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Phylogenetic community composition of P. densiflora seedlings from each habitat. An MCC tree was generated by Bayesian inference of an ITS2 sequence alignment from a total of 90 EMF species and one outgroup species (alignment length = 347 bp). The scale bar indicates 100 million years. The heatmap shows the relative abundance of each species in each habitat obtained by morphotype sequencing (Morph) and NGS. Differently colored blocks represent different habitats (brown = disturbed area, green = mature forest). Asterisks indicate significantly abundant species in each habitat detected by the CLAM test. Basidio = Basidiomycota, Asco = Ascomycota, Mucoro = Mucoromycota
Phylogenetic structure analysis at the seedling scale. The nearest taxon index (NTI) was estimated from the EMF community on each P. densiflora seedling. Two different datasets were used: (a) morphotype sequencing (n = 53) and (b) NGS (n = 79). The numbers in parentheses below the box plot indicate the number of seedlings investigated per sampling plot. Asterisks in the box plot indicate significant deviation from the null expectation (*p < 0.05, **p < 0.01, ***p < 0.001)
Spatial distribution analysis at the root tip scale. a Schematic diagram of the estimation of the species segregation index (SSI) from the map of all EMF root tips along the root system: “a linear network” indicates a root system of a P. densiflora seedling, and “a multitype point pattern” depicted by different colors indicates different species. The three nearest root tips were considered neighbors (k = 3) for every root tip i. j = k indicates k nearest neighbor of an i. b Examples of high and low SSI values. Scale bar = 5 mm. c SSI estimated from the EMF community on P. densiflora seedlings (n = 47). The numbers in parentheses below the box plot indicate the number of seedlings investigated per sampling plot. Asterisks in the box indicates significant deviation from the null expectation (*p < 0.05, **p < 0.01, ***p < 0.001)
Simple linear regression between nearest taxon index (NTI) and species segregation index (SSI). Analysis was performed for (a) 47 P. densiflora seedlings and (b) the seedlings per habitat, respectively (disturbed area: n = 25, mature forest: n = 22). Each circle indicates a P. densiflora seedling, and yellow filled circles indicate seedlings for which competitive displacement events were observed. The number of asterisks indicates the degree of significant relationship (*p < 0.05, **p < 0.01, ***p < 0.001)
Ectomycorrhizal fungi (EMF) form symbiotic relationship with the roots of host plants. EMF communities are composed of highly diverse species; however, how they are assembled has been a long-standing question. In this study, we investigated from a phylogenetic perspective how EMF communities assemble on Pinus densiflora seedlings at different spatial scales (i.e., seedling scale and root tip scale). P. densiflora seedlings were collected from different habitats (i.e., disturbed areas and mature forests), and their EMF communities were investigated by morphotype sequencing and next-generation sequencing (NGS). To infer assembly mechanisms, phylogenetic relatedness within the community (i.e., phylogenetic structure) was estimated and spatial distribution of EMF root tips was analyzed. The EMF communities on pine seedlings were largely different between the two habitats. Phylogenetically restricted lineages (Amphinema, /suillus–rhizopogon) were abundant in the disturbed areas, whereas species from diverse lineages were abundant in the mature forests (Russula, Sebacina, /tomentella–thelephora, etc.). In the disturbed areas, phylogenetically similar EMF species were aggregated at the seedling scale, suggesting that disturbance acts as a powerful abiotic filter. However, phylogenetically similar species were spatially segregated from each other at the root tip scale, indicating limiting similarity. In the mature forest seedlings, no distinct phylogenetic signals were detected at both seedling and root tip scale. Collectively, our results suggest that limiting similarity may be an important assembly mechanism at the root tip scale and that assembly mechanisms can vary across habitats and spatial scales.
