Materials and reagents

Biological materials

Maize seeds (Variety HQPM-5 from Indian Agriculture Research Institute, Pusa, New Delhi, India)
Serendipita indica fungus (strain DSM11827 gifted from Prof. Ajit Verma) (Varma, Kost, Rexer & Franken, 1997, European patent office, Muenchen, Germany. Patent No. 97121440.8-2105, Nov 1998)

Reagents

Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 1310-58-3)
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 7647-01-0)
Phenol (Sigma-Aldrich, catalog number: 108-95-2)
Lactic acid (Sigma-Aldrich, catalog number: 50-21-5)
Glycerol (Sigma-Aldrich, catalog number: 56-81-5)
Trypan blue (Sigma-Aldrich, catalog number: 72-57-1)
Glucose (Sigma-Aldrich, catalog number: 50-99-7)
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: 10043-52-4)
Ferrous chloride (FeCl₂) (Sigma-Aldrich, catalog number: 7705-08-0)
Calcium nitrate [Ca(NO₃)₂] (Sigma-Aldrich, catalog number: 13477-34-4)
Magnesium sulfate (MgSO₄.7H₂O) (Sigma-Aldrich, catalog number: 7487-88-9)
Potassium nitrate (KNO₃) (Sigma-Aldrich, catalog number: 7757-79-1)
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: 7778-77-0)
Boric acid (H₃BO₃) (Sigma-Aldrich, catalog number: 10043-35-3)
Manganese sulfate monohydrate (MnSO₄.H₂O) (Sigma-Aldrich, catalog number: 10034-96-5)
Copper sulfate (CuSO₄.5H₂O) (Sigma-Aldrich, catalog number: 7758-98-7)
Zinc sulfate (ZnSO₄.7H₂O) (Sigma-Aldrich, catalog number: 7733-02-0)
Ammonium molybdate [(NH₄)₂MoO₄] (Sigma-Aldrich, catalog number: 13106-76-8)
Sodium-hypochlorite (NaClO) (Sigma-Aldrich, catalog number: 7681-52-9)
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 7447-40-7)
Manganese(II) chloride (MnCl₂) (Sigma-Aldrich, catalog number: 7773-01-5)
Iron(II) sulfate heptahydrate (FeSO₄.7H₂O) (Sigma-Aldrich, catalog number: 7782-63-0)
Cobalt(II) chloride (CoCl₂) (Sigma-Aldrich, catalog number: 7646-79-9)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 7647-14-5)
Sucrose (Sigma-Aldrich, catalog number: 57-50-1)
Ethylenediaminetetraacetic acid ferric sodium salt (NaFeEDTA) (Sigma-Aldrich, catalog number: 15708-41-5)
Potassium iodide (KI) (Sigma-Aldrich, catalog number: 7681-11-0)
Manganese(II) chloride (MnCl₂) (Sigma-Aldrich, catalog number: 7773-01-5)
Sodium molybdate dihydrate (Na₂MoO₄.2H₂O) (Sigma-Aldrich, catalog number:10102-40-6)
Glycine (Sigma-Aldrich, catalog number: 56-40-6)
Ammonium phosphate dibasic [(NH₄)₂HPO₄] (Sigma-Aldrich, catalog number: 7783-28-0)
Iron(III) chloride (FeCl₃) (Sigma-Aldrich, catalog number: 7705-08-0)
Ethanol (Sigma-Aldrich, catalog number: 64-17-5)
Peptone (Sigma-Aldrich, catalog number: 73049-73-7)
Yeast extract (Sigma-Aldrich, catalog number: 8013-01-2)
Casamino acid (Sigma-Aldrich, catalog number: 65072-00-6)
Agar (Sigma-Aldrich, catalog number: 9002-18-0)
BD Difco^TM Bacto^TM Agar (Sigma-Aldrich, catalog number: DF0140-15-4)
Biotin (Sigma-Aldrich, catalog number: 7646-79-9)
Nicotinamide (Sigma-Aldrich, catalog number: 98-92-0)
Pyridoxal phosphate (Sigma-Aldrich, catalog number: 853645-22-4)
Aminobenzoic acid (Sigma-Aldrich, catalog number: 150-13-0)
Riboflavin (Sigma-Aldrich, catalog number: 83-88-5)
Thiamine hydrochloride (Sigma-Aldrich, catalog number: 67-03-8)
Pyridoxine hydrochloride (Sigma-Aldrich, catalog number: 58-56-0)
Nicotinic acid (Sigma-Aldrich, catalog number: 59-67-6)
Trypticase peptone (Sigma-Aldrich, catalog number: 91079-40-2)
Malt extract (Sigma-Aldrich, catalog number: 8002-48-0)
Myo-Inositol (Sigma-Aldrich, catalog number: 87-89-8)

