PRODUCTION AND EVALUATION OF NONRADIOACTIVE PROBES FOR THE DETECTION OF THE 2 CANDIDATUS-LIBEROBACTER SPECIES ASSOCIATED WITH CITRUS HUANGLONGBING (GREENING)

The production and evaluation of non-radioactive probes for the detect ion of 'Candidatus Liberobacter asiaticum' and 'Candidatus Liberobacte r africanum', the two bacterial species associated with citrus huanglo ngbing (greening) disease is described. Two DNA fagments, In 2.6 and A S 1.7, obtained previously from the beta operons of 'Candidatus Libero bacter asiaticum' and 'Candidatus Liberobacter africanum', respectivel y, were the starting materials for production of the two nonradioactiv e probes. These digoxigenin (DIG)-labelled probes were generated by PC R incorporation of DIG-11-dUTP, yielding In 1.7-DIG and AS 1.7-DIG. Pr obe In 1.7-DIG was hybridized with DNAs extracted from 24 field-collec ted samples in Ball (Indonesia). The membrane on which the DNAs were b lotted was first hybridized with radioactive probe P-32-ln 2.6. After the hybridization results were recorded, the radioactive probe was rem oved, and the membrane hybridized with DIG-labelled probe In 17-DIG. i dentical results were obtained for 23 samples. One sample was positive with the DIG-labelled probe and negative with the P-32-labelled probe . However, crosshybridization of In 1.7-DIG with DNA from L. africanum was higher than that obtained with the radioactive probe. This cross- hybridization could be eliminated by raising the temperature of the st ringent washing step. No field samples from Africa being available, pr obe AS 1.7-DIG was dot-blot hybridized against DNAs extracted from lea ves of greenhouse-kept citrus plants from different geographical origi ns and infected with one or other Liberobacter species. The data showe d that AS 1.7-DIG hybridized with L. africanum with a sensitivity to t hat of the radioactive probe. (C) 1997 Academic Press Limited.