modification of two-compartment petri dishes for inoculating arbuscular mycorrhizal fungi in superabsorbent polymer

Culturing Arbuscular Mycorrhizal Fungi Using SAP-AS for Enhanced Observation

Arbuscular mycorrhizal (AM) fungi (Glomeromycotina) are ancient plant root symbionts (~500 Ma1,2) that may have played an essential role in the colonization of terrestrial soils by tracheophytes. This long coevolution between AM fungi and tracheophytes places arbuscular mycorrhiza as a masterpiece of interkingdom mutualism. AM fungal hyphae significantly increase the ability of the host to forage for soil nutrients3, including nutrient carry-over to new hosts via mycorrhizal networks4. The hyphal network improves soil structure, and the production of glomalin could reduce soil erosion5. The transfer of part of the atmospheric carbon to the fungal root symbiont increases soil carbon sequestration6. Overall, AM fungi improve plant resilience to both abiotic and biotic stresses and have therefore, received considerable attention in agroecology7. Indeed, AM fungi-friendly agricultural management practices have the potential to reduce the use of chemical inputs for crop production and improve soil organic carbon content, which are important objectives that farmers need to integrate into their management practices in order to comply with national and international commitments regarding the transition to sustainable agricultural practices and the fight against climate change.
However, AM fungi are soil microscopic fungi, and their study is difficult due to their obligate biotrophy and rhizosphere distribution. Soil is one of the most difficult biotopes to study because of its opacity, the huge diversity of niches, and multi-trophic interactions at all scales. The isolation, propagation, and characterization of AM fungi are, therefore difficult. Until the middle of the 20th century, only AM fungal species forming sporocarps had been characterized8. However, the majority of AM fungal species produce non-sporocarpic spores ranging from ~20 &#956;m to ~500 &#956;m in diameter. The description of the soil wet sieving technique9 opened the way to describe these AM fungal species, and the rate of species description has increased since then. Nevertheless, AM fungi represent a small group of species compared to Dikarya.
Trap cultures, i.e., the inoculation with spores or an environmental soil sample containing AM fungal spores of a pot filled with autoclaved material such as turface&#160;and vermiculite and a sterilized seed of a host (leek, plantain), is one way to propagate AM fungi under controlled conditions10. However, the success of the inoculation can only be assessed by looking for the presence of arbuscules in root fragments after staining or by wet sieving a subsample or the entire pot to isolate spores. It is usually recommended not to disturb the system for at least 6-12 weeks before the analysis of the pot culture. This culture technique is suitable for propagating most known AM fungal species, but live observation of the fungal symbiont is not possible, and the success of inoculation is uncertain, especially when single spore cultures are attempted.
On the contrary, the in vitro propagation of AM fungi can be monitored live thanks to the transparency of the culture medium11, but this culture technique requires the availability of transformed roots and the presence of carbon in the culture medium to work in a sterile environment. The spores must be sterilized, and together with the association with a heterotrophic host, most of the known AM fungal species are not successfully propagated using this technique.
Therefore, the propagation of AM fungi using current techniques, although established and widely used in most laboratories, has some limitations for the study of AM fungi. Par&#233; et al. (2022)12 developed an in vivo technique using a transparent superabsorbent polymer (SAP) in combination with whole plants to propagate AM fungi. The technique, designed as an SAP-based autotrophic system (SAP-AS), is simple and inexpensive and combines the advantages of pot culture (association with an autotrophic host, non-sterile conditions) and in vitro cultures (transparent medium, live monitoring of symbiosis development). Here, we present a protocol explaining how to set up the cultures with single spore inoculation and use the SAP-AS for high-magnification observation of the extraradical mycelium. Specifically, we describe how to modify two-compartment Petri dishes, prepare the nutrient solution, prepare the superabsorbent polymer (SAP), prepare the seedlings, assemble the SAP-AS and inoculate with a single spore, pregerminate the spores, and live monitor the development of the symbiosis.

Author Spotlight: Enhancing AM Fungi Research with SAP-AS &amp;#8212; A Novel Technique for Single-Spore Cultures

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

Basically, all fungi are ancient and essential plant food symbionts, and they enhance the nutrient uptake of the host and are also key players in cell health. While cell microorganisms are difficult to study, our protocol allows for real time monitoring, digital observation, and efficient establishment of single spoke cultures. Currently, AM fungi are propagated in pot culture, in greenhouse condition, or in vitro in using Ri T-DNA transform root.Both technology have significantly contributed to the knowledge of those AM fungi in the last decade, but they have specific limitations. Assessment of successful establishment of the fungal partner relies on the presence of arbuscular in the roots or spore in the soil. In vitro propagation allows lives, more monitoring, but requires transform routes and sterile condition limiting its applicability to many AM fungal species.The pot culture or in vitro culture techniques used to study AMF have limitations in terms of live observation and successful propagation. To overcome these limitations, we have developed this super absorbent, polymer-based autotroph system, SAP-AS, which is a simple and inexpensive technique that combines the advantages of in vitro culture and pot culture. Our laboratory will focus on improving the establishment of new AM fungal species in single spore cultures and also studying the interaction we oversaw in microorganisms.This technique will allow us to study the competitive dynamics between different AM fungal species, high fault propagation mechanism, and nutrient uptake processes.

