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Sep 2017

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Plant Assays for Quantifying Ralstonia solanacearum Virulence
植物实验分析青枯雷尔氏菌的毒力   

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Abstract

Virulence assays are powerful tools to study microbial pathogenesis in vivo. Good assays track disease development and, coupled with targeted mutagenesis, can identify pathogen virulence factors. Disease development in plants is extremely sensitive to environmental factors such as temperature, atmospheric humidity, and soil water level, so it can be challenging to standardize conditions to achieve consistent results. Here, we present optimized and validated experimental conditions and analysis methods for nine assays that measure specific aspects of virulence in the phytopathogenic bacterium Ralstonia solanacearum, using tomato as the model host plant.

Keywords: Virulence assay (毒力检测), Bacterial wilt (青枯病), Vascular plant pathogen (维管植物病原体), Plant colonization (病毒的植物定植), Xylem sap (伤流液), R. solanacearum (青枯雷尔氏菌), Tomato (番茄)

Background

Ralstonia solanacearum is a soil-borne bacterium that causes bacterial wilt in a vast range of plants and continues to infect new hosts across the globe (Hayward, 1991; Elphinstone, 2005; Wicker et al., 2007; Genin, 2010; Weibel et al., 2016). As a result, R. solanacearum is among the most intensively studied phytopathogenic bacteria (Mansfield et al., 2012).

R. solanacearum can persist in soil or water reservoirs for long periods (Alvarez et al., 2008), and in the presence of a suitable host, it can enter the plant through wounds or lateral root emergence points (Denny, 2007). Thereafter it colonizes the water-transporting plant xylem vessels and thrives there. Massive production of exopolymeric substances (EPS) likely contributes to the clogging of the xylem channels, leading to blockage of water transport, followed by symptoms like wilting leaves, stunted growth, stem discoloration, and death. Molecular genetic studies revealed a consortium of many virulence factors that are required for pathogenesis and fitness in planta (Genin and Denny, 2012; Tran et al., 2016a and 2016b); recent in silico modeling (Peyraud et al., 2016) and in vivo transcriptomics and metabolomics (Jacobs et al., 2012; Khokhani et al., 2017; Lowe-Power et al., 2018) have further enhanced our understanding of how this bacterium switches from saprophytic to parasitic lifestyle.

To test hypotheses suggested by molecular data, researchers measure virulence on model host plants under controlled conditions. To be useful, such assays must be quantitative, biologically relevant, and replicable. We have developed or adapted several protocols to assess R. solanacearum interactions with tomato, a natural host and economically important crop plant. A naturalistic soil soak assay replicates many aspects of the infection process that occurs in the field. This assay quantifies the defects of mutant strains lacking virulence factors involved in the early phases of the disease such as sensing, invading, and colonizing host roots. For example, mutants lacking chemotaxis, swimming motility, extracellular plant cell wall-degrading enzymes, and type II and III secretion systems are all impaired in virulence following soil soak inoculation.

A petiole inoculation disease assay that introduces the pathogen directly into stem xylem vessels can identify traits that contribute to pathogen success in xylem vessels (Saile et al., 1997). Some mutants that are defective in virulence following soil soak inoculation have full wild-type virulence following cut-petiole inoculation into the stem; examples include mutants lacking motility and chemotaxis (Tans-Kersten et al., 2001; Yao and Allen, 2006). In another case, comparing results of these two assays revealed that extracellular DNA degradation, a trait initially thought to only play a role in interactions with host roots, was also critical for normal biofilm formation inside host xylem later in disease (Tran et al., 2016a and 2016b). When virulence traits are functionally redundant or make small contributions to pathogen success, neither soil soak nor petiole inoculation assays can reveal subtle differences between the wild-type and the target mutant strain (Macho et al., 2010). In those cases, we can use single or co-inoculation experiments to directly compare the growth of competing strains in planta (root and/or shoot colonization) and calculate their relative competitive fitness as a competitive index (CI). We also describe here the protocol to measure bacterial attachment to plant roots. Since R. solanacearum is a xylem-dwelling bacterium, it is important to understand how it affects and is affected by host plant xylem sap. Therefore, we provide a protocol for collection of xylem sap from healthy and infected tomato plants; this ex vivo sap can be used as a medium for bacterial growth curves or for metabolomic analyses.

Materials and Reagents

  1. Sterile conical flask 250 ml (Corning, PYREX®, catalog number: 4980-250 )
  2. Sterile 50 ml conical tubes (Stellar Scientific, catalog number: T50-100 )
  3. Flasks for preparing large volume of cultures (size depends on the experimental goals)
  4. Seedling tray (36-cell insert with holes) (J&P Park Acquisitions, Park Seed, catalog number: 96377 )
  5. Flat trays (greenhouse megastore, 11" W x 21.37" L x 2.44" D, CN-FL)
  6. 1.5 ml microcentrifuge tubes (Sigma-Aldrich, BRAND, catalog number: Z336769 )
  7. 8 cm wide pots (greenhouse megastore)
  8. Planting sticks (greenhouse megastore)
  9. Metal beads (2.4 mm) (OMNI, catalog number: 19-640 )
  10. Bead-beater tubes (USA Scientific, catalog number: 1420-9300 )
  11. Gosselin Square Polystyrene Petri Plate with 4 vents, 120 x 120 x 15.8 mm, Sterile (Corning, catalog number: BP124-05 )
  12. MicroporeTM Surgical Paper Tape (1 inch size) (3M, catalog number: 1530-1 )
  13. Aluminum foil (W.W. Grainger, catalog number: 16W479 )
  14. Paper towel (Singlefold Paper Towel, 9.1 x 10.25) (Cascades Pro, catalog number: H165 )
  15. WhatmanTM paper (Grade 1 Qualitative Filter Paper) (GE Healthcare, Whatman)
  16. Petri dish (Corning, Falcon®, catalog number: 351029 )
  17. 0.22-μm sterile filter (Merck, catalog number: SLGP033RS )
  18. 10 ml pipette (Disposable Polystyrene Serological Pipettes) (Corning, Falcon®, catalog number: 357551 )
  19. 1 ml syringe (New Sterile, Sealed, Tuberculin, Luer slip tube, No Needle, Disposable) (BD, catalog number: 9602 )
  20. 96-well half-area microplates (Corning, catalog number: 3697 )
  21. Soil mix, propagation mix, Sunshine® resilience silicon enriched, Re Plug and Seed Rsi (Sun Gro Horticulture, Lot-code: Q15322; SKU: 7263924)
    Ingredients:
    55-65% Canadian Sphagnum peat moss, vermiculite, dolomite lime, wetting agent;
    0.0001% Silicon dioxide (SiO2) from calcium silicate to increase root growth
  22. Ralstonia solanacearum strain from glycerol or water stock
  23. Bonny best wilt susceptible variety of Tomato seeds (Mountain valley seed) (stored at 4 °C)
  24. Sterile reverse osmolyzed water by Milli-Q system (SMQ)
  25. Agar (Fisher Scientific, catalog number: BP1423-2 )
  26. Bleach (Clorox Performance Bleach with CloroMax) (The Clorox Company, catalog number: 980042447 )
  27. 70% Ethanol (Diluting 100% Ethanol 200 proof) (Decon Labs, catalog number: 2716 )
  28. Glucose (Fisher Scientific, catalog number: D16-1 )
  29. Peptone (Fisher Scientific, catalog number: NC9931583 )
  30. Casamino acids (RPI, catalog number: C45000-5000.0 )
  31. Yeast extract (Fisher Scientific, catalog number: BP1422-2 )
  32. KOH (Fisher Scientific, catalog number: P250 10 )
  33. 1% 2,3,5-triphenyl tetrazolium chloride (TZC) (Sigma-Aldrich, catalog number: T8877 )
  34. KNO3 (Fisher Scientific, catalog number: P383 100 )
  35. KH2PO4 (Merck, Calbiochem, catalog number: 529568 )
  36. MgSO4 (MP biomedicals, catalog number: 150136 )
  37. Ca(NO3)2•4H2O (Fisher Scientific, catalog number: C109 )
  38. H3BO3 (Fisher Scientific, catalog number: A73 1 )
  39. MnCl2•4H2O (Sigma-Aldrich, catalog number: M8054 )
  40. ZnSO4•7H2O (Fisher Scientific, catalog number: Z68 )
  41. CuSO4•5H2O (VWR, BDH, catalog number: BDH9312 )
  42. (NH4)6Mo7O24 (Sigma-Aldrich, catalog number: A1343 )
  43. FeSO4•7H2O (Fisher Scientific, catalog number: I146 3 )
  44. Na2EDTA (Fisher Scientific, catalog number: S311
  45. Casamino acid-peptone-glucose (CPG) agar (see Recipes)
  46. CPG broth (see Recipes)
  47. Modified Hoagland's solution (see Recipes)

Note: All the chemicals were purchased from Sigma-Aldrich, Fisher Scientific, or other chemical companies.