Epiphytic orchids are commonly found in exposed environments, which plausibly lead to different root fungal community structures from terrestrial orchids. Until recently, few studies have been conducted to show the fungal community structure during the growth of a photosynthetic and epiphytic orchid in its natural growing site. In this study, the Vanda falcata (commonly known as Neofinetia falcata), one of Japan’s ornamental orchids, was used to characterize the fungal community structure at different developmental stages. Amplicon sequencing analysis showed that all development stages contain a similar fungal community: Ascomycota dominate half of the community while one-third of the community belongs to Basidiomycota. Rhizoctonia-like fungi, a polyphyletic basidiomycetous fungal group forming mycorrhizas in many orchids, exist even in a smaller portion (around one-quarter) compared to other Basidiomycota members. While ascomycetous fungi exhibit pathogenicity, two Ceratobasidium strains isolated from young and adult plants could initiate seed germination in vitro. It was also found that the colonization of mycorrhizal fungi was concentrated in a part of the root where it directly attaches to the phorophyte bark, while ascomycetous fungi were distributed in the velamen but never colonized cortical cells. Additionally, the root parts attached to the bark have denser exodermal passage cells, and these cells were only colonized by mycorrhizal fungi that further penetrated into the cortical area. Therefore, we confirmed a process that physical regulation of fungal entry to partition the ascomycetes and mycorrhizal fungi results in the balanced mycorrhizal symbiosis in this orchid.
Schematic overview of the experiment setup showing the artificial micro-landscapes we generated which differed either in relation to habitat connectance (experiment 1) or habitat heterogeneity (experiment 2). Dotted lines in experiment 1 present the minimal distances that AMF had to cross to reach another insert, which was around 2 cm in the high-connectance (a), 2 cm, or 70 cm in the medium-connectance (b), and 55 cm in the low-connectance treatment (c). In the second experiment, the micro-landscapes consisted of eight habitat patches differing in fertilization (yellow compartments indicate P-addition) with constant distances of 20 cm between each, arranged in four treatments, (a) an aggregated treatment, (b) an overdispersed treatment, and (c) two controls different in relation their fertilization state. Seeds of the host plant Medicago lupulina were sown at a density of 4 seeds/cm.² to standardize population density among inserts
Proportion of AMF root colonization: (a) total and (b) arbuscular colonization across three levels of habitat connectance (experiment 1). (c) Functional root colonization represents the sum of arbuscules and coils per root length and (d) the functional ratio represents functional over total AMF colonization. Data points in light to darker blue represent the habitat connectance levels “high,” “medium,” and “low.” Four inserts (= habitat patches) within each of the four replicates resulted in n = 16 counts per treatment. Habitat connectance affected arbuscular and functional colonization, and marginally affected total colonization and the functional ratio (see inserted F-statistics and full ANOVA model results in Appendix II: Tests 1.1, 1.3, 1.5, and 1.6)
Proportion of AMF root colonization: (a) total and (b) arbuscular colonization across three levels of habitat heterogeneity (experiment 2). (c) Functional root colonization represents the sum of arbuscules and coils per root length and (d) the functional ratio represents functional over total AMF colonization. Green points indicate the mixed overdispersed (OV), orange the mixed aggregated (AG), purple the unfertilized control (C–no P), and pink the P-fertilized control treatment (C-P). Eight inserts (= habitat patches) within each of the four replicates resulted in n = 32 data points per treatment, or n = 48 in the case of the aggregated treatment which had six replicates. Habitat heterogeneity affected arbuscular and functional colonization (see inserted F-statistics and full ANOVA model results in the Appendix II: Tests 2.3 and 2.5). The two control levels were considered one in the ANOVA models and compared to the two mixed habitats
Despite their ubiquity in terrestrial ecosystems, arbuscular mycorrhizal fungi (AMF) experience dispersion constraints and thus depend on the spatial distribution of the plant hosts. Our understanding of fungal-plant interactions with respect to their spatial distributions and implications for the functioning of the symbiosis remain limited. We here manipulated the location of habitat patches of Medicago lupulina in two experiments to explore the responses of AMF root colonization and extraradical hyphae. We tested the specific hypothesis that AMF-plant habitats high in connectance would stimulate root colonization and induce denser functional root colonization (colonization rate of arbuscules plus coils) because of higher propagule availability between nearby host plant patches (experiment 1). In experiment 2, we anticipated similar responses in mixed habitats of different soil fertility, namely phosphorus-fertilized or unfertilized soil, and anticipated a higher density of extraradical hyphae in the soil connecting the habitats with increased functional root colonization. In agreement with our hypothesis, we found the highest total and functional root colonization in unfragmented micro-landscapes, describing landscapes that occur within a spatial scale of a few centimeters with the AMF-plant habitats positioned adjacent to each other. In the second experiment, overdispersed micro-landscapes promoted functional root colonization. This study provides experimental evidence that the spatial distribution of habitats can determine AMF abundance at the microscale.
