Toward a standardized approach to measure AM fungal traits BOX2
Standardizing the quantification of mycorrhizal fungal traits can be useful to optimize data quality, reduce bias, facilitate comparability, and reproducibility among studies. This methodological uniformity can enhance the robustness of meta-analyses and promote collaboration among researchers, thereby advancing our ecological understanding of AM fungi. What follows is a proposal, based on the authors’ expert opinions, for the standardization of trait-measurement experiments. Researchers may have very good reasons to not comply with some or all of these recommendations in their work, and we want to make clear that this would not mean that the data lack value in the context of understanding AM fungal life histories. As long as deviations from proposed methods are documented along with the data, they would certainly still hold value.
a) Pot Size and Type: Measuring AM fungal traits might result in experiments with many experimental units. Therefore, pot size and substrate (see below) are two items that should be considered to make the experiment feasible. We propose the use of pots with volumes greater than 2 kg , when mesh bags are used. If inoculated containers or rotative cores are chosen, then containers (4 cm diameter x 20.5 cm deep) with conical open bottoms are recommended (Weremijewicz & Janos, 2019).
b) Soil texture: Production of AM fungal external mycelium and sporulation are certainly affected by soil texture, as it determines the number of pores and available free space for fungi to grow. Inert media like sand: expanded clay could be used to standardize the substrate. These media have the advantage of not containing AM fungal propagules. However, they hardly represent the common habitat of AM fungi. Therefore, extrapolating AM fungal trait measurements obtained using these media to field soil conditions might be challenging. We propose using a sterilized loam soil in standard experiments to measure AM fungal traits. If a loam soil is not available, adding quartzite sand or coarse river sand to the soil is suggested to bring the texture closer to a loam soil.
c) Soil sterilization: Autoclave 121oC (for as long as needed; adjust by placing a rod with autoclave tape to the center) repeated twice with a 24h interval. However, if budget and access to infrastructures allow, gamma radiation is a useful alternative that limits chemical alteration of organic matter and downstream consequences on dissolved organic carbon and aggregate stability (Berns et al. , 2008).
d) Nutrient solution: Plant nutrition is an important aspect to be considered as it impacts the establishment of the mycorrhizal symbiosis. We suggest the use of a modified Hoagland’s solution for monocots to provide the minimum amount of macro and micronutrients for the host plant (SupplementaryMaterial).
e) Typically a microbial wash to introduce and standardize non-mycorrhizal soil organisms is prepared by mixing all the tested AM fungal inocula with water and passing the resulting slurry through a 20 µm sieve to be added to the test pots (Ames et al. , 1987). This procedure is arguably the hardest to standardize. We suggest including a control treatment, without a microbial wash to ascertain the role of microorganisms on AM fungal traits.
f) Host plant: Mycorrhizal host plants vary widely in their growth habits (e.g., grasses, trees, forbs), growth rates, and root architecture, which affect the amount of root colonization and spread of hyphae in the soil. Host preference is also another factor to be considered, as it impacts AMF sporulation (Bever et al. , 1996). We suggest the use of Sorghum × drummondii (Sudan grass) as a standard host because a) it has been widely used to grow and maintain a vast array of AMF germplasm in culture collections (Morton et al. , 1993), b) it has a fasciculated root system that provides space for root colonization, and c) it is mycorrhizal dependent.
In addition to these recommendations, metadata should include temperature, soil/substrate type, pH, soil moisture content, soil fertility, light intensity and experiment duration.
Table 1: Key traits, their hypothesized function, and methods for trait measurement
Fungal and fungal mycorrhizal traits
Hypothesized symbiotic effects
AM fungi
Plant
Soil*
 
Qualitative/quantitative (unit)
References - methods on how to measure the traits
References**
Spores
 
 
 
 
 
 
 
Number
-fitness/competitive ability
-dispersal
-carbon storage
x
 
x
x
 
 
 
 
 
x
Quantitative: number of spores/g soil
Spores/meter of mycelia
Spores extracted by wet-sieving ((Gerdemann & Nicolson, 1963)) and sucrose gradient centrifugation (Ba, 1982) and counted under dissecting microscope
 
(Bever et al., 1996), (Chaudhary et al., 2020)
size - diameter
-fitness/competitive ability
-dispersal
-energy to support hyphal growth in absence of host
-carbon storage
-resistance to abiotic and biotic stress
x
 
x
x
 
 
x
x
 
 
 
 
 
 
 
 
 
 
x
x
Quantitative: size measured in µm
Spore diameter measured intact in water using a dissecting or optical microscope
((Morton, 1995, 1996))
(Chaudhary et al., 2020; Deveautour et al., 2020)
 
 
germination rate
% of total
-fitness/competitive ability
-carbon storage
x
 
 
 
x
Quantitative: % of germination
(Douds & Schenck, 1991), Spores over filter paper in a soil-filled Petri plate (Koske, 1981)
 