Modification of Two-Compartment Petri Dishes for Inoculating Arbuscular Mycorrhizal Fungi in Superabsorbent Polymer

To begin, use the rotary tool and the appropriate carving bit to drill two irrigation holes in the top of the lid. On the side, drill a one-centimeter opening. Use the electric rotary tool to drill a notch in the side of the Petri dish bottom for the plant stem.Next, using the electric rotary tool, equipped with the appropriate drill bits, drill a 30-millimeter rectangular notch down to the bottom of the Petri dish. For high-resolution observation, drill circular holes approximately 25 millimeters in diameter in the bottom of the Petri dish. Ensure the holes are not drilled directly under the lid's irrigation holes.Using the metal guide and paperclip, wedge the piece of the nylon mesh filter membrane in place and press it firmly onto the plastic barrier on the root or notch side. Then, align the top of the membrane with the top of the barrier. Using the pyrography kit, cut along the edge of the metal guide to melt and seal the membrane to the bottom of the Petri dish and the plastic barrier separating the two compartments.With the metal guide and paperclips still in place, use tweezers to carefully remove excess nylon mesh filter membrane. Under the dissecting microscope, verify that the nylon mesh filter membrane is properly sealed to the plastic to prevent root penetration into the HIFULL compartment. Then, apply the silicone sealant along the edge of the circular holes on the outside of the Petri dish bottom.Place the cover slip on the outside of the Petri dish bottom and gently press for perfect adhesion.

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Serie: Modifikation von zweiteiligen Petrischalen zur Inokulation von arbuskulären Mykorrhizapilzen in superabsorbierendem Polymer

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the rhizosphere. Typically, AM fungi are cultured under in vivo conditions in pot culture with an autotrophic host or under in vitro conditions with Ri Transfer-DNA transformed roots (heterotrophic host) in a Petri dish. Additionally, the cultivation of AM fungi in pot culture occurs in an opaque and non-sterile environment. In contrast, in vitro culture involves the propagation of AM fungi in a sterile, transparent environment. The superabsorbent polymer-based autotrophic system (SAP-AS) has recently been developed and shown to combine the advantages of both methods while avoiding their respective limitations (opacity and heterotrophic host, sterility). Here, we present a detailed protocol for easy preparation, single spore inoculation, and observation of AM fungi in SAP-AS. By modifying the Petri dishes, high-resolution photographic and video observations were possible on living specimens, which would have been difficult or impossible with current in vivo and in vitro techniques.

Here, we describe a simple and inexpensive technique for inoculating and observing arbuscular mycorrhizal fungi in superabsorbent polymer-based autotrophic systems.

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the ...

Cultivating Arbuscular Mycorrhizal-Fungi Using Super Absorbent Polymer-Based Autotrophic System (SAP-AS)

To begin, weigh five grams of dry super absorbent polymer or SAP, and mix it with 500 milliliters of MMS1 nutrient solution. Approximately one week before assembly, germinate plantago lanceolata seeds on blotting paper moistened with MMS1 nutrient solution in a Petri dish. Then incubate the Petri dish in the dark at room temperature.To start assembling the SAP-AS system, place the seedling stem in the notch on the side of the Petri dish with the root inward and the cotyledons or leaves and stems outward. Cover the roots with approximately 1.5 to two grams of vermiculite. Afterward, hydrate the root compartment with eight milliliters of MMS1 nutrient solution.Clear a vermiculite-free space along the nylon mesh filter on the side of the root compartment, and add five grams of hydrated SAP. Then add 15 grams of hydrated SAP to the highfill compartment. Now align the side notches of the lid and the Petri dish bottom to avoid damaging the stem and close the lid.Then seal the Petri dish with a strip of parafilm, joining the base and lid. Weigh the Petri dish and record its average weight to determine when the SAP-AS needs rehydration. Then stack the Petri dishes to optimize space.Use an opaque cover with an opening on the side to keep the seedlings in the dark. Using a flame candle or a Bunsen burner, heat the glass pipette and stretch the glass tube as it melts to separate it. Under a stereo microscope, break the tip of the glass pipette to reduce its internal diameter.Now open the Petri dish and locate where the spore is to be placed. Clear a space among the five grams of hydrated sap along the nylon mesh filter membrane to expose a section of a root. Under the dissecting microscope, pick up a spore in a liquid suspension with the extruded pipette, while pipetting some liquid before collecting the spore.After that, carefully deposit the spore on the root. Then replace the sap over the root fragment to maintain a moist microenvironment for the root and spore. After 24 hours, if needed, irrigate the root compartment, taking care not to wash off the spore.Align the side notches of the lid with the bottom of the Petri dish to avoid damaging the seedling stem and close the Petri dish with the lid.