Production of Arbuscular Mycorrhizal (AM) Fungal Inoculum and Phenotypic Evaluation of Rice and AM Symbiosis Under Saline Conditions

Summary

This article describes a protocol for generating arbuscular mycorrhizal (AM) fungi inoculum to investigate AM-enhanced salt stress tolerance in rice.

Abstract

Rice (Oryza sativa L.) is a vital food crop for more than half of the global population. However, its growth is severely impacted by saline soils, which present a significant challenge to crop production worldwide. Arbuscular mycorrhizal (AM) fungi, which form mutualistic symbiotic relationships with over 90% of agricultural plants and 80% of terrestrial plant species, have been shown to enhance the salt tolerance of rice plants. AM fungi are obligate symbionts that cannot complete their life cycle without a host root. Therefore, effectively utilizing plants to produce AM fungal inoculum is crucial for advancing research in this field. In this study, we present a series of robust methods that begin with generating sand inoculum containing spores of Rhizophagus irregularis using Allium tuberosum L. These methods include inoculating rice seedlings with the sand inoculum, analyzing the growth phenotype of mycorrhizal rice, and quantifying fungal colonization levels using trypan blue staining under salt stress. These approaches can efficiently generate AM fungal inoculum for further investigation into how AM symbiosis enhances the salinity tolerance of rice.

Introduction

Saline soil is a significant obstacle to crop production worldwide^1,2,3. Recent studies indicate that up to 50% of cultivated land will be degraded by 2050 due to salinization⁴. Salt-affected soils primarily cause toxicity in plants due to the accumulation of sodium (Na⁺) and chloride (Cl⁻) ions in plant tissues. These ions, which dominate saline soils, are also the most harmful to plants^5,6,7. For example, sodium inhibits many cytosolic enzyme activities⁸. Salt stress also affects photosynthetic efficiency and induces changes in ionic toxicity, osmotic pressure, and cell wall structure, collectively leading to the accumulation of reactive oxygen species (ROS)^{9,10,11,12,13}.

Arbuscular mycorrhizal (AM) symbiosis is an endosymbiotic association between fungi of the phylum Glomeromycota and plant roots, which evolved approximately 400-450 million years ago with the emergence of early land plants^14,15. Over 80% of vascular plants can be colonized by AM fungi¹⁶. This mutualistic relationship enhances plant nutrient uptake from the soil, thereby improving growth and stress tolerance^17,18,19,20. For example, during salt stress, AM fungi can maintain ion balance and help enhance water and nutrient availability, antioxidant activity, photosynthetic efficiency, and secondary metabolite production for plants^2,21,22,23. Additionally, AM symbiosis prevents excessive Na+ uptake and transport from roots to shoots, promoting the absorption of essential cations such as K⁺, Mg²⁺, and Ca²⁺. This process increases the Mg²⁺/Na⁺ or K⁺/Na⁺ ratio in plants under saline conditions^{23,24,25,26,27,28,29}.

Rice (Oryza sativa L.), a crucial food crop for over half of the global population, belongs to the family Gramineae (Poaceae) and is highly susceptible to salt stress³⁰. Studies have also highlighted the role of AM fungi in enhancing salt stress tolerance in rice^31,32,33. For instance, the AM fungus Claroideoglomus etunicatum improves the CO₂ fixation efficiency of rice (Oryza sativa L. cv. Puntal) under salt stress³¹. Moreover, the expression of key rice transporter genes associated with vacuolar sodium sequestration and Na+ recirculation from shoots to roots is enhanced in AM-colonized plants under salt stress³². Additionally, upland rice plants inoculated with Glomus etunicatum display enhanced photosynthetic capacity, elevated osmolyte production, improved osmotic potential, and greater grain yield under saline conditions³³. Our previous research also demonstrated that mycorrhizal rice (Oryza sativaL. cv. Nipponbare) exhibited better shoot and reproductive growth, a notably higher K⁺/Na⁺ ratio in the shoot, and improved reactive oxygen species (ROS) scavenging capacity due to AM symbiosis³⁴. These findings all demonstrate the positive impact of AM symbiosis on salt stress tolerance in rice through phenomic approaches. However, the experimental methods have not been published in video format.

