Name: Production of Arbuscular Mycorrhizal (AM) Fungal Inoculum and Phenotypic Evaluation of Rice and AM Symbiosis Under Saline Conditions (Video) | JoVE Journal
Uploaded: 2025-03-14T14:00:00.000+00:00
Duration: 7 min 43 s
Description: Learn to produce arbuscular mycorrhizal fungal inoculum and evaluate rice-AM symbiosis under saline conditions. This protocol demonstrates enhanced salt tolerance through improved root colonization and biomass.

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5 minutes

We acknowledge Yun-Hsin Chen establishing the system for investigating AM-enhanced salt stress tolerance in rice, and Kai-Chieh Chang establishing the system to generate sand inoculum. This work was supported by grants from the National Science and Technology Council, Taiwan (NSTC 113-2326-B-002 -008 -MY3).

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.
<ol>
	<li>Wash sand with tap water and autoclave it.</li>
	<li>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.</li>
	<li>Grow the garlic chives in the chamber with a 16-h/8-h day/night cycle at 23.5 &#176;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.</li>
	<li>Starting from 2 wpi, fertilize the garlic chives twice a week with 80 mL half-strength Hoagland solution containing 25 &#181;M KH2PO4. Fertilize once a week with 80 mL water.</li>
	<li>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 &#176;C.</li>
</ol>
2. Trypan blue staining to check fungal colonization level
<ol>
	<li>Incubate root pieces for 30 min at &#62;90 &#176;C in 10% KOH. Remove the KOH.</li>
	<li>Rinse the root pieces with double-distilled water (ddH2O) three times.</li>
	<li>Incubate the root pieces with 0.3 M HCl for 15 min to 2 h. Remove the HCl.</li>
	<li>Add 1 mL of 0.1% trypan blue and incubate the samples for 8 min at &#62;90 &#176;C.</li>
	<li>Wash the root pieces with 50% acidic glycerol. Transfer 10 root pieces onto slides and add a drop of 50% acidic glycerol.</li>
	<li>Seal the coverslips and slide with nail polish.</li>
	<li>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.</li>
</ol>
3. Inoculation of rice seedlings with sand inoculum and salt stress treatment
<ol>
	<li>Remove the hull (husk) from rice seeds.</li>
	<li>Sterilize the seeds with 70% ethanol (EtOH) for 4 min and 30 s.</li>
	<li>Place the rice seeds into a centrifuge tube. Add 3% bleach (prepared with sterile dH2O) and shake for 30 min.</li>
	<li>Remove the bleach and wash the seeds with sterile dH2O 3-4 times inside the laminar flow hood.</li>
	<li>Grow the seeds in half-strength Murashige-Skoog (1/2 MS) medium containing 0.8% agar at 30 &#176;C in the dark for 5 days.</li>
	<li>Grow the rice seedlings with a 12-h day/night cycle at 30/28 &#176;C and 70% air humidity for 2 days.</li>
	<li>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).</li>
	<li>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 &#181;M of KH2PO4.</li>
	<li>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).
	<ol>
		<li>For the non-saline condition, water the plants with half-strength Hoagland solution containing 25 &#181;M of KH2PO4&#160;on Tuesday and with water for the rest of the week.</li>
		<li>For the saline condition, water the plants with half-strength Hoagland solution containing 25 &#181;M of KH2PO4&#160;on Tuesday, with 150 mM of NaCl on Monday, Wednesday, and Friday, and with water for the rest of the week.</li>
	</ol>
	</li>
	<li>At 8 wpi, harvest the plants to measure their fresh weight. Place the plants in a 70 &#176;C oven for 2 days to measure the dry weight. Analyze the fungal colonization level by trypan blue staining.</li>
</ol>

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</ol>

The authors declare that they have no conflicts of interest.

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 ...

Saline soil is a significant obstacle to crop production worldwide1,2,3. 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&#8722;) ions in plant tissues. These ions, which dominate saline soils, are also the most harmful to plants5,6,7. 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 plants14,15. 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 tolerance17,18,19,20. 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 plants2,21,22,23. 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 conditions23,24,25,26,27,28,29.
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 rice31,32,33. 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 propagation37,38. 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.

