In Vitro Hyphal Branching Assay Using Rhizophagus irregularis

Materials and reagents

Biological materials

1. Rhizophagus irregularis DAOM197198 (4,000 spores/mL) (PremierTech)

1. Acetone (FUJIFILM WAKO Pure Chemical, CAS number: 67-64-1)

2. Ethanol (99.5%) (FUJIFILM WAKO Pure Chemical, CAS number: 64-17-5)

3. Gastrografin for oral/enema use (Bayer, 597.3 g/L amidotrizoic acid, CAS number: 117-96-4)

4. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (FUJIFILM WAKO Pure Chemical, CAS number: 10034-99-8)

5. Potassium nitrate (KNO₃) (FUJIFILM WAKO Pure Chemical, CAS number: 7757-79-1)

6. Potassium chloride (KCl) (FUJIFILM WAKO Pure Chemical, CAS number: 7447-40-7)

7. Potassium dihydrogen phosphate (KH₂PO₄) (FUJIFILM WAKO Pure Chemical, CAS number: 7778-77-0)

8. Calcium nitrate tetrahydrate [Ca(NO₃)₂·4H₂O] (FUJIFILM WAKO Pure Chemical, CAS number: 13477-34-4)

9. Sucrose (FUJIFILM, CAS number: 57-50-1)

10. Fe(III)-EDTA (NaFeEDTA·3H₂O) (DOJINDO, CAS number: 15708-41-5)

11. Potassium iodide (KI) (FUJIFILM WAKO Pure Chemical, CAS number: 7681-11-0)

12. Manganese chloride tetrahydrate (MnCl₂·4H₂O) (FUJIFILM WAKO Pure Chemical, CAS number: 13446-34-9)

13. Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (FUJIFILM WAKO Pure Chemical, CAS number: 7446-20-0)

14. Boric acid (H₃BO₃) (FUJIFILM WAKO Pure Chemical, CAS number: 10043-35-3)

15. Copper sulfate pentahydrate (CuSO₄·5H₂O) (FUJIFILM WAKO Pure Chemical, CAS number: 7758-98-7)

16. Disodium molybdate(VI) dihydrate (Na₂MoO₄·2H₂O) (FUJIFILM WAKO Pure Chemical, CAS number: 10102-40-6)

17. Potassium hydroxide (KOH) (FUJIFILM WAKO Pure Chemical, CAS number: 1310-58-3)

18. Glycine (FUJIFILM WAKO Pure Chemical, CAS number: 56-40-6)

19. Thiamine hydrochloride (FUJIFILM WAKO Pure Chemical, CAS number: 67-03-8)

20. Pyridoxine hydrochloride (FUJIFILM WAKO Pure Chemical, CAS number: 58-56-0)

21. Nicotinic acid (FUJIFILM WAKO Pure Chemical, CAS number: 59-67-6)

22. Myo-inositol (FUJIFILM WAKO Pure Chemical, CAS number: 87-89-8)

23. Phytagel (Sigma-Aldrich, CAS number: 71010-52-1)

24. rac-GR24 (StrigoLab, CAS number: 76974-79-3)

1. M medium (see Recipes) [16]

2. Calcium stock of M medium (see Recipes)

3. 1,000× vitamin stock of M medium (see Recipes)

4. 0.3% phytagel containing 3 mM MgSO₄·7H₂O (see Recipes)

1. M medium

   Reagent	Final concentration	Quantity or Volume
   MgSO4·7H2O	2.97 mM	731 mg
   KNO3	0.79 mM	80 mg
   KCl	0.87 mM	65 mg
   KH2PO4	0.035 mM	4.8 mg
   Sucrose	10% (w/v)	10,000 mg
   NaFeEDTA·3H2O	0.022 mM	9.18 mg
   KI	0.0045 mM	0.75 mg
   MnCl2·4H2O	0.030 mM	6 mg
   ZnSO4·7H2O	0.0092 mM	2.65 mg
   H3BO3	0.024 mM	1.5 mg
   CuSO4·5H2O	0.00052 mM	0.13 mg
   Na2MoO4·2H2O	9.92 nM	0.0024 mg
   Phytagel	4% (w/v)	4,000 mg
   H2O	n/a	1,000 mL
   Total (optional)	n/a	1,000 mL

   Note: Adjust pH at 5.5 using KOH solution and autoclave at 121 °C for 20 min. n/a, not applicable.