AM fungi are obligate symbionts that require a host root to complete their life cycle, making the use of plants to produce AM fungal inoculum crucial for research progress³⁵. A substrate-based production system, where AM fungi are grown in substrates like vermiculite or sand and spores are collected for inoculum³⁶, offers a cost-effective solution for large-scale AM fungal inoculum production. The efficiency of spore production depends on plant compatibility and growth, which affect fungal colonization and propagation^37,38. However, this method is often time-consuming, with traditional approaches taking up to 120 days and yielding low spore production. Recent improvements have reduced the production period to 90 days using maize as the host plant under LED light conditions³⁹. However, a robust method is presented for generating sand inoculum containing spores of Rhizophagus irregularis using Allium tuberosum L. within 10 weeks. This sand inoculum can be used to analyze the growth phenotype of mycorrhizal rice and quantify fungal colonization levels using trypan blue staining under salt stress. These approaches efficiently generate AM fungal inoculum for further investigation into how AM symbiosis enhances the salinity tolerance of rice.

Protocol

The details of the reagents and the equipment used in this study are listed in the Table of Materials.

1. Generation of sand inoculum containing spores of Rhizophagus irregularis using Allium tuberosum L.

1. Wash sand with tap water and autoclave it.
2. Add 2/3 of the sand to a pot (top diameter 14.7 cm, bottom diameter 11.5 cm, height 13 cm). Add 1,000 spores of AM fungi Rhizophagus irregularis. Cover with a thin layer of sand. Add 30 seeds of garlic chives (Allium tuberosum L.) and cover the seeds with sand.
3. Grow the garlic chives in the chamber with a 16-h/8-h day/night cycle at 23.5 °C (55% relative humidity). During the first week (1-week post-inoculation, wpi), cover the garlic chives with alumina paper to block light and water them three times a week.
4. Starting from 2 wpi, fertilize the garlic chives twice a week with 80 mL half-strength Hoagland solution containing 25 µM KH₂PO₄. Fertilize once a week with 80 mL water.
5. After 10 weeks, harvest the roots of garlic chives for trypan blue staining to assess the level of fungal colonization. If the colonization level exceeds 70%, stop watering the garlic chives until the sand is dry (about 5 weeks). Put all sand inoculum into a plastic bag and store it in a fridge at 4 °C.

2. Trypan blue staining to check fungal colonization level

1. Incubate root pieces for 30 min at >90 °C in 10% KOH. Remove the KOH.
2. Rinse the root pieces with double-distilled water (ddH₂O) three times.
3. Incubate the root pieces with 0.3 M HCl for 15 min to 2 h. Remove the HCl.
4. Add 1 mL of 0.1% trypan blue and incubate the samples for 8 min at >90 °C.
5. Wash the root pieces with 50% acidic glycerol. Transfer 10 root pieces onto slides and add a drop of 50% acidic glycerol.
6. Seal the coverslips and slide with nail polish.
7. Examine 10 fields of view of each root under a microscope to record the presence of fungal structures. Calculate the fungal colonization level as a percentage.
   NOTE: 50% acidic glycerol: Prepare by mixing glycerol and 0.3 M HCl in a 1:1 ratio. 0.1% trypan blue: Dissolve 100 mg of trypan blue in a mixture of 2:1:1 lactic acid, glycerol, and ddH₂O.