Equipment

  1. P1000 pipette (Eppendorf, catalog number: 3120000062 )
  2. P200 pipette (Eppendorf, catalog number: 3120000054 )
  3. P10 pipette (Eppendorf, catalog number: 3120000020 )
  4. Forceps (VWR, catalog number: 470018-952)
    Manufacturer: Dunrite Instruments, catalog number: 141001 .
  5. Scalpel (Bard-Parker® SafeSwitchTM Reusable Scalpel Handle, Size 3 L) (Aspen Surgical, catalog number: ST-1013LNS )
  6. Sharp razor blade (Carbon Steel Razor Blades) (Azpack, catalog number: YSJ-762-Q )
  7. Incubator (6M Lab Incubator) (Precision Scientific, catalog number: 31487 )
  8. Benchtop Shaker (Thermo Fisher Scientific, model: MaxQTM 4000 )
  9. Growth chamber with the following conditions:
    Light intensity: 300-500 μmol/m2•sec-1
    12 h, light, 28 °C
    12 h, dark, 28 °C
    50-70% humidity
    ~500 ppm CO2 measured
  10. Powerlyzer® 24 homogenizer (MO BiO Laboratories, catalog number: 13155 )
  11. Centrifuge (15 amp version) (Eppendorf, model: 5810 R )
  12. Centrifuge (Eppendorf, model: 5417 R )
  13. Spectrophotometer (UV/Vis) (Beckman Coulter, model: DU 730 )
  14. Vortex mixer (Vortex-Genie 2) (Scientific industries, catalog number: SI-0246 )
  15. Biosafety cabinet (The Baker, SterilGard®, model: SG403A-HE )
  16. Balance (Roche Diagnostics, model: 05942861001 )
  17. Autoclave (Vacuum Steam Sterilizer) (Getinge, model: 533LS-E )

Software

  1. PRISM Graphpad software

Procedure

This section includes subsections from Procedure A to Procedure I:

  1. Preparing the bacterial culture for soil soak (depending on the goal of the experiment, this subsection can be followed by Procedures C-F or C-E-G or C-G)
  2. Sowing and transplanting of tomato plants–this subsection describes how to grow tomato plants to ensure reproducible results for soil soak or petiole inoculation (C or D) which can be followed by measuring disease progress (F) or competition and bacterial colonization (E and/or G). 
  3. Soil soak inoculation–pouring a dilute bacterial suspension into pots containing unwounded tomato plants, which closely resembles the natural root infection process. Infection by this method is stochastic. This procedure can be followed by Procedure E or F or G or H, or E-G.
  4. Petiole inoculation–this alternative inoculation procedure is less natural but allows on to determine if a particular gene contributes to R. solanacearum success in the host stem. Petiole inoculation also ensure synchronized infection. (This procedure can be used for Procedure E or F or G or H, or E-G.) 
  5. Competition assay–to quantify relative competitive fitness of wild-type and mutant R. solanacearum, we measure bacterial colonization in stems (Procedure G) from 3 to 7 days post inoculation or later (depending on the experimental goals). 
  6. Disease progress curve–this procedure is used to quantify bacterial wilt disease development following soil soak (Procedure C) or petiole inoculation (Procedure D). 
  7. Bacterial colonization–this procedure is used to measure the ability of bacteria to colonize the roots (early stages of infection) or stem (later stages of infection). This method quantified R. solanacearum population size in planta following soil soak (Procedure C) or petiole inoculation (Procedure D) or competition assay (Procedure E). 
  8. Xylem sap collectionex vivo tomato xylem sap can be used for either metabolomic analyses or as a medium for bacterial growth. Generally plants are infected by naturalistic soil soak method (Procedure A-B-C). 
  9. Root attachment assay–to quantify the ability of mutants vs. wild-type bacteria to attach to tomato seedling roots, one of the very first steps in R. solanacearum infection of plants. 


  1. Preparing the bacterial culture for soil soak
    1. Streak out Ralstonia solanacearum from water stock or glycerol stock on a CPG + TZC plate and let it grow in a 28 °C static incubator for 48 to 72 h.
      Note: Make sure the morphology of the colonies is of wild-type (WT) and not spontaneous phcA- like mutant (Khokhani et al., 2017).
    2. Once you see the isolated colonies on the plate, pick one colony to inoculate 100 ml of CPG broth (with appropriate antibiotics) in a 250-ml sterile flask and culture overnight (18-20 h) in a shaker-incubator at 220 rpm, 28 °C.
    3. Centrifuge (6,900 x g, 5 min) the overnight culture in a 50 ml conical tube twice to pellet all 100 ml of cell culture. Discard the supernatant.
      Note: When you decant the supernatant, some of the EPS tends to come off too and that is a good sign that the culture is not contaminated with spontaneous phcA like mutants that do not make wild-type level of EPS.
    4. Resuspend the cell pellet in 20 ml of sterile MilliQ (SMQ) water using a P1000 pipette, then add the remaining 30 ml of SMQ water and mix well by vortexing.
    5. Measure the optical density (λ = 600 nm, in a spectrophotometer) of the overnight cell culture by diluting it 10-fold in SMQ by pipetting 100 μl of resuspended cell culture in 900 μl of SMQ water in a 1.5 ml Eppendorf tube.
      Example: If the OD600 is 0.5 after diluting, the actual OD600 is 5.0.
    6. Calculate the volume of original cell suspension required to generate the desired volume of bacterial suspension at OD600 = 0.1, which corresponds to about 108 CFU/ml.
      For one biological replicate that has 10 technical replicates, we do the following math:
      For soil-soak inoculation, we add 5 x 109 cells per pot (50 ml of 0.1 OD600 cell suspension). So overall, we need 10 x 50 ml = 500 ml of cell suspension. Therefore, for 500 ml of 0.1 OD600 culture, we need 10 ml of 5.0 OD600 resuspended cell culture and the remaining 490 ml of SMQ water.
      Note: Make a little extra inoculum so that you do not run out of cells for the last pot due to pipetting error. Generally, three biological replicates, each containing 14-20 technical replicates (plants) per strain are preferred for statistical analysis.
    7. Check the OD600 again to make sure it is around 0.1 and dilution plate to confirm final inoculum cell density.
      Note: Please see the attached Excel sheet for calculation.

  2. Sowing and transplanting tomato seeds
    1. Add dry potting mix into a seedling tray with a gentle press.
    2. Place 4-5 seeds of Bonny Best (bacterial wilt-susceptible variety) in each cell.
      Note: We have determined that tomato cv. Moneymaker is also similarly susceptible to bacterial wilt.
    3. Cover them with a thin (1-3 mm) layer of potting mix as shown in Figure 1.


      Figure 1. Sowing the tomato seeds in multi-pot-tray

    4. Carefully water the soil until the soil is wet and not flooded.
    5. Incubate the trays in the growth chamber at 28 °C with 12 h light/12 h dark cycles.
      Note: High light intensity in the growth chamber is essential for good disease development.
    6. Water the soil without flooding the tray (water should not flow out of the tray). Try to water the plants every morning around the same time.
    7. Stop watering the seedling tray on the 13th day after planting so that the soil gets a little dry. This allows the soil to fall off seedlings easily with a little shaking and makes it easy to transfer the seedlings to bigger pots. If the soil is wet, many roots can break even with gentle shaking.
    8. On the 14th day (Figure 2), transfer the seedlings from the seedling tray to bigger pots (Figure 3) by shedding away soil carefully (not to break the roots as much as possible). To remove soil, gently tap root ball as soil falls away.


      Figure 2. Seedling growth on day 14th of planting seeds


      Figure 3. Transplantation pots with soil mix ready for transfer of 14-day old tomato seedlings

    9. Fill 8-cm diameter plastic pots with potting mix. It is important to not compress the soil to avoid creating an anaerobic condition in the pots.
    10. Water the soil until it is wet but not flooded and use your index finger or a Sharpie to make a hole in the center of the pot.
    11. Put seedlings onto the pots and when all pots have seedlings, put each seedling into a hole and gently press the soil around the base of the seedling so it is well-supported and will not fall over after being watered.
    12. Let the tomato plants grow in the bigger pots for another week till they are 21 days old. The first true leaf should be fully expanded and the second true leaf should be opening.

  3. Soil soak inoculation assay
    The following protocol has been used previously (Huang and Allen, 2000; Tans-Kersten et al., 2001; Colburn-Clifford et al., 2010).
    1. Stop watering the plants one or two days before inoculation, depending on the soil humidity. This will allow the soil to soak up all the bacterial culture poured into it. Be careful not to over dry, as R. solanacearum survival decreases in dry conditions.
    2. On the day before the inoculation of tomato plants, start an overnight culture of Ralstonia solanacearum as mentioned above in Step A1.
    3. On the day of inoculation follow Steps A2 to A7.
    4. Once you prepare the bacterial cultures, perform dilution plating of the inoculum and incubate the CPG plates for about 48 h to count the colonies. This will confirm that the OD600 was around 0.1 and that the bacterial cells were not contaminated or killed during the preparation.
    5. To blind-rate plants (preferred procedure), ask a colleague to code your inoculum flasks (e.g., with colored tape) and remove the identifying strain label from the flask. Colleague can keep the strain code list until you are ready to analyze data after the experiment is complete.
    6. Measure 50 ml of bacterial suspension using 50 ml conical tubes and pour this into each pot. You can do the same thing for all the target strains you aim to test for virulence, but use only one tube for each bacterial strain. Make sure to put planting sticks into the pots to label the plants for future disease rating (Figure 4). Do not wound plants.


      Figure 4. Plant pots with planting sticks ready for inoculation

    7. Check soil moisture within a few hours. For consistent results, the soil should not be desiccated for several days following inoculation. Dry soil will reduce bacterial viability and delay disease onset.
    8. After inoculation, water the plants following day with Hoagland’s solution. For the remaining 13 days use tap water to water the plants.
    Note: If disease does not develop well, the following variables can be manipulated: a) Plant age. Use younger plants (18 or 19 days after planting); b) Humidity. Ensure growth chamber humidity is at least 65%; c) Inoculum density. Typically, by 14 days post-inoculation wild-type R. solanacearum causes a mean disease index of 3.5 or more after inoculation with 50 ml of a 1 x 108 CFU suspension (OD600 = 0.1). Inoculum can be increased to OD600 = 0.2 if necessary.

