Materials and Reagents

Micro centrifuge tube 1.5 ml (Tarsons, catalog number: 500010 )
Petri dish 90 mm (Tarson, catalog number: 460050 )
Petri dish 60 mm (Tarson, catalog number: 460061 )
Microtips 200-1,000 μl (Tarson, catalog number: 521020 )
Microtips 2-200 μl (Tarson, catalog number: 521010 )
Microtips 0.2-10 μl (Tarson, catalog number: 521000 )
Absorbent cotton wool (Bengal Surgicals Limited, India/Azpack, catalog number: 12356477 )
Tissue paper roll (Solimo/Kimberly-Clark WypAll L10 Paper Wipes, catalog number: 13478218 )
Polygrid micro tube stand (Tarson, catalog number: 205110 )
Plastic tray (Agrawal Plastic and Packaging Industries)
Cotton plugs (Prepared in laboratory)
Spray bottle (Abdos, catalog number: P11191 )
Inoculating loop (Himedia, catalog number: LA014 )
Hand gloves (Kimtech, catalog number: 97613 )
Spatula (Himedia, catalog number: LA007 )
Parafilm (Himedia, catalog number: LA017 )
HiDispo^TM Bag-12 (Himedia, catalog number: PW040 )
Tomato seeds var. Pusa Ruby (JAI Kisan Seeds Private Limited, India)
R. solanacearum antagonizing bacteria (Staphylococcus warneri GL1, Bacillus velezensis GL3, B. velezensis GL5 and B. velezensis GMC2) isolated in authors laboratory from the endosphere of Gnetum gnemon
R. solanacearum F1C1 strain (provided by Prof. S.K. Ray, Tezpur university)
70% ethanol (Himedia, catalog number: MB106-500ML )
1% NaOCl (Himedia, catalog number: AS102-100ML )
Sterile distilled water
Peptone (Himedia, catalog number: RM001-500G )
Acicase (Himedia, catalog number: CR013-500G )
Glucose (Himedia, catalog number: MB037-500G )
Nutrient broth (Himedia, catalog number: M002-500G )
Glycerol (Himedia, catalog number: AS100-500ML )
Agar powder (Himedia, catalog number: CR301-500G )
Casamino acid peptone glucose broth (see Recipes)

Equipment

Beaker (Borosil, catalog number: BRL_1060D21 )
Erlenmeyer flask 250 ml (Borosil, catalog number: 4980021 )
Glass beaker (Borosil, catalog number: 1000D121 )
Forceps (Tarsons, catalog number: 486000 )
Spirit lamp/alcohol burner lamp (Himedia, catalog number: LA275 )
Growth Chamber (Hipoint, model: 740FHLED )
Orbital shaking incubator (Remi, model: CIS-24 plus )
Refrigerated centrifuge (Sigma model: 2-16KL )
Autoclave (Optics, model: 50 litres )
Water purification system (Sartorius, model: Arium mini Plus UV )
Pipettor (Eppendorf Research Plus, catalog numbers: 3120000020 , 3120000046 , 3120000062 )
Weighing balance (Shimadzu, model: BL220H )
Laminar air flow cabin (Cleanair Systems, model: CAH 1200 )
Eppendorf Biophotometer (Eppendorf, model: D30 )

Software

SPSS 20.0
Microsoft Office Excel 2016

Procedure

Tomato seedling preparation
1. Surface sterilize the tomato seeds by treating with 70% ethanol for 5 min followed by 1% NaOCl for 5 min and ﬁve washes with sterile water under laminar air flow cabin.
2. Take the seeds in a glass beaker with sterile water, stir gently and wait for 10 min.
3. Discard the seeds floating on surface of water after 10 min to maximize the number of potentially viable seeds.
4. Keep the beaker with the remaining seeds at 28 °C, in the dark for 24 h.
5. Wash the soaked seeds three times with sterile water.
6. Prepare a three-layered germination bed–sterile tissue paper on top, 3 cm thick sterile cotton in the middle and a polythene layering (HiDispoTM Bag) at the bottom. Place this bed in a plastic tray (30 × 20 cm²) and soak the bed with sterile water (Video 1).
  
  Video 1.How to prepare germination bed
7. Spread the seeds uniformly on the bed and place this germination tray in a growth chamber conditioned at 28 °C, 75% relative humidity and 12 h of photoperiod (light intensity 450 μmol m^-2 s^-1).
8. Spray sterile water on a regular basis to sustain the growth of seedlings.
9. Five days old seedlings are used for priming with R. solanacearum antagonizing bacterium.

