*2.9. Biological Assays in Tomato Seedlings*

Germination of tomato seedlings and preparation of bacterial inoculum were prepared as described by Singh et al. [22]. Freshly grown *R. solanacearum* was inoculated into 50 mL BG media broth with 0.5% glucose and incubated at 28 ◦C and 150 rpm for 24 h. The bacterial cultures were obtained by centrifugation at 4000 rpm and 4 ◦C for 15 min and were then resuspended in an equal volume of sterile distilled water to obtain a concentration of approximately 10<sup>9</sup> cfu mL<sup>−</sup>1. Strain NEAU-HV9 was cultured in ISP 2 broth on rotary shaker for 3 days at 28 ◦C and centrifuged at 10,000 rpm. Subsequently, cell pellets were diluted in 0.85% (*w*/*v*) NaCl solution and adjusted to 107, 108 or 10<sup>9</sup> cfu mL<sup>−</sup>1. Root inoculation of *R. solanacearum* in tomato seedlings was carried out as described by Singh et al. [22]. About 15 to 20 mL of *R. solancearum* inoculum was taken in a sterile container. Tomato seedlings (6 to 7 days old) were picked one at a time from the germinated seedling tray and then the roots of each seedling were dipped in the bacterial inoculum (up to the root-shoot junction). Four treatments were established as follows: TR 1 (tomato seedlings were pre-inoculated with suspension (107, 108 or 109 cfu mL<sup>−</sup>1) of strain NEAU-HV9 and then inoculated with *R. solanacearum)*; TR 2 (tomato seedlings were pre-inoculated with *R. solanacearum* and then transferred to microfuge tubes with the addition of 1 to 1.5 mL of sterile water and active fraction, where the final treatment concentrations were 1 × MIC and 2 × MIC, respectively); CK 1 (tomato seedlings were inoculated with sterile water); and CK 2 (tomato seedlings only were inoculated with *R. solanacearum*). For all of the treatments, the root-dip inoculated seedlings were transferred to an empty 1.5 mL sterile microfuge tube. After approximately 5 minutes, 1 to 1.5 mL of sterile water was added to the tube. All the inoculated seedlings, along with the controls, were transferred to a growth chamber maintained at 28 ◦C with 75% relative humidity (RH) and a 12-h photoperiod. Seedlings were analyzed for disease progression after 7 days. Sets of 4 seedlings were recruited in each dilution inoculation, and each assay was performed in triplicate.
