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Bioassay of Xanthomonas albilineans Attachment on Sugarcane Leaves
采用生物测定法研究甘蔗叶白纹黄单胞菌的附着   

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Abstract

Sugarcane (interspecific hybrids of Saccharum species) is an economically important crop that provides 70% of raw table sugar production worldwide and contributes, in some countries, to bioethanol and electricity production. Leaf scald, caused by the bacterial plant pathogen Xanthomonas albilineans, is one of the major diseases of sugarcane. Dissemination of X. albilineans is mainly ensured by contaminated harvesting tools and infected stalk cuttings. However, some strains of this pathogen are transmitted by aerial means and are able to survive as epiphytes on the sugarcane phyllosphere before entering the leaves and causing disease. Here we present a protocol to estimate the capacity of attachment of X. albilineans to sugarcane leaves. Tissue-cultured sugarcane plantlets were immersed in a bacterial suspension of X. albilineans and leaf attachment of X. albilineans was determined by two methods: leaf imprinting (semi-quantitative method) and leaf washing/homogenization (quantitative method). These methods are important tools for evaluating pathogenicity of strains/mutants of the sugarcane leaf scald pathogen.

Keywords: Attachment(附着), Leaf imprinting(叶印记), Leaf scald(叶烫伤), Pathogenicity(致病性), Phyllosphere(叶际), Sugarcane(甘蔗), Tissue culture(组织培养), Xanthomonas albilineans(白纹黄单胞菌)

Background

The mechanisms that govern the interactions between X. albilineans and its host plant (the sugarcane) are not well known. Albicidin, a phytotoxin produced by albilineans, is the only molecular factor which has been demonstrated to play a role in pathogenicity of this pathogen (Birch, 2001). However, pathogenicity of X. albilineans doesn’t completely depend on albicidin. Albicidin-deficient mutants are still able to colonize efficiently the sugarcane stalk and to produce disease symptoms (Birch, 2001; Rott et al., 2011). Studies using full grown sugarcane are space and time consuming. Bioassays using miniaturized plants (tissue-cultured plants) or detached leaf bioassays can be very useful because they are less space consuming and they allow the study of plant-pathogen interactions in controlled environments. In vitro propagation of plants is widely used to rapidly propagate disease-free planting material under controlled conditions (Kumar and Reddy, 2011). Additionally, leaf imprinting has been widely used to study the ecology of bacteria associated with the phyllosphere (Hirano and Upper, 2000; Yadav et al., 2010). However, to our knowledge, these techniques have never been associated to decipher pathogenicity of bacterial plant pathogens. To identify additional pathogenicity factors of X. albilineans, especially factors involved in the early phases of infection (epiphytic phase), we developed a new miniaturized bioassay using tissue cultured sugarcane plantlets. Attachment of X. albilineans to sugarcane leaves under axenic condition was reproduced (Fleites et al., 2013; Mensi et al., 2016). This bioassay will permit the rapid testing of leaf attachment capacity of wild type and mutant strains of the pathogen causing leaf scald disease, but also of other bacteria colonizing the sugarcane leaf canopy.

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, GosselinTM, 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, BactoTM, 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, BactoTM, 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 NT])
        Where,
        Ni is the number of leaf imprints with score i (i is the individual scale),
        NT 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.

简介

甘蔗(蔗糖种类的种间杂交种)是一种经济上重要的作物,其在世界范围内提供70%的生食糖生产,并在一些国家对生物乙醇和电力生产作出贡献。由细菌植物病原体Xanthomonas albilineans引起的叶片烫伤是甘蔗的主要疾病之一。传播X。 albilineans主要由受污染的收获工具和感染的茎秆确保。然而,这种病原体的一些菌株通过空中装置传播,并且能够在进入叶子并引起疾病之前作为甘蔗叶片上的附生植物生存。在这里,我们提出一个协议来估计X的附件容量。白皮书到甘蔗叶。将组织培养的甘蔗小植物浸入X的细菌悬浮液中。白皮书和X附件。通过两种方法确定了白皮书:叶片印记(半定量方法)和叶洗/匀浆(定量方法)。这些方法是评估甘蔗叶烫伤病原体菌株/突变体致病性的重要手段。