Solutions

Sterile water (1 L)
Double-distilled water (DI water) (1 L)
Half-strength modified Hoagland solution (1 L) (see Recipes)
Ethanol (1 L) 70% (w/v) (see Recipes)
Sodium hypochlorite (NaClO) solution (1 L) 0.75% (w/v) (see Recipes)
0.8% Bacto agar (500 mL) (see Recipes)
Lactophenol (50%) (see Recipes)
Trypan blue (100 mL) (see Recipes)
10% KOH (100 mL) (see Recipes)
Aspergillus modified medium (AMM) or Kaefer (KF) medium (1 L) (see Recipes)
MN-Agar medium (1L) (see Recipes)

Recipes

Half-strength modified Hoagland solution (1 L)

Reagent	Final concentration	Quantity
Macronutrients
Ca(NO₃)₂·4H₂O	4 mM	944.5 mg
MgSO₄·7H₂O	2 mM	492.9 mg
KNO₃	6 mM	606.6 mg
KH₂PO₄	1 mM	136.08 mg
Micronutrients
H₃BO₃	45 μM	2.78 mg
MnSO₄·4H₂O	20 μM	3.01 mg
CuSO₄·5H₂O	0.4 μM	99.87 mg
ZnSO₄·7H₂O	0.7 μM	201.32 mg
(NH₄)₂MoO₄·2H₂O	0.2 μM	41.18 mg
H₂O		1,000 mL
Total		1,000 mL

70% ethanol (1 L)
Reagent Quantity
Ethanol (absolute) 700 mL
H₂O 300 mL
Total 1,000 mL

0.75% sodium-hypochlorite (NaClO) (bleach) solution (100 mL)
Reagent Quantity
Sodium hypochlorite solution (6%) 12.5 mL
ddH₂O 87.5 mL
Total 100 mL

0.8% Bacto agar (500 mL)
Reagent Quantity
Bacto agar 4g
ddH₂O 500 mL
Total 500 mL

Lactophenol (50%)
Reagent Quantity
Phenol 150 mL
ddH₂O 150 mL
Lactic acid 125 mL
Glycerol 125 mL
Total 600 mL

Trypan blue (100 mL)
Reagent Quantity
Trypan blue 0.1 g
Lactophenol 100 mL
Total 100 mL

10% KOH (100 mL)
Reagent Quantity
KOH 10 g
ddH₂O 100 mL
Total 100 mL

Aspergillus modified medium/Kaefer medium (1 L)

