Chemiluminescence-based quantification of the colonization rates of Lotus japonicus roots by arbuscular mycorrhizal fungi

Simple evaluation of the levels of root colonization by arbuscular mycorrhizal (AM) fungi is challenging for conventional histochemical staining because most roots show an endogenous background signal. Herein, we report a chemiluminescence-based novel method for the quantification of the relative colonization rates of AM fungi. Lotus japonicus seedlings were grown in two types of soils with different inoculum potential [i.e., the former vegetation was maize (host) or buckwheat (non-host)]. Indigenous AM fungi colonizing roots were labelled with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), which specifically targets the N-acetylglucosamine polymers of fungal cell walls. After the roots were removed from the soil and rinsed with water, all procedures were conducted in 24-well plates. AM fungi were detected by chemiluminescence in the presence of enhanced chemiluminescence (ECL) reagents, and the signal strengths were measured by image analyses. Post-staining of AM fungal structures in the presence of 3,3'-diaminobenzidine (DAB) and H2O2 (DAB staining) revealed that even small colonization events (e.g., hyphopodia) could be detected, whereas the background signals observed in DAB staining were eliminated in the chemiluminescence method. Chemiluminescence thus provides both high sensitivity and high specificity. The chemiluminescence-based colonization levels of roots grown in maize soil were significantly higher than those of roots grown in buckwheat soil, demonstrating the feasibility for high-throughput quantification of the relative colonization rates of L. japonicus seedlings by AM fungi.