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A Method for Radioactive Labelling of Hebeloma cylindrosporum to Study Plant-fungus Interactions
一种用于研究植物-真菌相互作用的圆形孢子粘花菇放射性标记方法   

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Abstract

In order to quantify P accumulation and P efflux in the ectomycorrhizal basidiomycete fungus Hebeloma cylindrosporum, we supplied 32P to mycelia previously grown in vitro in liquid medium. The culture had four main steps that are 1) growing the mycelium on complete medium with P, 2) transfer the mycelia into new culture solution with or without P, 3) adding a solution containing 32P and 4) rinsing the mycelia before incubation with or without plant. The main point is to rinse very carefully the mycelia after 32P supply in order to avoid overestimation of 32P efflux into the medium.

Keywords: Culture in vitro(体外培养), Phosphate availability(磷酸盐利用度), 32P labelling(32P标记), Ectomycorrhizal fungi(外生菌根真菌)

Background

It is well known that the association between mycorrhizal fungi and plants improves the P nutrition of the host-plant (reviewed by Smith and Read, 2008; Plassard and Dell, 2010; Cairney, 2011; Smith et al., 2015). This positive effect has been attributed primarily to phosphate (Pi) uptake by the fungal cells exploring the soil far from the roots, allowing the exploration of a large volume of soil beyond the depletion zone formed around actively absorbing roots (Smith and Read, 2008; Cairney, 2011; Smith et al., 2015). However, to benefit to the host plant, absorbed Pi has to be transported from the fungal cells exploring soil towards those in close contact with the host cells. In ectomycorrhizal symbiosis, these exchanges are thought to take place in a territory called the ‘Hartig net’ inside the ectomycorrhizal roots (Smith and Read, 2008; Cairney, 2011). In the Hartig net, the fungal cells colonize the walls of cortical cells but there is no direct communication between the two plasma membranes, meaning that P has to be released from the fungal cells via a yet unknown mechanism. Taken together, this knowledge indicates that the ability of the fungus to take up P in external hyphae and to release P is therefore an important feature of the fungal species for its positive effect on plant P nutrition. Here, we developed a methodology using 32P labelling to follow the net 32P accumulation and release by an ectomycorrhizal fungal species cultivable in vitro, without its host plant (Torres-Aquino et al., 2017). This method could be used with other fungal or plant material.

Materials and Reagents

  1. Sterile plastic Petri dish, 90 mm (Dominique DUTSCHER, Gosselin, catalog number: 688302 )
  2. Sterile plastic Petri dish, 35 mm (Corning, Falcon®, catalog number: 351008 )
  3. Liquid scintillation vial, 6 ml with cap (PerkinElmer, catalog number: 6000592 )
  4. Liquid scintillation vial, 20 ml with cap (PerkinElmer, catalog number: 6008117 )
  5. Waste bags for radionuclides (SCIE-PLAS, catalog number: RRP-BAG17 )
  6. Protective paper for bench (Dominique DUTSCHER, Benchguard, catalog number: 090277 )
  7. Liquid scintillation cocktail Ultimagold (PerkinElmer, catalog number: 6013329 )
  8. Nichrome wire, stainless steel, round, 22 gauge, 0.64 mm diameter (suppliers for electronic cigarettes)
  9. Aluminium screw cap, 40 mm with rubber liner (VWR, SPV, catalog number: 215-2690 )
  10. Multi-Purpose Silicone for kitchen or bathroom, 280 ml (Castorama, Rubson)
  11. 60 ml Luer-lock syringes (Dominique DUTSCHER, Omnifix, catalog number: 921016 )
  12. Teflon PTFE microtube, 1.15 mm and 1.75 mm for internal (int) and external (ext) diameter (diam), respectively (Dominique DUTSCHER, PTFE, catalog number: 091932 )
  13. Needles 18 G 0.9 x 40 mm (Dominique DUTSCHER, BD MicrolanceTM 3, catalog number: 301300 )
  14. Tubing, int diam 1.14 mm (Dominique DUTSCHER, Silicone, catalog number: 4906591 )
  15. Tubing, int diam 3.17 mm (Dominique DUTSCHER, Silicone, catalog number: 4906600 )
  16. Microtubes, 1.5 ml (Dominique DUTSCHER, Eppendorf, catalog number: 033511 )
  17. Valve Luer polycarbonate one way (Cole-Parmer, catalog number: EW-30600-01 )
  18. Sterile syringe filters for air, 0.2 µm, 6.4 cm diam (Labomoderne, Midisart, catalog number: RS3320 )
  19. Autoclavable Polypropylene bag, 3 L, non-printed (Dominique DUTSCHER, catalog number: 140230 )
  20. Tips 1,200 µl for pipet (Dominique DUTSCHER, Sartorius, catalog number: 077200B )
  21. Home-made syringe holder
  22. Home-made needle holder for aeration
  23. Folding skirted caps, 14.9 mm diam (Dominique DUTSCHER, catalog number: 110602 )
  24. Paper for sterilization (Dominique DUTSCHER, catalog number: 006950 )
  25. Hebeloma cylindrosporum (ectomycorrhizal basidiomycete) (laboratory’s own collection, available upon request)
  26. Manganese(II) sulfate monohydrate (MnSO4·H2O) (Sigma-Aldrich, catalog number: M7899-500G )
  27. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z0251-100G )
  28. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768-500G )
  29. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: C8027-500G )
  30. Sodium molybdate dihydrate (Na2MoO4·2H2O) (Sigma-Aldrich, catalog number: M1651-100G )
  31. Potassium nitrate (KNO3) (Sigma-Aldrich, catalog number: P8291-1KG )
  32. Sodium phosphate monobasic monohydrate (NaH2PO4·H2O) (Sigma-Aldrich, catalog number: 71504-250G-M )
  33. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 63138-250G )
  34. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333-500G )
  35. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080-500G )
  36. Ferric ammonium citrate (Sigma-Aldrich, catalog number: RES20400-A702X )
  37. D-glucose (Sigma-Aldrich, catalog number: G8270-1KG )
  38. Agar-agar (Sigma-Aldrich, catalog number: A7002-500G )
  39. KH232PO4 in water (PerkinElmer, catalog number: NEX055002MC )
  40. 2-N-morpholino-ethanesulfonic acid, 4-morpholineethanesulfonic acid monohydrate (MES) (Sigma-Aldrich, catalog number: 69892-500G )
  41. Tris(hydroxymethyl)aminomethane (TRIS) (Sigma-Aldrich, catalog number: T1378-500G )
  42. 1 N sulfuric acid solution (EMD Millipore, catalog number: 1.09072.1000 )
  43. Trace elements (see Recipes)
  44. Mineral salt base solutions (see Recipes)
  45. Thiamine solution (see Recipes)
  46. N6 complete liquid solution (see Recipes)
  47. Solid N6 complete liquid solution (see Recipes)
  48. Interaction medium (IM) (see Recipes)

