An Efficient Inoculation Technique to Assess the Pathogenicity of Pantoea Species Associated to Bacterial Blight of Rice

Figure 1. Typical Pantoea-induced symptoms developed on leaves inoculated with either P. stewartii or P. agglomerans using the leaf infiltration method (front leaf) compared to the absence of symptoms on rice leaves inoculated using the leaf clipping method (back leaf)

Materials and Reagents

1. Latex gloves
2. 2 ml micro-centrifuge tubes (Dominique DUTSCHER, catalog number: 0 33297 )
3. 1 liter plastic pots
4. 50 ml Falcon tube (Sigma, catalog number: CLS430828 )
5. 500 ml wash bottle
6. Plates 96 wells (VWR, catalog number: 735-0083 )
7. 10, 20, 200, and 1,000 μl tips (Eppendorf, catalog number: 2231300008 )
8. Paper towel
9. Petri dishes
10. Inoculating loop
11. Marker
12. Parafilm
13. Fertilizer (urea and NPK 15-15-15 beaded and pelletized)
14. Blunt-ended (needleless) syringes 
15. Sterilized field Soil
16. Brand^® UV cuvettes macro, chamber volume 2.5-4.5 ml (Sigma, catalog number: Z637157 )
17. Icebox 
18. Plant materials: Rice seeds, including control accessions (susceptible, partially and highly resistant)
19. Pantoea spp. Strains
20. Hydrochloric acid (HCl)
21. Alcohol
22. Sterilized ultra-pure water
23. Peptone (EuroMedex, catalog number: P3300 )
24. Beef extract (Merck, CAS no. 68990-09-0)
25. Sucrose (Merck, CAS no. 57-50-1)
26. Glucose (Sigma, CAS no. 50-99-7)
27. Yeast extract (Merck, CAS no. 8013-01-2)
28. Tryptone (Merck, CAS no. 91079-40-2)
29. Glutamic acid (Merck, CAS no. 56-86-0)
30. Peptone Sucrose Agar (PSA plates) (see Recipes)
31. 10 N HCl (see Recipes)
32. 70% ethanol (see Recipes)
33. Inoculum (see Recipes)
    

Equipment

1. Tweezers
2. Autoclave machine
3. Nethouse, greenhouses/screenhouses/growth chambers
4. Mortars and pestles
5. MilliQ sterile ultrapure water system
6. Laminar flow cabinet
7. 10, 20, 200, and 1,000 μl pipettes
8. -20 °C or deep freezer
9. 28 °C growing incubator
10. Spectrophotometer
11. Vortexer
12. pH meter
13. Microcentrifuge
14. Precision balance
    

Procedure

1. Rice plant growth conditions
   1. Grow rice plants under semi-controlled environment greenhouses/screenhouses/growth chambers with the following conditions during: approximately 12 h of light, 25 ± 5 °C (day) and 20 ± 5 °C (night), and 75% relative humidity.
      Note: Use the following recommended rice varieties as checks: C101A51, Azucena, Sahel 108, Moroberekan.
   2. Make 3 holes in 20 cm diameter pots containing autoclaved (121 °C for 20 min) field soil or pre-conditioned potting compost.
   3. Sow 3 surface disinfected seeds per hole, use 3 holes per pots and 3 pots/rice accession (27 seeds/variety/replication).
   4. About 30-35 days after sowing, inoculate the plants as described below.
   5. Apply urea (recommended quantity for the rice varieties used) after inoculation for optimal plant growth.
      
      
      
2. Inoculum preparation and plant inoculation (see Video 1)
   1. Streak the bacterial strains kept at -20 °C or -80 °C freezer on freshly prepared PSA agar plates and incubate them at 28 °C for 1-3 days. Use a strain diagnosed as Pantoea spp. as control.
   2. Then, scrape off the cells from the plates and suspend them in MilliQ sterile ultrapure water.
   3. Adjust the concentration of this suspension at 10⁸ cells/ml (OD = at 600 nm) using a spectrophotometer.
   4. Then, when plants are 4-5-week-old, use a needleless syringe to pump inoculum and infiltrate 1 to 2 ml of the bacterial suspension inoculum into 3-6 youngest fully expanded leaves using the needleless syringe. Infiltrate leaves at about 3-5 cm below the tip of the leaves (Figure 2).
      