Location of the study sites and plots in Southern Spain: Sierra Sur de Jaén park (referred as Jaén, triangle), and Sierras de Cazorla, Segura y las Villas natural park (referred as Segura, square)
Schematic workflow for the inference of deterministic and stochastic processes through the community phylogenetic (βNTI, expressed in absolute values) and compositional (RCBray) turnover indices
Structure of ectomycorrhizal fungal communities associated with the plant species of this study: Cistus albidus, yellow; Quercus faginea, pink; and Quercus ilex, brown. Fungal community composition was analysed by non-multidimensional scaling (stress = 0.29) on the Bray–Curtis dissimilarity matrix. Strength and direction of vectors indicate the relative weight of occurring fungal orders in structuring ECM fungal communities (correlation significance: ‘***’ p < 0.005, ‘**’ p < 0.01, ‘*’ p < 0.05, ‘.’ p < 0.1)
Effects of environmental variables on the phylogenetic turnover (βNTI) of ectomycorrhizal fungal communities. Distance-based redundant analysis (F2,89 = 3.37, p < 0.01) using significant principal components (PC) after forward selection (arrows). PCA 4 significantly correlated with the phylogenetic distance between C. albidus (yellow) and Quercus spp. (Q. faginea in pink and Q. ilex in brown) (see Table S3). Ellipses enclose βNTI values for each plant species (plotted by using the standard errors with ordiellipse function, vegan R package). PCA 9 significantly correlated with the narrowest decomposed spatial variable, indicating a spatially structured unmeasured environmental variable affecting ectomycorrhizal fungal phylogenetic turnover βNTI
Percentage of ectomycorrhizal fungal community turnover explained by different assembly processes across habitat scales (region, site, plot nested in site and host plant species nested in plot and site). Differences across scales for each assembly process were assessed via a χ.² test. Significant results are highlighted in bold (p < 0.05)
The assembly of biological communities depends on deterministic and stochastic processes whose influence varies across spatial and temporal scales. Although ectomycorrhizal (ECM) fungi play a key role in forest ecosystems, our knowledge on ECM community assembly processes and their dependency on spatial scales is still scarce. We analysed the assembly processes operating on ECM fungal communities associated with Cistus albidus L. and Quercus spp. in Mediterranean mixed forests (Southern Spain), for which root tip ECM fungi were characterized by high-throughput sequencing. The relative contribution of deterministic and stochastic processes that govern the ECM fungal community assembly was inferred by using phylogenetic and compositional turnover descriptors across spatial scales. Our results revealed that stochastic processes had a significantly higher contribution than selection on root tip ECM fungal community assembly. The strength of selection decreased at the smallest scale and it was linked to the plant host identity and the environment. Dispersal limitation increased at finer scales, whilst drift showed the opposite pattern likely suggesting a main influence of priority effects on ECM fungal community assembly. This study highlights the potential of phylogeny to infer ECM fungal community responses and brings new insights into the ecological processes affecting the structure and dynamics of Mediterranean forests.
Mitospore formation on pure cultures of Tuber japonicum on MMN agar medium. a, b Mycelial colonies of FFPRI 460542 (a) and FFPRI 460543 (b) strains. Arrows indicate conidiophores. c Structures of conidiophores in the strain FFPRI 460542. d Conidiophores on the colony of the FFPRI 460516 strain. e Holoblastic conidiogenesis in the strain FFPRI 460542. f Obovate and cylindrical to bacilliform mitospores in the S75-1 strain. g, h Cryo-scanning electron micrographs for conidiogenesis in the strain FFPRI 460542. Bars indicate 1 cm (a, b), 50 µm (c), 100 µm (d), 10 µm (e), and 5 µm (f–h), respectively
The members of the genus Tuber are Ascomycota that form ectomycorrhizal associations with various coniferous and broadleaf tree species. In the teleomorphic stage, the species of the genus produce fruit bodies known as true truffles. Recent studies have discovered mitosporic structures, including spore mats, of several Tuber species on forest soils, indicating the presence of a cryptic anamorphic stage or an unknown reproductive strategy. Here, we report in vitro mitospore formation on the mycelium of T. japonicum, which belongs to the Japonicum clade, collected in several regions in Japan. Twenty of the 25 strains formed mitospores on modified Melin–Norkrans agar medium, indicating that mitospore formation is likely a common trait among strains of T. japonicum. The fungus forms repeatedly branched conidiophores on aerial hyphae on colonies and generates holoblastic mitospores sympodially on the terminal and near apical parts and/or occasionally on the middle and basal parts of the conidiogenous cells. Mitospores are hyaline and elliptical, obovate, oblong, or occasionally bacilliform, with a vacuole and often distinct hilar appendices. Formation of mitospores by T. japonicum in vitro is useful in understanding the functions of mitospores in the genus Tuber under controlled environmental conditions.