Equipment

  1. Sample bottles,120 ml (VWR, SPV, catalog number: SPVAGO2246 )
  2. Glass bottles, 1,000 ml, ISO borosilicate, graduated (Dominique DUTSCHER, catalog number: 046415 )
  3. Glass bottles, 2,000 ml, ISO borosilicate, graduated (Dominique DUTSCHER, catalog number: 046416 )
  4. Two pairs of stainless steel straight tweezers Wironit, Brucelles type, 130 mm (Dominique DUTSCHER, catalog number: 491037 )
  5. A scalpel handle for blade 20 to 25 (Dominique DUTSCHER, catalog number: 3740004 )
  6. Surgical blade sterile N°21 (Dominique DUTSCHER, catalog number: 132521 )
  7. A standalone burner (Dominique DUTSCHER, catalog number: 071109 )
  8. Butane gas cartridge for the burner (Dominique DUTSCHER, catalog number: 060415 )
  9. A nail, a hammer, scissors and cutting pliers
  10. Incubator with controlled temperature set at 25 °C
  11. Autoclave
  12. Laminar flow cabinet
  13. Cork-borer, 7.5 mm diameter (Dominique DUTSCHER, catalog number: 942783 )
  14. Aquarium air-pumps (SuperFish, model: air-box Nr.4 )
  15. Shield, fixed 15° angle, flat base, Beta; 530 x 350 mm shield; 350 x 300 mm base (upright x horizontal) (SCIE-PLAS, catalog number: RPP-S15L )
  16. Midi-box with hinged lid, Beta; external dimensions: 80 x 185 x 105 mm; internal dimensions: 60 x 165 x 85 mm (height x width x depth) (SCIE-PLAS, catalog number: RPP-B6 )
  17. Bin for beta wastes on the bench, 3.3 L capacity (SCIE-PLAS, catalog number: RPP-B17 )
  18. Liquid scintillation Counter TRI-CARB 4910TR (PerkinElmer, catalog number: A491000 )

Software

  1. Microsoft Excel for calculations
  2. Statistica 7.1 (StatSoft Inc., Tulsa, OK, USA) for statistical analysis

Procedure

  1. Preparation of equipment
    1. Glass jars for liquid cultures
      The mycelia are grown in glass jars (120 ml) with an aluminium cork previously equipped with a nichrome wire enabling to suspend the fungal inoculum at the surface of the liquid solution (see Figure 1).
      Preparation: first pierce the centre of an aluminium cap with a nail and a hammer. Then, cover the hole with a small amount of silicone paste and allow the paste to dry for 24 h. Cut the rubber seal in its centre with scissors and place it inside the cap.
      Meanwhile, cut the nichrome wire with cutting pliers in fragments 15 cm long whose one end is curved to form a kind of hook. Fill each jar with 40 ml of liquid nutrient medium. Take a nichrome wire and slip it into the hole of the cap equipped with its rubber seal through the silicone paste, as centred as possible. Screw the cap onto the glass jar and set the height of the wire just above the nutritive solution. Autoclave the jars at 121 °C for 20 min and allow them to cool before use.


      Figure 1. Equipment used to grow mycelia of H. cylindrosporum. It is made of a 120 ml-glass jar containing 40 ml of a complete nutrient liquid medium. A. Each jar is inoculated by one plug or disk of fungal mycelium placed on a nichrome wire. B. Aspect of the mycelium grown on the surface of medium in unshaken conditions.

    2. Preparation of syringe holders, connectors for aeration and syringes
      60 ml-syringes are used to rinse each mycelium before measuring either fungal 32P accumulation and/or 32P efflux from the labelled fungus into the interaction medium (IM) (see Recipes). This rinsing step is essential to avoid overestimation of 32P accumulation and/or efflux. Typically, we used 6 fungal replicates per treatment and we built homemade equipment (Figures 2 and 3) for easy handling of the mycelia.
      1. Preparation of syringe holders (Figure 2):
        Cut the different pieces of wood according to the dimensions given in Figure 2. For example, to hold 6 syringes, the top piece is 37 cm long x 6 cm wide and the one underneath is 32 cm long x 6 cm wide. Adjust and maintain the two pieces together to drill the holes in a two-by-two alignment. Afterwards, assemble all the pieces together as shown in Figure 2.


        Figure 2. Homemade holders to support syringes for incubation of mycelia in interaction medium

      2. Preparation of connectors (Figure 3)
        First, take an 18 G needle with a pair of tweezers, heat its collar over a flame to soften the glue and pull it with another pair of clamps. After cooling, adjust a small piece of silicone tube (1.14 mm internal diameter) of about 3 cm long on the top of the needle. Take a skirt cap and stitch 6 needles into its top and close its bottom by a 1 ml pipette tip previously cut at its finest end. If necessary, add silicone paste to fragile places to strengthen the airproof of the system. To the cut end of the blue pipette tip, add a piece of silicone tube (3.17 mm internal diameter) about 20 cm long that will be plugged later into a sterile air filter placed between the air pump and the connector (see Figure 3). Finally, place the whole system [connector + PTFE tubes + large diameter silicone tube] in an autoclavable bag and sterilize it by autoclaving (115 °C, 40 min).


        Figure 3. Homemade connector to aerate syringes during the incubation of mycelia in interaction medium

      3. Preparation of syringes: remove the plunger of each syringe and discard it. Using a hot nail, drill a hole at the top of the syringe opposite to the graduations giving the volume. This hole will be used to place the microtube for aeration inside the syringe. Place all drilled syringes in an autoclavable bag and sterilize it by autoclaving (115 °C, 40 min).

