Development of a DNA-Based Real-Time PCR Assay To Quantify Allorhizobium vitis Over Time in Grapevine (Vitis vinifera L.) Plantlets

Allorhizobium vitis is the primary causal pathogen of grapevine crown gall disease. Because this endophytic bacterium can survive as a systemic latent (symptomless) infection in grapevine, detecting and monitoring its development in planta is of great importance. In plant bacteria studies, plate counting is routinely used as a simple and reliablemethod to evaluate the bacterial population level in planta. However, isolation techniques are time-consuming and present some disadvantages such as the risk of contamination and the need for fresh samples for research. In this study, we developed a DNA-based real-time PCR assay that can replace the classical method to monitor the development of Allorhizobium vitis in grapevine plantlets. Primers targeting Allorhizobium vitis chromosomic genes and the virulent tumor-inducing plasmid were validated. The proposed quantitative real-time PCR technique is highly reliable and reproducible to assess Allorhizobium vitis numeration at the earliest stage of infection until tumor development in grapevine plantlets. Moreover, this low-cost technique provides rapid and robust in planta quantification of the pathogen and is suitable for fundamental research to monitor bacterial development over time.

Automated summary

An efficient DNA-based real-time PCR assay was developed to monitor the development of Allorhizobium vitis in grapevine plantlets. The novel technique targets chromosomal genes and the virulent tumor-inducing plasmid of the pathogen, providing reliable and reproducible quantification from early infection to tumor formation in plants. This low-cost method is suitable for fundamental research and offers a rapid and robust quantification of the pathogen in planta.