   5. Perform the infiltration on the underside of the leaves by pressing the mouth of the syringe on the leaf surface.
      Note: The gloved fingers should gently block and support the opposite side of the leaf and gently press the plunger. Take care not to crush the leaf. Also, be sure not to overlap the midrib as this will raise the syringe and break the necessary seal.
   6. Then, gently wipe the leaf surface with a sterilized paper towel to remove the excess liquid from the leaf surface.
   7. Finally, hold the leaf briefly against the light to confirm that infiltration was successful as such infiltrated spots will show water-soaking features. Use sterile distilled water as a negative control.
      
      
      
      Video 1. Peptone Sucrose Agar (PSA plates) preparation, plating and growth of bacteria, inoculum preparation and inoculation of rice plants
      
      
      Figure 2. Rice plants aged 4-5 weeks after sowing that were just inoculated (note that the inoculated leaves that are bending)
      
      
      
3. Symptoms progression
   After inoculation, monitor symptom development on a daily basis.
   Note: In susceptible reactions, a spectacular water-soaking will appear 4-5 days after inoculation (DAI) at the infiltration spots of the leaves while it is much weaker in resistant reaction. About 8 DAI, the resulting necrotic lesions at the inoculation site will increase lengthwise along the principal veins towards to the tip of the leaf. These lesions later expand and turn from straw yellow to light brown color and ultimately develop into typical blight symptoms at 15 to 21 DAI (Figure 3).
   
   
   Figure 3. Scoring of the Pantoea blight symptom progression on rice plants inoculated at 35 days after sowing using a scale we developed. A = 2-4 DAI: Water-soaking at the inoculation point. B = 5-7 DAI: Yellow necrotic lesions at the inoculation points spreading in both directions (length at width). C = 10-12 DAI: Yellow necrotic lesions at the inoculation points increasing lengthwise along the principal veins towards to the tip of the leaf. D = 14-17 DAI: Ascending asymmetrical yellow necrosis of the tips of the leaves on 0.5 to 1 cm. E = 21-23 DAI: Presence of symptoms on three leaf areas, i) a grey necrotic area (at the tips of the leaves); ii) green area, iii) a yellowish area (which separates area i from ii). Area ii can evolve and areas i and ii can partially merge. F = 28-30 DAI: Total grey necrotic leaf area spreading from the tip of the leaf to the inoculation point.
   
   
4. Symptoms scoring
   Pathogenicity assessment was made as follow: record the disease severity at 14 and 21 DAI according to the symptom severity scale below (Figure 4):
   1. Water-soaking at the inoculation point.
   2. Yellow necrotic lesions at the inoculation point spreading in both directions (length and width).
   3. Yellow necrotic lesions at the inoculation point increasing lengthwise along the principal veins and towards to the tip of the leaf.
   4. Ascending asymmetrical yellow necrosis of 0.5 to 1 cm on the tip of the leaf.
   5. Presence of symptoms on three leaf areas, i) grey necrotic area (on the end of the leaf); ii) green area, iii) yellowish area (which separates area i from area ii). Green area can evolve and partially merge with the necrotic area.
   6. Total grey necrotic leaf area seen from the tip of the leaf to the inoculation point.
   7. Total necrotic greyish/brown leaf with leaf curling.
      Accessions are then classified for resistance as follow:
      •Resistant (R)
      •Moderate Resistant (MR)
      •Moderate Susceptible (MS)
      •Susceptible (S)
      Resistance/susceptibility phenotypes of plants from pure lines will be more or less the same compared to those of mixed seeds. In all cases, select the most representative response for each accession.
        Pathogenicity of the isolates were classified as non-, moderate and fully pathogenic. As regards to their virulence, they were classified as avirulent when they induced only R and MR pathogenicity phenotypes and virulent when they induced MS and S ones.
      