背景 管理X之间相互作用的机制。 albilineans 和它的宿主植物(甘蔗)不是公知的。 Albicidin是由白念珠菌产生的植物毒素,是已被证明在这种病原体的致病性中发挥作用的唯一分子因子(Birch,2001)。然而,X的致病性。白皮书不完全依赖于albicidin。缺乏Albicidin的突变体仍然能够有效地定殖甘蔗茎并产生疾病症状(Birch,2001; Rott等人,2011)。使用全长甘蔗的研究是空间和耗时的。使用小型化植物(组织培养的植物)或分离的叶生物测定法的生物测定可能是非常有用的,因为它们的空间消耗更少,并且允许在受控环境中研究植物 - 病原体相互作用。 体外植物的繁殖被广泛用于快速传播控制的条件(Kumar和雷迪,2011)下无病种植材料。此外,叶片印迹已广泛用于研究与叶片相关的细菌的生态学(Hirano and Upper,2000; Yadav等人,2010)。然而,据我们所知,这些技术从未被相关的破译细菌植物病原体的致病性。识别X的其他致病因素。特别是涉及感染早期阶段(附生期)的因素,我们开发了使用组织培养的甘蔗小植株的新型小型化生物测定。附件X。白藜芦醇在无菌条件下的甘蔗叶被转载(Fleites等人,2013; Mensi等人,2016)。这种生物测定可以快速检测引起叶片烫伤病的病原体的野生型和突变菌株的叶片附着能力,也可以对其他定殖在甘蔗叶冠上的细菌进行检测。