2. Calcium stock of M medium

   Reagent	Final concentration	Quantity or Volume
   Ca(NO3)2·4H2O	1.22 M	2,880 mg
   H2O	n/a	10 mL
   Total (optional)	n/a	10 mL

   Note: Sterilize the stock using a 0.45 μm filter and store at room temperature (23–25 °C) until use. n/a, not applicable.




3. 1,000× vitamin stock of M medium

   Reagent	Quantity or Volume
   Glycine	30 mg
   Thiamine hydrochloride	1 mg
   Pyridoxine hydrochloride	1 mg
   Nicotinic acid	5 mg
   Myo-inositol	500 mg
   H2O	10 mL
   Total (optional)	10 mL

   Note: Sterilize the mixture using a 0.45 μm filter and store at -30 °C until use.




4. 0.3% phytagel containing 3 mM MgSO₄·7H₂O

   Reagent	Final concentration	Quantity or Volume
   MgSO4·7H2O	3 mM	739.4 mg
   Phytagel	0.3% (w/v)	3,000 mg
   H2O	n/a	1,000 mL
   Total (optional)	n/a	1,000 mL

   Note: No need to adjust pH. Autoclave at 121 °C for 20 min. n/a, not applicable.

Laboratory supplies

1. 24-well plates (TrueLine, catalog number: TR5002)

2. 1.5 mL microcentrifuge tube (BIO-BIK, catalog number: CF-0150)

3. 15 mL centrifuge tube (Labcon, catalog number: 3132-345)

4. 50 mL centrifuge tube (Labcon, catalog number: 3181-345)

5. 10 mL disposable serological pipette (ASONE, catalog number: 2-4131-14)

6. Cell strainer (40 μm mesh) (Falcon, catalog number: 352340)

7. 1 mL needleless plastic syringe (TERUMO, catalog number: SS-01T)

8. 0.45 μm PTFE filter (Shimadzu, catalog number: GLCTD-HPTFE1345)

9. Inspection film tape (aglis, catalog number: LP0002K)

Equipment

1. Electronic pipette (Labnet, model: FASTPETTE Pro)

2. Laminar flow hood (SANYO, model: MCV-B131S)

3. Centrifuge (KUBOTA, model: 3740)

4. Swing rotor for 50 mL centrifuge tubes (KUBOTA, model: SD-242)

5. Autoclave (TOMY SEIKO, model: LSX-300)

6. Vacuum concentrator (Thermo Fisher Scientific, model: Savant SpeedVac DNA130)

7. Plant growth chamber (NIPPON MEDICAL & CHEMICAL INSTRUMENTS, model: LH-411S)

8. Stereomicroscope (Olympus, model: SZX16) equipped with a digital camera (Olympus, model: DP-26)

9. Imaging software (Olympus, model: CellSens standard v1.18)

Software and datasets

1. Microsoft Office Excel 10

2. R v4.2.0 (https://www.r-project.org/)

Procedure

1. Spore separation from inoculum

   To observe and count hyphal branches, fragments of fungal hyphae should be removed from the R. irregularis spore suspension in advance. This step builds upon the protocol developed previously [23]. Maintain the suspension and collected spores axenic by working on a laminar flow food.

   1. Prepare 8%, 16%, 32%, and 50% (v/v) Gastrografin solutions in sterile 50 mL centrifuge tubes. Invert the tubes to mix the dense and thick reagents with sterile distilled water.

   2. In a new 50 mL centrifuge tube, pour each Gastrografin solution very gently using an electronic pipette equipped with a 10 mL disposable pipette as follows:

      1. First, pour 10 mL of 50% Gastrografin, followed by 5 mL of 32%, 16%, and 8% Gastrografin. Do not disturb the layers.

      2. Second, load 5 mL of spore suspension onto the surface of 8% Gastrografin softly. The spore concentration of the final solution can be changed.

