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In Vitro Hyphal Branching Assay Using Rhizophagus irregularis.

Takaya Tominaga¹, Hironori Kaminaka²

¹Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Nara, Japan.

|August 30, 2024

Summary

Researchers developed a new in vitro method to study hyphal branching in Rhizophagus irregularis, a key arbuscular mycorrhizal (AM) fungus. This assay quantifies the effects of plant hormones like strigolactones on AM fungal symbiosis.

Keywords:

AM symbiosis Axenic culture Hyphal branching assay Rhizophagus species branching factor

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Area of Science:

Plant-Microbe Interactions
Mycology
Plant Physiology

Background:

Arbuscular mycorrhizal (AM) fungi, symbiotic Glomeromycotina, enhance plant growth, particularly in low-phosphate soils, by facilitating nutrient uptake.
Plant hormones called strigolactones (SLs) are known to induce hyphal branching in AM fungi.
While Gigaspora species are common in assays due to simple branching, Glomeraceae fungi like Rhizophagus irregularis, with complex branching, are also widely used.

Purpose of the Study:

To develop and validate an in vitro method for producing and quantifying hyphal branches of the model AM fungus Rhizophagus irregularis.
To adapt existing bioassay methods for better applicability to hydrophilic compounds and optimize them for R. irregularis.

Main Methods:

Developed an in vitro assay where Rhizophagus irregularis spores are placed between gels, allowing controlled diffusion of chemicals to germinating spores.
Modified a previously established method to enhance its suitability for hydrophilic compounds.
Optimized the system specifically for counting hyphal branches of R. irregularis.

Main Results:

Successfully reproduced the known positive effect of a synthetic strigolactone on R. irregularis hyphal branching.
Demonstrated the utility of the assay for quantifying the physiological effects of various chemicals on R. irregularis.

Conclusions:

The developed in vitro hyphal branching assay is effective for studying Rhizophagus irregularis.
This method provides a valuable tool for assessing the impact of synthesized chemicals and plant metabolites on AM fungal symbiosis.
The assay requires a minimum of 12 days for completion.