DETECTION OF PHOMOPSIS VITICOLA IN GRAPEVINE CANES USING MOLECULAR AND CLASSICAL TECHNIQUES
DL WHISSON1, B BRANT1, BE STUMMER2 and RWA SCHEPER2
1South Australian Research and Development Institute (SARDI), Waite Precinct, GPO Box 397, Adelaide, SA 5001, Australia; 2Department of Crop Protection and CRC for Viticulture, The University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia
Background and objectives
Materials and methods
P. viticola isolates were prepared as described previously  except that the infected canes were not surface sterilized. Pycnidia were produced in vitro on PDA or oatmeal agar. The number of nuclei in dormant and germinating conidia of taxon 1 and 2 was determined by UV fluorescence following treatment with Hoechst Dye 33258.
The infection and colonization process was studied by inoculating taxon 1 or taxon 2 conidia onto the second internode of micropropagated grapevine plantlets followed by incubation in a 20oC/16oC, 12 h light/dark cycle. Infection was observed by removing the epidermal layer and staining with lactoglycerol aniline blue. After lesions appeared, stem sections were excised and (a) the DNA extracted or (b) hand-sectioned, stained with KOH-aniline blue and observed for fluorescence.
DNA isolated from mycelia of taxon 1 and 2 was cloned into a plasmid vector using established methods. The clones were tested for species and taxon specificity, and for their ability to detect genetic variation among isolates using Southern analysis. DNA was extracted from canes suspected of being infected with P. viticola and the taxon-specific DNA probes were used to detect the fungus in a slot-blot format. DNA prepared from P. viticola mycelia, disease-free grapevine canes and several fungi, including Botrytis cinerea, was used as controls.
Results and conclusions
Taxon 1 conidia are smaller than those of taxon 2 and contain distinctive large guttules at either end. Hoechst staining revealed that conidia of both types contain a single nucleus which divides during germ tube production, with the eventual inclusion of a pair of nuclei in each hyphal compartment.
Conidia were observed to penetrate the cuticle directly and the plant produced papillae in response. Micropropagated plantlets displayed lesions 6 days after inoculation and subcuticular hyphae were observed in asymptomatic stem sections above the point of inoculation.
The DNA libraries contained clones specific to each taxon, as well as clones that identified variation among isolates . Several taxon 1 and taxon 2 specific multi-copy probes detected the fungus in approximately 50 ng of total grapevine DNA from canes suspected of being infected. No homology was found to DNA prepared from other fungi or grapevine tissue. The probes appear to provide the sensitivity required for a DNA-based diagnostic test, but may be inadequate for low or initial infection. The development of a PCR-based assay using the specific clones may enhance the sensitivity while maintaining the specificity.