   3. Centrifuge the tubes at 500× g for 10 min at room temperature in a swing rotor for 50 mL centrifuge tubes.

      Note: Decelerate the rotor slowly if possible.

   4. Collect the supernatant, including the upper three layers, using the electronic pipette (Figure 1).

      Note: R. irregularis spores must be floating in the 16% Gastrografin layer, and hyphal fragments should be precipitated to the bottom of the tube. Spore density differs among AM fungal species. In the case of R. clarus, spores float in the 32% Gastrografin layer.

      
      

      
      Figure 1. Isolation of Rhizophagus irregularis spores from the inoculum

   
   

   5. Pour the supernatant onto a sterile cell strainer and rinse the collected spores with 10 mL of sterile distilled water three times.

   6. Put the cell strainer on a Petri dish filled with sterile distilled water and move the spores to a new 15 mL centrifuge tube.

      Note: Ideally, adjust spore concentration to approximately 2,000 spores/mL.

   7. Keep the spore suspension at 4 °C in the dark.




2. Preparation of germinating R. irregularis spores and chemical treatment

   1. Add 1 mL of sterile calcium stock of M medium and 1 mL of 1,000× vitamin stock of M medium (see Recipes) to 1 L of autoclaved M medium.

   2. Pour 350 μL of M medium into each well of a sterile 24-well plate and keep the plate still until the gel solidifies.

   3. Dilute the purified spore suspension to approximately 1,000 spores/mL with sterile distilled water.

   4. Drop an 8 μL aliquot of the purified spore suspension onto the center of each M medium.

   5. After placing spores on the media, check if approximately 8 spores are placed on the medium center using a stereomicroscope. Adjust the number under a laminar flow condition if one well contains more or fewer spores. In case of more spores, remove extra spores by sucking them with a 200 μL micropipette.

   6. Open the 24-well plate in the laminar flow hood for approximately 5 min to evaporate excessive water (Figure 2).

      Note: The spores move around on the medium surface if too much water remains.

   7. Pour 150 μL of 0.3% phytagel containing 3 mM MgSO₄·7H₂O (see Recipes). The gel should be cooled sufficiently in advance to allow it to be touched (approximately 40 °C). The 0.3% phytagel should be poured gently into the perimeter of the M medium using a micropipette (see Figure 2).

      
      

      
      Figure 2. In vitro culture of Rhizophagus irregularis spores and chemical treatment. A. R. irregularis spores (approximately 8 spores) are placed on the center of each well filled with M medium. After drying off excess water, cool 0.3% phytagel with 3 mM MgSO₄·7H₂O is poured gently into the perimeter of the M medium to cover the spores. B. To exchange the solvent of rac-GR24 (positive control) from acetone to distilled water, the acetone stock is centrifuged in vacuo. The residues are redissolved in distilled water and sterilized using the 0.45 μm PTFE filter. Images show germinating R. irregularis spores (arrowheads) treated with H₂O (C) and 100 nM GR24 (D) for 5 days. E. The number of hyphal branches in R. irregularis treated with H₂O and 100 nM GR24. Dots and error bars represent individual values and the standard deviation, respectively (n > 15). The E-value was lower than 0.05 in the Wilcoxon rank-sum test.

   
   

   8. Close the 24-well plate lid and seal it with two layers of inspection film to prevent the dehydration of the gels.

   9. Place the plate horizontally in a growth chamber at 25 °C and incubate it for 5 days in the dark.

      Note: In our condition, R. irregularis spores usually germinate in this period.

   10. After 5 days, treat 200 μL of chemicals diluted in sterile distilled water onto each gel containing germinating spores. Use the positive control rac-GR24 diluted in acetone as follows:

       1. Add an 8 μL aliquot of 10 mM rac-GR24 in a 1.5 mL tube.

       2. Centrifuge the tube in a vacuum concentrator at 35 °C for 5 min. If acetone is left in the tube, adjust the evaporation time to remove the solvent completely.

