Bioassay of Xanthomonas albilineans Attachment on Sugarcane Leaves

Materials and Reagents

1. Sterile scalpels blades (Swan Morton, catalog numbers: n° 11 and n° 24 )
   
2. Sterilized pipette tips
   200 µl (Thermo Fischer Scientific, Fischer Scientific, catalog number: 02-681-2 )
   1,000 µl (Thermo Fischer Scientific, Fischer Scientific, catalog number: 02-681-4 )
   
3. Sterile plastic loops (Greiner Bio One, catalog number: 731171 )
   
4. Falcon 15 ml conical centrifuge tubes (SARSTEDT, catalog number: 62.554.502 )
   
5. Soft tissue (Orapi Hygiène, catalog number: 186 )
   
6. Disposable, sterile splinter removers/tweezers – 11.1 cm. (4 ¼ in.) (TSIC Solution, catalog number: UTIL-1037 )
   
7. 90 x 15 mm Petri dishes (Corning, Gosselin^TM, catalog number: BP93B-15 )
   
8. 1.5 ml microcentrifuge tube (SARSTEDT, catalog number: 72.690.001 )
   
9. Disposable pellet pestle for 1.5 ml centrifuge tube (Kimble Chase Life Science and Research Products, catalog number: 749521-1500 )
   
10. Sugarcane plantlets (cultivar CP68-1026 susceptible to leaf scald disease of sugarcane)
    
11. Xanthomonas albilineans wild type strains and mutants affected in pathogenicity (grown for 4 to 5 days on Wilbrink medium + appropriate antibiotics); for characteristics of mutants, see Fleites et al. (2013) and Mensi et al. (2016)
    
12. Sterile distilled water
    
13. Tween 20 (Sigma-Aldrich, catalog number: P2287 )
    
14. Sucrose (Merck Millipore, catalog number: 107687 )
    
15. Peptone (BD, Bacto^TM, catalog number: 211677 )
    
16. Potassium phosphate, dibasic, trihydrate (K₂HPO₄·3H₂O) (EMD Millipore, Calbiochem®, catalog number: 529567 )
    
17. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (EMD Millipore, catalog number: 105886 )
    
18. Sodium sulfite (Na₂SO₃) (EMD Millipore, catalog number: 106657 )
    
19. Agar (BD, Bacto^TM, catalog number: 214010 )
    
20. Potassium bromide, KBr (Sigma-Aldrich, catalog number: P0838 )
    
21. Benomyl (Sigma-Aldrich, catalog number: 381586 )
    
22. Cycloheximide (Sigma-Aldrich, catalog number: C1988 )
    
23. Ethanol (Sigma-Aldrich, catalog number: 32294 )
    Note: This product has been discontinued.
    
24. Cephalexin (Sigma-Aldrich, catalog number: C0675000 )
    
25. Novobiocin (Sigma-Aldrich, catalog number: 1475008 )
    
26. Kasugamycin (Sigma-Aldrich, catalog number: 19408-46-9 )
    
27. Ammonium nitrate (NH₄NO₃) (Sigma-Aldrich, catalog number: A3795 )
    
28. Potassium nitrate (KNO₃) (Sigma-Aldrich, catalog number: P8291 )
    
29. Calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O)  (Sigma-Aldrich, catalog number: C2786 )
    
30. Magnesium sulfate heptahydrate (MgSO₄·7H₂O)  (Sigma-Aldrich, catalog number: 63138 )
    
31. Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5655 )
    
32. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
    
33. Boric acid (H₃BO₃) (Sigma-Aldrich, catalog number: B6768 )
    
34. Manganese(II) sulfate monohydrate (MnSO₄·H₂O) (Sigma-Aldrich, catalog number: M7899 )
    
35. Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Sigma-Aldrich, catalog number: Z1001 )
    