Truffle cultivation has drawn more and more attention for its high economic and ecological values in the world. To select symbionts suitable for cultivation purposes, we conducted greenhouse-based mycorrhization trials of two Tuber species (T. formosanum and T. pseudohimalayense) with five broad-leaved tree species (Corylus yunnanensis, Quercus aliena var. acutiserrata, Q. acutissima, Q. robur, Q. variabilis) and one conifer species (Pinus armandii). Axenically germinated seedlings of all tree species were either inoculated, or not, with spore suspensions of these two truffles in the greenhouse. Eight months after inoculation, T. formosanum or T. pseudohimalayense ectomycorrhizae were successfully formed on these six tree species, as evidenced by both morphological and molecular analyses. All selected trees showed good receptivity to mycorrhization by both fungi, with average colonization rates visually estimated at 40-50%. Plant growth, photosynthesis, and nutrient uptake were assessed 2 years after inoculation and were mainly affected by host species. Mycorrhization by both fungi significantly improved P uptake of the hosts, and the interaction between truffle species and host plant species had significant effects on leaf water and leaf K concentrations. In addition, a significantly negative correlation between leaf Ca and leaf C concentration was found across all the seedlings. In addition, mycorrhization had slightly increased plant stem and canopy, but had no significant effects on plant photosynthesis. Overall, these results suggest that the effects of these two Tuber ECMF on plant growth and nutrient acquisition depend on the identity of the host species. Moreover, all selected plant species could be symbiotic partners with either T. pseudohimalayense or T. formosanum for field cultivation purposes.
Schematic diagrams of four experimental designs to quantify the activity of the arbuscular mycorrhizal (AM) pathway(s) of uptake: a regular pot containing a specialised hyphal compartment (HC) with labelled soil, e.g. Svenningsen et al. (2018) (a), a split-pot which can be used with or without a HC if one side contains AM fungal inoculum and isotope and the other does not, e.g. Grønlund et al. (2013) (b), a cross-pot with hyphal compartment side arm(s) containing isotope and differential P supply, e.g. Cavagnaro et al. (2005) (c), and a split-plate that allows for detailed and precise tracing experiments including with quantum dots, e.g. Whiteside et al. (2019) (d). Each of these examples uses dual labelling of phosphorus but also could be used with a single isotope/quantum dot or hyphal compartment; the design is dependent on the research question
Arbuscular mycorrhizal (AM) fungi colonise plant roots, and by doing so forge the ‘mycorrhizal uptake pathway(s)’ (MUP) that provide passageways for the trade of resources across a specialised membrane at the plant–fungus interface. The transport of nutrients such as phosphorus (P), nitrogen and zinc from the fungus, and carbon from the plant, via the MUP have mostly been quantified using stable or radioactive isotope labelling of soil in a specialised hyphae-only compartment. Recent advances in the study of AM fungi have used tracing studies to better understand how the AM association will function in a changing climate, the extent to which the MUP can contribute to P uptake by important crops, and how AM fungi trade resources in interaction with plants, other AM fungi, and friend and foe in the soil microbiome. The existing work together with well-designed future experiments will provide a valuable assessment of the potential for AM fungi to play a role in the sustainability of managed and natural systems in a changing climate.