<table><tbody><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;6&lt;/sub&gt;Mo&lt;sub&gt;7&lt;/sub&gt;O&lt;sub&gt;24&lt;/sub&gt;.4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>FERAK</td><td>12054-85-2</td><td>half-strength Hoagland solution</td></tr><tr><td>Bleach</td><td>Gaulix</td><td>Gaulix-2108</td><td>rice sterilization&amp;nbsp;</td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;.4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>13477-34-4</td><td>half-strength Hoagland solution</td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;.5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>7758-99-8</td><td>half-strength Hoagland solution</td></tr><tr><td>EtOH</td><td>Honeywell</td><td>67-63-0</td><td>rice sterilization&amp;nbsp;</td></tr><tr><td>Fe-citrate</td><td>Sigma</td><td>3522-50-7</td><td>half-strength Hoagland solution</td></tr><tr><td>Garlic chives seeds</td><td>KNOWN-YOU SEED Co., LTD.</td><td>V-015</td><td>Allium tuberosum L. seeds</td></tr><tr><td>Glycerol</td><td>J.T.Baker</td><td>56-81-5</td><td>Trypan blue staining</td></tr><tr><td>HCl</td><td>Sigma</td><td>7647-01-0</td><td>Trypan blue staining</td></tr><tr><td>KCl</td><td>Merck</td><td>&amp;nbsp;7447-40-7</td><td>half-strength Hoagland solution</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>7646-93-7</td><td>half-strength Hoagland solution</td></tr><tr><td>KNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Avantor</td><td>7757-79-1</td><td>half-strength Hoagland solution</td></tr><tr><td>KOH</td><td>Honeywell</td><td>1310-58-3</td><td>Trypan blue staining</td></tr><tr><td>Lactic acid</td><td>Sigma</td><td>50-81-7</td><td>Trypan blue staining</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>10034-99-8</td><td>half-strength Hoagland solution</td></tr><tr><td>MnSO&lt;sub&gt;4&lt;/sub&gt;.H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Honeywell</td><td>10034-96-5</td><td>half-strength Hoagland solution</td></tr><tr><td>MS salts</td><td>PhytoTech</td><td>M404</td><td>half-strength Murashige&amp;ndash;Skoog (1/2 MS) medium</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;B&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;.10H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>1330-43-4</td><td>half-strength Hoagland solution</td></tr><tr><td>NaCl</td><td>Bioshop</td><td>7647-14-5</td><td>salt stress treatment</td></tr><tr><td>NaOH</td><td>J.T.Baker</td><td>1310-73-2</td><td>half-strength Murashige&amp;ndash;Skoog (1/2 MS) medium</td></tr><tr><td>Rhizophagus irregularis spore</td><td>Premier Tech</td><td>L-ASP-A</td><td>AM fungal spore (MycoriseASP, Premier Tech, Rivi&amp;egrave;re-du-Loup, Qu&amp;eacute;bec, Canada )</td></tr><tr><td>Sucrose</td><td>Bioshop</td><td>57-50-1</td><td>half-strength Murashige&amp;ndash;Skoog (1/2 MS) medium</td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>72-57-1</td><td>Trypan blue staining</td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Avantor</td><td>7446-20-0</td><td>half-strength Hoagland solution</td></tr></tbody></table>

production of arbuscular mycorrhizal (am) fungal inoculum and phenotypic evaluation of rice and am symbiosis under saline conditions

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

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.

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...

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

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 ...

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JoVE Journal

Biology

Begin with rice seedlings pre-germinated under dark, aseptic conditions to ensure uniform sprouting.Transfer these seedlings to a flask containing a nutrient-rich, semi-solid medium pre-inoculated with a test bacterium.As the plant grows, the roots release chemotactic compounds into the surrounding medium.These compounds stimulate bacterial proliferation, leading to adhesion, multiplication, and colony formation on the root surface.As the roots develop, the bacteria enter the plant through...

Root-Mediated Endophytic Colonization of Beneficial Bacteria in Rice

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Establishment of Rice Plant Leaf Infection through Fungal Inoculation via Wounding

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Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Most plants within communities likely are interconnected by arbuscular mycorrhizal (AM) fungi, but mediation of plant interactions by them has been investigated primarily by growing plants with versus without mycorrhizas. We present a method to manipulate common mycorrhizal networks among mycorrhizal plants to investigate their consequences for plant interactions.

Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

The protocol here describes the methods for the assessment of the arbuscular mycorrhizal colonization patterns and strategy in roots for two species: Zea mays and Festuca rubra. The use of the MycoPatt method permits the calculation of parameters, the conversion of mycorrhizal structures into digital data, and the mapping of their real position in roots.

Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays

This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy equipment.

Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone

Here, we describe a simple and inexpensive technique for inoculating and observing arbuscular mycorrhizal fungi in superabsorbent polymer-based autotrophic systems.

Inoculating and Observing Arbuscular Mycorrhizal Cultures on Superabsorbent Polymer-Based Autotrophic Systems

Learn to produce arbuscular mycorrhizal fungal inoculum and evaluate rice-AM symbiosis under saline conditions. This protocol demonstrates enhanced salt tolerance through improved root colonization and biomass.

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

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

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