02023nam a2200217 a 450000100080000000500110000800800410001910000210006024501050008126000390018630000100022550000180023552013970025365000340165065000140168465000120169865000230171065300090173365300430174265300200178510519872009-12-11       1997    bl uuuu  m   00u1 u    #d1 aGUIMARAES, P. M.  aDevelopment of a DNA probe and characterisation of Curtobacterium flaccumfaciens pv. flaccumfaciens.  aLondon: University of Londonc1997  a224p.  aPh.D. Thesis.  aBacterial wilt of beans is a seedborne disease caused by the plant pathogenic coryneform bacteria Curtobacterium flaccumfaciens pv. flaccumfaciens. In order to construct a DNA probel for the identification of this organism, a chromosomal DNA library was built in the plasmid vector pGEM-7zf(+). The "Shot-gun" method was used for the construction of the DNA library in conjunction with a differential hybridisation technique. One of the clones (pPMP-26 D), after subsequent digestion with restriction endonucleases EcoRI/SacI, yielded a fragment of approximately 230 bp (pPMP-26 D) which hybridised specifically to C. f.pv. flaccumfaciens and not to any of the other eight-two coryneforms or bean pathogens tested in Southern, colony and dot-blots. Thisfragment was subcloned for use as a probe for the detection and identification of this organism. The uniqueness of the probe was confirmed by DNA sequencing. In addition, chemotaxonomic (fatty acid analysis) and genomic fingerpriting technique (ITS profiling, PCR of repetitive DNA and macrorestriction analysis by PFGE) were examined for the identification of profiles which allowed the differentiation of the pathovarswithin the species. The most discriminatory results were obtained by the use of pulsed-field gel electrophoresis analysis of HindIII  and XbaI  digests, Which produced markedly different patterns for each of the four C.  aCurtobacterium flaccumfaciens  agermplasm  aFeijão  aPhaseolus Vulgaris  aBean  aGermoplasma: Patogeno: Identificação  aIdentifications