Black ink staining protocol: A cost-effective substitute in quantifying arbuscular mycorrhizal colonization in plant roots

Abstract

Arbuscular mycorrhizal (AM) fungi, ubiquitously distributed across diverse terrestrial ecosystems, establish symbiotic associations with the majority of vascular plants, fulfilling essential physiological and ecological functions. Mycorrhizal development represents the initiation of host-fungus interactions and serves as a metric for assessing mutualistic efficacy. However, mycorrhizal detection underscores the urgent need to develop cost-effective, efficient, and environmentally benign dyestuff. Therefore, wild-collected and laboratory-grown roots of Medicago sativa were selected. Six reagents including black ink, red ink, acid fuchsin, trypan blue, Sudan IV, and aniline blue were evaluated in conjunction with computer vision techniques to identify optimal one. Concurrently, root characteristics were quantified, and interrelationships among root traits, image quality, and colonization indices were analyzed to unravel the mechanism of their interactions. The findings demonstrated that wild roots exhibited pronounced lignification, achieving a mycorrhizal colonization rate of 100 %, which was better than the two laboratory groups. And the fungal community displayed a markedly greater colonization intensity compared to the Claroideoglomus etunicatum. Evaluation of the six reagents revealed distinct staining efficacy, with significant variations in image clarity, gray-level co-occurrence matrix (GLCM) indices, and colonization parameters across treatments. Specifically, aniline blue proved ineffective, while Sudan IV showed selective binding. Notably, black ink in glacial acetic acid achieved optimal mycorrhizal detection efficacy. Moreover, correlation matrix identified microscopic image quality as critical determinant of quantification accuracy, influenced by both reagent types and root properties, and AvgDiam exerted the most substantial impact (|R| > 0.75).