36. Potassium iodide (KI) (Sigma-Aldrich, catalog number: P8166 )
    
37. Ammonium molybdate tetrahyddrate ((NH₄)₆Mo₇O₂₄·4H₂O) (Sigma-Aldrich, catalog number: M1019 )
    
38. Copper(II) nitrate trihydrate (Cu(NO₃)₂·3H₂O) (Sigma-Aldrich, catalog number: 61194 )
    
39. Iron(II) sulfate heptahydrate (FeSO₄·7H₂O) (EMD Millipore, catalog number: 103965 )
    
40. Na₂EDTA·2H₂O (Sigma-Aldrich, catalog number: E5134 )
    
41. Nicotinic acid (Sigma-Aldrich, catalog number: N4126 )
    
42. Pyridoxol hydrochloride (Sigma-Aldrich, catalog number: P6280 )
    
43. Myo-inositol (Sigma-Aldrich, catalog number: I7508 )
    
44. Thiamine dichloride (Sigma-Aldrich, catalog number: T1270 )
    
45. Phytagel (Sigma-Aldrich, catalog number: P8169 )
    
46. Wilbrink medium (WM) (see Recipes)
    
47. Wilbrink Selective Davis (WSD) medium (see Recipes)
    
48. Macronutrients (see Recipes)
    
49. Micronutrients (see Recipes)
    
50. Ferric EDTA (see Recipes)
    
51. Fuji vitamins (see Recipes)
    
52. Nutritive medium for growth of sugarcane plantlets (see Recipes)
    

Equipment

1. Growth chamber
   
2. 200 x 20 mm Pyrex test tubes with cap (Legallais, catalog number: 761224 )
   
3. 200 mm long tweezers with blunt tips (VWR, catalog number: 82027-436 )
   
4. Micropipettes 
   20-200 µl (Eppendorf, catalog number: 3120000054 )
   100-1,000 µl (Eppendorf, catalog number: 3120000062 )
   
5. Scalpels (Swan Morton, catalog numbers: N° 3G S/S and N° 4G S/S )
   
6. Incubator for microbiology (Memmert, model: B40 )
   Note: This product has been discontinued.
   
7. Benchtop vortex (Scientific Industries, model: Vortex Genie 2 )
   
8. Spectrophotometer (Eppendorf Biophotometer)
   
9. 250 or 500 ml wide neck Erlenmeyer flasks (Borosilicate glass) (Duran, catalog number: 21 226 36 or 21 226 44 )
   
10. Laminar flow cabinet or sterile hood
    
11. Autoclave
    

Software

1. Package R, version 2.14.1 (R Development Core Team)

Procedure

1. Preparation of tissue-cultured sugarcane plantlets
   1. Tissue cultured plantlets of sugarcane cultivar CP68-1026 originating from apex tissue are propagated and maintained in a growth chamber at 28 °C with 12 h light.
   2. Four weeks prior to inoculation, young secondary tillers are transferred into new 200 x 20 mm test tubes containing the nutritive medium for tissue cultured plantlets, as follows:
   1. Using 200 mm long tweezers, remove the tissue cultured-plantlet from the tube and place it on a glass plate under a sterile hood, separate young secondary tillers from the primary tiller with a scalpel and sterile blades.
   2. Using 200 mm long tweezers, place each secondary tiller in a new 200 x 20 mm test tube containing the nutritive medium for sugarcane plantlets. Secondary tillers may vary in size but only tillers that were 150-200 mm long were transferred for future inoculation. All transferred plantlets must have two to four unfolded leaves.
      
   3. Incubate the test tubes in a growth chamber at 28 °C with 12 h of light for four weeks.
      
      
2. Bacterial inoculum preparation
   1. Five days before plant inoculation, streak out each strain of X. albilineans to be tested from -80 °C distilled water stocks using a sterilized blade or a pipet tip onto WM agar plates with appropriate antibiotics (for mutant strains) and let the cultures grow for 5 days in an incubator at 28 °C.
   2. For each strain, suspend bacterial colonies from the agar plate with a sterile polypropylene loop in 10 ml of sterile distilled water in a 15 ml Falcon conical centrifuge tubes (to obtain a turbid suspension). Vortex the bacterial suspension manually or gently with a benchtop vortex and then measure its optical density at 600 nm (OD₆₀₀) using a spectrophotometer.
   3. To prepare the stock suspension, adjust the bacterial suspension with sterile distilled water to obtain an OD₆₀₀ = 0.25-0.30 which corresponds to 10⁹ Colony Forming Units (CFU)/ml.
   4. To prepare the inoculum at 10⁷ CFU/ml, dilute 5 ml of the bacterial suspension calibrated at 10⁹ CFU/ml in 495 ml of sterile distilled water in a 500 ml Erlenmeyer flask.
      