Antagonistic bacterial inoculum preparation
1. Culture the R. solanacearum antagonizing bacterium individually in nutrient broth medium at 28 ± 2 °C in shaking condition (120 rpm) for 24 h in the dark.
2. Centrifuge the culture broth at 430 x g for 10 min at room temperature and discard the supernatant. Resuspend the cell pellet in sterile water without vortexing.
3. Repeat the above step two more times and adjust the cell density of final suspension to ~10⁸ CFU ml^-1 (OD = 0.4). About 10 ml R. solanacearum antagonizing bacterial cell suspension is sufficient to prime 30 seedlings.

Seedling root bacterization with antagonistic bacterium
1. Uproot 5 days old tomato seedlings from the germination tray without damaging the root system.
2. Put 30 seedlings in a Petri dish (60 mm diameter, 10 mm height) with 10 ml of R. solanacearum antagonizing bacterial cell suspension. Ensure the contact of the root with the bacterial suspension (Figure 1).
  
  Figure 1. Seedling root bacterization
3. Keep the Petri dish with seedlings in a growth chamber conditioned at 28 °C, 75% relative humidity, and 12 h of photoperiod for 16 h (light intensity 450 μmol m^-2 s^-1).
4. Treat 30 seedlings with sterile water to serve as a negative control group, which will be inoculated later with R. solanacearum (CK+R in Table 1).
5. Mock inoculate another 30 seedlings with sterile water to act as control group to nullify the natural death of seedlings in hydroponic conditions without being exposed to any bacteria (CK+W in Table 1).
6. After 16 h of priming with antagonizing bacteria, seedlings are ready for R. solanacearum inoculation.
  
  Table 1. The types of treatment groups required in this method with description and purpose

R. solanacearum inoculum preparation
1. Culture R. solanacearum in casamino acid-peptone-glucose (CPG) broth at 28 ± 2 °C in shaking condition (120 rpm) for 24 h in the dark.
2. Centrifuge the culture broth at 430 x g for 10 min at room temperature and discard the supernatant. Resuspend the cell pellet in sterile water without vortexing.
3. Repeat the above step two more times and adjust the cell density of final suspension to ~10⁸ CFU ml^-1.

Inoculation of R. solanacearum in antagonistic bacteria primed seedlings
1. Take out the seedlings from the Petri dish and keep them in another sterile, dry Petri dish before R. solanacearum inoculation. Use these seedlings for inoculation immediately within 5 min, otherwise seedlings may dry out.
2. Inoculate the seedlings with R. solanacearum by root-dip inoculation method described by Singh et al. (2018) . Briefly, take the R. solanacearum suspension in a wide mouth tube and immerse the seedling in the inoculum upto the root shoot junction for a few seconds.
3. Seedlings of the group CK+W should not be inoculated with R. solanacearum. It is kept as control.
4. Place the inoculated seedlings in 1.5 ml microcentrifuge tube and then add 1 ml sterile water into it. Use one seedling per microcentrifuge tube.
5. Place the microcentrifuge tube in a rack and keep inside a growth chamber conditioned at 28 °C, 75% relative humidity, and 12 h of photoperiod for 10 days (light intensity 450 μmol m^-2 s^-1). Add an equal amount of sterile water to the tubes on a regular basis to sustain the viability of seedlings.
6. A seedling is considered wilted when the leaves turn yellow to dark brown and the shoot bends at a sharp angle along the side of the microcentrifuge tube (Figure 2).
  
  Figure 2. Effect of different treatment on tomato seedlings after 7 days of R. solanacearum inoculation. CK+W: neither inoculation with antagonizing bacterium nor R. solanacearum, GL1+R: inoculation with antagonizing bacterium Staphylococcus warneri GL1 followed by R. solanacearum inoculation, and CK+R: mock inoculation with sterile water followed by R. solanacearum inoculation. This figure is reprinted (with slight modification) from original research article Agarwal et al. (2020) with permission from Elsevier.
7. Keep records of wilted seedlings on a daily basis from 1^st day to 10^th day post R. solanacearum inoculation at an interval of 24 h.
8. A stepwise demonstration of the protocol is given in Figure 3.