A single reverse dot-blot hybridisation test to detect asymptomatic stem canker fungus infections from field samples

Pathogenic fungi of stone fruit were isolated from lesions on trees from the Western Cape.  The genera included were Botryosphaeria, Diaporthe and Leucostoma.  The isolates were cultured and their DNA was isolated.  PCR was performed to amplify the complete internal transcribed spacer (ITS) regions of the ribosomal DNA.  Analyses of the sequence data were done.  From these, oligonucleotides were designed for the detection of the 3 different stem canker fungi at the genus level.  These oligonucleotides were in the ITS I region for Diaporthe and Botryosphaeria, and the ITS II region for Leucostoma.  Dot blot hybridisation assays of the respective genera demonstrated and confirmed the specificity of each oligonucleotide.  Subsequently, these oligonucleotides were blotted onto nitrocellulose membranes and probed with DIG-dUTP PCR amplified ITS-DNA.  These probes hybridised to their respective genus-specific oligonucleotides at 50^oC in a reverse dot blot hybridisation (RDBH) assay.  Unrelated fungi did not cross hybridise when probed against the genus-specific oligonucleotides.  Optimisation of conditions for field testing of isolates yielded labelled ITS-DNA for mixed apple and fungal DNA, while no hybridisation signals were obtained for subsequent RDBH assays.  PCR amplification and hybridisation signals were negative for the ITS-DNA from total DNA of inoculated trees.  Further optimisation of the PCR and RDBH assays from field samples is therefore required.