       3. After evaporating the solvent, add 800 μL of distilled water to the tube.

       4. In the laminar flow hood, sterilize the 100 nM rac-GR24 using a 1 mL needleless syringe with a 0.45 μm PTFE filter.

       5. Pour 200 μL of the 100 nM rac-GR24 solution onto the gels.

   11. Seal plates twice with inspection film tape and place them in the same growth chamber at 25 °C. Incubate plates for 7–10 days in the dark.




3. Quantification of R. irregularis hyphal branching

   1. Under a stereomicroscope, count the number of hyphal branches except for that of the initially elongating hypha (Figure 3). Remove the 24-well plate lid if it becomes misty during observation.

      Notes:

      1. Count hyphal branches from the runner hyphae (see Figure 3A, B).

      2. Ignore the short hyphae formed at the boundary of the subtending and runner hyphae. The hyphae often show vigorous branching without any chemical treatment (Figure 3C, D).

      3. Basically, the hyphal branches of a single spore are considered to be one biological replicate. If multiple spores are tied with a subtending hypha, it is counted as one germinating spore (see Figure 3B).

   2. Check the differences among treatments using a statistical calculation (see Data analysis).

      
      

      
      Figure 3. Hyphal structures and growth patterns of Rhizophagus irregularis. A, B. Count the number of hyphal tips emerging from runner hyphae to quantify the hyphal branching. When you find multiple spores tied with a subtending hypha (B, right drawing), count them as one biological replicate. C, D. Germinating spores with highly branching hyphal clusters near the subtending hyphae. Exclude these spores from your quantification. Arrows indicate germinating R. irregularis spores drawn in (B) and (D).

Data analysis

Data can be analyzed using Microsoft Excel. We usually prepare three or four wells containing germinating spores for each treatment. For statistical analysis, R is used to perform Wilcoxon’s rank-sum test with a Bonferroni correction in the case of multiple comparisons. Approximately 20 spores per treatment at least should be assessed. The p-value cutoff is 0.05.

Validation of protocol

This protocol or parts of it has been used and validated in the following research article:

1. Tominaga et al. [24]. Monoterpene glucosides in Eustoma grandiflorum roots promote hyphal branching in arbuscular mycorrhizal fungi. Plant Physiology (Figure 3).

General notes and troubleshooting

General notes

1. Hyphal fragments should be removed as much as possible because some hyphal branches will emerge from them and this will mask the germinating spores.

2. Many spores, i.e., exceeding 8, and a longer (more than 10 days) incubation period can make hyphal branches indistinguishable. In addition, R. irregularis likely develops more hyphal branches when the spores are crowded in a medium, although the reasons remain unclear.

3. Exposing spores and hyphae to air influences R. irregularis growth and branching. The AM fungal spores exhibit straight hyphal elongation and moderate hyphal branching, although we are unsure why.

Troubleshooting

Problem 1: Spores do not germinate well.

Possible cause: The spore suspension includes many dead spores. Alternatively, phytagel is insufficiently cooled.

Solution: Increase the number of spores or prepare a new spore suspension. Make sure to pour sufficiently cooled phytagel onto the spores.

Problem 2: Cannot count the hyphal branches due to a complex branching pattern.

Possible cause: Each well may contain too many germinating spores, or the incubation period might be unnecessarily long.

Solution: Use fewer spores and shorten the period of chemical treatment. Instead, prepare more wells to assess sufficient spores (>10). Importantly, a germinating spore showing massive branches around subtending hypha without a long runner hypha can be ignored from the quantification. We observe germinating spores with traceable hyphal elongation and branching.

Acknowledgments

This protocol was developed by modifying a previous method [1]. The authors thank Dr. Hiromu Kameoka (China Academy of Science, China) for useful discussions on the in vitro culture of R. irregularis. This work was supported by the Japan Society for the Promotion of Science (JSPS) (KAKENHI grant numbers 20J21994 and 23KJ1578). The graphical abstract and figures were created using Biorender.com.

Competing interests

The authors declare that they have no competing financial or non-financial interests.

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