A Quick Method for Screening Biocontrol Efficacy of Bacterial Isolates against Bacterial Wilt Pathogen Ralstonia solanacearum in Tomato

Materials and Reagents

1. Micro centrifuge tube 1.5 ml (Tarsons, catalog number: 500010 )

2. Petri dish 90 mm (Tarson, catalog number: 460050 )

3. Petri dish 60 mm (Tarson, catalog number: 460061 )

4. Microtips 200-1,000 μl (Tarson, catalog number: 521020 )

5. Microtips 2-200 μl (Tarson, catalog number: 521010 )

6. Microtips 0.2-10 μl (Tarson, catalog number: 521000 )

7. Absorbent cotton wool (Bengal Surgicals Limited, India/Azpack, catalog number: 12356477 )

8. Tissue paper roll (Solimo/Kimberly-Clark WypAll L10 Paper Wipes, catalog number: 13478218 )

9. Polygrid micro tube stand (Tarson, catalog number: 205110 )

10. Plastic tray (Agrawal Plastic and Packaging Industries)

11. Cotton plugs (Prepared in laboratory)

12. Spray bottle (Abdos, catalog number: P11191 )

13. Inoculating loop (Himedia, catalog number: LA014 )

14. Hand gloves (Kimtech, catalog number: 97613 )

15. Spatula (Himedia, catalog number: LA007 )

16. Parafilm (Himedia, catalog number: LA017 )

17. HiDispo^TM Bag-12 (Himedia, catalog number: PW040 )

18. Tomato seeds var. Pusa Ruby (JAI Kisan Seeds Private Limited, India)

19. 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

20. R. solanacearum F1C1 strain (provided by Prof. S.K. Ray, Tezpur university)

21. 70% ethanol (Himedia, catalog number: MB106-500ML )

22. 1% NaOCl (Himedia, catalog number: AS102-100ML )

23. Sterile distilled water

24. Peptone (Himedia, catalog number: RM001-500G )

25. Acicase (Himedia, catalog number: CR013-500G )

26. Glucose (Himedia, catalog number: MB037-500G )

27. Nutrient broth (Himedia, catalog number: M002-500G )

28. Glycerol (Himedia, catalog number: AS100-500ML )

29. Agar powder (Himedia, catalog number: CR301-500G )

30. Casamino acid peptone glucose broth (see Recipes)

Equipment

1. Beaker (Borosil, catalog number: BRL_1060D21 )

2. Erlenmeyer flask 250 ml (Borosil, catalog number: 4980021 )

3. Glass beaker (Borosil, catalog number: 1000D121 )

4. Forceps (Tarsons, catalog number: 486000 )

5. Spirit lamp/alcohol burner lamp (Himedia, catalog number: LA275 )

6. Growth Chamber (Hipoint, model: 740FHLED )

7. Orbital shaking incubator (Remi, model: CIS-24 plus )

8. Refrigerated centrifuge (Sigma model: 2-16KL )

9. Autoclave (Optics, model: 50 litres )

10. Water purification system (Sartorius, model: Arium mini Plus UV )

11. Pipettor (Eppendorf Research Plus, catalog numbers: 3120000020 , 3120000046 , 3120000062 )

12. Weighing balance (Shimadzu, model: BL220H )

13. Laminar air flow cabin (Cleanair Systems, model: CAH 1200 )

14. Eppendorf Biophotometer (Eppendorf, model: D30 )

Software

1. SPSS 20.0

2. Microsoft Office Excel 2016

Procedure

1. 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.




2. 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.




3. 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




4. 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.




5. 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.

   
   

   
   Figure 3. Schematic diagram representing biocontrol efficacy assay for R. solanacearum antagonizing bacteria

   
   

   6. Calculation of biocontrol efficacy

      1. Calculate disease severity index (DSI) using the formula:

         

      2. Calculate biocontrol efficacy (BE) using the formula:

         

      3. Calculate the DSI in the groups, and BE of antagonizing bacterium from the records on 10^th day post R. solanacearum inoculation.

   Data analysis

   1. Perform One-Way ANOVA on the calculated DSI and BE, with P value less than 0.05, between-treatments in order to test the performances of biocontrol bacteria in the treatment groups.

   2. To further investigate the nature of the differences between the control and different treatment means, perform Duncan Multiple Range Test (DMRT) after a statistically significant ANOVA.

   3. Perform descriptive statistics (mean and standard error) on the calculated DSI and BE across all the treatment groups.

   4. Data (Figure 4) can be found in the original research article titled “Endophytes from Gnetum gnemon L. can protect seedlings against the infection of phytopathogenic bacterium Ralstonia solanacearum as well as promote plant growth in tomato”; doi:10.1016/j.micres.2020.126503, published in Microbiological Research ( Agarwal et al., 2020 ).

      
      

      
      Figure 4. Biocontrol efficacy of bacterial isolates and respective disease severity index in treatment groups. CK+R: only R. solanacearum treated control, GL1+R: Staphylococcus warneri GL1+R. solanacearum, GL3+R: Bacillus velezensis GL3+R. solanacearum, GL5+R: B. velezensis GL5+R. solanacearum, GMC2+R: B. velezensis GMC2+R. solanacearum. Different lower-case letters represent significant differences between treatments (one-way ANOVA, P < 0.05) according to Duncan's multiple range tests. This figure is reprinted (with slight modification) from original research article Agarwal et al. (2020) with permission from Elsevier.

   Notes

   1. Confirm the antagonistic ability of bacterial isolate against R. solanacearum by any of the standard methods such as agar well diffusion, disc diffusion, or spot on lawn assay with at least three repetitions prior to start this in planta biocontrol efficacy assay.

   2. Calculate the age of seedlings from the day of putting the seeds on the germination tray.

   3. The culture media, and incubating conditions for optimal growth can be different for different R. solanacearum antagonizing bacteria. This change will not have any effect further, since the cell number is adjusted for all isolates and media contamination is removed from the bacterial cells after repeated wash with sterile water.

   4. In the initial root bacterization stage, take some additional seedlings in order to compensate any damage/death of seedlings while handling.

   5. The number of seedlings in treatment groups can be increased as per convenience and to ensure the biocontrol efficacy of bacterial isolates, repeat this assay at least three times.

   6. This assay cannot be conducted for more than 10 days post R. solanacearum inoculation in tomato, as seedlings (control group-1, Table 1) are unable to sustain after this time period in sterile water without any supplements. The age up to which the seedlings can sustain in sterile water is a subject of consideration before trying this assay on other hosts.

   Recipes

   1. Casamino acid peptone glucose broth (pH 6.5-7.0) (100 ml)

      100 mg Acicase

      1 g Peptone

      500 mg Glucose

      Add distilled water up to 100 ml volume and autoclave at 121 °C under 15 psi pressure for 20 min

   Acknowledgments

   The authors would like to thank Prof. Suvendra Kumar Ray, Tezpur University for providing R. solanacearum F1C1 strain used in this study. The authors are thankful to DST, Govt. of India for providing DST-FIST Support to Department of Botany, Gauhati University, where this research work was carried out. Facilities created using SERB, Govt of India sponsored ECR award project (grant number: ECR/2017/000710) awarded to NA was utilized for carrying out the research work.

   Competing interests

   The authors declare no conflict of interests.

   
   

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