A simple and low-cost technique to initiate single-spore cultures of arbuscular mycorrhizal fungi using a superabsorbent polymer

Abstract

Most species of arbuscular mycorrhizal fungi (AMF) are propagated with a host plant in a pot culture. However, the soil matrix makes it difficult to monitor the establishment and development of the symbiosis. In vitro culturing using Ri T-DNA transformed roots provides a clear medium and a sterile environment which offsets the constraints of the soil matrix. Nevertheless, the sterile conditions and the Ri T-DNA transformed roots provide very different growing conditions compared to a pot culture. Transparent soil based on superabsorbent polymer (SAP) has the potential of combining the advantages of current in vivo and in vitro culture methods without the constraints associated with either technique (opacity and sterility). Here we describe a SAP-based autotrophic culture as an alternative to current in vivo and in vitro culture methods. This system using two-compartment Petri dishes makes it easy to initiate single-spore cultures and to monitor fungal propagation. The SAP-based autotrophic system allowed the establishment of single-spore cultures of seven species (Diversispora varaderana, Funneliformis geosporus, Gigaspora rosea, Racocetra fulgida, Rhizophagus irregularis, R. intraradices and Sclerocystis sp.) from six genera and three families. Cultures were maintained over several months under non-sterile conditions. The Petri dishes avoid the problem of cross contamination and they can be stacked for space optimization. The grains of SAP colonized with new spores were used as inoculum to initiate new cultures in the SAP-based system. The SAP-based autotrophic culture method is a low-cost and low-tech approach, which makes the study of AMF much more accessible.

Rhizophagus irregularis, the model fungus in arbuscular mycorrhiza research, forms dimorphic spores

“…The superabsorbent polymer (SAP)‐based autotrophic system was used for symbiotic cultures, (Paré et al ., 2022). The polymer was a poly(acrylamide‐co‐acrylic acid) potassium salt, and it is marketed as HORTA‐SORB® MD Granule by Horticultural Alliance (Sarasota, FL, USA).…”

“…The mother spores of R. cf fasciculatus together with their respective daughter spores were then transferred to microscope slides for observation by light microscopy. The superabsorbent polymer (SAP)-based autotrophic system was used for symbiotic cultures, (Par e et al, 2022). The polymer was a poly(acrylamide-co-acrylic acid) potassium salt, and it is marketed as HORTA-SORB ® MD Granule by Horticultural Alliance (Sarasota, FL, USA).…”

Inoculating and Observing Arbuscular Mycorrhizal Cultures on Superabsorbent Polymer-Based Autotrophic Systems

“…Install nylon mesh filter membrane. 2022) 12 showed that the conditions on SAP were relatively neutral (6.7 to 7.4).…”

“…The modified twocompartment Petri dishes allow the cultivation of different AM fungal species. Paré et al12 have propagated seven different AMF species from six genera and three families.Modification of two-compartment Petri dishes is easily done with inexpensive tools. The cost and quantity of materials (vermiculite, SAP, Pasteur pipette, etc.)…”

Arbuscular Mycorrhizal Fungi as Biostimulant and Biocontrol Agents: A Review

“…They use either Ri T-DNA root-transformed organs [266] placed into bioreactors comprising a solid support [267], airlift bioreactor [268] or small containers [269]. Whole plants can also be used with aerial organs outside a Petri dish while AMF and roots are in the Petri dish filled with a solid medium [270,271]. These in vitro techniques are performed in completely sterile conditions and should make it possible to produce large quantity, high quality and contaminant-free AMF inoculums [259,272].…”