  4. Petiole inoculation assay
    The following protocol has been used in (Brown and Allen, 2004; Dalsing and Allen, 2014).
    Note: Follow all the steps mentioned in Procedure B and water the plants for all 21 days of plant growth.
    1. On the day before inoculation, start 5 ml overnight CPG cultures of R. solanacearum.
    2. On the day of inoculation, water the plants (now 21-day old) first in the morning and then proceed to inoculation.
    3. Next day, spin down 1 ml of the overnight culture, and resuspend the pellet in 1 ml of SMQ water.
    4. Measure the OD600 and dilute the bacterial suspension in SMQ H2O to make 2 ml of OD600 = 0.1 bacterial cell suspension.
    5. Before moving forward, confirm the OD600 by spectrophotometer and dilution plating as described in Step A6.
    6. Dilute the 0.1 OD cell suspension to OD600 = 0.001.
    7. To infect the plant, cut off the first true leaf near the base of the petiole with a sharp razor blade. The petiole should be cut diagonally, parallel to the floor, to create a horizontal surface where a droplet of bacteria can be applied without running down the stem (Figure 5).


      Figure 5. Petiole inoculation of 21-day old tomato plant. The blue arrow indicates the horizontal stump of cut petiole where 2 μl of bacterial cell suspension is placed.

    8. Use a 10 ml pipette to carefully place 2 μl of OD600 = 0.001 cell suspension (equivalent to 2,000 cells) on the cut petiole surface. One can change the number of cells depending on the experimental goals. The droplet will be quickly absorbed by the plant, indicating that the bacteria were pulled into the xylem.
    9. You can cut all the petioles of plants in one tray and then apply the bacteria to each plant. For example, 10 plants at a time.

  5. Competition assay to measure relative competitive fitness of bacterial strains in planta
    The following protocol has been used in (Yao and Allen, 2006; Lowe et al., 2015).
    1. To measure the competitive fitness of the target strain relative to another strain (usually the wild-type) (Yao and Allen, 2006), each strain must carry a different antibiotic resistance. For example if the wild-type strain is kanamycin-resistant and the target mutant strain is gentamicin-resistant.
      Note: To assure that antibiotic resistance marker gene does not add a fitness cost to the marked strain, compare the in planta growth of marked strains to growth of the unmarked parent strain.
    2. Prepare fresh plates of the marked strains on CPG plates supplemented with the required antibiotics for overnight culture.
    3. Grow the plants as mentioned in Procedures B and C.
    4. To inoculate the plants (on 21st day of plant growth), separately prepare 0.1 OD600 cultures of the two competing strains. Check the OD600 using spectrophotometer.
    5. Combine the two strains in a 1:1 ratio of bacterial population. Again, dilution plate to check the actual number of cells.
    6. Add 50 ml of this mixed bacterial suspension to each pot for naturalistic soil soak competition, or 2 μl of the same suspension to the cut petiole for petiole inoculation competition.
    7. To check the root or stem colonization, sample the plants over 3 to 7 days post inoculation. From here, follow Procedure G.
    Note: Please see the attached Excel sheet for calculation.

  6. Measuring Disease Progress, and creating a disease progress curve
    1. Before rating plants, water the plants first, wait for 1 h, and then record the disease by looking for wilted leaves. We record the disease on the scale from 0 to 4. If no leaves are wilted, the plant is scored as Disease Index (DI) = 0; if 24% or less of the leaf area is wilted we score that as DI = 1; if 25-49% is wilted, DI = 2; 50-74%, DI = 3; and 75 -100%, DI = 4.
    2. We rate the disease symptoms until 14 days post inoculation. Wilt symptoms normally begin to appear 3-4 days post petiole inoculation or 4-5 days post soil-soak inoculation.

  7. Measuring bacterial colonization of tomato roots and stems
    The protocol for root colonization has been used in (Lowe et al., 2015), and stem colonization has been used in (Dalsing et al., 2015; Lowe-Power et al., 2016).
    1. Sample collection depends on the experimental goal. Please refer to published articles where the sampling time is guided by the objective of the study.
    2. For root colonization, take the plants out of pots and gently shake off as much soil as possible. 
    3. Put the plant in a container filled with water and let the roots soak and wash gently until all the soil comes off. Repeat the procedure for all the plants per treatment. (Figures 6A-6C)
    4. Wash roots for 15 sec in bleach, then successively for 15 sec in 2 fresh water baths and dry for 30 sec on a tissue (Figures 6D-6F).
    5. Cut off the roots at the crown junction (Figure 6G).
    6. Collect the roots and cut them into small pieces with a razor blade to ensure that each sample contains roots from every region (Figure 6H).
    7. Place up to 300 mg of the cut roots into a prepared bead-beater tube containing 4 small metal beads and 700 μl of SMQ water.
    8. For stem colonization, use a razor blade to harvest 1 cm of stem surrounding the petiole inoculation site (Figure 6I).


      Figure 6. Steps for root (A-H) and stem colonization (I) quantification

    9. Place up to 100 mg of cut stem into a prepared grinding tube containing 4 metal beads and 900 μl of SMQ water.
    10. Grind the tissue using a Powerlyzer® bead-beater machine with the following settings:
      Speed = 2,200 rpm, Time = 1 min 30 sec, Dwell = 4 min, Cycles = 2, Total time = 7 min
    11. Serially dilute the homogenates 10-fold in 96-well plates by mixing 20 μl of each dilution with 180 μl of SMQ. For root samples: make five successive 1:10 dilutions per sample (100-10-4); for stem samples: make seven successive 1:10 dilutions per sample (100-10-6).
    12. Plate three 10-μl drops of each dilution onto CPG plates (with appropriate antibiotics). Dry the plates by opening the lid in a Biosafety Cabinet for approximately 15 min.
    13. Incubate the plates at 28 °C for 36-48h and count the colonies. Colonies should not be allowed to grow together.
      Note: Normalize the final population size by the ratio of actual bacterial numbers in the original individual inoculum, as determined by dilution plating of the inoculum. Please see the attached Excel sheet for calculation.

  8. Xylem sap collection by root pressure
    The following protocol is adapted from Goodger et al. (2005) and was used in Khokhani et al. (2017) and Lowe-Power et al. (2018).
    For a basic experiment, we prepare 9 plants per treatment per time point (sampling is destructive). Sap will be combined into 3 pools, each containing sap from 3 plants. Occasionally sap cannot be collected from a plant due to uneven cutting, so it is prudent to prepare additional plants. Root pressure accumulates overnight when plants are in well-watered soil. Root pressure decreases over the course of the day. Therefore, sap collection should occur within several hours of light onset and plants must be well watered the evening before sap collection. Sap exudation rate depends on many variables, including soil moisture content, plant size, and disease state. In the experiments described in Lowe-Power et al. (2018), we harvested median volumes of 473 μl and 280 μl from healthy and wilt-symptomatic (disease index 1) plants that were 26-31 days old (Figure 7B).
    Preparations:
    1. One tube per plant to grind tissue to determine colonization (4 metal beads and 900 μl SMQ water) 
    2. One tube per plant for the xylem sap collection (number, color code, etc.), pre-weighed, pre-cooled on ice. If possible, arrange all tubes and plants in the same parallel to avoid cross-contaminating samples.
    3. One tube per plant to sterilize the sap.
    4. One tube per pool.
    5. Set timer to 33 min.
    6. Detop the tomato plants with a sharp razor blade to yield a flat stump surface. If the stump surface is angled, sap will likely drip down the stem. For soil soak inoculated plants, cut ~1 cm above cotyledons. For cut-petiole inoculated plants, cut just above the inoculation site. Generally, it works well to detop plants in batches of 5 with 5 min intervals between batches.
    7. Allow the first droplet of sap to accumulate for ~2 ½ min. Then gently blot the stump dry with a tissue. Use a P200 pipette to wash the stump with sterile SMQ water, then blot dry again, to minimize cytosolic contamination.
    8. Collect xylem sap for 30 min, always using a fresh tip and pipetting the xylem sap of each plant into the corresponding tube, which is kept on ice. The sap should accumulate as a bubble due to surface tension (Figure 7A). If the stump looks wet, but sap is not accumulating, it is covertly dripping down the stem. Gently blotting the stump dry may restore surface tension to exuded sap and allow it to accumulate. We have included the estimation of xylem sap volume collected from the tomato plants (Figure 7B).


      Figure 7. Collection of xylem sap. A. Xylem sap on detopped tomato plant. B. Sample dataset showing xylem sap volume from healthy and infected plants. Each dot represents the volume of sap collected from one plant.

    9. Keep the top of the plants to take samples to quantify each plant’s bacterial colonization (collect a ~50-100 mg slice from the lowest part of the stem), as described above.
    10. Weigh each tube again to determine the xylem sap volume you gained from each plant (density is similar to water, so 1 mg = 1 μl).
    11. Pools should be composed of xylem sap from plants producing high, medium and low amount of xylem sap. For pooling, combine equal volumes of xylem sap of each plant (e.g., 100 μl). Filter-sterilize the xylem sap from each plant into a fresh 1.5 ml tube using a 1 ml syringe and a 4 mm diameter 0.22 μm filter (4 mm size filters reduce sample loss).
    12. This xylem sap can be used as medium for growth curve assays or for metabolite analysis. Bacterial growth assays can be performed with 50 μl of sap in Corning 96-well half-area microplates. The amount of sap needed for metabolite analysis should be empirically determined based on the concentration of target metabolites and sensitivity of the detection method.