      
3. Sugarcane plantlets inoculation by immersion
   1. For inoculation, use plantlets with two to four fully unfolded leaves (Figure 1).
      
      
      Figure 1. Sugarcane plantlet exhibiting three fully expanded leaves in a test tube
      
      
   2. Use sterile tweezers to immerse the leaves of the tissue-cultured plantlets into the bacterial suspension adjusted at 10⁷ CFU/ml for 30-40 sec (Figure 2). During immersion of leaves, make gentle circular movements of the Erlenmeyer flask in order to optimize the contact of the bacterial suspension with the sugarcane leaf surfaces.
      
      
      Figure 2. Immersion of leaves of a sugarcane plantlet for 30-40 sec in a X. albilineans suspension adjusted at 10⁷ CFU/ml
      
      
   3. After immersion, remove excess of bacterial suspension by dragging the leaves across the edge of the wall of the Erlenmeyer flask: no visible droplet should be seen on the leaf surfaces. Then, replace the inoculated plantlets in the same test tubes containing the same nutritive medium (using sterile tweezers).
   4. Keep inoculated plantlets in a growth chamber at 28 °C with 12 h of light for 14 days.
   5. Inoculate at least six (for leaf imprinting assay, cf. D.1) to 15 (for leaf washing and tissue homogenization assay, cf. D.2) sugarcane plantlets per strain of X. albilineans and distribute them randomly in the growth chamber.
   6. Inoculate control plantlets with only sterile distilled water.
      
      
4. Determination of X. albilineans leaf attachment
   Leaf attachment of X. albilineans can be determined using two methods: 1. imprints of sugarcane leaves on selective medium (semi-quantitative method) and 2. leaf washing and tissue homogenization (quantitative method).

   1. Determination of capacity of X. albilineans leaf attachment by leaf imprinting
   1. Fourteen days after immersion, remove the inoculated sugarcane plantlets (one by one) from the test tubes and put them on a sterile soft tissue (Figure 3). Use a scalpel with a sterile blade to remove leaves (remove and imprint only non-withered leaves).
      
      
      Figure 3. Removal of the sugarcane plantlet from the test tube in an aseptic work area (laminar flow cabinet). A. Remove the inoculated sugarcane plantlet from the test tube using long sterile tweezers, 14 days after immersion and B. put it on a sterile soft tissue.
      
      
   2. Using disposable sterile tweezers, gently place detached leaves on WSD medium in 90 x 15 mm plates and apply a soft pressure with the tweezers to imprint the lower and then the upper leaf surface on the agar medium (Figure 4). Leaves for imprinting may vary in length but must have a green color. If the length of the leaf exceeds the plate diameter, cut the leaf into two or three fragments before performing the imprint. The quality of the imprinted leaf area can be observed by trans-illumination using day or artificial light.
      
      
      Figure 4. Imprint the inoculated sugarcane leaf (upper and lower surfaces) on WSD medium using disposable sterile tweezers
      
      
   3. Place agar plates in an incubator at 28 °C for 5 days.
   4. Five days after incubation, examine plates for presence or absence of X. albilineans colonies in the imprint area. Density of X. albilineans colonies growing in the imprint area can vary between absences of colonies to confluent growth of bacteria in the imprint area (Figure 5).
      
      
      Figure 5. Growth of colonies of X. albilineans in imprints of sugarcane leaves on WSD medium, 14 days after immersion of leaves in a bacterial suspension adjusted at 10⁷ CFU/ml. a. Imprint of the upper surface of leaf n1; b. Imprint of the lower surface of leaf n1; c. Imprint of the upper surface of leaf n2; d. Imprint of the lower surface of leaf n2. Leaf n1 is the first leaf from the top of the sugarcane plantlet that is completely unfolded. Leaf n2 is the leaf immediately below leaf n1 (Reprinted from Fleites et al., 2013).
      