Main pathways of secondary plant metabolism resulting in the production of alkaloids, phenolics, saponins, and terpenes (in gray, green, pink, and brown shaded portions, respectively) mentioned in this review. Examples of upregulated compounds or classes of compounds in medicinal plants associated with AMF are highlighted with green type. This figure is modified from Dos Santos et al. (2021)
Non-nutritional and nutritional factors influencing the production of secondary metabolites (i.e., terpenoids, phenolics, and flavonoids) in AMF-colonized plants. Non-nutritional factors (leftside in orange): AMF colonization results in the activation of plant defense mechanisms with the production of phenolics and flavonoids. Change in phytohormone levels, such as jasmonic acid (JA), gibberellic acid (GA3), and 6-benzylaminopurine (BAP), increases the number and size of glandular trichomes and leads to transcriptional activation of sesquiterpenoid biosynthetic gene expression. AMF induce the production of signaling molecules, such as nitric oxide, salicylic acid (SA), and hydrogen peroxide, which influence the activation of key enzymes such as l-phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS), for the biosynthesis of phenolic compounds. Nutritional factors (rightside in blue): AMF colonization increases plant nutrients and water uptake leading to increased plant growth and leaf biomass. This results in enhanced plant photosynthetic capacity and increased production of photosynthates which are precursors of different secondary metabolites. Increased leaf biomass leads to an increased density of glandular trichomes in which terpenoids are synthesized and stored. This figure is adapted with permission from Springer Nature Customer Service Centre GmbHS: Springer Nature, Phytochemistry Reviews. Insight into the mechanisms of enhanced production of valuable terpenoids by arbuscular mycorrhiza (Kapoor et al. 2017). We thank Evangelia Tsiokanou (National and Kapodistrian University of Athens, Greece) for graciously providing the picture of the plant used in this figure
(a) Morus alba trees cultivated in aeroponic conditions and (b) close-up view of Morus alba roots grown aeroponically (Chajra et al. 2020). (c) Anchusa officinalis associated with Rhizophagus irregularis MUCL 41,833 growing in a semi-hydroponic cultivation system and (d) close-up view of a plant (UCLouvain, greenhouse). (e) Plant-based bioreactor system for the mass production of AMF as described in Declerck et al. (2009) (WO/2009/ 090,220)
(a) A 145-mm mycorrhizal donor plant in vitro culture system. (i) The donor plant is Crotalaria spectabilis growing in a root compartment (RC) in close association with the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833 and (ii) the receiver plants are Alkanna tinctoria growing under a lid in a hyphal compartment (HC) in which only a profuse, active extraradical mycelium network proliferates; (b) close-up view of extensive development of extraradical mycelium and spores in the HC; (c) a 90-mm half-closed arbuscular mycorrhizal plant in vitro culture system allowing the growth of the roots of Lithospermum erythrorhizon in close association with R. irregularis MUCL 41833; (d) close-up view of the reddish roots due to shikonin production; (e) a 90-mm root organ culture in vitro system allowing the growth of Ri T-DNA transformed A. tinctoria hairy root (Rat et al. 2021) in assocation with R. irregularis MUCL 41833 in the RC; (f) close-up view of the red AMF spores produced in the RC (arrows). We thank Alicia Varela Alonso (Institut für Pflanzenkultur, Germany) for graciously providing the pictures c and d and Angélique Rat (Ghent University, Belgium) for providing the Alkanna tinctoria hairy roots used in this figure. The system (a) starts with a donor plant (Crotalaria spectabilis) introduced into the RC of a bi-compartmented system (a small Petri dish indicated by a dashed circle (RC) (90 mm diameter)) placed in a large Petri dish (HC) (145 mm diameter). A hole is made in both Petri dishes allowing the shoot to extend outside the system. Approximately 500 spores from an AMF in vitro culture are placed in contact with the roots. The roots and AMF are kept in the dark during the whole growth period, while shoots remain under light. Once the donor plant is well colonized, the extraradical mycelium starts to cross the partition wall separating the RC from the HC, developing profusely in the HC. At that time, one or several receiver micropropagated plants (Alkanna tinctoria) are placed in the HC with their roots in contact with the extraradical mycelium. The plants are planted inside the HC under a lid. Briefly, the base of a cylinder (150 mm high, 100 mm diameter) matches a hole made in the lid of the 145-mm Petri dish. The cylinder top is glued to a 100-mm Petri dish lid. The culture dishes containing the A. tinctoria plants are sealed and covered, up to the base of the cylinder, by black plastic bags. The systems are incubated in a growth chamber to allow plant and AMF growth (detailed procedures of this system can be found in Lalaymia and Declerck (2020)). For system (c), homogenously chopped agar containing AMF propagules from an AMF in vitro culture is inoculated to the newly growing roots of a micropropagated seedling of Lithospermum erythrorhizon. After a few days, the new hyphae growing from the spores colonize the roots of L. erythrorhizon. In system (e), fine root structures of Ri T-DNA transformed Alkanna tinctoria hairy roots are cut and placed in the RC part of a bi-compartmental Petri dish. Chopped agar containing AMF propagules is spread on the young parts of the hairy roots. After a few days, new hyphae growing from spores colonize the A. tinctoria hairy root, producing new spores and extensive mycelium after several months. All these three techniques should be conducted under a laminar flow hood with sterilized laboratory materials