Reagent	Quantity
Glucose	20 g
Peptone	2 g
Yeast Extract	1 g
Casamino acid	1 g
Vitamin stock solution	1 mL
Macro-element stock solution	50 mL
Micro-element stock solution	2.5 mL
CaCl₂ (0.1 M)	1 mL
FeCl₂ (0.1 M)	1 mL
Agar	10 g
pH (Adjust with 1 N HCl)	6.5
ddH₂O	944.5 mL
Total	1,000 mL
Macro-elements stock
KCl	10.4 g
MgSO₄·7H₂O	10.4 g
KH₂PO₄	30.4 g
ddH₂O	1,000 mL
Total	1,000 mL
Micro-elements stock
ZnSO₄·7H₂O	22 g
H₃BO₃	11 g
MnCl₂·4H₂O	5 g
FeSO₄·7H₂O	5 g
CoCl₂·6H₂O	1.6 g
CuSO₄·5H₂O	1.6 g
ddH₂O	1,000 mL
Total	1,000 mL
Vitamin stock
Biotin	5 mg
Nicotinamide	50 mg
Pyridoxal Phosphate	10 mg
Aminobenzoic Acid	10 mg
Riboflavin	25 mg
ddH₂O	100 mL
Total	100 mL
0.1 M FeCl₂
FeCl₂	1.62 g
ddH₂O	100 mL
Total	100 mL
0.1 M CaCl₂
CaCl₂	1.11 g
ddH₂O	100 mL
Total	100 mL

MN-Agar medium (1 L)

Reagent	Quantity
NaCl	23.4 mg
KNO₃	80 mg
Ca(NO₃)₂·4H₂O	288 mg
Sucrose	10 g
NaFeEDTA	8 mg
KI	0.8 mg
MnCl₂·4H₂O	6 mg
Na₂MoO₄·2H₂O	0.0024 mg
Glycine	3 mg
KH₂PO₄	272.2 mg
(NH₄)₂HPO₄	40.8 mg
CaCl₂	81.6 mg
MgSO₄·7H₂O	731 mg
FeCl₃	583.9 mg
Thiamine hydrochloride	67.4 mg
Pyridoxine hydrochloride	67.4 mg
Nicotinic acid	0.5 mg
Trypticase peptone	1 g
Glucose	10 g
Malt extract	50 g
Myo-Inositol	50 mg
Bacto agar	10 g
KCl	65 mg
H₃BO₃	1.5 mg
MnSO₄·H₂O	6.0 mg
ZnSO₄·7H₂O	2.7 mg
CuSO₄·5H₂O	0.2 mg
pH	5.8
Total	1,000 mL

Notes:

All the stock solutions are stored at 4 °C and the vitamin stock at -20 °C. The stock of FeSO₄·7H₂O is prepared separately.
Filter sterilize the vitamin stocks.

Laboratory supplies

Gloves (Genesee Scientific, catalog number: 44-100M)
Lab coat (Fisher Scientific, catalog number: 19-181-570)
Pipettes of different sizes (Fisher Scientific, catalog number: 13-690-029)
Tip boxes (Fisher Scientific, catalog number: 01-670-713)
1 L Glass bottles (Fisher Scientific, catalog number: 13951L)
100 mL Glass bottles (Fisher Scientific, catalog number: 06-414-1A)
Petri dishes 20mm (Fisher Scientific, catalog number: 08-757-099)
Petri dishes 150mm (Fisher Scientific, catalog number: 50-403-868)
Plastic trays (Fisher Scientific, catalog number: 11-394-455)
Blotting papers (Fisher Scientific, catalog number: 09-301-199)
Germination paper (Fisher Scientific, catalog number: NC1466201)
Spreader (Fisher Scientific, catalog number: 14-665-230)
Beaker (Fisher Scientific, catalog number: 07-250-056)
250 mL flask (Fisher Scientific, catalog number: 10-040F)
Surgical blade (Fisher Scientific, catalog number: 22-079-774)
Muslin cloth ( Amazon.in, item model number: HAZC017639)
Nail paint (Amazon.in, item model number: CC4407)

Equipment

Laminar flow hood (Thermo Scientific^TM, catalog number: 1323TS)
Light microscope (Leica Microscope, Type 020-518.500, Germany and Nikon Eclipse Ti)
Autoclave (Thymol autoclave, India, product code: TAI-902)
Incubator shaker (Multitron Incubator Shaker, HT-Infors, Switzerland)
Glass house (School of Life Sciences, Jawaharlal Nehru University, New Delhi India)

Software and datasets

Microsoft Office Excel 10
GraphPad Prism 8

Procedure

Preparation of stock solutions
1. To ensure the successful execution of all procedures outlined in this protocol and the proper maintenance of the S. indica culture, it is necessary to prepare the listed stock solutions mentioned above, and then prepare working solutions and mixtures in sterile water and store in glass bottles at room temperature, as required during the different steps.
2. Sterilize the KF medium using the autoclave at 121.6 °C under 15 psi for 20 min before plating.