  2. Preparation of fungal cultures
    Note: This is done in sterile conditions, in a laminar flow cabinet. The cork-borer and the clamps are flame-sterilized and allowed to cool before use.
    1. Step 1: Growing new cultures in solid medium from fungal stock cultures
      For experiments, we use stock cultures renewed approximately every 6 months as sources of fungal material. These fungal stock cultures are grown in the dark at 25 °C on agar nutrient medium (solid N6 medium, see Recipes below) in 90 mm Petri dishes. The nitrogen (N) source in the medium (nitrate) is adapted to this fungal species as it does not grow well on ammonium, in contrast to most ectomycorrhizal species.
      At the time of experiments, it is necessary to establish new fungal cultures by transferring one agar plug (7.5 mm diameter) on the centre of a new Petri dish (90 mm diameter) containing 25 ml of solid N6 complete medium, the mycelium face down on the nutritive medium to enhance the growth of the fungus. New cultures are grown for 3 weeks, in the dark, at 25 °C in an incubator.
    2. Step 2: Growing the fungus in glass jars from solid cultures
      First, prepare the fungal plugs with the cork-borer by punching holes in the 3-week old mycelium agar culture.
      Then, unscrew the lid of a jar and hold it in one hand; take a pair of clamps in the other hand and remove a plug of fungal culture from the solid medium; prick the plug on the tip of the hook of the nichrome wire. Screw the lid properly on the jar and put the fungal plug just on the surface of the medium by slipping the nichrome wire (Figure 1A). The fungus is grown at 25 °C for two weeks.
    3. Step 3: Growing the fungus in glass jars from liquid cultures for labelling experiments
      The 2-week old mycelia grown in liquid medium are then used to produce the fungal material for labelling experiments. For that, disks of the fungus are punched from the mycelium and placed on the tip of the hook of the nichrome wire, as described in step B2. This procedure avoids the presence of agar-agar in the fungal culture, as it can interfere with labelling experiments by accumulating 32P from the labelled solution and releasing 32P into the non-labelled solution. The agar-free mycelia are grown for 7 days, at 25 °C, without shaking.

  3. P pre-treatments
    1. Preparation of nutritive solution
      Prepare +P and -P nutritive liquid media for fungal pre-treatment that are N6 medium with P (supplied as NaH2PO4 1 mM, see Recipes) or without P (by omitting the addition of NaH2PO4 in the N6 medium), 40 ml per mycelium. Prepare additional N6 medium without P (100 ml per mycelium) to rinse the mycelia (see Figure 4B).
    2. Preparation of jars
      Pour 40 ml of liquid medium, either +P or -P, in new jars. Screw lids without nichrome wire on the jars and autoclave them as described before. Pour also additional -P N6 medium in 1 L bottles and autoclave.
    3. Transfer of mycelia to new media
      Bring the flasks containing the seven-day-old mycelia into the flow laminar cabinet and the mycelium is withdrawn from the medium by slipping the nichrome wire up through the lid. The nutritive medium is allowed to drip from the fungus before the lid is unscrewed (Figure 4A).
      1. For the -P treatment only, rinse each mycelium in -P medium by transferring it successively into a 35 mm-Petri dish containing 20 ml of fresh -P medium (Figure 4B) for 3 min. This operation is repeated four times, meaning that 5 Petri dishes are prepared for each mycelium as illustrated in Figure 5. This step is essential to eliminate the carry-over solution that would otherwise contaminate the -P solution with orthophosphate (Pi). After rinsing, transfer each mycelium into a new flask containing 40 ml of -P medium and allow them to grow for 5 additional days at 25 °C, in the dark, to get P-depleted mycelia.
      2. For the +P treatment, transfer the mycelia just allowed to drip dry into new flasks containing 40 ml of +P medium, without rinsing. They are allowed to grow under the same conditions as -P conditions.


        Figure 4. Transfer of the mycelia to new media. A. the nichrome wire is first pulled through the lid to allow the solution to drip from the fungus before the lid is unscrewed. B. Then the lid is unscrewed and the mycelium rinsed five times in 20 ml P-deficient medium contained in 35 mm-Petri dishes.


        Figure 5. Picture of Petri dishes arranged in the laminar flow cabinet for rinsing the mycelia

  4. Labelling with 32P
    1. Safety precautions: take care to perform the experiments following safety precautions for the handling of Radioactive materials that are principally (i) wear gloves, a lab coat and a dosimeter to control the radiation dose received by the body, (ii) place the radioactive solutions or material behind plexiglas screens, (iii) use pipette tips with filters, (iv) throw away the radioactive solutions or materials in specific containers, (v) keep these containers behind Plexiglass screens, (vi) eliminate the waste after a period of time of at least 10 times the decay period of 32P namely 140 days and only if the radioactivity of the waste is not higher than twice the background (generally 0.8 to 1.6 counts per second).
    2. Prepare a set of jars with lid without nichrome wire filled with 40 ml of +P medium and autoclave them at least the day before the labelling experiment. Place a Plexiglass screen and protective paper on the bench and open the jars behind the Plexiglas screen. Label the medium with 32P orthophosphate by quickly adding KH232PO4. Dilute the radio-labelled source so as to obtain an initial activity of 3.3 x 104 Bq per µmol Pi in the +P medium. Homogenize the solution by hand shaking of the jars and take a subsample (150 µl) to measure the actual radioactivity in the medium. Store the subsample in 1.5 ml Eppendorf tubes at -20 °C, in a Plexiglass box. Use 50 µl for radioactivity measurement.
    3. Bring the jars with mycelia next to the jars with labelled medium. Allow the fungus to drip dry (as in Figure 4A). Put the lid with the fungus in the jar with the radio-labelled medium, screw it and slip the nichrome wire down until the fungus touches the solution surface. Allow the fungus to incubate for 16 h at 25 °C, in the dark.

  5. Rinsing the mycelia in syringes
    1. Place the syringes in the holders on the bench, and equip them with a PTFE microtube for aeration (Figure 6). In each syringe, pour 60 ml of interaction medium (IM) and place a collection vessel under the tap. Plug the connectors for aeration into an air pump and check whether or not IM is well aerated. However, turn off the air pump during the rinsing of mycelia. Place the Plexiglass screens in front of the holders.
    2. Bring the jars with mycelia in the labelled solution behind the screen, one jar in front of one syringe. Allow the labelled solution to drip from each mycelium (as in Figure 4A) for about 5 min. Unscrew the cap, slip the nichrome wire with the fungus down from the cap and place it into a syringe. Bend the nichrome wire so as to hang the mycelium at a level corresponding to 20 ml (see Figure 6).
    3. After all the mycelia are suspended in the syringes, open the tap to allow the IM to flow into the collection vessel placed under the syringe. Close the tap and pour about 30 ml of new IM in each syringe. After 5 min, open the tap again to allow the IM to flow into the collection vessel. Repeat the same operation four times, with the tap open after 10, 15, 20 and 40 min after addition of new IM. On the whole period, each mycelium is rinsed with 150 ml of IM. At each rinsing time, collect 1 ml of IM and placed it into 6 ml liquid scintillation vial to measure radioactivity.
    4. After the 90 min-rinsing period, you can sample 6 mycelia to measure their fresh weight and their 32P contents. You can also use the mycelia for incubation with plant to measure fungal 32P efflux in the IM over time (see Becquer et al., 2017).


