Analysis of the type IV fimbrial-subunit gene fimA of Xanthomonas hyacinthi: Application in PCR-mediated detection of yellow disease in hyacinths

A sensitive and specific detection method was developed for Xanthomonas hya cinthi; this method was based on amplification of a subsequence of the type IV fimbrial-subunit gene fimA from strain S148, The fimA gene was amplifie d by PCR with degenerate DNA primers designed by using the N-terminal and C -terminal amino acid sequences of trypsin fragments of FimA. The nucleotide sequence of fimA was determined and compared with the nucleotide sequences coding for the fimbrial subunits in other type IV fimbria-producing bacter ia, such as Xanthomonas campestris pv, vesicatoria, Neisseria gonorrhoeae, and Moraxella bovis, In a PCR internal primers JAAN and JARA, designed by u sing the nucleotide sequences of the variable central and C-terminal region of fimA, amplified a 226-bp DNA fragment in all X. hyacinthi isolates. Thi s PCR was shown to be pathovar specific, as assessed by testing 71 Xanthomo nas pathovars and bacterial isolates belonging to other genera, such as Erw inia and Pseudomonas. Southern hybridization experiments performed with the labelled 226-bp DNA amplicon as a probe suggested that there is only one s tructural type IV fimbrial-gene cluster in X hyacinthi. Only two Xanthomona s translucens pathovars cross-reacted weakly in PCR, Primers amplifying a s ubsequence of the fimA gene of X. campestris pu, vesicatoria (T, Ojanen-Reu hs, N, Kalkkinen, B. Westerlund-Wikstrom, J, van Doorn, K. Haahtela, E.-L, Nurmiaho-Lassila, K. Wengelink, U. Bonas, and T. K, Korhonen, J. Bacteriol. 179: 1280-1290, 1997) were shown to be pathovar specific, indicating that the fimbrial-subunit sequences are more generally applicable in xanthomonad s for detection purposes. Under laboratory conditions, approximately 1,000 CFU of X. hyacinthi per mi could be detected. In inoculated leaves of hyaci nths the threshold was 5,000 CFU/ml, The results indicated that infected hy acinths with early symptoms could be successfully screened for X hyacinthi with PCR.