  9. Root attachment to tomato seedlings
    The following protocol has been used in Tran et al. (2016b) and Khokhani et al. (2017).
    1. Surface sterilize the tomato seeds by soaking them in 2% bleach for 5 min, followed by 70% ethanol for 5 min, then six rinses with SMQ water.
    2. With the help of sterile forceps in a biosafety cabinet, transfer the sterilized seedlings by spreading them out on to 1% agar Petri plates overlaid with a sterile WhatmanTM filter paper.
    3. Seal the Petri plates with MicroporeTM tape and cover with aluminum foil to exclude light.
    4. Incubate the plates at 28 °C for 4 days. In the meantime, prepare 1% agar square plates, CPG + TZC + required antibiotic, bead-beater tubes with 4 metal beads and 300 μl of SMQ water.
    5. On the 4th day, mark the back of the square plates with sharpie as shown here (Figure 8).


      Figure 8. Square plates marked with 2 cm area in which the roots of tomato seedlings are arranged

    6. In the biosafety cabinet, arrange the tomato seedlings so that their roots span the 2-cm length (from the root tip). Approximately 40 tomato seedlings can be accommodated in one square plate, with 20 on each side.
    7. Prepare the bacterial inoculum as described in Steps D2- D6.
    8. Dilution plate the inoculum and incubate the plates at 28 °C to confirm inoculum density.
    9. Next, use a micropipette to carefully distribute 10 μl of the OD600 = 0.001 bacterial suspension (~10,000 cells) over the 2-cm length of each root in the square plate.
    10. Incubate the square plates at room temperature for 2 h.
    11. After two hours, use a sterile scalpel to cut out the 2-cm length indicated by the sharpie marks for all 40 roots.
    12. Transfer all the cut roots to a Petri dish containing SMQ water and swirl the plate to remove loosely attached bacterial cells. After the first rinse, transfer all the roots to another Petri plate containing SMQ water and rinse again.
    13. Next, transfer the roots on to a new paper towel and gently pat dry the roots. Transfer 4 roots at one time.
    14. After pat-drying, transfer each group of 4 roots into a prepared bead-beater tube containing metal beads and 300 μl SMQ water. For 40 roots, you will have 10 technical replicates per biological replicate per treatment.
    15. Place the bead-beater tubes containing cut roots along with metal beads in the Powerlyzer® using the following settings: Speed = 2,200 rpm, Time = 1 min 30 sec, Dwell = 4 min, Cycles = 2, Total time = 7 min.
    16. After grinding, plate the homogenates along with their 1:10, 1:100, 1:1,000 serial dilutions for each sample.
    17. After 48 h, count the colonies to determine the number of bacterial cells in the original inoculum and the number of bacteria attached to the roots.
    18. Calculate the percent attachment by dividing the number of bacterial cells recovered from root by the number of cells present in the inoculum, per cm of roots.
      Note: Please see the attached Excel sheet for calculation.

Data analysis

Figure 9 represents a disease progress assay from Dalsing et al. (2015). Plants were rated daily for 14 days using the 0 to 4 disease index as described above. Data presented are mean results from 3 to 4 independent assays, each containing 10 plants per strain. Error bars indicate standard errors of the means. Disease progress curves of the ΔaniA and ΔhmpX mutants were significantly different from those of the WT strain (P < 0.001, repeated measures ANOVA, PRISM Graphpad software).
  The protocols presented here generate quantitative data that demand thoughtful, nuanced analysis. Fortunately, a recent paper (Schandry, 2017) offers a detailed practical guide to analyzing data on R. solanacearum infection, including the relevant R code.


Figure 9. Sample disease progress graphs from one of the virulence assays in Dalsing et al. (2015)

Notes

  1. Calibrate your pipettes regularly, especially the P10 used for dilution plating. 
  2. With every new experiment, use a new bottle of SMQ water to avoid any contamination. 
  3. Check your strain stocks regularly for spontaneous phcA mutants. 
  4. Do not inoculate using colonies from CPG plates that have grown for more than 4 days after streaking out from water or freezer stocks. “Old” colonies have often lost virulence.

Recipes

  1. Casamino acid-peptone-glucose (CPG) agar (1 L)
    5 g Glucose
    10 g Peptone
    1 g Casamino acids
    1 g Yeast extract
    Adjust to pH 6.5-7.0 with 1 M KOH
    16 g Agar
    Autoclave at 121 °C, 20 min
    1 ml of 1% 2,3,5-triphenyl tetrazolium chloride (TZC) (dissolved in water, filter-sterilized and added into CPG before pouring the plates)
  2. CPG broth (1 L)
    5 g Glucose
    10 g Peptone
    1 g Casamino acids
    1 g Yeast extract
    Adjust to pH 6.5-7.0 with KOH
    Autoclave
  3. Modified Hoagland's solution
    Mixture
    1.1 M KNO3
    0.2 M KH2PO4
    0.92 M MgSO4
    For watering, use 4 ml/L water
    Calcium Nitrate
    1.29 M Ca(NO3)2•4H2O
    For watering, use 5 ml/L water
    Micronutrients
    0.4626 M H3BO3 (Boric acid)
    0.0915 M MnCl2•4H2O (Manganese Chloride)
    0.0077 M ZnSO4•7H2O (Zinc Sulfate)
    0.0032 M CuSO4•5H2O (Copper Sulfate)
    0.0007 M (NH4)6Mo7O24 (Molybdic acid)
    For watering, use 4 ml of the stock/L water
    Iron
    FeSO4•7H2O (Ferrous Sulfate) 25.02 g/18 L of water
    Na2EDTA (Ethylenediaminetetraacetic acid) 33.48 g/18 L
    Dissolve the above in 4 L of deionized RO water. Heat to 80 °C for one hour. Let cool to room temperature and add deionized RO water to 18 L
    For watering, use 4 ml/L water

Acknowledgments

We thank Patrizia Ricca for capturing the images of soil soak, root and stem colonization. We also thank all the authors of whose papers have been cited for adapting and/or modifying their protocols to perform the respective experiments.

Competing interests

The authors declare no conflict of interest.