      
      Figure 4. Scale developed and used to score symptom severity
      
      
5. Assessment of bacterial population
   At 7, 14 and 21 DAI, bacterial populations can be assessed as a function of colony-forming units (cfu) recovered from the inoculated leaves. In practice, cut 1 cm² leaf pieces 2 cm below and above the inoculation points and make two corresponding samples. Ground these pieces in distilled sterile water (1 ml per leaf piece) using sterilized mortar and pestle or automatic ball mills. Make a 10 fold dilution of the resulting leaf saps and plate in triplicate on PSA or Pantoea Genus specific Agar (PGSA) described by Kini et al. (2019). Count the colonies on the three plates of each dilution and convert the data into cfu per leaf. Finally, perform, Analysis of Variances (ANOVA) on the collected data and calculate a correlation function.
   

Notes

1. Other media are also suitable for culturing Pantoea spp. and include nutrient broth or NB (per liter: beef extract, 3 g; peptone, 5 g; sucrose, 10 g), glucose yeast extract or GYE (per liter: glucose, 20 g; yeast extract, 10 g), and Tryptone Sucrose or TS (per liter: Tryptone, 10 g; sucrose, 10 g; glutamic acid, 1 g). Solidification of each medium is achieved by supplementation with 15 g agar/L.
2. To avoid contamination and ensure purity of bacterial strains, the PGSA medium can be used to select and purify the bacteria strains.
3. The genus Pantoea is composed of about 27 species. Before inoculation, ensure that the species of the strains used is known. To this end, a multiplex PCR (Kini et al., 2018) scheme can therefore be used. 
4. Certain Pantoea spp. are quarantine pests in several countries (Health et al., 2018). So, the manipulation of such species may be restricted by biosafety measures. Always, manipulate the bacteria in accordance with your country’s legislation.
5. Resistance or susceptibility depends on specific interactions between the rice plant and Pantoea spp. The choice of resistant or susceptible control accessions may also depend on the species and races of Pantoea spp. and the objectives of the experiment to be carried out.
6. Plants of different stages may be used for different purposes, such as measuring plant disease resistance that is developmentally regulated.
7. For easiest syringe inoculum infiltration in leaves, maintain rice plants at high humidity and inoculate at the beginning of the light cycle as this keeps stomata open.
   

Recipes

1. Peptone Sucrose Agar (PSA plates) (see Video 1)
   
   1. In 1 L of MilliQ water, add 10 g peptone, 10 g sucrose, 16 g agar, and 1 g glutamic acid
   2. Adjust pH to 7.1 ± 0.2 using 1 M KOH and NaOH buffers
   3. Autoclave at 121 °C for 20 min
   4. Let the medium cool down but not solidify, then pour it into Petri dishes
2. 10 N HCl
   To prepare 1,000 ml of 10 M HCl, proceed as follow:
   
   1. Poor 83 ml d’ HCl in a test tube
   2. Complete to 1,000 ml of water
3. 70% ethanol
   Using Gay-Lussac dilution table and 96° ethanol available in the laboratory:
   
   1. Poor 40.85 ml of this ethanol were poured in an test tube
   2. Complete to 100 ml with distilled sterile water
4. Inoculum
   
   1. Collect bacterial colonies in 50 ml Falcon tube containing 45 ml of MilliQ water
   2. Then, mix and take 2 ml inoculum in a Brand^® UV cuvettes macro, chamber volume 2.5-4.5 ml and introduce it in a spectrophotometer and take the reading at OD₆₀₀
   3. Then, adjust the inoculum by diluting it in MilliQ sterile water
   4. For each dilution, make sure the concentration is of 10⁸ CFU/ml

Acknowledgments

This protocol was developed from procedures used previously in different laboratories (Yang and Bogdanove, 2013) for inoculation and virulence assay for bacterial blight and bacterial leaf streak of rice. Dr. Kossi Kini benefited a PhD scholarship from the French Research Institute for Sustainable Development (IRD) within the program “Allocation de Recherche pour une Thèse au Sud” (ARTS), a grant from the International Foundation for Science (IFS) and financial support from the Ministry of Foreign Affairs (Japan) and the Global Rice Science Partnership (GRiSP) through AfricaRice. All these institutions are gratefully acknowledged.

Competing interests

Authors have no competing interest.

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