A Whole-Plant Culture Method to Study Structural and Functional Traits of Extraradical Mycelium

An in vivo whole-plant bi-dimensional experimental system has been devised and tested with different host plants to obtain extraradical mycelium (ERM) produced by different isolates of arbuscular mycorrhizal fungi (AMF). In this system, a plantlet is inoculated with AMF to establish mycorrhizal symbiosis and, after colonization, newly formed extraradical hyphae and spores are removed. Then, the mycorrhizal root system is wrapped in a nylon net and placed between two membranes in a Petri dish, allowing ERM to grow on the membrane surfaces. Such extraradical hyphae may be used for in situ morphometric analyses or harvested for molecular and biochemical assays; in the latter case, when reassembled, the whole-plant system can allow new mycelial harvests up to a maximum plant lifespan of 6 months. In this experimental system, which was tested with diverse host plant species and lines, values of explored membrane surface areas and densities of ERM showed large variations, and their length ranged from 9.7 ± 2.0 to 120.1 ± 14.1 m per plant, depending on host and AMF identity. Across the different plant-AMF combinations tested, the whole-plant system produced 2.0 ± 0.6 to 8.3 ± 3.3 mg of ERM fresh biomass per plant per harvest. This experimental system can be used for a wide range of AMF and host plant species, either establishing arbuscular mycorrhizas or other mycorrhizal interactions. ERM produced and collected in the whole-plant system is suitable for morphological, physiological, and molecular analyses, facilitating studies on the different aspects of mycorrhizal symbiotic interactions.