Background

Ralstonia solanacearum is the causal agent of bacterial wilt disease in more than 250 plant species including important crops, such as tomato, potato, pepper, and eggplant, among others, and is considered one of the most dangerous plant pathogens in the world (Mansfield et al., 2012). R. solanacearum is a soil-borne pathogen that enters plants through the roots, using wounds, root tips, and secondary root emergence points as penetration sites; it then progresses via the root cortex, finally reaching the vascular system (Xue et al., 2020). In susceptible plants, R. solanacearum moves through the xylem vessels to colonize the whole plant; eventual replication may lead to populations up to 10¹⁰ bacteria per gram of plant tissue in diseased plants, which will block water and nutrient flow in the vascular system and eventually cause plant wilting and death (Peeters et al., 2013). Multiple experimental methods to assess R. solanacearum virulence in plants under laboratory conditions have been described previously (Morel et al., 2018). The most widely used assay relies on soil-drenching of plant roots with R. solanacearum suspensions, followed by observation of the resulting wilting symptoms over time (Morel et al., 2018). Other assays involve bypassing the root penetration process by introducing the bacterial suspensions directly into plant tissues (either leaves or stems) and provide more accurate assessments of bacterial replication (Macho et al., 2010; Morel et al., 2018; Yu and Macho, 2021).

In this protocol, we provide a detailed description of a method aimed at quantitating bacterial fitness in tomato xylem vessels by direct injection into the stem using a microsyringe; given the subsequent fast bacterial replication and plant colonization, we assume that a substantial proportion of this inoculum reaches the xylem vessels. At the desired time after inoculation, samples are taken from 2 cm above the inoculation site by collecting xylem sap from an excised stem section. Bacterial quantitation in this xylem sap provides a clean measurement of bacterial fitness in the plant vascular system. Like other Gram-negative bacterial pathogens, R. solanacearum requires a type III secretion system to inject type III effector (T3E) proteins into host cells and cause disease. Since T3E activities often contribute redundantly to the development of disease, it is generally difficult to determine their involvement in virulence; however, we have recently used this method to detect the virulence attenuation of different R. solanacearum knockout mutants lacking individual effectors (Xian et al., 2020; Yu et al., 2020) and to analyze bacterial pathogenicity following chemical treatment of plant tissues (Wang et al., 2021), revealing a simple, versatile, and powerful assay for the study of R. solanacearum-plant interactions.