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 cyto....

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....

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