Maintenance and growth of fungal species (S. indica)
1. Prepare 25 mL of KF-agar medium in a 100 mL glass bottle and autoclave it.
2. Add the vitamin stock into this KF-agar medium at a temperature of 40 °C (kept in a water bath) and mix gently to avoid bubble formation.
3. Pour this KF-agar medium into a 90 mm Petri dish in a laminar flow hood.
4. Allow 20–30 min to complete the solidification of the KF media in the Petri dish inside the laminar flow hood. Note: To improve solidification and prevent water droplet accumulation inside the lids, it is recommended to keep the Petri dish lid open.
5. Inoculate a single ball/colony of S. indica from the liquid KF medium (the sample has already been incubated in a shaker incubator at 30 ± 2 °C for 7–9 days with shaking at 100 rpm) or cut a slice sized 5 × 5 mm of pregrown S. indica on a solid dish culture using a sterilized surgical blade and place this slice of KF-agar-containing S. indica culture on the center of the 90 mm fresh KF medium Petri dish.
  Note: A fine pressure can be applied onto the slice to make sure the S. indica slice can make full contact with the fresh KF medium.
6. Incubate the dishes at 28–30 °C incubator in darkness for 5–7 days for full growth.
7. After incubation, observe the growth of the S. indica by visualizing the grey-colored, cotton-like growth (see the right panel of Figure 1).
  
  Figure 1. Schematics showing how to inoculate the S. indica on a solid KF-agar 90 mm Petri dish
8. Now, S. indica is ready for the preparation of chlamydospores and further colonization study with maize seedlings.
  Note: For liquid broth medium growth, S. indica was maintained routinely on solidified Aspergillus (Aspergillus niger) modified medium (Hill and Kafer, 2001). Growth of S. indica was studied in 250 mL culture flasks with constant shaking at 100 rpm, and at 30 ± 2 °C for 7–9 days in a metabolic shaker (Multitron Incubator Shaker, HT-Infors, Switzerland).

Maize seed surface sterilization and germination
1. Take the required quantity of desired functional maize seeds (we used the HQPM5 variety from IARI, New Delhi, India).
2. Take approximately 120 seeds (in our case) and dip them into 100 mL of autoclaved DD water for 1 h using a 250 mL beaker.
3. Wash the maize seeds with detergent (teepol or Triton X-100) using 3–5 drops in 50 mL of water.
4. Repeat washing of the maize seeds with autoclaved DD water by changing the water approximately 5 times for a total of 10 min.
5. Sterilize the maize seeds by soaking them in a 250 mL beaker containing 70% ethanol for 40 s, manually swirling it.
6. Remove the ethanol and wash five times with autoclaved DD water for a total of 10 min.
7. Again, sterilize the maize seeds by soaking them in a 100 mL beaker containing 50 mL of 6% NaOCl (final concentration 0.75%) for 1 min, manually swirling it.
8. Wash the maize seeds with autoclaved DD water six times for 10 min.
  Note: In this final washing, make sure there is no remaining NaOCl.
9. Transfer the maize seeds into a new beaker and heat it in a water bath at 60 °C for 5 min.
10. Place the maize seeds in water agar dishes/germination sheets using sterile forceps, spacing them 1 inch apart for germination at 28 °C in the dark for 3 days (10–15 seeds per dish).
  Note: Use bi size (150 mm) glass or plastic Petri dishes for making 0.8% water agar dishes (0.8% Bacto Agar, Difco, Detroit, MI, USA).