Medicinal plants are an important source of therapeutic compounds used in the treatment of many diseases since ancient times. Interestingly, they form associations with numerous microorganisms developing as endophytes or symbionts in different parts of the plants. Within the soil, arbuscular mycorrhizal fungi (AMF) are the most prevalent symbiotic microorganisms forming associations with more than 70% of vascular plants. In the last decade, a number of studies have reported the positive effects of AMF on improving the production and accumulation of important active compounds in medicinal plants. In this work, we reviewed the literature on the effects of AMF on the production of secondary metabolites in medicinal plants. The major findings are as follows: AMF impact the production of secondary metabolites either directly by increasing plant biomass or indirectly by stimulating secondary metabolite biosynthetic pathways. The magnitude of the impact differs depending on the plant genotype, the AMF strain, and the environmental context (e.g., light, time of harvesting). Different methods of cultivation are used for the production of secondary metabolites by medicinal plants (e.g., greenhouse, aeroponics, hydroponics, in vitro and hairy root cultures) which also are compatible with AMF. In conclusion, the inoculation of medicinal plants with AMF is a real avenue for increasing the quantity and quality of secondary metabolites of pharmacological, medical, and cosmetic interest.
Ectomycorrhizal (ECM) fungi improve the host plant’s tolerance to abiotic and biotic stresses. Cenococcum geophilum (Cg) is among the most common ECM fungi worldwide and often grows in saline environments. However, the physiological and molecular mechanisms of salt tolerance in this fungus are largely unknown. In the present study, 12 isolates collected from different ecogeographic regions were used to investigate the mechanism of salt tolerance of Cg. The isolates were classified into four groups (salt-sensitive, moderately salt-tolerant, salt-tolerant, and halophilic) based on their in vitro mycelial growth under 0, 50, 125, 250, and 500 mM NaCl concentrations. Hence, the Na, Ca, P, and K concentrations of mycelia and the pH of the culture solution were determined. Compared with salt-tolerant isolates, treatment with 250 mM NaCl significantly increased the sodium concentration and decreased the potassium concentration of salt-sensitive isolates. RNA-sequencing and qRT-PCR analysis were conducted to identify differentially expressed genes (DEGs) involved in transmembrane transport and oxidoreductase activity pathways. The hydrogen peroxide concentration and activities of peroxidase and superoxide dismutase in mycelia were determined, and the accumulation and scavenging of reactive oxygen species in the salt-sensitive isolates were more active than those in the salt-tolerant isolates. The results supply functional validations to RNA-seq and qRT-PCR analysis. This study provides novel insights into the salt-stress response of Cg isolates and provides a foundation for elucidation of the salt-tolerance mechanism of ECM fungi.
Arbuscular mycorrhizal (AM) fungi form a root endosymbiosis with many agronomically important crop species. They enhance the ability of their host to obtain nutrients from the soil and increase the tolerance to biotic and abiotic stressors. However, AM fungal species can differ in the benefits they provide to their host plants. Here, we examined the putative molecular mechanisms involved in the regulation of the physiological response of Medicago truncatula to colonization by Rhizophagus irregularis or Glomus aggregatum, which have previously been characterized as high- and low-benefit AM fungal species, respectively. Colonization with R. irregularis led to greater growth and nutrient uptake than colonization with G. aggregatum. These benefits were linked to an elevated expression in the roots of strigolactone biosynthesis genes (NSP1, NSP2, CCD7, and MAX1a), mycorrhiza-induced phosphate (PT8), ammonium (AMT2;3), and nitrate (NPF4.12) transporters and the putative ammonium transporter NIP1;5. R. irregularis also stimulated the expression of photosynthesis-related genes in the shoot and the upregulation of the sugar transporters SWEET1.2, SWEET3.3, and SWEET 12 and the lipid biosynthesis gene RAM2 in the roots. In contrast, G. aggregatum induced the expression of biotic stress defense response genes in the shoots, and several genes associated with abiotic stress in the roots. This suggests that either the host perceives colonization by G. aggregatum as pathogen attack or that G. aggregatum can prime host defense responses. Our findings highlight molecular mechanisms that host plants may use to regulate their association with high- and low-benefit arbuscular mycorrhizal symbionts.