      Figure 6. Complete device used to rinse the mycelia after the labelling period. A. Syringe containing the interaction medium; B. Polycarbonate valve; C. Collection vessel; D. Mycelium; E. Nichrome wire; F. Microtube Teflon PTFE for individual aeration; G. Home-made connector for gathering 4 to 7 Teflon tubes; H. Sterile filter; I. Air pump.

  6. Radioactivity measurements
    1. Add 4 ml of scintillation liquid into each 6 ml-vial containing IM or labelled medium, close the vial and mix it several times upside down by hand. Make blank samples by pouring 4 ml of scintillation liquid in a vial. Measure the radioactivity using a Liquid Scintillation Counter, following manufacturer’s instructions.
    2. To measure radioactivity in the fungus, blot each mycelium between filter paper sheets and record its fresh weights. Depending on the size of the mycelium, put it in a 6 ml- or a 20 ml-scintillation vial and add 4 or 12 ml of scintillation cocktail for counting, respectively.
    3. Correct raw data for decay occurring during radioactivity measurement using the equation written in Excel:
      cpmt0 = Exp(Ln(cpmt-cpmbackground)) + (0.00003366099 x t), with
      cpm = counts per minute
      t0 = time of the beginning of experiment
      t = time of cpm measurement of the sample in counter in minutes
      cpmbackground = cpm of solvent (blank sample)
      cpmt = radioactivity of the sample at time t
      cpmt0 = radioactivity of the sample at time t0 corresponding to the first measurement. This value is taken as the reference one for all measurements.
    4. Transform cpm in Becquerel (Bq) using the formula:
      1 Bq= 1 cpm x 0.033
    5. Calculate the specific activity (SAi) of phosphate supplied to the fungus (replicatei) as follows, assuming that the concentration of phosphate is 1 mM:
      SAi (Bq/µmole Phosphate) = [(cpmt0 of labelled medium/volume used for measurement (ml)) x 0.033]
    6. Calculate the amount of P taken up by the fungusi (replicate i) during the exposure to 32P as follows:
      P accumulation in fungusi (µmoles) = (cpmt0 in the fungusi x 0.033)/SAi

Data analysis

Express amounts of 32P (in Bq) released by the mycelia or accumulated in the fungus either per mycelium or per gram of fresh weight. You can also express the data as µmoles of phosphate accumulated by the fungus during the 16 h incubation period and calculate the corresponding uptake rate of Pi. If you compare several treatments, test the normality of data using the Kolmogorov Smirnov test and, where necessary, transform the data either square root or log10 prior to analysis to meet the assumptions of ANOVA. To compare the effect of treatments, use an ANOVA analysis.

Notes

  1. As the growth of the fungus in liquid medium may vary, we prepare up to 4 additional culture flasks inoculated with the fungus to discard those with poor growth.
  2. Aeration provided through PTFE microtubes could collapse because of liquid meniscus. They can be pulled out from the microtubes by branching a 60 ml syringe completely filled with air and strongly pushing the air through the microtubes.
  3. The height of the microtubes in the syringes may need to be adjusted to allow the air to exit. If they are too deep in the medium, the air will not flow.

Recipes

  1. Trace elements (1,000 ml)
    3.08 g MnSO4·H2O
    4.41 g ZnSO4·7H2O
    2.82 g H3BO3
    0.98 g CuSO4·5H2O
    0.29 g Na2MoO4·2H2O
    Add ddH2O to 1,000 ml, store at 4 °C
  2. Mineral salt base solutions (100 ml each)


  3. Thiamine solution (100 ml)
    0.01 g Thiamine-HCl
  4. N6 complete liquid solution (1,000 ml)
    1. Add in a 1 L-beaker approximately 500 ml of deionized water and the following volumes of mineral base solutions:
      6 ml KNO3
      1 ml NaH2PO4
      1 ml MgSO4
      4 ml KCl
      0.5 ml CaCl2
      0.5 ml ferric ammonium citrate
    2. Add also trace elements 0.2 ml and thiamine solution 0.5 ml
    3. Complete the volume to 1 L and check the pH which should be adjusted to 5.5
    4. Finally, add 5 g of D-glucose and shake until complete dissolution
    5. This solution is distributed in glass jars
  5. Solid N6 complete liquid solution (1,000 ml)
    Take two 1 L glass bottles:
    1. Add 7.5 g of Agar-agar and pour 500 ml of liquid N6 medium in each bottle
    2. Sterilize the medium at 121 °C for 20 min
    3. Pour the cooled medium (55-60 °C) in Petri dishes 90 mm diameter
  6. Interaction medium (3,000 ml)
    1. Add in a 3 L-beaker approximately 1.5 L of deionized water and the following volumes of mineral base solutions:
      0.6 ml MgSO4
      1.5 ml CaCl2
      3.2 g of MES (final concentration of 5 mM)
      1.82 g of TRIS (final concentration of 5 mM)
    2. Complete to 3 L with deionized water and adjust the pH to 5.5 with 1 N H2SO4
    3. Pour 1.5 L of medium into 2 L glass bottles and sterilize by autoclaving (20 min, 121 °C)

Acknowledgments

This research was supported by INRA (France) through annual funding devoted to their researchers and a fellowship through a Contract for Young Scientist (CJS) granted to Adeline Becquer, and by CONACYT (Mexico) through a Ph-D fellowship granted to Margarita Torres-Aquino. The protocol is adapted from our previous work (Torres-Aquino et al., 2017). We also thank the three anonymous reviewers for their comments that helped us to improve the protocol.

References

  1. Becquer, A., Torres-Aquino, M., Le Guernevé, C., Amenc, L.K., Trives-Segura, C., Staunton, S., Quiquampoix, H. and Plassard, C. (2017). Establishing a symbiotic interface between cultured ectomycorrhizal fungi and plants to follow fungal phosphate metabolism. Bio Protoc 7(20): e2577.
  2. Cairney, J. W. G. (2011). Ectomycorrhizal fungi: the symbiotic route to the root for phosphorus in forest soils. Plant Soil 344: 51-71.
  3. Plassard, C. and Dell, B. (2010). Phosphorus nutrition of mycorrhizal trees. Tree Physiol 30(9): 1129-1139.
  4. Smith, S. E. and Read, D. J. (2008). Mycorrhizal symbiosis. 3rd edition. Academic Press.
  5. Smith, S. E., Anderson, I. C. and Smith, F. A. (2015). Mycorrhizal associations and phosphorus acquisition: from cells to ecosystems. Annual Plant Reviews 48: 409-440.
  6. Torres-Aquino, M., Becquer, A., Le Guerneve, C., Louche, J., Amenc, L. K., Staunton, S., Quiquampoix, H. and Plassard, C. (2017). The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31 P NMR spectroscopy study. Plant Cell Environ 40(2): 190-202.