关键字:附着, 叶印记, 叶烫伤, 致病性, 叶际, 甘蔗, 组织培养, 白纹黄单胞菌

材料和试剂

  1. 无菌解剖刀刀片(Swan Morton,目录号:n°11和n°24)
  2. 灭菌移液器吸头
    200微升(Thermo Fischer Scientific,Fischer Scientific,目录号:02-681-2)
    1000μl(Thermo Fischer Scientific,Fischer Scientific,目录号:02-681-4)
  3. 无菌塑料环(Greiner Bio One,目录号:731171)
  4. Falcon 15 ml锥形离心管(SARSTEDT,目录号:62.554.502)
  5. 软组织(OrapiHygiène,目录号:186)
  6. 一次性无菌剥离器/镊子 - 11.1厘米。 (4¼in。)(TSIC解决方案,目录号:UTIL-1037)
  7. 90×15mm培养皿(Corning,Gosselin TM,目录号:BP93B-15)
  8. 1.5ml微量离心管(SARSTEDT,目录号:72.690.001)
  9. 1.5ml离心管一次性丸粒杵(Kimble Chase Life Science and Research Products,目录号:749521-1500)
  10. 甘蔗苗(品种CP68-1026易受甘蔗叶片烫伤)
  11. 野生型菌株和致突变体的突变体(Wilbrink培养基+适当的抗生素生长4至5天);对于突变体的特征,参见Fleites等人(2013)和Mensi等人。(2016)
  12. 无菌蒸馏水
  13. 吐温20(Sigma-Aldrich,目录号:P2287)
  14. 蔗糖(Merck Millipore,目录号:107687)
  15. 蛋白胨(BD,Bacto TM ,目录号:211677)
  16. 磷酸氢钾,二元,三水合物(K 2/2 HPO 4·3H 2 O)(EMD Millipore,Calbiochem,目录号:529567) br />
  17. 七水硫酸镁(MgSO 4·7H 2 O)(EMD Millipore,目录号:105886)
  18. 亚硫酸钠(Na 2 SO 3)(EMD Millipore,目录号:106657)
  19. 琼脂(BD,Bacto TM ,目录号:214010)
  20. 溴化钾,KBr(Sigma-Aldrich,目录号:P0838)
  21. Benomyl(Sigma-Aldrich,目录号:381586)
  22. 环己酰亚胺(Sigma-Aldrich,目录号:C1988)
  23. 乙醇(Sigma-Aldrich,目录号:32294)
    注意:本产品已停产。
  24. 头孢氨苄(Sigma-Aldrich,目录号:C0675000)
  25. 新生霉素(Sigma-Aldrich,目录号:1475008)
  26. 春日霉素(Sigma-Aldrich,目录号:19408-46-9)
  27. 硝酸铵(NH 4 NO 3)(Sigma-Aldrich,目录号:A3795)
  28. 硝酸钾(KNO 3)(Sigma-Aldrich,目录号:P8291)
  29. 硝酸钙四水合物(Ca(NO 3 3)2·4H 2 O)(Sigma-Aldrich,目录号:C2786)
  30. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:63138)
  31. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  32. 氯化钾(KCl)(Sigma-Aldrich,目录号:P5405)
  33. 硼酸(H 3 O 3 BO 3)(Sigma-Aldrich,目录号:B6768)
  34. 硫酸锰(II)一水合物(MnSO 4·H 2 O)(Sigma-Aldrich,目录号:M7899)
  35. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Sigma-Aldrich,目录号:Z1001)
  36. 碘化钾(KI)(Sigma-Aldrich,目录号:P8166)
  37. 钼酸铵四氢化物((NH 4))6 Mo 7 O 24·4H 2/> O)(Sigma-Aldrich,目录号:M1019)
  38. 硝酸铜(II)三水合物(Cu(NO 3 3)2·3H 2 O)(Sigma-Aldrich,目录号:61194) br />
  39. 硫酸铁(II)七水合物(FeSO 4·7H 2 O)(EMD Millipore,目录号:103965)
  40. Na 2 EDTA·2H 2 O(Sigma-Aldrich,目录号:E5134)
  41. 烟酸(Sigma-Aldrich,目录号:N4126)
  42. 吡哆醇盐酸盐(Sigma-Aldrich,目录号:P6280)
  43. 肌醇(Sigma-Aldrich,目录号:I7508)
  44. 二氯化二硫化胺(Sigma-Aldrich,目录号:T1270)
  45. Phytagel(Sigma-Aldrich,目录号:P8169)
  46. Wilbrink介质(WM)(见配方)
  47. Wilbrink选择性戴维斯(WSD)培养基(见食谱)
  48. 大量营养素(见食谱)
  49. 微量营养素(见食谱)
  50. EDTA EDTA(参见食谱)
  51. 富士维生素(见食谱)
  52. 甘蔗苗生长营养培养基(见食谱)

设备

  1. 生长室
  2. 200 x 20毫米带盖的Pyrex试管(Legallais,目录号:761224)
  3. 200毫米长钝镊子(VWR,目录号:82027-436)
  4. 微量移液器
    20-200μl(Eppendorf,目录号:3120000054)
    100-1,000μl(Eppendorf,目录号:3120000062)
  5. 头盔(天鹅莫顿,目录号:N°3G S/S和N°4G S/S)
  6. 微生物培养箱(Memmert,型号:B40)
    注意:本产品已停产。
  7. 台式涡旋(Scientific Industries,型号:Vortex Genie 2)
  8. 分光光度计(Eppendorf Biophotometer)
  9. 250或500ml宽颈锥形瓶(硼硅酸盐玻璃)(Duran,目录号:21 226 36或21 226 44)
  10. 层流柜或无菌罩
  11. 高压灭菌器

软件

  1. 软件包R,版本2.14.1(R开发核心团队)

