Experimental approaches BOX 1
Various experimental approaches can be employed to investigate morphological and physiological AM fungal traits in semi-realistic conditions including soil or substrate and plant host(s). We identified five main approaches in the literature. Gazey et al. (1992) utilized asterile mesh bag approach to examine the association between the initiation of sporulation and external hyphae production in twoAcaulospora species. This technique involves the use of 25 µm mesh bags (2 cm wide, 10 cm across, and 10 cm long) containing 200 g of uninoculated, steamed soil. The mesh bags can be placed in pots anywhere along the soil profile. Data are gathered at weekly intervals from 3 to 8 weeks post-plant host germination within the ”sterile mesh bag,” encompassing external hyphae length, spore numbers. Outside the ”sterile mesh bag” host biomass and root colonization, among other variables, can be measured. This approach allows measuring mycorrhizal traits within a controlled sterile soil environment without the interference of propagules present in the original inoculum. The sterile soil should be free from AM fungal propagules and researchers should have controls with non-inoculated mesh bags. Furthermore, as samples are collected from a small soil volume inside the mesh bag, correlations between root colonization, spores, and external hyphae are more likely to represent realistic relationships among these traits. The temporal approach (weekly sampling) permits close monitoring of the entire AM fungal and plant life-cycles. Nonetheless, there are potential limitations to this approach, such as the requirement for propagules to infiltrate the mesh bag, which may be limiting for some taxa (e.g., (Thonar et al. , 2011)). Additionally, if many isolates or species are being analyzed concurrently, the entire experiment may be time-consuming and labor-intensive.
A second approach similar to the sterile mesh bag was used by (Jakobsenet al. , 1992a) to study the abundance of AM fungal hyphae in soil and P uptake into roots. Root compartment bags consisted of cylindrical (60 mm diameter) bags constructed using 25 µm nylon mesh and filled with AM fungal inoculum. These bags are placed in the center of 1.5 L pots, surrounded by steamed sterilized soil and pre-germinated seeds are transplanted into each bag. Thus, the host generated roots are confined within the compartment bag, while AM fungal hyphae are unrestricted to extend into the surrounding soil. After 25 days, root compartment bags can be transplanted into rectangular PVC boxes (L x W x H = 300 x 185 x 130 mm) surrounded by 7 kg of steamed dry soil. To measure hyphal growth, 10 mm diameter soil cores are obtained on five sampling dates at different distances from the root compartment. Similar to the sterile mesh bag method, the root compartment approach enables the measurement of hyphal length in an environment devoid of pre-existing mycorrhizal inoculum. By transplanting the root compartment into large rectangular boxes, this approach is very suitable to study distance and rate of spread of AM fungal external hyphae, allowing a direct comparison of these traits among fungal taxa. However, the experimental design requires a substantial quantity of soil for the setup. A similar approach uses a trap plant on the other end, which allows measurements of resource movement between hosts in a community (Mikkelsen et al. , 2008).
A third approach, named inoculated containers, was used by (Hart & Reader, 2002a) to establish the taxonomic basis for the observed variation in root colonization strategies among AMF families. Fungal biomass is initially measured based on ergosterol concentration to equalize the amount of inoculum at the onset of the experiment. However, we recommend using a different approach (e.g., fatty acids) as has been shown that AM fungi do not produce ergosterol (Olsson et al. , 2003; Olsson & Lekberg, 2022). Containers (4 cm diameter x 20.5 cm deep) are ⅔ filled with soil, then inoculated with soil inoculum containing spores, hyphae and colonized root fragments and sown with leek as a surrogate host. Following a 30 d period, the shoots are harvested, and the soil subjected to experimental treatments, including different hosts. Containers are harvested six times over a 12 w period to measure the extent of root and soil colonization. The inoculated container approach, using small-volume containers, allows the concurrent study of many isolates over time. The standardization of fungal biomass among species allows a direct final comparison among taxa. Given that the abundance of AM fungal external hyphae is assessed in the same container in which the inoculum was introduced, differentiating between newly produced hyphae and those present in the original inoculum is not feasible.
Direct measurements of physiological/chemical traits can require efficient separation of fungal hyphae from the growth substrate. (Zhanget al. , 2023b) employed hyphal in-growth bags , consisting of glass beads and fine silt and clay particles (as a nutrient source) and surrounded by fine nylon mesh (pore size = 38 um) based on the original design by (Neumann & George, 2005), to harvest enough mycelium so that carbon and nitrogen concentrations could be assessed following combustion, and phosphorus concentration following wet digestion, after eight weeks of incubation. In-growth bags were 2 cm wide and 10 cm long and filled with 40 g of the soil-glass bead mixture and buried lengthwise in the top 10 cm of each pot. The mesh size was sufficient to prevent in-growth of Medicago sativa roots but not root hairs of Festuca arundinacea , which requires a mesh size of 10 um to exclude (Zhang et al. , 2021). Soil particles in the bag can still stick to hyphae and need to be carefully removed before analysis. This method, while similar to the sterile mesh bag, is more suitable to study physiology.
Another approach to study traits associated with common mycorrhizal networks (CMNs) was proposed by (Johnson et al. , 2001) usingrotative cores . In this method, a slot (2 cm wide and 5 cm long) is made on the side of a conical container (270 ml) and covered with a 40 µm nylon mesh or hydrophobic membrane. Containers are fitted into polystyrene foam and assembled in pots or microcosms, depending on the study’s objective. The containers are then filled with soil and sand mixtures, inoculated with AM fungi and seeded with a host plant to establish the CMNs for 2-3 months. Following this period, CMN treatments can be established by either maintaining the containers’ positions throughout the experiment (undisturbed CMNs) or rotating them (one full rotation) periodically to physically sever the AM fungal hyphal network. This method is highly suitable for understanding the role of external mycelium and the impact of AM fungal mycelium network disruption on various soil (e.g., bacterial community structure, soil aggregation) and plant (e.g., biomass production, nutrient allocation) parameters (Babikova et al. , 2013). Although the preparation of modified containers is somewhat laborious, this approach enables the investigation of the role of CMNs in plant interactions across multiple species simultaneously.
The methods described above are appropriate for assessing fungal traits and their interrelationships; however, these are not the sole approaches available. The study of genetic traits typically requires the use of isolation techniques into in vitro culture see (Declerck et al. , 2010). For example, in vitro root-organ cultures methods have enabled major breakthroughs in the understanding of genetic and physiological traits such as nutrient exchange ratios (Cranenbroucket al. , 2005; Kiers et al. , 2011) and patterns of hyphal anastomosis between isolates in the same species (Giovannetti et al. , 1999). In addition, in vitro systems may be instrumental in investigating trait interactions between AM fungi and other microorganisms (Faghihinia et al. , 2023). Considering the vast amount of literature on mycorrhizal research and the diverse factors involved in experiments (e.g., plant hosts, soil type, fertilization regime, environmental conditions), a comprehensive understanding of traits for different AM fungal taxa has not been achieved. Given that AM fungi are influenced by soil parameters, plant hosts, and environmental conditions, we propose a set of standard items that should be considered in experiments aiming to measure AM fungal traits. We acknowledge that certain experimental items could be standardized, while others that are more challenging to standardize (e.g., soil type, lighting conditions) could be collected as metadata. By adopting this approach, experiments could be conducted in different laboratories using the same AM fungal taxa while modulating different environmental conditions (e.g.,soil disturbance, salinity, drought, CO2 concentration, temperature, light intensity) to examine the degree of conservation of AM fungal traits across systems and the validity of predictions based on taxonomic identity.