简介

为了量化外生菌根担囊菌真菌Hebeloma cylindrosporum中的P积累和P流出,我们向以前在体外生长的菌液提供了 32 P 中。 培养物有四个主要步骤:1)在具有P的完全培养基上培养菌丝体,2)将菌丝体转移到具有或不具有P的新培养溶液中,3)加入含有32和32的溶液 )在与或不与植物孵育之前冲洗菌丝体。 要点是在 32 P供应之后非常仔细地冲洗菌丝体,以避免过高估计P P。
【背景】众所周知,菌根真菌和植物之间的关联改善了宿主植物的P营养(由Smith和Read,2008; Plassard和Dell,2010; Cairney,2011; Smith等人,2015)。这种积极的作用主要是由于真菌细胞探索远离根部的土壤的磷酸盐(Pi)吸收,允许探索大量的土壤超过主要吸收根部周围形成的耗尽区(Smith and Read,2008; Cairney,2011; Smith ,2015)。然而,为了受益于宿主植物,吸收的Pi必须从探测土壤的真菌细胞转移到与宿主细胞紧密接触的细胞中。在外生菌根共生中,这些交流被认为发生在外生菌根内的“Hartig网”领域(Smith and Read,2008; Cairney,2011)。在Hartig网中,真菌细胞定居在皮质细胞壁上,但是两个质膜之间没有直接的连通,这意味着P必须通过一个未知的机制从真菌细胞释放出来。综上所述,这种知识表明,真菌在外部菌丝中摄取P并释放P的能力因此是真菌物种对植物P营养的积极作用的重要特征。在这里,我们开发了一种使用 32 P标记的方法,以便通过体外外来菌根真菌物种可培养的来追踪净积累和释放,不含其主机厂(Torres-Aquino等人,2017)。该方法可与其他真菌或植物材料一起使用。

关键字:体外培养, 磷酸盐利用度, 32P标记, 外生菌根真菌

材料和试剂

  1. 90毫米无菌塑料培养皿(Dominique DUTSCHER,Gosselin,目录号:688302)
  2. 35毫米无菌塑料培养皿(康宁,猎鹰,目录号:351008)
  3. 液体闪烁瓶,6毫升盖子(PerkinElmer,目录号:6000592)
  4. 液体闪烁瓶,20毫升盖(PerkinElmer,目录号:6008117)
  5. 放射性核素废物袋(SCIE-PLAS,目录号:RRP-BAG17)
  6. 台式保护纸(Dominique DUTSCHER,Benchguard,目录号:090277)
  7. 液体闪烁鸡尾酒Ultimagold(PerkinElmer,目录号:6013329)
  8. 镍铬合金线,不锈钢,圆形,22号,直径0.64毫米(电子香烟供应商)
  9. 铝螺丝帽,橡胶衬套40毫米(VWR,SPV,目录号:215-2690)
  10. 多功能硅胶厨房或浴室,280毫升(Castorama,Rubson)
  11. 60 ml Luer-lock注射器(Dominique DUTSCHER,Omnifix,目录号:921016)
  12. Teflon PTFE微管,分别为内径(int)和外径(直径)为1.15 mm和1.75 mm(Dominique DUTSCHER,PTFE,目录号:091932)
  13. 针18 G 0.9 x 40 mm(Dominique DUTSCHER,BD Microlance TM 3,目录号:301300)
  14. 管道,直径1.14 mm(Dominique DUTSCHER,Silicone,目录号:4906591)
  15. 管,直径3.17毫米(Dominique DUTSCHER,Silicone,目录号:4906600)
  16. Microtubes,1.5 ml(Dominique DUTSCHER,Eppendorf,目录号:033511)
  17. 阀门Luer聚碳酸酯单向(Cole-Parmer,目录号:EW-30600-01)
  18. 空气无菌注射器过滤器,0.2μm,6.4 cm直径(Labomoderne,Midisart,目录号:RS3320)
  19. 高压灭菌聚丙烯袋,3L,未印刷(Dominique DUTSCHER,目录号:140230)
  20. 提示1,200μl用于移液器(Dominique DUTSCHER,Sartorius,目录号:077200B)
  21. 自制注射器支架
  22. 自制针架用于曝气
  23. 折叠裙边帽,14.9毫米直径(Dominique DUTSCHER,目录号:110602)
  24. 灭菌纸(Dominique DUTSCHER,目录号:006950)
  25. (octulum菌根菌纲)(ectomycorrhizal担子菌)(实验室自己的收集,可根据要求提供)
  26. 硫酸锰(II)一水合物(MnSO 4•H 2 O)(Sigma-Aldrich,目录号:M7899-500G)
  27. 硫酸锌七水合物(ZnSO 4•7H 2 O)(Sigma-Aldrich,目录号:Z0251-100G)
  28. 硼酸(H 3 3 BO 3)(Sigma-Aldrich,目录号:B6768-500G)
  29. 硫酸铜(II)五水合物(CuSO 4•5H 2 O)(Sigma-Aldrich,目录号:C8027-500G)
  30. 钼酸钠二水合物(Na 2 MoO 4•2H 2 O)(Sigma-Aldrich,目录号:M1651-100G)
  31. 硝酸钾(KNO 3)(Sigma-Aldrich,目录号:P8291-1KG)
  32. 磷酸二氢钠一水合物(NaH 2 PO 4•H 2 O)(Sigma-Aldrich,目录号:71504-250G-M) br />
  33. 硫酸镁七水合物(MgSO 4•7H 2 O)(Sigma-Aldrich,目录号:63138-250G)
  34. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333-500G)
  35. 氯化钙二水合物(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:C5080-500G)
  36. 柠檬酸铁铵(Sigma-Aldrich,目录号:RES20400-A702X)
  37. D-葡萄糖(Sigma-Aldrich,目录号:G8270-1KG)
  38. 琼脂(Sigma-Aldrich,目录号:A7002-500G)
  39. 在水中的KH 2 32 PO 4 (PerkinElmer,目录号:NEX055002MC)
  40. 2-N-吗啉代 - 乙磺酸,4-吗啉乙磺酸一水合物(MES)(Sigma-Aldrich,目录号:69892-500G)
  41. 三(羟甲基)氨基甲烷(TRIS)(Sigma-Aldrich,目录号:T1378-500G)
  42. 1N硫酸溶液(EMD Millipore,目录号:1.09072.1000)
  43. 微量元素(参见食谱)
  44. 矿物盐碱溶液(参见食谱)
  45. 硫胺素溶液(参见食谱)
  46. N6完全液体溶液(见配方)
  47. 固体N6完全液体溶液(见配方)
  48. 互动介质(IM)(见配方)