 
(Tommerup, 1983), (Maia & Yano-Melo, 2001)
germination timing
-fitness/competitive ability
-resistance to abiotic and biotic stress
 
x
 
x
 
x
 
x
Quantitative - % germination per unit of time
Qualitative - stratification needed
(Koske, 1981)
(Tommerup, 1984, 1985), (Koske et al., 1996) (Douds & Schenck, 1991) (Juge et al., 2002)
color
- dispersal
- palatability
- UV protection
- germination duration
 
x
x
 
 
 
Qualitative: color based on CMYK color chart
 
Quantitative: RGB color channels extracted from digitized images (JPG, TIF), calculation of luminance and saturation
Spores observed under a dissecting microscope and compared with color chart (Morton, 1996) or imaged and analyzed using computer software (Deveautour et al. 2020)
Deveautour et al. (2020),(Zanne et al., 2020)
ornamentation
-dispersal
-resistance to abiotic and biotic stress
x
x
 
 
x
Qualitative: type of ornamentation. Quantitative: size in µm
Spores mounted on slides and observed under microscope
Koske and Walker, 1985
Chaudhary et al. (2020)
 
wall thickness
- dispersal
-palatability
- carbon storage
- resistance to abiotic and biotic stress
x
x
x
 
 
 
x
Quantitative: thickness in µm
Spores mounted on slides and thickness measured under microscope Morton (1995, 1996)
(Pawlowska et al., 1999)
(Moore et al., 1985)
wall number
 
 
-dispersal
-resistance to abiotic and biotic stress
x
x
 
 
x
Quantitative: number of walls
Spores mounted on slides and observed under microscope Morton (1995, 1996)
(Walker, 1983)
sporocarps
-dispersal
x
 
x
Size and existence of fungal peridium on the sporocarp surface.
Sporocarps are measured under a microscope for size (Redecker et al., 2007) and existence of peridium (Schüßler et al., 2011)
(Mangan & Adler, 2002)
wall chemical receptors
- perception of host/soil environmental cues affecting germination
x
x
 
 
Knowledge gap (develop a method to identify specific receptors)
Knowledge gap (are there specific receptors on the spore wall that trigger germination?)
Spore nuclear content
-spore viability and germination
- colonization ability after dispersal
x
 
x
x
 
x
x
Number of nuclei per spore
Confocal microscopy, Flow cytometry
(Kokkoris et al., 2020, 2021)
(Bianciotto et al., 1995; Marleau et al., 2011)
 
Elemental composition
-Energy support for hyphal growth
x
 
x
Elemental composition
Proton-induced X-ray emission
(Hammer et al., 2011)
Extraradical Hyphae
 
 
 
 
 
 
 
length
– nutrient/water acquisition
-carbon storage
-soil aggregation
-plant productivity
-resistance to abiotic and biotic stress
x
 
x
x
 
x
x
 
x
x
x
x
x
 
x
x
x
x
Quantitative: hypha length in m/g dry soil
(Miller et al., 1995)
(Wilson et al., 2009)
(Johnson et al., 2015)
architecture (branching rate, anastomoses rate,
absorptive/runner hypha)
 
– nutrient/water acquisition
-carbon storage
-soil aggregation
-plant productivity
-resistance to abiotic and biotic stress
x
 
x
x
x
x
 
x
 
x
x
x
x
 
x
 
x
x
x
Qualitative
 
 
 
Quantitative: Using image analysis in in vitro systems
(Friese & Allen, 1991) or (Bago et al., 1998b) in monoxenic conditions
 
(Hammer et al., 2023)
Knowledge gap (e.g., is hyphal architecture akin to root architecture for nutrient acquisition? how does hyphal architecture influence soil aggregate stability?)
inter-host connection
- transfer of nutrient/water/signals among hosts
 -resistance to abiotic and biotic stress
 
x
 
 
x
 
x
 
 
x
 
x
Quantitative: amount of nutrient/signal transferred
Number of hosts connected by the same fungus
(Weremijewicz & Janos, 2019)
(Frey & Schüepp, 1993)
Knowledge gap (e.g., are there fungi that interconnect more hosts than others? can common mycorrhizal networks provide additional pathogen protection?)
hyphal diameter
–carbon storage
- resistance to abiotic and biotic stress
- palatability
x
x
 
x
 
x
 
x
 
 
 
Quantitative: in µm
(Friese & Allen, 1991)
(Klironomos & Kendrick, 1996)
growth rate
– nutrient/water acquisition
- carbon storage
- resistance to abiotic and biotic stress
x
 
x
x
x
 
x
x
x
 
x
Quantitative: hyphal growth in mm/day
(Schütz et al., 2022)
(Jakobsen et al., 1992b)
hyphal lifespan/turnover
- carbon storage
- nutrient/water acquisition
x
x
x
x
x
x
Qualitative
(Pepe et al., 2018)
(Pepe et al., 2018)
Genetic organization/hyphal fusion (homokaryon/dikaryon)
(note: also applies to spores)
-fitness
– nutrient/water acquisition
-carbon storage
-soil aggregation
-plant productivity
-resistance to abiotic and biotic stress
- hyphal network interconnectedness
x
x
 
x
x
x
x
 
 
x
 
x
 
x
x
x
x
 
 
x
 
 
 
x
x
 
 
 
 
x
Quantitative ddPCR: number of nuclei?
(Cornell et al., 2022)
(Cornell et al., 2022)
(Serghi et al., 2021)
Exudation rate/leakiness
-carbon storage
-influence soil pH and fertility
-soil aggregation
-resistance to abiotic and biotic stress
x
x
 
x
x
x
x
 
x
x
x
x
 
x
 
Quantitative: measure release of a molecule in µM
(Tawaraya et al., 2006)
(Tawaraya et al., 2006)
absorptive capacity
– nutrient/water acquisition
x
x
x
Quantitative: mol m-1 s-1 or % of nutrient taken up
(Frey & Schüepp, 1993), (Jakobsen et al., 1992a)
(Frey & Schüepp, 1993)
color
-resistance to abiotic and biotic stress
x
x
 
