Tomato Stem Injection for the Precise Assessment of Ralstonia solanacearum Fitness in Planta

Abstract

Ralstonia solanacearum is a soil-borne pathogen with worldwide distribution that causes bacterial wilt disease in more than 250 plant species. R. solanacearum invades plants through the roots, reaches the vascular system, and colonizes the whole plant by moving through the xylem, where it eventually replicates rapidly, causing plant death. Usual assays to measure the virulence of R. solanacearum under laboratory conditions rely on soil-drenching inoculation followed by observation and scoring of disease symptoms. Here, we describe a protocol to assess the replication of R. solanacearum following injection into tomato stems. This protocol includes four major steps: 1) growth of tomato plants; 2) R. solanacearum injection into tomato stems; 3) collection of tomato xylem samples and bacterial quantitation; and 4) data analysis and representation. This method bypasses the natural penetration process of the pathogen, thus minimizing variation associated with stochastic events during bacterial invasion, and provides a sensitive and accurate measurement of bacterial fitness inside xylem vessels.

Keywords: Bacterial wilt; Fitness; Ralstonia solanacearum; Stem injection; Tomato; Virulence.

Figure 1.. Growth of tomato seedlings in Jiffy pots.

A. Germination of tomato seeds (Solanum lycopersicum cv. Moneymaker) in a 1:2 mixture of standard potting soil and vermiculite. B. Transferring tomato seedlings to individual water-soaked jiffy pots containing a pre-packed soil mixture. C. Four-week-old tomato plants ready for Ralstonia inoculation.

Figure 2.. Preparation of R. solanacearum inoculum

Figure 3.. Stem injection and sampling of xylem sap.

A. Four-week-old tomato plants in Jiffy pots. B. Using a 10-μl microsyringe, inject 5 μl bacterial suspension into the stems of 4-week-old tomato plants. C. Transect the tomato stems with a sterile scalpel just below the cotyledon emergence site. D. Collect 2.5 μl xylem sap at the cutting site using a micropipette.

Figure 4.. Preparation of serial dilutions and bacterial spreading.

A. Dissolve the xylem sap containing Ralstonia in 1 ml sterile water in a sterile 1.5-ml microcentrifuge tube; dilute the suspensions by 10 to 10⁴ times. B. Spread the diluted bacterial suspensions (we tried to spread 10^-1 or 10^-3) on phi medium plates using standard procedures, as previously described (Yu and Macho, 2021). Incubate the plates upright at 28°C for 2 days and count the colonies on solid phi medium plates. Photos in B show the usual colony pattern after plating using a spiral plater (Yu and Macho, 2021).

Figure 5.. Representation of the growth of R. solanacearum GMI1000 wild-type, mutant, and complemented strain in tomato stems.

Different colors represent different strains, and horizontal bars represent mean values (n = 6 plants per strain). Data are represented as the mean ± SEM. Asterisks indicate significant differences (***P < 0.0001, t-test). The graph was plotted using GraphPad Prism 7.