设备

  1. 样品瓶120毫升(VWR,SPV,目录号:SPVAGO2246)
  2. 玻璃瓶,1000毫升,ISO硼硅酸盐,毕业(Dominique DUTSCHER,目录号:046415)
  3. 玻璃瓶,2000毫升,ISO硼硅酸盐,毕业(Dominique DUTSCHER,目录号:046416)
  4. 两对不锈钢直镊子Wironit,Brucelles类型,130 mm(Dominique DUTSCHER,目录号:491037)
  5. 刀片20至25的解剖刀手柄(Dominique DUTSCHER,目录号:3740004)
  6. 外科刀片无菌N°21(Dominique DUTSCHER,目录号:132521)
  7. 独立燃烧器(Dominique DUTSCHER,目录号:071109)
  8. 用于燃烧器的丁烷气瓶(Dominique DUTSCHER,目录号:060415)
  9. 钉子,锤子,剪刀和剪钳
  10. 温度设定在25°C的孵化器
  11. 高压灭菌器
  12. 层流柜
  13. 软木钻,直径7.5毫米(Dominique DUTSCHER,目录号:942783)
  14. 水族馆气泵(SuperFish,型号:机箱Nr.4)
  15. 盾,固定15°角,平底,Beta; 530 x 350 mm屏蔽; 350 x 300 mm底座(竖直x水平)(SCIE-PLAS,目录号:RPP-S15L)
  16. 带铰接盖的小箱,Beta;外形尺寸:80 x 185 x 105 mm;内部尺寸:60 x 165 x 85 mm(高x宽x深)(SCIE-PLAS,目录号:RPP-B6)
  17. Bin凳子上的beta废物,3.3升容量(SCIE-PLAS,目录号:RPP-B17)
  18. 液体闪烁计数器TRI-CARB 4910TR(PerkinElmer,目录号:A491000)

软件

  1. 用于计算的Microsoft Excel
  2. Statistica 7.1(StatSoft Inc.,Tulsa,OK,USA)进行统计分析

程序

  1. 准备设备
    1. 用于液体培养的玻璃罐
      菌丝体在玻璃瓶(120ml)中生长,其中先前配备有镍铬合金线的铝软木,以使真菌接种物悬浮在液体溶液的表面(参见图1)。
      准备:首先用钉子和锤子刺穿铝盖的中心。然后,用少量的硅胶膏覆盖孔,并使糊剂干燥24小时。用剪刀将橡胶密封圈中心切割,并将其放在盖子内。
      同时,用15厘米长的切割钳切割镍铬丝,其一端弯曲形成一种钩。每个罐装满40ml液体营养培养基。拿一个镍铬合金丝,并将其通过硅胶粘贴到装有橡胶密封垫的盖子的孔中,尽可能居中。将盖子拧到玻璃瓶上,并将电线的高度设置在营养溶液的正上方。将瓶子在121℃高压灭菌20分钟,然后使其冷却。


      图1.用于生长H的菌丝体的设备。 cylindrosporum 。 它由一个装有40毫升完整营养液体培养基的120毫升玻璃瓶制成。 A.每个罐子由放置在镍铬合金丝上的一个真菌菌丝体的塞子或盘接种。 B.培养条件下在培养基表面生长的菌丝体的方面
    2. 注射器支架,充气连接器和注射器的准备工作
      使用60ml注射器冲洗每个菌丝体,然后测量从标记的真菌到真菌培养基(IM)中的真菌


      32 P积累和/或 32 P流出(见食谱)。该冲洗步骤对于避免高估P积累和/或流出是至关重要的。通常,我们每次处理使用6个真菌重复,并且我们建立了自制设备(图2和3),以便于处理菌丝体。
      1. 注射器支架的准备(图2):
        根据图2中给出的尺寸切割不同的木材。例如,要保持6个注射器,顶部的长度为37厘米长x 6厘米,下面的长度为32厘米长×6厘米宽。调整并保持两个部件在一起,以双向对准钻孔。之后,将所有的部分组合在一起,如图2所示

        图2.支持用于在相互作用介质中孵育菌丝体的注射器的自制支架

      2. 连接器的准备(图3)
        首先,用一双镊子取18针针,将其衣领加热一段火焰,软化胶水并用另一对夹子拉。冷却后,调整针头顶部约3厘米长的小片硅胶管(内径1.14毫米)。拿一个裙帽,将6针缝合到其顶部,并通过1毫升移液管尖端将其底部关闭,先前在其最好的端部切割。如有必要,可将硅胶加入脆弱处,以加强系统的防风。在蓝色移液管尖端的切割端,加入约20厘米长的硅胶管(3.17毫米内径),随后将其插入空气泵和连接器之间的无菌空气过滤器(见图3)。最后,将整个系统[连接器+ PTFE管+大直径硅胶管]置于高压灭菌袋中,并通过高压灭菌(115°C,40分钟)灭菌。


        图3.在相互作用介质中培养菌丝体期间充气注射器的自制连接器

      3. 注射器的准备:取出每个注射器的柱塞并将其丢弃。使用热指甲,在与注射量相对应的刻度表相对的注射器顶部钻一个孔。该孔将用于将微管放置在注射器内进行通气。将所有钻孔的注射器放在高压灭菌袋中,并通过高压灭菌(115°C,40分钟)灭菌。

