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

• DOI: 10.1094/pdis-04-20-0732-re
• Publication Date: 2020-07-31T12:08:12Z
• Authors: Trong Nguyen-Huu; Jeanne Doré; Essaïd Aït Barka; Céline Lavire; Christophe Clément; Ludovic Vial; Lisa Sanchez
• Keywords: grapevine shoots; 0301 basic medicine; 03 medical and health sciences; [SDV]Life Sciences [q-bio]; Agrobacterium; Vitis; Allorhizobium vitis; DNA; real-time PCR; Real-Time Polymerase Chain Reaction; patho-gen detection

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 reliable method 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.