程序

  1. 组织培养甘蔗苗的制备
    1. 来自顶端组织的甘蔗品种CP68-1026的组织培养的小植株在28℃的培养室中传播并维持12小时光。
    2. 在接种前四周,将年轻的二次分蘖转移到含有组织培养的小植株的营养培养基的新的200×20mm试管中,如下:
      1. 使用200毫米长的镊子,从管中取出组织培养的小植物,并将其放置在无菌罩下的玻璃板上,用手术刀和无菌刀片分离来自初级耕耘机的年轻次生分蘖。
      2. 使用200毫米长的镊子,将每个二次耕耘机放在一个新的200 x 20毫米的试管中,其中包含甘蔗苗的营养介质。次要分蘖的大小可能有所不同,但只有150-200毫米长的分蘖被转移用于将来接种。所有转移的小植物必须有两到四个展开的叶子。
    3. 将试管在28℃的生长室中孵育12小时光照四周
  2. 细菌接种物准备
    1. 在植物接种前5天,连续染上每株X片段。使用灭菌叶片或移液管尖端的-80℃蒸馏水库进行试验,并用合适的抗生素(用于突变株)进行测试,并使培养物在28℃的培养箱中生长5天C。
    2. 对于每个菌株,用15ml Falcon锥形离心管中的10ml无菌蒸馏水中的无菌聚丙烯环将琼脂平板上的细菌菌落悬浮(以获得混浊悬浮液)。用台式涡旋手动或轻轻地旋转细菌悬浮液,然后使用分光光度计测量其在600nm(OD 600)的光密度。
    3. 为了制备原液悬浮液,用无菌蒸馏水调节细菌悬浮液,得到对应于10μL/ml菌落形成单位(CFU)/ml的OD 600/0.25-0.30 。
    4. 为了在10μg/ml CFU/ml下制备接种物,将5ml以10μg/ml CFU/ml校准的5ml细菌悬浮液稀释在495ml无菌蒸馏水中的500ml锥形瓶。

  3. 甘蔗苗通过浸泡接种
    1. 对于接种,使用2至4个完全展开的叶子的植物(图1)

      图1.在试管中展示三个完全膨胀的叶子的甘蔗苗

    2. 使用无菌镊子将组织培养的小植株的叶子浸入调节至10μg/ml CFU/ml的细菌悬浮液中30-40秒(图2)。在浸入叶子期间,使锥形瓶的温和循环运动,以优化细菌悬浮液与甘蔗叶表面的接触。


      图2.在X中,甘蔗苗的叶子浸泡30-40秒。调节为10μg/ml的白蛋白悬浮液 CFU/ml

    3. 浸泡后,通过将叶子拖过锥形瓶的壁边缘去除过量的细菌悬浮液:叶片表面上不应有可见的液滴。然后,将含有相同营养培养基的相同试管(使用无菌镊子)取代接种的小植株。
    4. 将接种的小植株在28℃的生长室中保持12小时,持续14天。
    5. 接种至少六个(用于叶印记测定,参见D.1)至15(对于叶洗涤和组织匀浆测定,参见D.2)每个X株菌株的甘蔗苗。并将它们随机分布在生长室中。
    6. 用无菌蒸馏水接种对照小植物。

  4. 确定X。 albilineans 叶子附件
    X附件的叶子。可以使用以下两种方法测定白蛋白:1,甘蔗叶在选择培养基上的印迹(半定量方法)和2.叶洗涤和组织匀浆(定量方法)。
    1. 确定X的容量。叶片印记的叶子附着物
      1. 浸泡14天后,从试管中取出接种的甘蔗小植物,并放入无菌软组织(图3)。使用带有无菌刀片的手术刀去除叶子(仅移除并印刷非枯叶)。



        图3.在无菌工作区域(层流柜)中从试管中取出甘蔗小植物。 A。使用长期无菌镊子从试管中取出接种的甘蔗小植物,浸泡14天后,将其放在无菌软组织上。

      2. 使用一次性无菌镊子,将分离的叶子轻轻放置在90 x 15 mm平板上的WSD培养基上,并用镊子施加软压,以打印琼脂培养基上的下部和上部叶片表面(图4)。用于印记的叶片可能长度不同,但必须具有绿色。如果叶片的长度超过板的直径,则在进行印记之前将叶切成两片或三片。印刷叶面积的质量可以通过使用日光或人造光的反照照观察


        图4.使用一次性无菌镊子在WSD培养基上印刷接种的甘蔗叶(上下表面)

      3. 将琼脂平板放在28℃的培养箱中5天。
      4. 培养5天后,检查板是否存在X。阿拉伯人殖民地在印记区域。 X的密度。在印记区域生长的白色念珠菌菌落可能因菌落缺失与印迹区域细菌繁殖增长而异(图5)。