  2. 真菌培养物的制备
    注意:这是在无菌条件下,在层流柜中完成的。将软木塞和夹具进行火焰灭菌,并在使用前让其冷却。
    1. 步骤1:从真菌培养物中培养固体培养基中的新培养物 对于实验,我们使用大约每6个月更新的库存培养作为真菌材料的来源。这些真菌存活培养物在25℃下在琼脂营养培养基(固体N6培养基,见下文的配方)中在黑暗中生长在90mm培养皿中。培养基(硝酸盐)中的氮(N)源适应于这种真菌物种,因为它们在铵中不能很好地生长,与大多数外生菌根类型相反。
      在实验时,有必要通过将一个琼脂塞(直径7.5毫米)转移到含有25毫升固体N6完全培养基的新培养皿(直径90毫米)的中心,建立新的真菌培养物,菌丝体向下在营养介质上增强真菌的生长。新培养物在黑暗中培养3周,在培养箱中培养25℃
    2. 步骤2:从固体培养物中生长玻璃罐中的真菌 首先,通过在3周龄的菌丝体琼脂培养物中冲孔,用软木钻孔器准备真菌塞。
      然后,拧下一个瓶子的盖子,一只手握住;另一方面拿一对夹子,从固体培养基中取出真菌培养物塞;将插头插入镍铬合金线钩的尖端。将盖子妥善地拧在瓶子上,并将真空塞子放在介质的表面上,方法是将镍铬合金丝滑动(图1A)。真菌在25℃下生长两周。
    3. 步骤3:从液体培养物中生长玻璃罐中的真菌用于标记实验
      然后将在液体培养基中生长的2周龄的菌丝体用于生产用于标记实验的真菌材料。为此,如步骤B2中所述,真菌盘从菌丝体上冲出并放置在镍铬合金线钩的尖端上。该方法避免在真菌培养物中存在琼脂,因为它可以通过从标记溶液中积聚 32 P而将干扰标记实验,并将 32 P释放到非 - 标签解决方案无琼脂的菌丝体在25℃下生长7天,不摇动
  3. P预处理
    1. 营养液的制备
      制备+ P和-P营养液体培养基,用于真菌预处理,其为N6培养基,P(作为NaH 2 PO 4提供1mM,参见食谱)或不含P (通过省略在N6培养基中加入NaH 2 PO 4),每个菌丝体40ml。准备额外的N6培养基,不含P(每个菌丝体100ml)冲洗菌丝体(见图4B)。
    2. 罐子的准备
      在新罐中倒入40毫升液体培养基,即+ P或-P。在瓶子上没有镍铬丝的螺旋盖,如前所述将其高压灭菌。在1升瓶中加入另外的P N6培养基,并进行高压灭菌。
    3. 将菌丝体转移到新媒体
      将含有七日龄菌丝体的烧瓶装入流层层室,通过将镍铬合金线向上滑过盖子从介质中取出菌丝体。允许营养介质在盖子拧下之前从真菌滴落(图4A)。
      1. 仅对于-P处理,通过将培养基中的每个菌丝体依次转移到含有20ml新鲜-P培养基(图4B)的35mm培养皿中3分钟,从而冲洗每个菌丝体。该操作重复四次,这意味着为每个菌丝体制备5个培养皿,如图5所示。该步骤对于消除否则会以正磷酸盐(Pi)污染-P溶液的结转溶液是必需的。漂洗后,将每个菌丝体转移到含有40毫升P培养基的新烧瓶中,并允许它们在25℃下在黑暗中再生长5天以得到P贫化的菌丝体。
      2. 对于+ P处理,将刚刚允许滴入干燥的菌丝体转移到含有40ml + P培养基的新烧瓶中,而不进行漂洗。允许它们在与-P条件相同的条件下生长。


        图4.将菌丝体转移到新介质。 A.首先将镍铬合金丝线穿过盖子,以在盖子拧下之前让溶液从真菌滴落。 B.然后拧下盖子,将菌丝体在含有35mm培养皿的20ml P缺陷培养基中冲洗5次。


        图5.布置在层流柜中用于冲洗菌丝体的培养皿图片

  4. 标签与 32 P
    1. 安全预防措施:请遵照以下安全措施进行实验:放射性物质的处理,主要是(i)戴手套,实验室外套和剂量计来控制身体接收的辐射剂量,(ii)放置放射性物质或材料在有机玻璃屏幕后面,(iii)使用带有过滤器的吸头,(iv)丢弃特定容器中的放射性溶液或材料,(v)将这些容器放在有机玻璃屏幕后面,(vi)在一段时间后消除废物是至少10倍衰变期的 32 P,即140天,只有当废物的放射性不高于背景的两倍(通常为每秒0.8到1.6次)时。
    2. 准备一套具有盖子的罐子,没有填充40ml + P培养基的镍铬合金线,并至少在标签实验前一天对其进行高压灭菌。将有机玻璃屏幕和防护纸放在工作台上,打开有机玻璃屏幕后面的罐子。通过快速添加KH 2 32 PO 4 标记具有 32 P正磷酸盐的介质。稀释放射性标记的来源,以便在+ P培养基中获得3.3×10 4个/μm2 Pq /μmolPi的初始活性。通过手摇摇瓶来均化溶液,并取一个子样品(150μl)来测量介质中的实际放射性。将子样品在-20°C的有机玻璃盒中储存在1.5 ml Eppendorf管中。使用50μl进行放射性测量。
    3. 将带有菌丝体的罐子放在带有标记介质的罐子旁边。让真菌滴干(如图4A所示)。将带有真菌的盖子放在带放射性标记的培养基的罐中,拧上它并将镍铬合金丝滑下,直到真菌接触溶液表面。允许真菌在25°C,黑暗中孵育16小时。

  5. 在注射器中冲洗菌丝体
    1. 将注射器放在长凳上的支架上,并装入PTFE微管进行曝气(图6)。在每个注射器中,倒入60 ml的相互作用介质(IM),并将收集容器放在水龙头下面。将连接器插入空气泵,并检查IM是否充分通风。但是,在清洗菌丝体时请关闭空气泵。将有机玻璃屏幕放在支架前。
    2. 将带有菌丝体的罐子放在屏幕后面的标签溶液中,一个瓶子在一个注射器前面。使标记的溶液从每个菌丝体滴落约5分钟(如图4A所示)。拧下盖子,将真菌丝从盖子上滑下,将其放入注射器中。弯曲镍铬合金线,以将菌丝悬挂在相当于20毫升的水平(见图6)。
    3. 将所有菌丝体悬挂在注射器中后,打开水龙头,使IM流入注射器下方的收集容器中。关闭水龙头,并在每个注射器中倒入约30 ml的新IM。 5分钟后再次打开水龙头,使IM进入收集容器。重复相同的操作四次,添加新的IM后10,15,20和40分钟后,开关即可打开。在整个期间,每个菌丝体用150ml的IM冲洗。在每个漂洗时间,收集1毫升IM,并将其放入6毫升液体闪烁瓶中以测量放射性
    4. 在90分钟漂洗期后,您可以对6个菌丝体进行采样,以测量其鲜重和它们的含量。您还可以使用菌丝体与植物一起孵化,以随时间测量IM中的真菌32P外排(参见Becquer等人,2017)。


      图6.标签期后用于冲洗菌丝体的完整装置。 :一种。含有相互作用介质的注射器; B.聚碳酸酯阀;收集船D.菌丝体镍铬丝F. Microtube聚四氟乙烯PTFE单独曝气; G.自制连接器,用于收集4到7个特氟纶管;无菌过滤器I.空气泵。