Qualitative: color described by CMYK model
(de la Providencia et al., 2005) using transformed roots and (Koske, 1981) using spores over filter paper on soil-filled Petri plate
Knowledge gap (are darker hyphae, more melanized, more resistant to fungivores?)
wall/membrane chemical composition
-resistance to abiotic and biotic stress
-nutrient/water acquisition
-fungal recognition (anastomosis)
-palatability
 
x
 
x
 
x
 
x
 
 
x
x
 
x
 
 
 
 
 
Quantitative: in µg
(Bethlenfalvay et al., 1981) for chitin.
(Frey et al., 1992, 1994) for chitin and ergosterol (Butler & Lachance, 1986) for melanin; (Harrison & Vanbuuren, 1995) for P transporters
(Deveautour et al., 2020)
 
pattern of anastomosis
- fungal recognition
- fitness
x
x
 
 
Quantitative: number of anastomosis per hyphal length (cm) or percentage of anastomosis (%)
(de la Providencia et al., 2005)
(de la Providencia et al., 2005)
Intraradical Hyphae
 
 
 
 
 
 
 
-hyphal thickness
-Resource flux/exchange
-resistance to abiotic and biotic stress
x
 
x
x
 
x
 
Quantitative: in µm
(Abbott, 1982)
Knowledge gap (e.g., are thicker hyphae more resistant to pathogens? Is there a tradeoff between nutrient transfer and biotic resistance in terms of hyphal thickness?)
-pattern of
colonization (localized / widespread)
-Resource flux/exchange
-resistance to abiotic and biotic stress
x
 
x
x
 
x
 
Qualitative
(Dickson, 2004)
(McGONIGLE et al., 1990)
(Abbott, 1982)
 
Knowledge gap (e.g., is resource exchange more/less efficient when colonization is localized or widespread?)
-rate of root colonization
-Resource flux/exchange
-resistance to abiotic and biotic stress
x
x
 
Quantitative: % of root colonization over time
(Dickson, 2004)
(Campo et al., 2020)
Arbuscules
 
 
 
 
 
 
 
- architecture (Paris/Arum type)
- resource flux/exchange
-resistance to abiotic and biotic stress
 
x
 
x
x
 
x
 
Qualitative
(Dickson, 2004)
(van Aarle et al., 2005)
- turnover rate
-resource flux/ exchange
x
x
 
Quantitative: number of days
(Alexander et al., 1989)), (Toth & Miller, 1984)
Knowledge gap (e.g., are some arbuscules more short lived than others? How does arbuscule turnover affect resource exchange?)
-number
-resource flux/ exchange
x
x
 
Quantitative: number of arbuscules
Quantification of Arbuscules Using Morphometric Cytology - (Toth, 1992)
 
Magnified  intersections  method - (McGONIGLE et al., 1990)
 
Image analysis - (Smith & Dickson, 1991)
 
Direct count - (Menge et al., 1978)
(Koch et al., 2017)
Vesicles
 
 
 
 
 
 
 
-size and form (globose/lobbed)
- carbon storage
- resistance to abiotic and biotic stress
x
x
x
x
 
Quantitative: in µm (for size). Qualitative (for form)
(Abbott, 1982)
Knowledge gap (e.g., are fungi with larger vesicles more resistant to stress?)
-number
- carbon storage
x
x
 
Quantitative: number per root length
(Abbott, 1982), (Menge et al., 1978)
(Kobae et al., 2016)
-chemical composition (C/lipid storage)
- carbon storage
x
x
 
Quantitative: % total lipids/ fatty acids 
(Jabaji-Hare et al., 1984)
(Jabaji-Hare et al., 1984)
Turnover rate
 
- carbon storage
x
x
x
Quantitative: number of days
Knowledge gap (adapt the method used for arbuscules)
Knowledge gap (e.g., are some vesicles more short lived than others? How does vesicle turnover affect C storage?)
Other genetic traits
 
 
 
 
 
 
 
Genome size
-reproductive rate
- survival
 
 
 
 
Whole genome sequencing
Flow cytometry
Sperschneider et al., 2023
 
 (Hosny et al., 1998)
GC content of the genome
- mycorrhizal host response
-host preference
x
 
x
 
x
x
 
Whole genome sequencing
Knowledge gap (missing genomes across phylogeny)
(Malar C et al., 2022)
*abiotic and/or biotic
**Examples/papers showing the relationship between measuring the trait its function. References might not necessarily represent best practice.