References

  1. Alvarez, B., Lopez, M. M. and Biosca, E. G. (2008). Survival strategies and pathogenicity of Ralstonia solanacearum phylotype II subjected to prolonged starvation in environmental water microcosms. Microbiology 154(Pt 11): 3590-3598.
  2. Brown, D. G. and Allen, C. (2004). Ralstonia solanacearum genes induced during growth in tomato: an inside view of bacterial wilt. Mol Microbiol 53(6): 1641-1660.
  3. Colburn-Clifford, J. M., Scherf, J. M. and Allen, C. (2010). Ralstonia solanacearum Dps contributes to oxidative stress tolerance and to colonization of and virulence on tomato plants. Appl Environ Microbiol 76(22): 7392-7399.
  4. Dalsing, B. L. and Allen, C. (2014). Nitrate assimilation contributes to Ralstonia solanacearum root attachment, stem colonization, and virulence. J Bacteriol 196(5): 949-960.
  5. Dalsing, B. L., Truchon, A. N., Gonzalez-Orta, E. T., Milling, A. S. and Allen, C. (2015). Ralstonia solanacearum uses inorganic nitrogen metabolism for virulence, ATP production, and detoxification in the oxygen-limited host xylem environment. MBio 6(2): e02471.
  6. Denny, T. (2007). Plant pathogenic Ralstonia species. In: Gnanamanickam, S. S. (Ed.). Plant-Associated Bacteria. Springer 573-644.
  7. Elphinstone, J. G. (2005). The current bacterial wilt situation: a global overview. In: Allen, C., Prior, P. and Hayward, A. C. (Eds.). Bacterial Wilt Disease and the Ralstonia solanacearun Species Complex. APS Press 9-28.
  8. Genin, S. (2010). Molecular traits controlling host range and adaptation to plants in Ralstonia solanacearum. New Phytol 187(4): 920-928.
  9. Genin, S. and Denny, T. P. (2012). Pathogenomics of the Ralstonia solanacearum species complex. Annu Rev Phytopathol 50: 67-89.
  10. Goodger, J. Q. D., Sharp, R. E., Marsh, E. L. and Schachtman, D. P. (2005) Relationships between xylem sap constituents and leaf conductance of well‐watered and water‐stressed maize across three xylem sap sampling techniques. J Exp Bot 56(419): 2389-2400. 
  11. Hayward, A. C. (1991). Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol 29: 65-87.
  12. Huang, Q. and Allen, C. (2000). Polygalacturonases are required for rapid colonization and full virulence of Ralstonia solanacearum on tomato plants. Physiol Mol PlantPathol 57(2):77-83.
  13. Jacobs, J. M., Babujee, L., Meng, F., Milling, A. and Allen, C. (2012). The in planta transcriptome of Ralstonia solanacearum: conserved physiological and virulence strategies during bacterial wilt of tomato. MBio 3(4): e00114-12.
  14. Khokhani, D., Lowe-Power, T. M., Tran, T. M. and Allen, C. (2017). A single regulator mediates strategic switching between attachment/spread and growth/virulence in the plant pathogen Ralstonia solanacearum. MBio 8(5): e00895-17.
  15. Lowe, T. M., Ailloud, F. and Allen, C. (2015). Hydroxycinnamic acid degradation, a broadly conserved trait, protects Ralstonia solanacearum from chemical plant defenses and contributes to root colonization and virulence. Mol Plant Microbe Interact 28(3): 286-297.
  16. Lowe-Power, T. M., Hendrich, C. G., von Roepenack-Lahaye, E., Li, B., Wu, D., Mitra, R., Dalsing, B. L., Ricca, P., Naidoo, J., Cook, D., Jancewicz, A., Masson, P., Thomma, B., Lahaye, T., Michael, A. J. and Allen, C. (2018). Metabolomics of tomato xylem sap during bacterial wilt reveals Ralstonia solanacearum produces abundant putrescine, a metabolite that accelerates wilt disease. Environ Microbiol 20(4): 1330-1349.
  17. Lowe-Power, T. M., Jacobs, J. M., Ailloud, F., Fochs, B., Prior, P. and Allen, C. (2016). Degradation of the plant defense signal salicylic acid protects Ralstonia solanacearum from toxicity and enhances virulence on tobacco. MBio 7(3): e00656-16.
  18. Macho, A. P., Guidot, A., Barberis, P., Beuzón and Genin, S. (2010). A competitive index assay identifies several ralstonia solanacearum type III effector mutant strains with reduced fitness in host plants. MPMI 23(9):1197-1205.
  19. Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Dow, M., Verdier, V., Beer, S. V., Machado, M. A., Toth, I., Salmond, G. and Foster, G. D. (2012). Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13(6): 614-629.
  20. Peyraud, R., Cottret, L., Marmiesse, L., Gouzy, J. and Genin, S. (2016). A Resource Allocation Trade-off between virulence and proliferation drives metabolic versatility in the plant pathogen Ralstonia solanacearum. PLoS Pathog 12(10): e1005939.
  21. Saile, E., McGarvey, J. A., Schell, M. A. and Denny, T. P. (1997). Role of extracellular polysaccharide and endoglucanase in root invasion and colonization of tomato plants by Ralstonia solanacearum. Phytopathology 87(12): 1264-1271.
  22. Schandry, N. (2017). A practical guide to visualization and statistical analysis of R. solanacearum infection data using R. Front Plant Sci 8: 623.
  23. Tans-Kersten, J., Huang, H. and Allen, C. (2001). Ralstonia solanacearum needs motility for invasive virulence on tomato. J Bacteriol 183(12): 3597-3605.
  24. Tran, T. M., MacIntyre, A., Hawes, M. and Allen, C. (2016b). Escaping underground nets: Extracellular DNases degrade plant extracellular traps and contribute to virulence of the plant pathogenic bacterium Ralstonia solanacearum. PLoS Pathog 12(6): e1005686.
  25. Tran, T. M., MacIntyre, A., Khokhani, D., Hawes, M. and Allen, C. (2016a). Extracellular DNases of Ralstonia solanacearum modulate biofilms and facilitate bacterial wilt virulence. Environ Microbiol 18(11): 4103-4117.
  26. Weibel, J., Tran, T. M., Bocsanczy, A. M., Daughtrey, M., Norman, D. J., Mejia, L. and Allen, C. (2016). A Ralstonia solancearum strain from Guatemala infects diverse flower crops, including new asymptomatic hosts Vinca and Sutera, and causes symptoms in geranium, mandevilla vine, and new host Afircan daisy (Osteospermum ecklonis). Plant Health Prog 17: 114-121.
  27. Wicker, E., Grassart, L., Coranson-Beaudu, R., Mian, D., Guilbaud, C., Fegan, M. and Prior, P. (2007). Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential. Appl Environ Microbiol 73(21): 6790-6801.
  28. Yao, J. and Allen, C. (2006). Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum. J Bacteriol 188(10): 3697-3708.

简介

毒力测定是研究体内微生物发病机制的有力工具。 良好的分析跟踪疾病发展,并结合定向诱变,可以识别病原体毒力因子。 植物的疾病发展对环境因素如温度,大气湿度和土壤水位极其敏感,因此标准化条件以获得一致的结果可能具有挑战性。 在这里,我们提出优化和验证的实验条件和分析方法的九个测定,测量植物病原细菌 Ralstonia solanacearum 的毒力的特定方面,使用番茄作为模型宿主植物。

【背景】 Ralstonia solanacearum 是一种土壤传播的细菌,在广泛的植物中引起细菌枯萎,并继续感染全球的新宿主(Hayward,1991; Elphinstone,2005; Wicker et al。 ,2007; Genin,2010; Weibel et al。,2016)。结果, R. solanacearum 是研究最深入的植物致病菌之一(Mansfield et al。,2012)。

R上。 solanacearum 可以长期存在于土壤或水库中(Alvarez et al。,2008),并且在合适的宿主存在下,它可以通过伤口或侧根进入植物出现点(Denny,2007)。此后,它在水输送植物木质部导管上定殖并在那里繁殖。大量生产外聚合物(EPS)可能会导致木质部通道堵塞,导致水分运输受阻,其次是枯萎病,生长发育迟缓,茎变色和死亡等症状。分子遗传学研究揭示了许多毒力因子的联合体,这些因子是植物的发病机制和适应性 (Genin和Denny,2012; Tran et al。,2016a和2016b);最近的 in silico 建模(Peyraud et al。,2016)和体内转录组学和代谢组学(Jacobs et al。 >,2012; Khokhani et al。,2017; Lowe-Power et al。,2018)进一步增强了我们对这种细菌如何从腐生生物转变为寄生生活方式的理解。

为了检验分子数据提出的假设,研究人员在受控条件下测量模型寄主植物的毒力。为了有用,这种测定必须是定量的,生物学相关的和可复制的。我们已经开发或改编了几种协议来评估 R. solanacearum 与番茄的相互作用,番茄是一种天然宿主和经济上重要的作物。自然土壤浸泡试验复制了在田间发生的感染过程的许多方面。该测定法量化了缺乏毒力因子的突变菌株的缺陷,所述毒力因子涉及疾病的早期阶段,例如感染,侵入和定殖宿主根。例如,缺乏趋化性,游动能力,细胞外植物细胞壁降解酶和II型和III型分泌系统的突变体在土壤浸泡接种后的毒力均受损。

将病原体直接引入茎木质部导管的叶柄接种疾病测定法可以鉴定导致木质部导管中病原体成功的特征(Saile et al。,1997)。土壤浸泡接种后毒力有缺陷的一些突变体在切叶柄接种到茎中后具有完全的野生型毒力;例子包括缺乏运动性和趋化性的突变体(Tans-Kersten et al。,2001; Yao and Allen,2006)。在另一个案例中,比较这两个分析的结果表明,细胞外DNA降解,最初被认为仅在与宿主根相互作用中起作用的特性,对于疾病晚期宿主木质部内正常生物膜形成也是至关重要的(Tran et al。,2016a和2016b)。当毒力性状在功能上是多余的或对病原体成功做出的贡献很小时,土壤浸泡和叶柄接种分析都不能揭示野生型和目标突变株之间的细微差异(Macho et al。,2010) 。在这些情况下,我们可以使用单一或共同接种实验直接比较竞争菌株在植物(根和/或芽定植)中的生长,并计算它们作为竞争指数的相对竞争适合度(CI) )。我们在这里还描述了测量细菌附着到植物根部的方案。自 R。 solanacearum 是木质部栖息的细菌,了解它如何影响宿主植物木质部汁液是很重要的。因此,我们提供了从健康和受感染的番茄植物中收集木质部汁液的方案;该离体汁液可用作细菌生长曲线或代谢组学分析的培养基。