Achlorophyllous, mycoheterotrophic plants often have an elaborate mycorrhizal colonization pattern, allowing a sustained benefit from external fungal root penetrations. The present study reveals the root anatomy and mycorrhizal pattern of eight mycoheterotrophic Thismia spp. (Thismiaceae), all of which show separate tissue compartments segregating different hyphal shapes of the mycorrhizal colonization, as there are intact straight, coiled and peculiarly knotted hyphae as well as degenerated clumps of hyphal material. Those tissue compartments in Thismia roots potentially comprise exo-, meso-and endoepidermae, and exo-, meso-and endocortices, although not all species develop all these root layers. Differences in details among species according to anatomy (number of root layers, cell sizes and shapes) and colonization pattern (hyphal shapes within cells) are striking and can be discussed as an evolutionary series towards increasing mycorrhizal complexity which roughly parallels the recently established phylogeny of Thismia. We suggest functional explanations for why the distinct elements of the associations can contribute to the mycorrhizal advantage for the plants and, thus, we emphasize the relevance of structural traits for mycorrhizae.
Total free amino acid nitrogen (fAA-N) content in soil from wet heath, mesic heath, mesic meadow, dry heath, and blanket bog, for control (C) and warming treatment by OTC (open top chamber, T). P values of the effect of site and warming treatment and their interaction. Different letters represent significant differences between sites
RDA analysis diagram, free amino acid content (µg∙g⁻¹ soil; error bars indicate 95% confidence interval). The sites are blanket bog, wet heath, mesic heath, mesic meadow, and dry heath. Treatment T is warming, and C is control (no treatment). The direction of mycorrhizal types ERM, ECM, and AM/NM by black arrows. Total variation is 73.6%, and explanatory variables account for 100.0%, where axis 1 explains 84.2% of the variation and axis 2 explains 11.2% of the variation. Red arrows represent the direction and strength of the individual amino acids: alanine (Ala), glycine (Gly), valine (Val), leucine (Leu), serine (Ser), theonine (The), proline (Pro), asparagine (Asn), aspartate (Asp), methionine (Met), glutamate (Glu), phenylalanine (Phe), tyrosine (Tyr)
The soil nitrogen (N) cycle in cold terrestrial ecosystems is slow and organically bound N is an important source of N for plants in these ecosystems. Many plant species can take up free amino acids from these infertile soils, either directly or indirectly via their mycorrhizal fungi. We hypothesized that plant community changes and local plant community differences will alter the soil free amino acid pool and composition; and that long-term warming could enhance this effect. To test this, we studied the composition of extractable free amino acids at five separate heath, meadow, and bog locations in subarctic and alpine Scandinavia, with long-term (13 to 24 years) warming manipulations. The plant communities all included a mixture of ecto-, ericoid-, and arbuscular mycorrhizal plant species. Vegetation dominated by grasses and forbs with arbuscular and non-mycorrhizal associations showed highest soil free amino acid content, distinguishing them from the sites dominated by shrubs with ecto-and ericoid-mycorrhizal associations. Warming increased shrub and decreased moss cover at two sites, and by using redundancy analysis, we found that altered soil free amino acid composition was related to this plant cover change. From this, we conclude that the mycorrhizal type is important in controlling soil N cycling and that expansion of shrubs with ectomycorrhiza (and to some extent ericoid mycorrhiza) can help retain N within the ecosystems by tightening the N cycle.