3. Inoculation of rice seedlings with sand inoculum and salt stress treatment

1. Remove the hull (husk) from rice seeds.
2. Sterilize the seeds with 70% ethanol (EtOH) for 4 min and 30 s.
3. Place the rice seeds into a centrifuge tube. Add 3% bleach (prepared with sterile dH₂O) and shake for 30 min.
4. Remove the bleach and wash the seeds with sterile dH₂O 3-4 times inside the laminar flow hood.
5. Grow the seeds in half-strength Murashige-Skoog (1/2 MS) medium containing 0.8% agar at 30 °C in the dark for 5 days.
6. Grow the rice seedlings with a 12-h day/night cycle at 30/28 °C and 70% air humidity for 2 days.
7. Transfer the rice seedlings into plastic tubes containing sterilized sand. Add either no inoculum (mock) or 5 mL of sand inoculum containing spores of Rhizophagus irregularis (Ri).
8. Water the rice plants with dH₂O 7 days a week for the first week after inoculation. Fertilize the plants every second day with a half-strength Hoagland solution containing 25 µM of KH₂PO₄.
9. At 5 weeks post-inoculation (wpi), treat one batch with 150 mM of NaCl (saline condition) and leave the other batch without NaCl (non-saline condition).
   1. For the non-saline condition, water the plants with half-strength Hoagland solution containing 25 µM of KH₂PO₄ on Tuesday and with water for the rest of the week.
   2. For the saline condition, water the plants with half-strength Hoagland solution containing 25 µM of KH₂PO₄ on Tuesday, with 150 mM of NaCl on Monday, Wednesday, and Friday, and with water for the rest of the week.
10. At 8 wpi, harvest the plants to measure their fresh weight. Place the plants in a 70 °C oven for 2 days to measure the dry weight. Analyze the fungal colonization level by trypan blue staining.

Results

The step-by-step workflow is shown in Figure 1.At 10 weeks post-inoculation (wpi), fungal structures such as vesicles and spores, which are characteristic of the late stage and AM symbiosis, were clearly observed inside the roots of garlic chives (Figure 2A). The levels of intraradical hyphae, arbuscule, vesicle, extraradical hyphae, and spore were 80%, 47%, 63%, 4%, and 1%, respectively, indicating the progression of fungal deve...

Discussion

There are a few tips regarding the preparation and usage of sand inoculum. First, from our experience, the colonization level of garlic chive should be higher than 70% (Figure 2C). Otherwise, the following inoculation on other plants, such as tomato and rice, will not successfully reach over 50% at 7 weeks post-inoculation (wpi) (Figure 2E). Second, the sand inoculum should be air-dried thoroughly before storage and kept inside a clean plastic bag in the fridge ...

Materials

Name	Company	Catalog Number	Comments
(NH4)6Mo7O24.4H2O	FERAK	12054-85-2	half-strength Hoagland solution
Bleach	Gaulix	Gaulix-2108	rice sterilization
Ca(NO3)2.4H2O	Sigma	13477-34-4	half-strength Hoagland solution
CuSO4.5H2O	Sigma	7758-99-8	half-strength Hoagland solution
EtOH	Honeywell	67-63-0	rice sterilization
Fe-citrate	Sigma	3522-50-7	half-strength Hoagland solution
Garlic chives seeds	KNOWN-YOU SEED Co., LTD.	V-015	Allium tuberosum L. seeds
Glycerol	J.T.Baker	56-81-5	Trypan blue staining
HCl	Sigma	7647-01-0	Trypan blue staining
KCl	Merck	7447-40-7	half-strength Hoagland solution
KH2PO4	Merck	7646-93-7	half-strength Hoagland solution
KNO3	Avantor	7757-79-1	half-strength Hoagland solution
KOH	Honeywell	1310-58-3	Trypan blue staining
Lactic acid	Sigma	50-81-7	Trypan blue staining
MgSO4.7H2O	Sigma	10034-99-8	half-strength Hoagland solution
MnSO4.H2O	Honeywell	10034-96-5	half-strength Hoagland solution
MS salts	PhytoTech	M404	half-strength Murashige–Skoog (1/2 MS) medium
Na2B4O7.10H2O	Sigma	1330-43-4	half-strength Hoagland solution
NaCl	Bioshop	7647-14-5	salt stress treatment
NaOH	J.T.Baker	1310-73-2	half-strength Murashige–Skoog (1/2 MS) medium
Rhizophagus irregularis spore	Premier Tech	L-ASP-A	AM fungal spore (MycoriseASP, Premier Tech, Rivière-du-Loup, Québec, Canada )
Sucrose	Bioshop	57-50-1	half-strength Murashige–Skoog (1/2 MS) medium
Trypan blue	Sigma	72-57-1	Trypan blue staining
ZnSO4.7H2O	Avantor	7446-20-0	half-strength Hoagland solution