关键字:毒力检测, 青枯病, 维管植物病原体, 病毒的植物定植, 伤流液, 青枯雷尔氏菌, 番茄

材料和试剂

  1. 无菌锥形瓶250毫升(Corning,PYREX ®,目录号:4980-250)
  2. 无菌50毫升锥形管(Stellar Scientific,目录号:T50-100)
  3. 用于制备大量培养物的烧瓶(尺寸取决于实验目标)
  4. 幼苗托盘(带孔的36个细胞插入物)(J&amp; P Park Acquisitions,Park Seed,目录号:96377)
  5. 平托盘(温室大型超市,11“W x 21.37”L x 2.44“D,CN-FL)
  6. 1.5 ml微量离心管(Sigma-Aldrich,BRAND,目录号:Z336769)
  7. 8厘米宽的盆(温室大型超市)
  8. 种植枝(温室大型超市)
  9. 金属珠(2.4 mm)(OMNI,目录号:19-640)
  10. 珠子打管(USA Scientific,目录号:1420-9300)
  11. Gosselin Square聚苯乙烯培养皿,带4个通风口,120 x 120 x 15.8 mm,无菌(Corning,目录号:BP124-05)
  12. Micropore TM 手术纸胶带(1英寸)(3M,目录号:1530-1)
  13. 铝箔(W.W.Grainger,目录号:16W479)
  14. 纸巾(单层纸巾,9.1 x 10.25)(Cascades Pro,目录号:H165)
  15. Whatman TM 论文(1级定性滤纸)(GE Healthcare,Whatman)
  16. 培养皿(Corning,Falcon ®,目录号:351029)
  17. 0.22-μm无菌过滤器(默克,目录号:SLGP033RS)
  18. 10 ml移液器(一次性聚苯乙烯血清移液器)(Corning,Falcon ®,目录号:357551)
  19. 1毫升注射器(新的无菌,密封,结核菌素,鲁尔滑管,无针,一次性)(BD,目录号:9602)
  20. 96孔半区域微孔板(Corning,目录号:3697)
  21. 土壤混合物,繁殖混合物,Sunshine ®韧性硅富集,Re Plug和Seed Rsi(Sun Gro园艺,批号:Q15322; SKU:7263924)
    成分:
    55-65%加拿大泥炭藓,蛭石,白云石,润湿剂;
    来自硅酸钙的0.0001%二氧化硅(SiO 2 )以增加根生长
  22. Ralstonia solanacearum 菌株来自甘油或水原液
  23. Bonny最好的枯萎病易感品种番茄种子(山谷种子)(储存在4°C)
  24. Milli-Q系统(SMQ)无菌反渗透水
  25. 琼脂(Fisher Scientific,目录号:BP1423-2)
  26. 漂白剂(Clorox Performance Bleach with CloroMax)(The Clorox Company,目录号:980042447)
  27. 70%乙醇(稀释100%乙醇200证明)(Decon Labs,目录号:2716)
  28. 葡萄糖(Fisher Scientific,目录号:D16-1)
  29. 蛋白胨(Fisher Scientific,目录号:NC9931583)
  30. 酪蛋白氨基酸(RPI,目录号:C45000-5000.0)
  31. 酵母提取物(Fisher Scientific,目录号:BP1422-2)
  32. KOH(Fisher Scientific,目录号:P250 10)
  33. 1%2,3,5-三苯基四唑氯化物(TZC)(Sigma-Aldrich,目录号:T8877)
  34. KNO 3 (Fisher Scientific,目录号:P383 100)
  35. KH 2 PO 4 (Merck,Calbiochem,目录号:529568)
  36. MgSO 4 (MP biomedicals,目录号:150136)
  37. Ca(NO 3 ) 2 •4H 2 O(Fisher Scientific,目录号:C109)
  38. H 3 BO 3 (Fisher Scientific,目录号:A73 1)
  39. MnCl 2 •4H 2 O(Sigma-Aldrich,目录号:M8054)
  40. ZnSO 4 •7H 2 O(Fisher Scientific,目录号:Z68)
  41. CuSO 4 •5H 2 O(VWR,BDH,目录号:BDH9312)
  42. (NH 4 ) 6 Mo 7 O 24 (Sigma-Aldrich,目录号:A1343)
  43. FeSO 4 •7H 2 O(Fisher Scientific,目录号:I146 3)
  44. Na 2 EDTA(Fisher Scientific,目录号:S311)&nbsp;
  45. 酪蛋白氨基酸 - 蛋白胨 - 葡萄糖(CPG)琼脂(见食谱)
  46. CPG肉汤(见食谱)
  47. 修改了Hoagland的解决方案(见食谱)

注意:所有化学品均购自Sigma-Aldrich,Fisher Scientific或其他化学公司。

设备

  1. P1000移液器(Eppendorf,目录号:3120000062)
  2. P200移液器(Eppendorf,目录号:3120000054)
  3. P10移液器(Eppendorf,目录号:3120000020)
  4. 镊子(VWR,目录号:470018-952)
    制造商:Dunrite Instruments,目录号:141001。
  5. 手术刀(Bard-Parker ® SafeSwitch TM 可重复使用的手术刀手柄,尺寸3 L)(Aspen Surgical,目录号:ST-1013LNS)
  6. 锋利的剃须刀片(碳钢剃刀刀片)(Azpack,目录号:YSJ-762-Q)
  7. 孵化器(6M Lab Incubator)(Precision Scientific,目录号:31487)
  8. 台式振荡器(Thermo Fisher Scientific,型号:MaxQ TM 4000)
  9. 生长室具有以下条件:
    光强度:300-500μmol/ m 2 •sec -1
    12小时,光照,28°C
    12小时,黑暗,28°C
    湿度50-70%
    测量~500ppm CO 2
  10. Powerlyzer ® 24均质器(MO BiO Laboratories,目录号:13155)
  11. 离心机(15安培型)(Eppendorf,型号:5810 R)
  12. 离心机(Eppendorf,型号:5417 R)
  13. 分光光度计(UV / Vis)(Beckman Coulter,型号:DU 730)
  14. 涡旋混合器(Vortex-Genie 2)(科学产业,目录号:SI-0246)
  15. 生物安全柜(The Baker,SterilGard ®,型号:SG403A-HE)
  16. Balance(Roche Diagnostics,型号:05942861001)
  17. 高压灭菌器(真空蒸汽灭菌器)(Getinge,型号:533LS-E)

软件

  1. PRISM Graphpad软件

程序

本节包括从程序A到程序I的小节:

  1. 准备细菌培养物进行土壤浸泡(取决于实验目标,本小节可以遵循程序C-F或C-E-G或C-G)
  2. 播种和移植番茄植物 - 本小节描述了如何种植番茄植物以确保土壤浸泡或叶柄接种(C或D)的可重复结果,然后可以测量疾病进展(F)或竞争和细菌定植(E和/或G)。&nbsp;
  3. 土壤浸泡接种 - 将稀释的细菌悬浮液倒入含有未受伤的番茄植物的盆中,这与自然根感染过程非常相似。这种方法的感染是随机的。该程序可以是程序E或F或G或H,或E-G。
  4. 叶柄接种 - 这种替代接种程序不太自然,但允许确定特定基因是否有助于 R. solanacearum 在宿主茎上成功。叶柄接种也可确保同步感染。 (此程序可用于程序E或F或G或H,或E-G。)&nbsp;
  5. 竞争测定 - 量化野生型和突变体 R的相对竞争适合度。 solanacearum ,我们测量接种后3至7天或之后(取决于实验目标)茎中的细菌定植(程序G)。&nbsp;
  6. 疾病进展曲线 - 该程序用于量化土壤浸泡(程序C)或叶柄接种(程序D)后的青枯病发展。&nbsp;
  7. 细菌定植 - 该程序用于测量细菌定殖根(感染的早期阶段)或干细胞(感染的后期阶段)的能力。该方法量化了 R.土壤浸泡(程序C)或叶柄接种(程序D)或竞争试验(程序E)后,植物中
  8. Xylem sap collection - 离体番茄木质部汁液可用于代谢组学分析或作为细菌生长的培养基。通常植物受自然土壤浸泡法感染(程序A-B-C)。&nbsp;
  9. 根附着分析 - 量化突变体与野生型细菌附着于番茄幼苗根部的能力,这是 R的最初步骤之一。 solanacearum 植物感染。&nbsp;


  1. 准备细菌培养土壤浸泡
    1. 在CPG + TZC板上从水原液或甘油原液中划出 Ralstonia solanacearum ,并使其在28°C静态培养箱中生长48至72小时。
      注意:确保菌落的形态是野生型(WT)而不是自发的phcA样突变体(Khokhani等,2017)。
    2. 一旦你看到平板上的分离菌落,挑选一个菌落在250毫升无菌烧瓶中接种100毫升CPG肉汤(含有适当的抗生素),在摇床培养箱中以220转/分钟培养过夜(18-20小时), 28°C。
    3. 将过夜培养物在50ml锥形管中离心(6,900 x g ,5分钟)两次,以沉淀所有100ml细胞培养物。丢弃上清液。
      注意:当您倒出上清液时,一些EPS也会脱落,这是一个很好的迹象,表明培养物没有被自发的phcA污染,如不会产生野生型EPS的突变体。
    4. 使用P1000移液管将细胞沉淀重悬于20ml无菌MilliQ(SMQ)水中,然后加入剩余的30ml SMQ水并通过涡旋混合均匀。
    5. 通过在1.5ml Eppendorf管中的900μlSMQ水中移取100μl重悬的细胞培养物,在SMQ中将其稀释10倍,测量过夜细胞培养物的光密度(λ= 600nm,在分光光度计中)。
      示例:如果稀释后OD 600 为0.5,则实际OD 600 为5.0。
    6. 计算在OD 600 = 0.1时产生所需体积的细菌悬浮液所需的原始细胞悬浮液的体积,其对应于约10 8 CFU / ml。
      对于一个具有10个技术重复的生物复制品,我们进行以下数学计算:
      对于土壤浸泡接种,我们每盆加入5×10 9个细胞(50ml 0.1 OD 600 细胞悬浮液)。总的来说,我们需要10 x 50 ml = 500 ml的细胞悬液。因此,对于500ml的0.1OD 600 培养物,我们需要10ml的5.0OD 600 重悬细胞培养物和剩余的490ml SMQ水。
      注意:加一点额外的接种物,这样你就不会因为移液错误而耗尽最后一个罐子的细胞。通常,每个菌株含有14-20个技术重复(植物)的三个生物重复用于统计分析。
    7. 再次检查OD 600 以确保其大约为0.1和稀释板以确认最终的接种细胞密度。
      注意:请参阅附Excel表用于计算。

  2. 播种和移栽番茄种子
    1. 将干燥的灌封混合物加入幼苗托盘中,轻轻按压。
    2. 在每个细胞中放置4-5粒Bonny Best(易受细菌枯萎病的品种)的种子。
      注意:我们已确定番茄cv。赚钱的人也同样容易受到青枯病的影响。
    3. 用一层薄(1-3毫米)的灌封混合物覆盖它们,如图1所示。


