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.