      
   5. To estimate Extent of Leaf Attachment (ELA) of X. albilineans, give a score ranging from 0 to 6 to each leaf imprint (lower and upper surfaces) using the following scale (Figure 6):
      0 = 0 to 5 five colonies in the leaf imprint,
      1 = 6 to 50 colonies in the leaf imprint,
      2 = more than 50 colonies and no confluent growth,
      3 = confluent growth of bacteria in less than 10% of the leaf imprint,
      4 = confluent growth of bacteria in 10% to 40% of the leaf imprint,
      5 = confluent growth of bacteria in 41% to 80% of the leaf imprint,
      6 = confluent growth of bacteria in 81% to 100% of the imprint.
      
      
      Figure 6. Score used to estimate Extent of Leaf Attachment (ELA). 0 = 0 to 5 five colonies in the leaf imprint, 1 = 6 to 50 colonies in the leaf imprint, 2 = more than 50 colonies and no confluent growth, 3 = confluent growth of bacteria in less than 10% of the leaf imprint, 4 = confluent growth of bacteria in 10% to 40% of the leaf imprint, 5 = confluent growth of bacteria in 41% to 80% of the leaf imprint, 6 = confluent growth of bacteria in 81% to 100% of the leaf imprint (Reprinted from Mensi et al., 2016).
      
      
   6. Calculate ELA for each inoculated plantlet as follows:
      ELA = 100 ([1 x N₁ + 2 x N₂ + 3 x N₃ + 4 x N₄ + 5 x N₅ + 6 x N₆]/[6 x N_T])
      Where,
      N_i is the number of leaf imprints with score i (i is the individual scale),
      N_T is the total number of leaf imprints per plantlet.
   7. Calculate average ELA for each bacterial strain or mutant tested using all ELA values obtained for the six inoculated plantlets.
      
   2. Determination of X. albilineans leaf attachment by leaf washing and tissue homogenization.
      For this bioassay, 15 sugarcane plantlets were inoculated per strain. At each sampling time (1 h, 7 and 14 days), five plantlets and two leaves per plantlet were individually used to determine attachment capacity of X. albilineans. The experiment is repeated independently at least two times. Leaf washing is performed to detach and quantify bacteria that are located on the leaf surface, whereas leaf homogenization is performed to isolate and quantify bacteria from protected areas (such as stomatal chambers) or from inside the leaf.
   1. Leaf washing
   1. At 1 h, 7 and 14 days after immersion of sugarcane leaves in the bacterial suspension, use sterile tweezers to remove plantlets from the test tubes, put them on sterile soft tissue (Figure 3) and cut two leaves from each plantlet (using a scalpel with a sterile blade). Collect only inoculated and non-withered leaves.
   2. Cut a 7 cm long fragment from each leaf (Figure 7A) and immerse each fragment individually in a 1.5 ml micro-centrifuge tube containing 1 ml of sterile distilled water with 0.005% Tween 20 (Figure 7B). Note that each leaf fragment is analyzed separately.
      
      
      Figure 7. Steps describing leaf washing and tissue homogenization. A. Cut a 7 cm long fragment from two leaves of each inoculated plantlet. B. For leaf washing, immerse each leaf fragment individually in a 1.5 ml micro-centrifuge tube containing 1 ml of sterile distilled water and 0.005% Tween 20. C. For tissue homogenization, use a pestle made for micro-centrifuge tubes and homogenize the leaf fragment in the same tube, each tube containing 820 μl of wash water after removal of 180 μl for bacterial counts. D. Homogenized leaf tissue.
      
      
   3. Vortex the tubes vigorously for 10 sec to wash leaf fragments.
   4. Use 100 μl of each wash water to make 10 and 100 fold dilutions.
   5. Gently drop three or four times 20 μl of each undiluted, 10 and 100-fold diluted wash water on WSD plates.
   6. Place the agar plates in an incubator at 28 °C for 6 days (Figure 8).
      
      
      Figure 8. Growth of X. albilineans colonies in drops of undiluted (A), 10 (B) and 100 times (C) diluted wash water or homogenized tissue, 6 days after deposition on WSD plates
      
      
   2. Leaf homogenization after leaf washing
   1. Using a pestle made for 1.5 ml micro-centrifuge tubes, homogenize each leaf fragment in the remaining wash water (820 μl after removal of 180 μl in A. for determination of bacterial populations) for 5 min (Figures 7C and 7D).
   2. Use 100 μl of each homogenized tissue suspension to make 10 and 100 fold dilutions.
   3. Gently drop three or four times 20-μl of undiluted, 10 and 100 times diluted homogenized tissue on WSD plates. Place the agar plates in the incubator at 28 °C for 6 days.
      