      图1.将番茄种子播种在多锅托盘中

    4. 小心地给土壤浇水,直到土壤潮湿而不被淹没。
    5. 将培养皿在28°C的生长室中孵育12小时光照/ 12小时黑暗循环。注意:生长室中的高光强度对于良好的疾病发展至关重要。
    6. 在没有淹没托盘的情况下给土壤浇水(水不应流出托盘)。每天早上大约在同一时间给植物浇水。
    7. 在种植后的第13天日停止给幼苗托盘浇水,使土壤稍微干燥。这使得土壤很容易轻微地从幼苗上脱落,并且很容易将幼苗转移到较大的盆中。如果土壤湿润,许多根可以轻轻摇动甚至收支平衡。
    8. 在第14天天(图2),将幼苗从幼苗托盘转移到较大的盆中(图3),仔细地脱落土壤(尽可能不破坏根部)。为了去除土壤,在土壤掉落时轻轻敲打根球。


      图2.第14天的幼苗生长 种植种子


      图3.带有土壤混合物的移植罐准备转移14日龄番茄幼苗

    9. 用灌封混合物填充直径8厘米的塑料盆。重要的是不要压缩土壤以避免在盆中产生厌氧条件。
    10. 给土壤浇水,直到它被弄湿但没有被淹没,用食指或Sharpie在锅的中心开一个洞。
    11. 将幼苗放在盆中,当所有盆都有幼苗时,将每个幼苗放入一个洞中,轻轻按压幼苗基部周围的土壤,使其得到良好的支撑,并在浇水后不会翻倒。
    12. 让西红柿在较大的盆中生长一周,直到它们21天大。第一片真叶应该完全展开,第二片真叶应该打开。

  3. 土壤浸泡接种试验
    先前已使用以下方案(Huang和Allen,2000; Tans-Kersten 等人,2001; Colburn-Clifford 等人,2010)。
    1. 根据土壤湿度,在接种前一天或两天停止给植物浇水。这将使土壤吸收倒入其中的所有细菌培养物。小心不要过度干燥,如 R.在干燥条件下,solanacearum 存活率下降。
    2. 在接种番茄植物的前一天,开始如上述步骤A1中所述的 Ralstonia solanacearum 的过夜培养物。
    3. 在接种当天,按照步骤A2至A7进行。
    4. 准备好细菌培养物后,对接种物进行稀释平板培养,并将CPG平板培养约48小时以计数菌落数。这将证实OD 600 约为0.1,并且在制备期间细菌细胞未被污染或杀死。
    5. 要对植物进行盲目评估(首选程序),请同事对您的接种瓶(例如,用彩色胶带)进行编码,并从烧瓶中取出识别菌株标签。同事可以保留应变代码列表,直到您准备好在实验完成后分析数据。
    6. 使用50ml锥形管测量50ml细菌悬浮液,并将其倒入每个罐中。您可以针对所有目标菌株进行同样的测试,以检测其毒力,但每种细菌菌株仅使用一个管。确保将种植棒放入盆中以标记植物以用于未来的疾病评级(图4)。不要伤害植物。


      图4.种植盆准备接种的盆栽

    7. 几个小时内检查土壤水分。为了获得一致的结果,接种后几天土壤不应干燥。干燥的土壤会降低细菌活力,延缓疾病发作。
    8. 接种后,用Hoagland溶液在一天后给植物浇水。剩余的13天使用自来水给植物浇水。
    注意:如果疾病发育不良,可以操纵以下变量:a)植物年龄。使用较年轻的植物(种植后18或19天); b)湿度。确保生长室湿度至少为65%; c)接种密度。通常,接种后14天,野生型R. solanacearum在接种50 ml 1 x 10 8 后,导致平均疾病指数为3.5或更高 CFU悬浮液(OD 600 = 0.1)。如果需要,接种物可以增加到OD 600 = 0.2。

  4. 叶柄接种试验
    以下协议已用于(Brown和Allen,2004; Dalsing和Allen,2014)。
    注意:按照程序B中提到的所有步骤,在植物生长的所有21天内给植物浇水。
    1. 在接种前一天,开始5ml过夜的 R的CPG培养物。雷尔氏菌。
    2. 在接种当天,首先在早晨给植物(现在21日龄)浇水,然后进行接种。
    3. 第二天,旋转1ml过夜培养物,并将沉淀重悬于1ml SMQ水中。
    4. 测量OD 600 并稀释SMQ H 2 O中的细菌悬浮液,以制备2ml OD 600 = 0.1细菌细胞悬浮液。
    5. 在向前移动之前,通过分光光度计和稀释电镀确认OD 600 ,如步骤A6中所述。
    6. 将0.1OD细胞悬浮液稀释至OD 600 = 0.001。
    7. 为了感染植物,用锋利的剃刀刀片切除叶柄底部附近的第一片真叶。叶柄应对角切割,平行于地面,以形成一个水平表面,在这里可以施加一滴细菌而不会沿着茎干向下移动(图5)。


      图5. 21日龄番茄植株的叶柄接种。蓝色箭头表示切割叶柄的水平残端,其中放置了2μl细菌细胞悬浮液。

    8. 使用10ml移液管在切割的叶柄表面上小心地放置2μlOD 600 = 0.001细胞悬浮液(相当于2,000个细胞)。可以根据实验目标改变细胞数量。液滴很快被植物吸收,表明细菌被拉入木质部。
    9. 您可以在一个托盘中切割植物的所有叶柄,然后将细菌应用于每个植物。例如,一次有10个工厂。

  5. 竞争试验测定植物中细菌菌株的相对竞争适合度
    以下协议已用于(Yao和Allen,2006; Lowe et al。,2015)。
    1. 为了测量目标菌株相对于另一种菌株(通常是野生型)的竞争适应性(Yao和Allen,2006),每种菌株必须具有不同的抗生素抗性。例如,如果野生型菌株是卡那霉素抗性的并且目标突变菌株是庆大霉素抗性的。
      注意:为了确保抗生素抗性标记基因不会增加标记菌株的适应性成本,比较标记菌株的植物生长与未标记亲本菌株的生长。
    2. 在补充有所需抗生素的CPG平板上制备标记菌株的新鲜平板用于过夜培养。
    3. 如程序B和C中所述种植植物。
    4. 为了接种植物(在植物生长的第21天),分别制备两种竞争菌株的0.1OD 600 培养物。使用分光光度计检查OD 600 。
    5. 将两种菌株以1:1的细菌群体比例组合。再次,稀释板检查实际细胞数。
    6. 向每个罐中加入50ml这种混合细菌悬浮液用于自然土壤浸泡竞争,或者将2μl相同的悬浮液加入到切割的叶柄中用于叶柄接种竞争。
    7. 为了检查根或茎定植,在接种后3至7天对植物进行取样。从这里开始,按照程序G.
    注意:请参阅随附的Excel表格进行计算。

  6. 衡量疾病进展,并创造疾病进展曲线
    1. 在对植物进行评级之前,先给植物浇水,等待1小时,然后通过寻找枯萎的叶子来记录疾病。我们以0到4的等级记录疾病。如果没有叶子枯萎,植物的评分为疾病指数(DI)= 0;如果24%或更少的叶面积枯萎,我们得分为DI = 1;如果25-49%枯萎,DI = 2; 50-74%,DI = 3;和75-100%,DI = 4。
    2. 我们将疾病症状评定到接种后14天。枯萎症状通常在叶柄接种后3-4天或土壤浸泡接种后4-5天开始出现。

  7. 测量番茄根和茎的细菌定植
    根系定殖的方案已被用于(Lowe et al。,2015),并且已经使用了茎定植(Dalsing et al。,2015; Lowe-Power et al。,2016)。
    1. 样品采集取决于实验目标。请参阅已发表的文章,其中抽样时间以研究目标为指导。
    2. 对于根系定植,将植物从盆中取出并轻轻摇晃尽可能多的土壤。&nbsp;
    3. 将植物放入装满水的容器中,让根部浸泡并轻轻洗净,直至所有土壤脱落。对每个处理的所有植物重复该过程。 (图6A-6C)
    4. 在漂白剂中洗涤根部15秒,然后在2个淡水浴中连续洗涤15秒并在组织上干燥30秒(图6D-6F)。
    5. 切断胎冠连接处的根部(图6G)。
    6. 收集根部并用剃刀刀片将其切成小块,以确保每个样品含有来自每个区域的根(图6H)。
    7. 将最多300毫克的切根放入含有4个小金属珠和700μlSMQ水的制备的珠搅拌器管中。
    8. 对于茎的定植,使用剃刀刀片在叶柄接种部位周围收获1厘米的茎(图6I)。


      图6.根(A-H)和茎定植(I)定量的步骤

    9. 将最多100毫克切割的茎放入含有4个金属珠和900μlSMQ水的制备的研磨管中。
    10. 使用Powerlyzer ®打珠机打磨组织,并进行以下设置:
      速度= 2,200rpm,时间= 1分30秒,停留时间= 4分钟,循环= 2,总时间= 7分钟
    11. 通过将20μl每种稀释液与180μlSMQ混合,在96孔板中将匀浆系列稀释10倍。对于根样品:每个样品连续进行5次1:10稀释(10 0 -10 -4 );对于茎样品:每个样品连续进行7次1:10稀释(10 0 -10 -6 )。
    12. 将三种10μl各稀释液滴入CPG板(含有适当的抗生素)。通过在生物安全柜中打开盖子约15分钟来干燥板。
    13. 将板在28℃孵育36-48小时并计数菌落。不允许殖民地一起成长。
      注意:将最终种群大小标准化为原始个体接种物中实际细菌数量的比例,通过接种物的稀释平板测定。请参阅随附的Excel表格进行计算。

