This will allow rapid, sensitive and specific detection of the pathogen in groves or from commercial shipments of citrus. In addition, oligonucleotide primers were designed, based on the nucleotide sequence of the 572-bp probe. The primers are effective in the amplification of DNA from the bacterium; thereby increasing both the specificity and sensitivity of detection methods.
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