Understanding dynamics of Rhizophagus irregularis ontogenesis in axenically developed coculture through basic and advanced microscopic techniques.

Detailed information on structural changes that occur during ontogenesis of Rhizophagus irregularis in axenically developed coculture is limited. Our study aims to investigate the series of events that occur during mycorrhizal ontogenesis under axenic condition through basic and advanced microscopic techniques followed by comparison among these to identify the suitable technique for rapid and detailed analysis of mycorrhizal structures. Three stages were identified in mycorrhizal ontogenesis from initiation (preinfection stage of hyphae; its branching, infection and appressoria formation; epidermal opening; and hyphal entry), progression (arbuscular development; hyphal coils and vesicles) to maturity (extraradical spores). Scanning electron microscopy was found to be an efficient tool for studying spatial three-dimensional progression. Adding to the advantages of advanced microscopy, potential of autofluorescence to explore the stages of symbiosis nondestructively was also established. We also report imaging of ultrathin sections by bright field microscopy to provide finer details at subcellular interface. Owing to the merits of nondestructive sampling, ease of sample preparation, autofluorescence (no dye required), no use of toxic chemicals, rapid analysis and in depth characterization confocal laser scanning microscopy was identified as the most preferred technique. The method thus developed can be used for detailed structural inquisition of mycorrhizal symbiosis both in in planta and in an in vitro system.