  6. 放射性测量
    1. 在含有IM或标记培养基的每个6ml小瓶中加入4ml闪烁液,关闭小瓶并用手倒置数次。通过在小瓶中倒入4毫升闪烁液制成空白样品。按照制造商的说明,使用液体闪烁计数器测量放射性。
    2. 为了测量真菌中的放射性,请在滤纸之间吸收每个菌丝体并记录其新鲜重量。根据菌丝体的大小,将其放入6 ml或20 ml闪烁瓶中,并分别加入4或12 ml闪烁鸡尾酒进行计数。
    3. 使用Excel中编写的方程式来确定放射性测量期间发生衰变的原始数据:
      cpm t0 = Exp(Ln(cpm t -cpm background ))+(0.00003366099 x t),带有
      cpm =每分钟计数
      t0 =开始实验的时间
      t =计数器中样品的cpm测量时间(分钟)
      cpm 背景 =溶剂的cpm(空白样品)
      cpm =样品在时间t的放射性
      在对应于第一测量的时间t0的样品的放射性为cpm 。该值作为所有测量的参考值。
    4. 在Becquerel(Bq)中使用以下公式转换cpm:
      1 Bq = 1 cpm x 0.033
    5. 假设磷酸盐的浓度为1mM,计算提供给真菌(复制品)的磷酸盐的比活性(重复次数):
      (Bq /μmole磷酸盐)=用于测量的标记培养基/体积的体积(ml)×[0.033]
    6. 计算在暴露于 32 P期间由真菌(复制品i)摄取的P的量如下:
      真菌中的P积累(μmole)=(cpm x 0.033)/ SA

数据分析

由菌丝体释放或在菌体中累积的每个菌丝体或每克鲜重的表达量 32 P(在Bq中)。您还可以在16小时孵化期间将数据表达为真菌积累的磷酸盐的数量,并计算出Pi的相应摄取率。如果您比较了几种治疗方法,则使用Kolmogorov Smirnov检验来测试数据的正常性,并在必要时将数据转换为平方根或log10,然后进行分析以满足ANOVA的假设。为了比较治疗效果,使用ANOVA分析。

笔记

  1. 由于液体培养基中真菌的生长可能不同,我们准备最多4个接种真菌的培养瓶,以丢弃生长不良的培养瓶。
  2. 通过PTFE微管提供的曝气可能由于液体弯液面而倒塌。通过分配一个完全充满空气的60 ml注射器,并将空气强力推入微管,可以将它们从微管中拉出。
  3. 注射器中的微管的高度可能需要调整以允许空气离开。如果它们在介质中太深,空气就不会流动。

食谱

  1. 微量元素(1000毫升)
    3.08g MnSO 4•H 2 O
    4.41g ZnSO 4•7H 2 O
    2.82g H 3 BO 3
    0.98g CuSO 4•5H 2 O
    0.29g Na 2 MoO 4•2H 2 O
    将ddH 2 O加入到1000 ml中,保存在4°C
  2. 矿物盐碱溶液(各100毫升)


  3. 硫胺素溶液(100 ml)
    0.01克硫胺素-HCl
  4. N6完全液体溶液(1000毫升)
    1. 加入1L烧杯中约500ml去离子水和以下体积的矿物溶液:
      6ml KNO 3
      1ml NaH 2 PO 4
      1ml MgSO 4
      4 ml KCl
      0.5ml CaCl 2
      0.5毫升柠檬酸铁铵/ /
    2. 加入微量元素0.2 ml和硫胺素溶液0.5 ml
    3. 完成体积至1升,并检查pH值应调整为5.5
    4. 最后加入5g D-葡萄糖,摇匀至完全溶解
    5. 这种解决方案分布在玻璃罐中
  5. 固体N6完全液体溶液(1000毫升)
    拿两个1升玻璃瓶:
    1. 加入7.5g琼脂,并在每个瓶子中倒入500ml液体N6培养基
    2. 在121℃下灭菌20分钟
    3. 将冷却的介质(55-60°C)倒入90 mm直径的培养皿中
  6. 相互作用介质(3000 ml)
    1. 加入3LL烧杯中约1.5L去离子水和以下体积的矿物溶液:
      0.6ml MgSO 4
      1.5ml CaCl 2
      3.2g MES(终浓度为5mM)
      1.82克TRIS(终浓度为5 mM)
    2. 用去离子水完成3升,并用1NH 2 SO 4将pH调节至5.5。
    3. 将1.5升培养基倒入2升玻璃瓶中,并通过高压灭菌(20分钟,121℃)消毒

致谢

这项研究得到INRA(法国)的支持,通过为研究人员提供的年度资金和通过授予Adeline Becquer的年轻科学家合同(CJS)和CONACYT(墨西哥)通过授予玛格丽塔•托雷斯 - 阿基诺。该协议根据我们以前的工作(Torres-Aquino等人,2017)改编。我们还感谢三位匿名评审员的意见,帮助我们改进了协议。

参考

  1. Becquer,A.,Torres-Aquino,M.,LeGuernevé,C.,Amenc,L.K.,Trives-Segura,C.,Staunton,S.,Quiquampoix,H.and Plassard,C。(2017)。 建立培养的外生菌根真菌和植物之间的共生接口,以遵循真菌磷酸盐代谢。生物原菌 7(20):e2577。
  2. 凯恩,J. W. G.(2011)。 外生菌根真菌:森林土壤中磷的根系共生途径。 a>植物土壤 344:51-71。
  3. Plassard,C.和戴尔,B。(2010)。 菌根营养的磷营养。树生理学30 / 9):1129-1139。
  4. Smith,S.E。和Read,D.J。(2008)。 菌根共生第3版。 学术出版社。
  5. Smith,S.E.,Anderson,I.C。和Smith,F.A。(2015)。 菌根协会和磷获取:从细胞到生态系统。 年度植物评论 48:409-440。
  6. Torres-Aquino,M.,Becquer,A.,Le Guerneve,C.,Louche,J.,Amenc,L.K.,Staunton,S.,Quiquampoix,H.and Plassard,C.(2017)。 寄主植物Pinus pinaster对外生菌根真菌Hebeloma borrosporum的磷酸盐外排和多磷酸盐代谢发挥特殊的影响:放射性示踪剂,细胞学染色和31 P NMR光谱研究。植物细胞环境40(2):190-202。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Becquer, A., Torres-Aquino, M., Le Guernevé, C., Amenc, L. K., Trives-Segura, C., Staunton, S., Quiquampoix, H. and Plassard, C. (2017). A Method for Radioactive Labelling of Hebeloma cylindrosporum to Study Plant-fungus Interactions. Bio-protocol 7(20): e2576. DOI: 10.21769/BioProtoc.2576.
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