An improved method for bright-field imaging of arbuscular mycorrhizal fungi in plant roots

Easy observation methods to assess the colonization levels of arbuscular mycorrhizal (AM) fungi in plant roots are crucial for studying the biology of AM symbiosis and for considering agricultural use. Many AM studies employ Trypan Blue (TB) coupled with lactic acid to stain AM fungal structures as bright-field images; however, TB staining can be difficult to use owing to its noxiousness and high viscosity. Here, we report the development of an easy method for visualizing AM fungal structures as bright-field images using 3,3′-diaminobenzidine (DAB). Wheat germ agglutinin (WGA)-conjugated horseradish peroxidase (HRP), which specifically targets N-acetylglucosamine polymers and detects AM fungal cell walls, penetrated the cortical layers of 10% potassium hydroxide (KOH)-treated soybean roots and stained AM fungal mycelia in the presence of DAB and hydrogen peroxide (H₂O₂). Comparison between DAB and TB staining of soybean (Glycine max L.) roots suggested that the intactness of root systems and image contrast using DAB staining were superior. Background signals in stele observed by DAB staining were negligible as compared with those observed by WGA-fluorescein isothiocyanate staining. DAB staining, which combines the advantages of TB (easy bright-field imaging) and WGA-fluorophore (specific and high-quality) staining, provides a robust imaging method for macro- and micro-level analyses of AM roots and is applicable to at least six crops: soybean, onion (Alium cepa L.), potato (Solanum tuberosum L.), maize (Zea mays L.), rice (Oryza sativa L.), and sunflower (Helianthus annuus L.).