   3. Determination of bacterial population densities
   1. Count colonies for each dilution and each of the three-four replications after 6 days of growth; calculate the average number of colonies and correct for dilutions. For example, 15 colonies on the 10^-1 dilution plate correspond to 7,500 CFU present in the leaf section. Convert to log₁₀ CFU per leaf section for the wash water or homogenized tissue.
   2. For wash water and for homogenized tissue, calculate average log₁₀ CFU for each bacterial strain or mutant tested using all values obtained for the six inoculated plantlets.
      

Data analysis

1. Data were analyzed using the statistical software package R, version 2.14.1 (R Development Core Team) as described by Mensi et al. (2016). Extent of Leaf Attachment (ELA) values are the combination of means of three independent experiments with six replicates (plantlets) per strain or mutant in each experiment. Note that two to four leaves were imprinted per plantlet. Although there can be significant variability between means of ELA between plantlets, all data are used for statistical analysis.
   
2. For the determination of leaf attachment by leaf washing and leaf homogenization, data are from 20 leaves and combined from two separate experiments of five plantlets each (two leaves sampled per inoculated sugarcane plantlet and five plantlets used at each sampling time). Individual population data (CFU/ml) are transformed using the log10 function (y = log₁₀ [(CFU/ml) + 1]) before calculation of population means. When appropriate (few nil data), individual log transformed data can be used for mean comparison by variance analysis.
   
3. Data obtained by leaf imprinting (semi-quantitative method) and leaf washing (quantitative method) are correlated and yield similar information as they are both linked to bacterial populations present on the leaf surface. In contrast, data obtained by leaf homogenization are linked to bacteria that cannot be easily detached from the leaf surface and that are located in protected leaf areas or inside the leaf. Leaf imprinting is recommended for rapid and easy screening of bacterial strains for leaf attachment capacity. Leaf washing/homogenization is recommended to obtain more precise and quantitative data, and additional information regarding location of bacteria on or in the leaf.
   

Notes

1. Bacterial suspension preparation, plantlet sugarcane immersion, and foliar imprints are performed under a laminar flow cabinet (sterile conditions).
   
2. The bacterial suspension used to inoculate sugarcane plantlets is prepared freshly (just before the inoculation procedure). The bacterial suspension is prepared with only a few single colonies to obtain the desired bacterial concentration. The number of colonies used will depend on the size of the colonies. The bacterial strains stored in the freezer at minus 80 °C are each issued from a single colony.
   
3. Choose carefully the sugarcane leaves for imprinting or washing; we choose only green and non-withered leaves.
   
4. The area of confluent growth was estimated visually using a template with 10% leaf area increments.
   
5. Sugarcane cultivar CP68-2016 was used herein, but other cultivars susceptible to sugarcane leaf scald can also be tested.
   Non-specific adhering was not observed with the bacterial strains used herein. However, it can be a good idea to identify and include a bacterial species not adapted to sugarcane as a negative control.
   

Recipes

1. Wilbrink medium (WM) (1 L)
   10 g sucrose
   5 g peptone
   0.50 g K₂HPO₄·3H₂O
   0.25 g MgSO₄·7H₂O
   0.05 g Na₂SO₃
   15 g agar
   Add distilled water to make up 1 L
   Adjust pH to 6.8-7.0
   Autoclave 20 min at 121 °C
   
2. Wilbrink Selective Davis (WSD) medium (1L)
   Same composition as WM medium but supplemented with:
   5 g KBr and 0.004 g Benomyl (fungicide) before heat sterilization
   After autoclaving, add:
   0.1 g cycloheximide dissolved in 500 μl ethanol
   0.025 g cephalexin dissolved in 500 μl sterile distilled water
   0.03 g novobiocin dissolved in 500 μl ethanol
   0.05 g kasugamycin dissolved in 500 μl sterile distilled water
   