        图5. X的菌落生长。在将叶子浸入调节至10μg/ml CFU/ml的细菌悬浮液中的14天后,在WSD培养基上的甘蔗叶印记中的albilineans 。叶片上表面的印记n1; b。叶片下表面的印记n1; C。叶n2上表面的印记; d。叶n2下表面的印记。叶n1是完全展开的甘蔗小植物顶部的第一叶。叶n2是叶n1正下方的叶子(转载自Fleites等人,2013)。

      5. 估计X的叶附件(ELA)的范围。使用以下尺度对每个叶片印记(下表面和上表面)给出0到6的分数(图6):
        0 = 0到5个殖民地的叶片印记,
        1 =叶片印记中的6至50个菌落,
        2 =超过50个殖民地,没有汇合增长,
        3 =细菌繁殖生长不到叶片印记的10%,
        4 =细菌繁殖的10%〜40%的叶片印记,
        5 =细菌生长在41%〜80%的叶片印记,
        6 =细菌在81%至100%的印迹中汇合生长。


        图6.用于估计叶附件(ELA)的范围的分数。 0 =叶片印记中的0至5个5个菌落,1 =叶片印记中的6至50个菌落,2 =超过50个菌落,没有汇合生长,3 =细菌的汇合生长小于10%叶片印记,4 = 10%〜40%叶片细菌繁殖生长,5 =细菌共培养41%〜80%叶片印迹,6 =细菌繁殖生长81%〜100%的印记(转载自Mensi等人,2016)。

      6. 计算每个接种的小植株的ELA如下:
        ELA = 100([1×N 1 + 2×N 2 + 3 x < 4> 3 + 4 x 3 + 5 x 5 6×N 6 ]/[6×N ])
        哪里,
        > N 是每个小植株的叶片印记总数。
      7. 使用为六个接种的小植株获得的所有ELA值计算每个细菌菌株或突变体的平均ELA
    2. 确定X。通过叶子洗涤和组织匀浆,叶片附着物 对于这种生物测定,每个菌株接种15个甘蔗小植株。在每个采样时间(1 h,7和14天),每个小植株分别使用5个小植株和2个叶来确定X的附着能力。 albilineans 。实验重复独立至少两次。进行叶洗以分离和定量位于叶表面的细菌,而进行叶匀浆以分离和定量来自保护区(如气孔室)或叶内的细菌。
      1. 叶洗
        1. 在甘蔗叶浸入细菌悬浮液后1 h,7和14 d,使用无菌镊子从试管中取出小植株,将其放入无菌软组织(图3),并从每个小植物上切下两片叶子(使用手术刀用无菌刀片)。只收集接种和未枯叶的叶子。
        2. 从每个叶子切割7厘米长的片段(图7A),并将每个片段分别浸入含有1毫升无菌蒸馏水和0.005%吐温20的微量离心管中(图7B)。请注意,每个叶片片段分别进行分析


          图7.描述叶子洗涤和组织均质化的步骤。A.从每个接种的小植株的两个叶子上切下7厘米长的片段。 B.对于叶子洗涤,将每个叶片片段单独浸入含有1ml无菌蒸馏水和0.005%吐温20的1.5ml微量离心管中。C.对于组织匀浆,使用用于微量离心管的研杵,并使叶片在相同的管中,每个管含有820μl洗涤水,去除180μl用于细菌计数。 D.匀浆叶组织。

        3. 大力旋转管子10秒以洗涤叶片。
        4. 使用100μl的每个洗涤水进行10和100倍稀释。
        5. 在WSD平板上轻轻滴入20μl每个未稀释的10和100倍稀释的洗涤水。
        6. 将琼脂平板放在28℃的培养箱中6天(图8)

