Crown gall disease, caused by Allorhizobium vitis and Agrobacterium tumefaciens, poses a significant threat to grapevines and other horticultural crops. These soil-borne bacteria can persist for extended periods, complicating vineyard management. Traditional methods for pathogen detection, such as plating, are hindered by the high microbial diversity in soil, and existing semi-selective media often fail to distinguish tumourigenic from non-tumourigenic strains. This study aimed to validate DNA-based methods for detecting tumourigenic Al. vitis and A. tumefaciens strains in inoculated soils and develop a rapid bioassay to assess pathogenicity in grapevines. Approximately 50 g of garden soil was inoculated with 10³ to 10⁷ cells of known tumourigenic strains of Al. vitis (K309) and A. tumefaciens (Y1A-9). Non-inoculated and sterilised soils inoculated with 10⁷ cells of each strain served as negative and positive controls. Total DNA was extracted using the DNeasy Power Soil Kit (Qiagen, USA), and four different PCR protocols (Eastwell et al. 1995; Sawada et al. 1995; Szegedi & Bottka 2002; AWRI unpublished) were used to detect tumourigenic strains. A rapid bioassay using detached grapevine canes was developed to confirm the bacterial strains’ ability to induce tumours in grapevine host. One-year-old dormant Shiraz canes (2-nodes) were surface-sterilised, wounded, and inoculated with 10⁷ CFU of each strain, with a 0.9% NaCl solution as a negative control. Three replicate canes were used for each treatment and the experiment was repeated twice. The inoculated canes were incubated in a moist environment for 3–4 weeks in a glasshouse. PCR results showed that two of the four protocols effectively detected the target tumourigenic strains. The protocol specific to Al. vitis detected up to 10³ CFU/50 g of soil, while the A. tumefaciens primers detected up to 10⁵ CFU/50 g of soil. No amplification was observed in non-inoculated soil. Sterilised soil samples inoculated with the tumourigenic strains showed strong positive amplification. In the bioassay, both reference strains caused significant tumour formation on the detached grapevine canes 3-weeks after inoculation, while no tumours were observed on canes inoculated with the 0.9% NaCl solution. These results demonstrate that PCR-based detection methods, combined with a rapid bioassay, provide diagnostic laboratories with a reliable and efficient means of detecting tumourigenic Al. vitis and A. tumefaciens strains
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