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 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.
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 μm to ~500 μm 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 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. Paré 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.
1. Modification of two-compartment Petri dishes
NOTE: The materials required for this step are listed in the Table of Materials.
2. Preparation of 1 L of nutrient solution mMS-1
3. Preparation of SAP
NOTE: The materials required for this step are listed in the Table of Materials.
4. Preparation of seedlings
NOTE: The materials required for this step are listed in the Table of Materials.
5. Assembly and management of SAP-AS
NOTE: The materials required for this step are listed in the Table of Materials.
6. Inoculation of the SAP-AS with a single spore
NOTE: The materials required for this step are listed in the Table of Materials.
7. Spore germination on SAP (optional)
NOTE: The materials required for this step are listed in the Table of Materials.
8. Live monitoring of symbiosis development
NOTE: The materials required for this step are listed in the Table of Materials.
The SAP-AS is a simple and inexpensive technique for culturing AM fungi and observing the development of intraradical and extraradical fungal structures. Here, we provided a detailed protocol to help users set up SAP-AS and we introduced two modifications compared to the original description of the SAP-AS.
First, the presence of SAP on the root compartment along the Nitex membrane facilitates the inoculation of the system with a single spore (Figure 1).
Figure 1: Single spore inoculation of the SAP-AS. (A) Inoculation with a single spore of Rhizophagus irregularis. (B) Inoculation with a single spore of Funneliformis mosseae. The white arrows indicate the point of inoculation. Scale bars: 500 µm. Please click here to view a larger version of this figure.
It is easy to identify an area with a few roots where the spore can be deposited. The exact location of the spore can be marked on the surface of the Petri dish lid and checked periodically for germination and colonization of the roots. The presence of hydrated grains of SAP provides a moist environment conducive to spore germination. The inoculation on the roots next to the Nitex membrane allows rapid development of the symbiosis with the extraradical mycelium to quickly access the hyphal compartment and forage for nutrients (Figure 2). The ability to observe whether the single spore placed at the inoculation point germinates or not also allows us to identify and remove unsuccessful cultures and replace them.
Second, the coverslips at the bottom of the Petri dish allow high-resolution live imaging of the AM-fungal symbiosis (Figure 2B,C) and, in particular, of the cytoplasmic flow within the extraradical mycelium (Figure 2D).
Figure 2: Inoculated SAP-AS. (A) SAP-AS inoculated with a single spore of Rhizophagus irregularis (DAOM 197198). The host plant is Plantago lanceolata. (B) Cluster of spores observed through the coverslip in the hyphal compartment under a stereomicroscope. Scale bar: 500 µm. (C) High-resolution photograph of the spore cluster observed through the coverslip under a light microscope at 10x magnification. Scale bar: 100 µm (D) High-resolution photograph of hyphae observed through the coverslip under a light microscope at 100x magnification. Scale bar: 10 µm. Please click here to view a larger version of this figure.
Users can attempt to germinate AM fungal spores on SAP hydrated with mMS-1 nutrient solution to guarantee the SAP-AS are inoculated only with viable spores (Figure 3). However, this was tested only with commercial spores of R. irregularis, and the success of spore germination on SAP hydrated with mMS-1 nutrient solution may vary significantly with other AM fungal species.
Figure 3: Germination of R. irregularis spores in 12-well plate. Scale bar: 500 µm. Please click here to view a larger version of this figure.
Finally, because SAP-AS is an in vivo, non-sterile technique for propagating AMF, the Petri dish can be manipulated on the bench and opened to sample colonized roots, free spores, or colonized grains of SAP (Figure 4). Intraradical and extraradical fungal structures can be stained in a manner similar to AMF propagated in pot or root organ cultures (Figure 4C).
Figure 4: Extraradical and intraradical AM fungal structures extracted from the SAP-AS. (A) Free spores of R. irregularis observed in the root compartment. Scale bar: 200 µm. (B) Spores of R. irregularis stained in SAP. Scale bar: 500 µm. (C) Stained root fragments showing the intraradical hyphae and arbuscules. Scale bars: 50 µm, 100 µm. Please click here to view a larger version of this figure.
Supplementary Figure 1: Comparison of the thickness of the various tools available to manipulate AM fungal spores. Please click here to download this File.
Supplementary Figure 2: Stacked SAP-AS. Please click here to download this File.
Inoculation is the most critical step in the protocol, and the extruded glass Pasteur pipettes proved to be an excellent tool for accurately manipulating single spores of AM fungi while preserving their integrity. The extruded glass Pasteur pipettes are easily shaped using the flame of a candle or Bunsen burner, and the opening can be adjusted under the stereomicroscope to match the size of the spore being pipetted. It is important to manipulate the spores with tools adapted to the size of AM fungal spore (Supplementary Figure 1) and to inoculate the SAP-AS when the host plant roots are long enough to reach the Nitex membrane where the spore is deposited.
The SAP-AS are easy to adapt to the experiment requirements. Larger Petri dishes can be used, with multi-compartments, to monitor, for example, the interaction between closely relative strains or between different species of AMF or to modify the chemical (pH) or biological environment (introduction of nematodes, bacteria, fungi). Various mycotrophic host plants can also be used to provide the AM fungi with photosynthates. The nutrient solution mMS-1 was derived from the minimal (M) medium recipe described by Bécard and Fortin (1988)15 minus the sucrose, vitamins, and bacto agar to limit carbon sources. However, the SAP-AS can be supplemented with various nutrient solutions, depending on the experiment objectives.