          图8. X的增长。 (A),10(B)和100倍(C)稀释的洗涤水或均质组织中的白蛋白
          菌落在WSD平板上沉积6天后
          br />
      2. 叶洗后叶均匀
        1. 使用为1.5ml微量离心管制成的研杵,将剩余洗涤水中的每个叶片片段均匀化(在A.中去除180μl后测定细菌群体中的每个叶片片段)5分钟(图7C和7D)。
        2. 使用100μl每个匀浆的组织悬浮液制备10和100倍稀释液。
        3. 在WSD平板上轻轻滴入20μl未稀释,10和100倍稀释的均质组织三或四次。将琼脂平板置于培养箱中28℃下6天
      3. 细菌群体密度的测定
        1. 计数每个稀释的菌落,并在生长6天后进行三次重复的每一次;计算平均菌落数并校正稀释液。例如,10个 - 1 稀释板上的15个菌落对应于叶片部分中的7,500个CFU。转换为洗涤水或均质组织的每个叶片的日志 10 CFU。
        2. 对于洗涤水和均匀的组织,使用所获得的所有值计算每种细菌菌株或突变体的平均日志 10 CFU六个接种的小植株

数据分析

  1. 使用由Mensi等人(2016)描述的统计软件包R,版本2.14.1(R Development Core Team)分析数据。叶片附着(ELA)值的范围是每个实验中每个菌株或突变体的三个独立实验与六个重复(小植株)的手段的组合。请注意,每个小植物印有2到4个叶子。尽管植物之间的ELA手段之间可能存在显着差异,但所有数据均用于统计分析
  2. 为了通过叶子洗涤和叶匀浆测定叶子附着,数据来自20个叶子,并且从两个分别的5个小植株的实验组合(每个接种的甘蔗小植物采集两个叶子,在每个采样时间使用5个小植株)。在计算总体平均值之前,使用log10函数(y = log 10 /(CFU/ml)+ 1])转化个体群体数据(CFU/ml)。适当时(少数数据),单独日志转换数据可用于方差分析的平均比较。
  3. 通过叶片印迹(半定量方法)和叶洗(数量方法)获得的数据相关,并产生与叶片表面上存在的细菌群体相关的相似信息。相比之下,通过叶匀浆获得的数据与不能容易地从叶表面分离并位于受保护的叶片区域或叶内的细菌相关联。推荐叶片印迹快速,方便地筛选细菌菌株的叶片附着能力。建议采用叶子清洗/均质化方式获得更精确和定量的数据,以及关于叶片上或叶中细菌定位的其他信息。

笔记

  1. 在层流柜(无菌条件)下进行细菌悬浮液制备,甘蔗浸种和叶面印迹。
  2. 用于接种甘蔗小植物的细菌悬浮液新鲜(刚好在接种程序之前)制备。细菌悬浮液仅用几个单个菌落制备,以获得所需的细菌浓度。使用的菌落数量将取决于菌落的大小。储存在冷藏库中的菌株在零下80摄氏度下分别从单个菌落中发出。
  3. 甘蔗叶仔细选择印刷或洗涤;我们只选择绿色和无枯叶。
  4. 使用具有10%叶面积增量的模板目视估计汇合生长的面积。
  5. 本文使用甘蔗品种CP68-2016,但也可以测试其他对甘蔗叶烫伤敏感的品种。
    本文使用的细菌菌株未观察到非特异性粘附。但是,确定并包括不适应甘蔗的细菌种类作为阴性对照是一个好主意

