A NOVEL TECHNIQUE THAT CAN IDENTIFY OR DETECT ALL THE KEY FUNGAL PATHOGENS OF A CROP IN A SINGLE ASSAY. CA Levesque, CE Hariton and Y Ahn Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summeriand, B. C., Canada, VOH 1ZO Background and objectives The reverse dot blot technique involves hybridization of a probe made from unknown sample DNA to oligonucleotides specific to a species or a group of species bound to a solid support membrane. In the medical field, a reverse dot-blot system was developed to identify, in a single test, several possible mutations related to cystic fibrosis [1]. A similar approach recently designed to identify some oomycete fungi is now being expanded to study the many fungal species causing diseases in cranberry[2]. The taxonomy andlor the disease cycles are poorly understood for several of these fungi. An approach that could detect and identify the key fungi of this pathosystem would not only assist epidemiological studies, but would help to improve integrated disease management strategies. The main objective of this study is to develop a reverse dot blot hybridization system that could be used for rapid identification or detection of cranberry fungi from field samples. A secondary objective was to develop a ribosomal DNA sequence database for the key cranberry fungi. Materials and methods The following steps were used to develop a reverse dot blot membrane for cranberry fungi: i) amplify and Sequence RDNA Internal Transcribed Spacers (ITS) for all key cranberry fungal species; ii) search Genbank for closest ITS sequence matches and align all sequences; iii) identify and design species specific oligonucleotides; iv) synthesize oligonucleotides with 5' amino linker; v) blot oligonucleotides on nylon membrane. Samples to be used as hybridization probes were prepared the following way: i) pure cultures were obtained from collections and from fungi isolated from twigs, leaves, stems or berries; ii) fungi isolated from infected plant tissues were identifed by morphology (those fungal isolates that did not sporulate on culture media were sequenced as described above); iii) DNA was extracted with Fast-Prep (Savant/BiolOl) from pure cultures or directly from halves of the different tissue samples. For the reverse dot-blot assay, the ITS 1 & 11 were amplified and labelled simultaneously with alkaline labile digoxygenin using universal primers ITS5 and ITS4 for pure cultures, or a modified ITS1-F primer and ITS4 for amplification from plant DNA extracts. Hybridization, detection and erasing of membranes for re-use were performed as previously described [2]. Results and conclusions The closest matching sequences in Genbank were found for all cranberry fungi that were sequenced. Several putative species or group specific oligonucleotides were produced and tested. Several isolates obtained from infected tissue did not sporulate on artificial media but were identified as common cranberry pathogens by DNA sequencing and reverse dot blot hybridization. Pure cultures of the species of Allantophomopsis, Alternaria, Botrytis, Colletotrichum, Gloesporium, Penicillium, Pestalotia/Pestalotiopsis, Phomopsis, Phyllosticta, Physalospora and Synchronoblastia that cause cranberry diseases were identified. Quite often, more than one oligonucleotide was designed and tested to find at least one that hybridized to give a strong signal with no cross-reactivity. Oligonucleotides were designed that could differentiate between very closely related species such as Phyllosticta vaccinii
  • and P. elongata
  • , Physalospora vaccinii
  • and P. salishensis
  • (formerly classified as white strain of P. vaccinii
  • ), Ailantophomopsis cytisporea
  • and A. lycopodina
  • , or Colletotrichum acutatum
  • and C. gloeosporioides
  • . It was also possible to detect and identify fungi directly from tissues of cranberry fruits. In many instances, more than one fungus appeared to be present in a single berry half. Hybridization results using probes made from different cranberry fungi or infected berries demonstrated that reverse dot blot hybridization can be used for both identification and detection of fungal pathogens of cranberry. References 1. Kawasaki ES, Saiki R, Erlich H, 1993. Methods in Enzymology 218, 369-381. 2. Levesque CA, Hariton CE, de Cock AWAM, 1998. Phytopathology 88, (in press).