The propagation of AM fungi in SAP-AS requires regular watering. The limited volume of vermiculite and SAP exposes the roots and the AM fungus to fluctuations in humidity, especially in standard two-compartment Petri dishes (10 cm diameter). The ability to expand and, therefore, the transparency of the SAP grains decreases over time. In fact, the presence of cations from the nutrient solution progressively limits the expansion of the acrylate network and requires the replacement of the SAP after months. In addition, green algae and mold may grow over time if Petri dishes are not properly protected from light or overwatered.
To date, seven AM fungal species have been successfully cultivated in SAP-AS, which is far below the number of AM fungal species that can be propagated in pot cultures. However, both the biotic and abiotic conditions in SAP-AS are very similar to those in pot cultures, and it is likely that other AM fungal species should be able to propagate in SAP-AS. Direct inoculation of spores in SAP-AS probably provides environmental conditions closer to those of the rhizosphere in a natural soil due to the presence of root exudates and/or bacteria, if non-sterile spores/seeds are used for inoculation. This should be preferred to inoculation with germinated spores. In addition, the conditions that trigger spore germination within AMF are still poorly understood, therefore SAP hydrated with mMS-1 nutrient solution may not be adapted to germinate spores of other AM fungal species. Spore germination on SAP hydrated with mMS-1 nutrient solution was tested to specifically select viable spores for the inoculation step using only R. irregularis inoculum.
Inoculation and monitoring of the development of the AM symbiosis are easily performed in SAP-AS. The modified two-compartment Petri dishes allow the cultivation of different AM fungal species. Paré et al.12 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.) and reagents (mMS-1) required to prepare and maintain the SAP-AS are limited, allowing a large number of SAP-AS to be managed at minimal cost. The ability to stack the SAP-AS also significantly reduces the footprint of AM fungal cultures compared to pot cultures. For example, 50 SAP-AS (5 stacks of ten) can fit on a 1 m long shelf (Supplementary Figure 2). These features make the SAP-AS a simple and inexpensive technique that is compatible with teaching AM symbiosis in high school lab courses or at the undergraduate level in universities. Colonized grains of SAP can be used to inoculate new SAP-AS or pot cultures.
The presence of a coverslip at the back of the bottom of the Petri dish allows high-resolution photography and video of the extraradical fungal structures. The cytoplasmic flow can be easily studied under living conditions that are very close to natural conditions. This is of great importance for studies of AM fungi functions related to their mycelia (nutrients, water transport, soil structure, etc.) and for studies of hyphal morphogenesis.
AMF complete their biological cycle in plant roots and within the rhizosphere. The study of soil microorganisms is complex due to the inherent difficulty of observing the soil environment. The main objective of the SAP-AS is to recreate an environment as similar as possible to the rhizosphere for the propagation of AM fungi while maintaining the ability to observe the development of AM fungi in great detail. Because this implies non-sterile conditions, the amount of available carbon should be limited to avoid the proliferation of saprotrophic microorganisms. The knowledge of the behavior of AM fungi in the rhizosphere is still extremely limited, and the SAP-AS offers the possibility to make detailed comparisons between species regarding their ability to forage in the extraradical environment, their spore production, and their root colonization. This can be further complicated by adding interactions with other soil species such as bacteria, nematodes, protists, and root fungal pathogens, and the knowledge of soil microbiota interactions can be improved thanks to the SAP-AS.
The authors have nothing to disclose.
We would like to thank the two anonymous reviewers for their suggestions. Funding for this research was provided by Agriculture and Agri-Food Canada (AAFC) under project J-002295 (Management and enhancement of AAFC's biological collections).
100 x 15 mm Stackable Bi-Plate | Kord Valmark | 1204U09 | https://www.thomassci.com/Laboratory-Supplies/Petri-Dishes/_/100-x-15-mm-Stackable-Bi-Plate |
12-well plate | Greiner Bio-one | 665180 | https://shop.gbo.com/en/row/products/bioscience/cell-culture-products/cellstar-cell-culture-multiwell-plates/665180.html |
18 mm round glass coverslips | Fisher Scientific | 12-545-100 | https://www.fishersci.com/shop/products/fisherbrand-cover-glasses-circles-11/12545100#?keyword=12-545-100 |
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Commercial or scientific blender or kitchen hand blender | kitchenaid | KHBV53DG | https://www.kitchenaid.ca/en_ca/countertop-appliances/hand-blenders/hand-blender-products/p.variable-speed-corded-hand-blender.khbv53dg.html |
Dremel 199 Carving Bit | Dremel | 2615000199 | https://www.dremel.com/ca/en/p/199-2615000199 |
Dry SAP medium granulometry 1–2 mm | HORTA-SORB MD | 00810085242789 | https://www.horticulturalalliance.com/product/horta-sorb-md-granule/ |
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Glass Pasteur pipettes 230 mm | Kimble | RK-25554-14 | https://www.coleparmer.ca/i/dwk-life-sciences-kimble-disposable-pasteur-pipettes-plugged-end-borosilicate-glass-230-mm-1000-cs/2555414 |
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Plantago lanceolata seeds | ecoumene | NA | https://www.ecoumene.com/produit/semences/herbacees/plantain-lanceole-bio/ |
Polyvinyl alcohol-lactic acid-glycerol (PVLG). | NA | NA | https://invam.ku.edu/recipes |
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