食谱

  1. Wilbrink介质(WM)(1升)
    10g蔗糖
    5克蛋白胨
    0.50g K 2 2 HPO 4·3H 2 O
    0.25g MgSO 4·7H 2 O→// 0.05g Na 2 SO 3
    15克琼脂
    加蒸馏水使1升
    将pH调节至6.8-7.0
    高压灭菌器在121°C 20分钟
  2. Wilbrink选择性戴维斯(WSD)媒介(1L)
    与WM培养基相同,但补充:
    加热灭菌前5g KBr和0.004g Benomyl(杀真菌剂) 高压灭菌后,加入:
    将0.1g放线菌酮溶解于500μl乙醇中 0.025g头孢氨苄溶解于500μl无菌蒸馏水中 0.03g新生霉素溶于500μl乙醇中 将0.05克春雷霉素溶于500微升无菌蒸馏水中
  3. 大量营养素(1升)
    10g NH 4,否3
    10 g KNO 3
    5g Ca(NO 3)2< 2> 2< 2< 2> 715mg MgSO 4·7H 2 O→// 3g KH 2 PO 4
    650毫克KCl
    加入蒸馏水至1升
  4. 微量营养素(100毫升)
    160mg H 3 BO 3//
    1,400mg MnSO 4·H 2 O
    380mg ZnSO 4·7H 2 O
    75 mg KI
    10 mg(NH 4)6 O 7 Mo 2 O 4·4H 2 O 2
    35mg Cu(NO 3 3)2·3H 2 O
    加入蒸馏水至100 ml
    将溶液在-20℃下储存在1.5ml微量离心管中
  5. 三氯化铁
    2.78g FeSO 4·7H 2 O 2/
    3.73g Na 2 EDTA·2H 2 O 500毫升蒸馏水
    分别溶解Na 2 EDTA和FeSO 4,并通过温和加热溶液将FeSO 4加入到Na 2 EDTA中
  6. 富士维生素
    50毫克烟酸(沸水)
    10mg吡哆醇盐酸盐
    10克肌醇蛋白
    10毫克二氯化二硫化胺(最后加入)
    加入蒸馏水至100 ml
    将溶液在-20℃下储存在1.5ml微量离心管中
  7. 用于甘蔗苗生长的营养培养基(1L)(改编自Chatenet ,2001)
    100毫升大量营养素
    1毫升微量营养素
    5毫升三氯化铁EDTA 2毫升富士维生素
    8克琼脂或4克Phytagel
    40克蔗糖
    890毫升蒸馏水
    pH 5.8
    在121°C高压灭菌20分钟

致谢

我们感谢Marie-JoséeDarroussat提供卓越的技术援助。 ImèneMensi得到了Cirad博士研究生的支持。这项工作得到美国农业部国家食品和农业研究所(项目Hatch/Rott FLA-BGL-005404)的支持。该协议是由Fleites等人(2013)和Mensi等人发表的工作改编而成。 (2016)。

参考文献

  1. Birch,RG(2001)。  xanthomonas albilineans 和疾病控制的抗病机制方法。 Mol Plant Pathol 2(1):1-11。
  2. Chatenet,M.,Delage,C.,Ripolles,M.,Irey,M.,Lockhart,BEL and Rott,P。(2001)。  通过顶端分生组织培养检测和生产无病毒甘蔗的甘蔗黄叶病毒 植物细胞 85(11):1177-1180。
  3. Fleet,LA,Mensi,I.,Gargani,D.,Zhang,S.,Rott,P.和Gabriel,DW(2013)。  Xanthomonas albilineans OmpA1似乎是功能模块化的,OMC和C样域都是叶片烫伤疾病所必需的甘蔗。分子植物微生物相互作用 26(10):1200-1210。
  4. Hirano,SS and Upper,CD(2000)。  细菌在叶生态系统中,重点是


    - 病原体,冰核和附生植物。 Microbiol Mol Biol Rev 64(3):624-653。
  5. Kumar,N。和Reddy,MP(2011)。 植物繁殖:A review。 J Forest Sci 27(2):61-72。 br />
  6. Mensi,I.,Daugrois,JH,Pieretti,I.,Gargani,D.,Fleites,LA,Noell,J.,Bonnot,F.,Gabriel,DW and Rott,P。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25962850"target ="_ blank">表面多糖和群体感应涉及黄单胞菌属的附着和存活白皮书在甘蔗叶上。 Mol Plant Pathol 17(2):236-246。
  7. Rott,P.,Fleites,L.,Marlow,G.,Royer,M.and Gabriel,DW(2011)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm .nih.gov/pubmed/21190440"target ="_ blank">通过转座子诱变鉴定木质部入侵病原体黄单胞菌中的新的候选致病因子。 Mol Plant Microbe互动 24(5):594-605。
  8. Yadav,R.K.P.,Kakamanoli,K.and Vokou,D。(2010)。 通过连续稀释电镀评估叶片上的细菌群体和叶印记法。 生态印象 17:47-52。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mensi, I., Daugrois, J. and Rott, P. (2017). Bioassay of Xanthomonas albilineans Attachment on Sugarcane Leaves. Bio-protocol 7(2): e2111. DOI: 10.21769/BioProtoc.2111.
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