Preparation of chlamydospores of S. indica
1. Take the fully grown S. indica KF dishes (6–10 days old) to prepare the chlamydospores.
  Note: Use the completely grown S. indica on a KF medium and open the dish in laminar air flow.
2. Take approximately 2–3 mL of 2% filter-sterilized glucose solution prepared in DD water and pour on the top surface of the fully grown S. indica KF dishes.
3. Spread the 2% glucose solution gently using a sterile plastic spreader and aim to just collect the chlamydospores in the solution phase.
  Note: Try not to disturb the bottom part of the S. indica-containing hyphae.
4. Collect the solution containing chlamydospores in 2 mL Eppendorf tubes.
5. Filter this solution using a sterile muslin cloth to remove the fungal hyphae and any part of solid medium, transferring the chlamydospores to another tube.
6. Centrifuge the S. indica spores (in Eppendorf tubes) at 3× g at 30 °C for 3–4 h.
7. The quality of the spores can be checked by observation under a light microscope using glass slides.
8. The number of S. indica chlamydospores can be maintained at 5 × 10⁵/mL using a hemocytometer for further colonization with maize seedlings.

Colonization of S. indica with maize plants
1. Take out the germinated maize seedlings from the water agar dishes or germination sheets (3–4 days old) and wash the roots with sterilized water, then dip the roots in S. indica chlamydospore suspensions (maintained at 5 × 10⁵ spores per mL) to start the inoculation.
  Note: Forceps can be used to dip the roots into the S. indica chlamydospore suspensions.
2. Transfer the inoculated maize seedlings with the chlamydospores of S. indica to the sterile MN-agar medium.
3. Grow the inoculated maize seedlings in the MN medium for a minimum of 7 days for colonization.
4. Alternatively, grow the inoculated maize seedlings in pots filled with a mixture of sterile sand and soil in the ratio of 3:1.
5. Grow the inoculated plants under controlled conditions in a glasshouse with an 8 h light (1,000 Lux)/16 h dark period at a temperature of 28 °C with a relative humidity of 60%–70%.
6. Supplement the plants weekly with half-strength modified Hoagland solution.
7. For control plants (without S. indica inoculated), mock inoculate with autoclaved DD water–mixed sand. Check the S. indica colonization at different time points under the light microscope. In our case, plant roots were harvested at different dpi (5, 10, 15, and 20 days) and were assessed for colonization by trypan blue staining as shown below.

Histochemical analysis
1. Harvest the plant roots at 5, 10, 15, and 20 days after inoculation, and choose approximately 10–20 random samples of the root system for colonization estimation.
  Note: Use small surgical scissors to cut the root sample from the maize plants.
2. To soften the root samples, treat them with 10% KOH solution for 15 min and then acidify them with 1 N HCl for 10 min. Finally, stain them with 0.02% trypan blue overnight (Narayan et al., 2021).
3. Samples were destained with 50% Lactophenol for 1–2 h. Keep the samples on a rotary shaker.
4. Cut the root samples into 1 cm small fragments using the surgical blade and mount them on glass slides.
  Note: Label the glass slides with sample number and root number (see Figure 2).
  
  Figure 2. Schematics showing a glass slide mounted with small fragments of the S. indica-colonized maize roots after the trypan blue staining, for microscopic study
5. Cover the root samples with the glass coverslips, pressing the coverslips gently to crush the roots, and seal slides using nail paint (see Figure 2)
6. Observe these slides under a light microscope to see the blue-stained and pear-shaped chlamydospores of S. indica under the different magnifications. Please see Figure 3 to see how chlamydospores of S. indica look like in a colonized state inside the root cortical cells, under light microscope observation.
  