3. Macronutrients (1 L)
   10 g NH₄NO₃
   10 g KNO₃
   5 g Ca(NO₃)₂·4H₂O
   715 mg MgSO₄·7H₂O
   3 g KH₂PO₄
   650 mg KCl
   Add distilled water to make up to 1 L
   
4. Micronutrients (100 ml)
   160 mg H₃BO₃
   1,400 mg MnSO₄·H₂O
   380 mg ZnSO₄·7H₂O
   75 mg KI
   10 mg (NH₄)₆Mo₇O₂₄·4H₂O
   35 mg Cu(NO₃)₂·3H₂O
   Add distilled water to make up to 100 ml
   Solution to be stored at -20 °C in 1.5 ml micro-centrifuge tubes
   
5. Ferric EDTA
   2.78 g FeSO₄·7H₂O
   3.73 g Na₂EDTA·2H₂O
   500 ml distilled water
   Dissolve Na₂EDTA and FeSO₄ separately and add FeSO₄ to Na₂EDTA by mild heating of the solution
   
6. Fuji vitamins
   50 mg nicotinic acid (in boiling water)
   10 mg pyridoxol hydrochloride
   10 g myo-inositol
   10 mg thiamine dichloride (to be added last)
   Add distilled water to make up to100 ml
   Solution to be stored at -20 °C in 1.5 ml micro-centrifuge tubes
   
7. Nutritive medium for growth of sugarcane plantlets (1 L) (Adapted from Chatenet et al., 2001)
   100 ml macronutrients
   1 ml micronutrients
   5 ml Ferric EDTA
   2 ml Fuji vitamins
   8 g agar or 4 g Phytagel
   40 g sucrose
   890 ml distilled water
   pH 5.8
   Autoclave for 20 min at 121 °C
   

Acknowledgments

We thank Marie-Josée Darroussat for excellent technical assistance. Imène Mensi was supported by a PhD fellowship from Cirad. The work is supported by the USDA National Institute of Food and Agriculture [project Hatch/Rott FLA-BGL-005404]. This protocol was adapted from work published by Fleites et al. (2013) and Mensi et al. (2016).

References

1. Birch, R. G. (2001). Xanthomonas albilineans and the antipathogenesis approach to disease control. Mol Plant Pathol 2(1): 1-11.
   
2. Chatenet, M., Delage, C., Ripolles, M., Irey, M., Lockhart, B. E. L. and Rott, P. (2001). Detection of Sugarcane yellow leaf virus in quarantine and production of virus-free sugarcane by apical meristem culture. Plant Dis 85(11): 1177-1180.
   
3. Fleites, L. A., Mensi, I., Gargani, D., Zhang, S., Rott, P. and Gabriel, D. W. (2013). Xanthomonas albilineans OmpA1 appears to be functionally modular and both the OMC and C-like domains are necessary for leaf scald disease of sugarcane. Mol Plant Microbe Interact 26(10): 1200-1210.
   
4. Hirano, S. S. and Upper, C. D. (2000). Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae-a pathogen, ice nucleus, and epiphyte. Microbiol Mol Biol Rev 64(3): 624-653.
   
5. Kumar, N. and Reddy, M. P. (2011). In vitro plant propagation: A review. J Forest Sci 27(2): 61-72.
   
6. Mensi, I., Daugrois, J. H., Pieretti, I., Gargani, D., Fleites, L. A., Noell, J., Bonnot, F., Gabriel, D. W. and Rott, P. (2016). Surface polysaccharides and quorum sensing are involved in the attachment and survival of Xanthomonas albilineans on sugarcane leaves. Mol Plant Pathol 17(2): 236-246.
   
7. Rott, P., Fleites, L., Marlow, G., Royer, M. and Gabriel, D. W. (2011). Identification of new candidate pathogenicity factors in the xylem-invading pathogen Xanthomonas albilineans by transposon mutagenesis. Mol Plant Microbe Interact 24(5): 594-605.
   
8. Yadav, R. K. P., Kakamanoli, K. and Vokou, D. (2010). Estimating bacterial population on the phyllosphere by serial dilution plating and leaf imprint methods. Ecoprint 17: 47-52.