  8. Xylem根据压力收集汁液
    以下协议改编自Goodger et al。(2005),用于Khokhani et al。(2017)和Lowe-Power 等。(2018)。
    对于基础实验,我们每个时间点每个处理准备9株植物(取样是破坏性的)。 Sap将合并为3个池,每个池含有3株植物的汁液。由于切割不均匀,偶尔不能从植物中收集汁液,因此准备额外的植物是明智的。当植物处于充分浇水的土壤中时,根压会在一夜之间积累。根部压力在一天中降低。因此,汁液收集应该在光照开始的几个小时内发生,并且植物必须在汁液收集之前的晚上充分浇水。树液渗出率取决于许多变量,包括土壤含水量,植物大小和疾病状态。在Lowe-Power 等人描述的实验中。 (2018),我们从26-31天的健康和枯萎症状(疾病指数1)植物中收获了473μl和280μl的中位数体积(图7B)。
    准备工作:
    1. 每株一管研磨组织以确定定植(4个金属珠和900μlSMQ水)&nbsp;
    2. 每株植物一管用于木质部汁液收集(数量,颜色代码,等),预先称重,在冰上预先冷却。如果可能,将所有管和植物排列在同一平行线上,以避免交叉污染样品。
    3. 每株一管对树液进行消毒。
    4. 每个游泳池一管。
    5. 将计时器设置为33分钟。
    6. 用锋利的剃刀刀片将番茄植物取出,以产生平坦的残端表面。如果树桩表面成角度,树液可能会从树干上滴下来。对于土壤浸泡接种的植物,在子叶上方切割约1厘米。对于切叶柄接种的植物,在接种部位正上方切割。一般来说,它可以很好地分批处理植物,每批次间隔5分钟。
    7. 让第一滴液体积聚~2½分钟。然后用纸巾轻轻擦干树桩。使用P200移液器用无菌SMQ水清洗残端,然后再次吸干,以尽量减少细胞溶质污染。
    8. 收集木质部汁液30分钟,始终使用新鲜的尖端并将每株植物的木质部汁液移液到相应的管中,该管保持在冰上。由于表面张力,树液应该作为气泡积聚(图7A)。如果树桩看起来很潮湿,但树液不会积聚,它会隐蔽地从树干上滴下来。轻轻地将树桩吸干干净可以恢复表面张力,使树液渗出并使其积聚。我们已经包括了从番茄植物中收集的木质部汁液量的估算(图7B)。


      图7.木质部汁液的收集。 A.去皮番茄植株上的木质部汁液。 B.样品数据集显示来自健康和受感染植物的木质部汁液体积。每个点代表从一个植物收集的汁液量。

    9. 如上所述,保持植物顶部采集样品以量化每株植物的细菌定植(从茎的最低部分收集~50-100mg切片)。
    10. 再次称量每个管以确定从每株植物获得的木质部汁液体积(密度类似于水,因此1 mg =1μl)。
    11. 池应由来自植物的木质部汁液组成,产生高,中和低量的木质部汁液。对于汇集,将每株植物的等体积木质部汁液(例如,100μl)组合。使用1ml注射器和4mm直径0.22μm过滤器(4mm大小的过滤器减少样品损失)将来自每个植物的木质部汁液过滤灭菌到新鲜的1.5ml管中。
    12. 该木质部汁液可用作生长曲线分析或代谢物分析的培养基。可以在Corning 96孔半区域微量培养板中用50μl树液进行细菌生长测定。代谢物分析所需的汁液量应根据目标代谢物的浓度和检测方法的灵敏度凭经验确定。

  9. 番茄幼苗的根附着物
    以下协议已在Tran et al。(2016b)和Khokhani et al。(2017)中使用。
    1. 将番茄种子浸泡在2%漂白剂中5分钟,然后用70%乙醇浸泡5分钟,然后用SMQ水冲洗6次,对番茄种子进行表面灭菌。
    2. 在生物安全柜中使用无菌镊子的帮助,将灭菌的幼苗通过将其铺展到覆盖有无菌Whatman TM 滤纸的1%琼脂培养皿上进行转移。
    3. 用Micropore TM 胶带密封培养皿并用铝箔覆盖以排除光。
    4. 将板在28℃孵育4天。同时,准备1%琼脂方板,CPG + TZC +所需抗生素,珠子打浆管,4个金属珠和300μlSMQ水。
    5. 在4 th 日,用锐利标记方形板的背面,如图所示(图8)。


      图8.标有2 cm区域的方形板,其中番茄幼苗的根部排列

    6. 在生物安全柜中,安排番茄幼苗,使其根长度为2厘米(从根尖)。大约40个番茄幼苗可以容纳在一个方形板中,每侧20个。
    7. 按步骤D2-D6所述制备细菌接种物。
    8. 稀释平板接种物并在28℃下孵育平板以确认接种物密度。
    9. 接下来,使用微量移液管小心地将10μlOD 600 = 0.001细菌悬浮液(~10,000个细胞)分布在方形板中每个根的2cm长度上。
    10. 在室温下孵育方板2小时。
    11. 两个小时后,使用无菌手术刀切下所有40根的尖锐标记所示的2厘米长度。
    12. 将所有切根转移到含有SMQ水的培养皿中并旋转平板以去除松散附着的细菌细胞。在第一次漂洗后,将所有根部转移到另一个含有SMQ水的培养皿中并再次冲洗。
    13. 接下来,将根部转移到新的纸巾上,轻轻拍干根部。一次转移4根。
    14. 在干燥后,将每组4根转移到制备的含有金属珠和300μlSMQ水的珠搅拌器管中。对于40个根,每个生物重复每次处理将有10个技术重复。
    15. 使用以下设置将包含切根的珠子打针管和金属珠放在Powerlyzer ®中:速度= 2,200 rpm,时间= 1分30秒,停留时间= 4分钟,周期= 2,总时间= 7分钟。
    16. 研磨后,将匀浆与它们的1:10,1:100,1:1,000连续稀释液一起用于每个样品。
    17. 48小时后,计数菌落以确定原始接种物中的细菌细胞数和附着于根部的细菌数。
    18. 通过将从根部回收的细菌细胞数除以每厘米根中接种物中存在的细胞数来计算附着百分比。
      注意:请参阅随附的Excel表格进行计算。

数据分析

图9表示来自Dalsing 等人的疾病进展试验(2015)。如上所述,使用0至4疾病指数每天评价植物14天。所呈现的数据是3至4个独立测定的平均结果,每个测定每个菌株含有10株植物。误差棒表示均值的标准误差。 Δ aniA 和Δ hmpX 突变体的疾病进展曲线与WT株显着不同( P <0.001,重复测量方差分析; ,PRISM Graphpad软件)。
&NBSP;此处介绍的协议生成定量数据,需要深思熟虑,细致入微的分析。幸运的是,最近的一篇论文(Schandry,2017)提供了分析 R数据的详细实用指南。 solanacearum 感染,包括相关的R代码。


图9.来自Dalsing等人的一种毒力测定的样本疾病进展图。 (2015)

笔记

  1. 定期校准移液器,尤其是用于稀释电镀的P10。&nbsp;
  2. 每次新实验,都要使用一瓶新的SMQ水,以避免任何污染。&nbsp;
  3. 定期检查你的菌株是否存在自发的 phcA 突变体。&nbsp;
  4. 不要使用从水或冷冻库存中出苗后生长超过4天的CPG板的菌落接种。 “老”殖民地经常失去毒力。

食谱

  1. 酪蛋白氨基酸 - 蛋白胨 - 葡萄糖(CPG)琼脂(1L)
    5克葡萄糖
    10克蛋白胨
    1克酪蛋白氨酸
    1克酵母提取物
    用1M KOH调节pH至6.5-7.0
    16克琼脂
    高压灭菌器在121°C,20分钟
    1毫升1%2,3,5-三苯基四唑氯化物(TZC)(溶解于水中,过滤灭菌并在浇注板前加入CPG)
  2. CPG肉汤(1升)
    5克葡萄糖
    10克蛋白胨
    1克酪蛋白氨酸
    1克酵母提取物
    用KOH调节至pH 6.5-7.0
    高压灭菌器
  3. 修改了Hoagland的解决方案
    混合物
    1.1 M KNO 3
    0.2 M KH 2 PO 4
    0.92 M MgSO 4
    浇水时,使用4毫升/升水
    硝酸钙
    1.29 M Ca(NO 3 ) 2 •4H 2 O
    浇水时,使用5毫升/升水
    微量营养素
    0.4626 M H 3 BO 3 (硼酸)
    0.0915 M MnCl 2 •4H 2 O(氯化锰)
    0.0077 M ZnSO 4 •7H 2 O(硫酸锌)
    0.0032 M CuSO 4 •5H 2 O(硫酸铜)
    0.0007 M(NH 4 ) 6 Mo 7 O 24 (钼酸)
    浇水时,使用4毫升的库存/ L水

    FeSO 4 •7H 2 O(硫酸亚铁)25.02 g / 18 L水
    Na 2 EDTA(乙二胺四乙酸)33.48 g / 18 L
    将上述溶解在4L去离子RO水中。加热至80°C一小时。冷却至室温,加入去离子RO水至18L
    浇水时,使用4毫升/升水

致谢

我们感谢Patrizia Ricca捕获土壤浸泡,根和茎定植的图像。我们还要感谢所有论文的作者,他们已经引用了这些论文来调整和/或修改他们的协议来执行各自的实验。

利益争夺

作者宣称没有利益冲突。

参考

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引用:Khokhani, D., Tuan, T. M., Lowe-Power, T. M. and Allen, C. (2018). Plant Assays for Quantifying Ralstonia solanacearum Virulence . Bio-protocol 8(18): e3028. DOI: 10.21769/BioProtoc.3028.
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