Project Number
P04000059 / GenUS 15/03
Project Title
Molecular detection of crown gall in grapevine nurseries and vineyards of South Africa
Project Leader
Langenhoven, W E
Burger, J T
Institution
University of Stellenbosch. Faculty of AgriSciences. Department of Genetics
Project Description
Objectives & Rationale
Crown gall, caused by Agrobacterium vitis, results in severe economic losses in grapevine cultivation worldwide. The bacterium survives in soil, on live and/or dead grapevine tissue, as well as the surface of roots, for years, thus providing a source of inoculum for new plants. The objective of the research is to develop the molecular protocols necessary for routine detection of A. vitis in South Africa and to determine the extent of the disease in the South African grape growing regions and nursery industries.
Methods
Pathogenicity assays were performed under glasshouse conditions to optimise the detection protocol from plant material. Primers for detection of Agrobacterium species as well as species-specific primers were evaluated for their effectiveness to detect pathogenic A. vitis. Three strategies were evaluated to determine which would be the most suitable starting material for PCR amplification for detection namely, DNA, grapevine crude sap and isolated bacteria. The protocol was then used to screen plant and soil samples obtained from nurseries in the Western Cape Province. 16S rDNA and recA gene sequencing was performed to assess species diversity in field samples.
Key Results
The key outcome of this project is a diagnostic protocol for the routine detection of crown gall from grapevine samples. The sequencing of the recA gene of Agrobacterium isolates from field samples indicated low levels of diversity between A. vitis isolates from field samples.
Conclusion and Discussion / Recommendations
This study permitted the development of a diagnostic protocol for the routine detection of grapevine crown gall pathogen, Agrobacterium vitis from plant material. The final protocol relies on bacterial isolation with subsequent PCR amplification of Agrobacterium-like colonies. Results showed that genetic diversity among A. vitis isolates compared in this study was low enough that existing PCR-based diagnostic protocols optimised in this study would be adequate to detect the vast majority of Agrobacterium strains in the regions included in the study. Although A. vitis could be detected when using total DNA from vineyard soil, grapevine plant material or crude grapevine sap as template for PCR detection of the pathogen, cell viability and as a consequence the potential for the presence for a viable inoculum source, could not be assessed. In addition, crude sap only produced consistent results for inoculated plants during the pathogenicity assay. Application of the protocol involving bacterial isolation on semi-selective medium followed by PCR analysis, to 42 plant and 28 soil samples collected from nurseries in 2018, indicated the presence of non-pathogenic A. vitis in 90% of samples, while pathogenic A. vitis was not as widespread, and not always detected in galls, but occasionally in the corresponding vine section due to the pathogens’ irregular distribution. Based on this it is recommended that detection of pathogenic A. vitis not be limited to the gall material, and that primers specific for A. vitis be used together with primers specific for the tms2 (tumour-inducing) gene found on the Ti plasmid of pathogenic forms. The use of the tms2 primers provides an indication of the potential risk of the pathogen detected. Although detection of pathogenic and non-pathogenic A. vitis was possible in soil samples, given that the results these analyses did not always correlate with those of corresponding vines, results from soil analyses need to be treated with caution, especially as pertains to false negatives. The devised protocol for detection from plants is ready to be implemented.
Given the longevity of Agrobacterium in plant and soil material, it must be emphasized that to prevent the spread of crown gall, farms and nurseries need to adhere to strict phytosanitary measures, with infected material to be removed from vineyards and destroyed.