  Figure 3. Blue-colored chlamydospores of S. indica inside the maize root cortical cells, viewed under a light microscope.
7. Count the colonized root segments for further calculation.
8. The distribution of chlamydospores within the root was taken as an index for studying colonization. Percent colonization was calculated for the inoculated plants according to the method described previously (Narayan et al., 2017 and 2021). Specifically, it was calculated using the following formula. The calculation involved the total number of randomly selected roots, the number of colonized root segments observed under a microscope, and the total number of root segments taken from a random sample to observe chlamydospores under a microscope. Both biological and technical repeats were combined to calculate the standard deviation and determine the significance of the data. Table 1 provides an example of percent colonization.
  
  Table 1. Percent colonization of S. Indica with maize plant root
  Time after inoculation (days) Percent colonization (%)
  5 7.5 ± 5
  10 27.5 ± 9.6
  15 45 ± 12.9
  20 70 ± 11.5

Data analysis

Data can be analyzed using Excel. In our case, we used three biological and three technical repeats. Each time we used 120 seeds of maize. Statistical tests like standard deviation and standard error analysis can be done by using sigma plot or GraphPad Prism 8.

Validation of protocol

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

Kundu et al. (2022). Piriformospora indica recruits host-derived putrescine for growth promotion in plants. Plant Physiology. (Figure 6, panel h).
Narayan et al. (2021). Sulfur transfer from the endophytic fungus Serendipita indica improves maize growth and requires the sulfate transporter SiSulT. Plant Cell. (Figure 1, panel c).
Verma et al. (2022). Functional characterization of a high‐affinity iron transporter ( PiFTR ) from the endophytic fungus Piriformospora indica and its role in plant growth and development. Environmental Microbiology. (Figure 6, panel c).
Narayan et al. (2017). Antioxidant enzymes in chickpea colonized by Piriformospora indica participate in defense against the pathogen Botrytis cinerea. Scientific Reports. (Figure 1).
Jogawat et al. (2013). Piriformospora indica rescues growth diminution of rice seedlings during high salt stress. Plant signaling & behavior. (Figure 1, panel b).
Kumar et al. (2009). Antioxidant enzyme activities in maize plants colonized with Piriformospora indica. Microbiology. (Figure 1).

General notes and troubleshooting

General notes

The colonization efficiency of S. indica may vary depending on the maize cultivar, growth conditions, and chlamydospore quality.
Overstaining can hide the chlamydospores with the root tissues.
To improve colonization efficiency, use a fresh culture of S. indica.
Limitations associated with this protocol:

The colonization efficiency of S. indica may vary depending on the maize cultivar, growth conditions, and inoculation methods, which may affect the reproducibility of the results.
The use of high-throughput molecular techniques, such as RNA sequencing or metabolomics, may require a large amount of plant tissue, which may limit the application of this protocol to small-scale experiments.
This protocol only focuses on the early stages of maize growth, and the long-term effects of S. indica on plant growth and stress tolerance are still unclear.

Troubleshooting (Table 2)

Table 2. Troubleshooting

Common problems	Troubleshooting
Low colonization efficiency	Check the viability of the chlamydospores by growing on a liquid broth or solid KF-agar dishes and compare with normal S. indica growth.
Low germination efficiency	Check the quality of seeds. Avoid using very old, dormant seeds.
Chlamydospores are not visible in the microscope	Stain chlamydospores with trypan blue for a longer time.
Blue-colored chlamydospores are hidden in root tissues	Try to destain the roots for a longer time using Lactophenol.
Contamination of other fungal species	Ensure all equipment and reagents are in sterile conditions.

Acknowledgments

Jawaharlal Nehru University, New Delhi, and University Grants Commission, New Delhi India, ICMR, New Delhi, India is highly acknowledged for the financial support. We also acknowledge the original research paper in which this protocol was described and validated was published by Narayan et al., 2021, The Plant Cell (doi: 10.1093/plcell/koab006) from School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

Competing interests

All the authors declare no conflict of interests.

References

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