Rhizophagus irregularis. A A sporocarp with yellow swollen structures at the soil surface (bar = 1 cm). B Yellow swollen structures (bar = 500 μm). C Cross section of a sporocarp showing spores and yellow swollen structures (bar = 500 μm). D, E Spores and obovate swollen structures (bar = 100 μm). F Spores (bar = 100 μm). G A cracked spore showing a subtending hypha with an open pore (bar = 10 μm). H Spores with a positive reaction to Melzer’s reagent (bar = 100 μm). SS, swollen structure; SP, spore; IW, inner wall; LW, laminated wall; OW, outer wall
Diversispora epigaea. A A sporocarp with white to orange interwoven hyphae at the soil surface (bar = 1 cm). B Orange interwoven hyphae covering a sporocarp (bar = 500 μm). C Orange interwoven hyphae (bar = 100 μm). D Cross section of a sporocarp showing spores (bar = 500 μm). E Spores (bar = 200 μm). F A swollen structure with hyphae (bar = 100 μm). G A cracked spore showing the spore wall (bar = 50 μm). H A cracked spore showing a subtending occluded hypha (bar = 50 μm). IF, interwoven hyphae; SP, spore; SS, swollen structure; OW, outer wall; LW, laminated wall
Maximum liklihood phylogenetic tree based on partial sequences of the HD2 region in the putative AM fungal MAT locus of Rhizophagus irregularis, including sequences of R. irregularis sporocarps obtained in this study. INSD accession numbers are given for all sequences. The tree is rooted with Rhizophagus clarus (KT946655). Bootstrap values (BS) with 1000 replications are shown at each node (only BS > 70% are shown). The scale bar indicates the number of substitutions per site
Ratio of SNPs (%) shared by 1–8 spores isolated from sporocarps. ARhizophagus irregularis CE1901, CE1903, and CE2001. BDiversispora epigaea CE2018, CE2022, and CE2105. The number of SNPs examined is shown parenthetically next to the sporocarp numbers
Hierarchical clustering analysis of the spores isolated from sporocarps of Rhizophagus irregularis and Diversispora epigaea based on polymorphic SNPs. A On 24 spores from three sporocarps of R. irregularis (CE1901, CE1903, and CE2001) with 86 polymorphic SNP sites. B On 24 spores from three sporocarps of D. epigaea (CE2018, CE2022, and CE2105) with 300 polymorphic SNP sites. Approximately unbiased p-value (au; red character) and bootstrap probability value (bp; green character) with 1000 replications are shown at each node
Some arbuscular mycorrhizal (AM) fungal species known to form sporocarps (i.e., aggregations of spores) are polyphyletic in two orders, Glomerales and Diversisporales. Spore clusters (sporocarp-like structures) often formed in pot cultures or in vitro conditions are supposed to be clonal populations, while sporocarps in natural habitats with a fungal peridium are morphologically similar to those of epigeous sexual (zygosporic) sporocarps of Endogone species. Thus, in this study, we explored the genetics of sporocarpic spores of two AM fungi with a view to possibilities of clonal or sexual reproduction during sporocarps formation. To examine these possibilities, we investigated single-nucleotide polymorphisms (SNPs) in reduced genomic libraries of spores isolated from sporocarps molecularly identified as Rhizophagus irregularis and Diversispora epigaea. In addition, partial sequences of the MAT locus HD2 gene of R. irregularis were phylogenetically analyzed to determine the nuclear status of the spores. We found that most SNPs were shared among the spores isolated from each sporocarp in both species. Furthermore, all HD2 sequences from spores isolated from three R. irregularis sporocarps were identical. These results indicate that those sporocarps comprise clonal spores. Therefore, sporocarps with clonal spores may have different functions than sexual reproduction, such as massive spore production or spore dispersal via mycophagy.
Many ectomycorrhizal (ECM) fungi produce commercially valuable edible sporocarps. However, the effects of nitrogen (N) application on ECM fungal sporocarp formation remain poorly understood. In this study, we investigated the effect of application of various N concentrations (0, 5, 25, 50, 100, and 200 mg/L) on the growth of Laccaria japonica mycelia in vitro for 1 month. The results showed that L. japonica mycelial biomass was highest in the 50 mg/L treatment and was significantly inhibited at N concentrations higher than 200 mg/L. Next, we investigated the effects of N application on mycorrhizal colonization and sporocarp formation in L. japonica colonizing Pinus densiflora seedlings in pots. The seedlings were watered with nutrient solutions containing 0, 5, 25, 50, or 100 mg N/L. The biomass, photosynthetic rate, and mycorrhizal colonization rates of the seedlings were measured at 45 days (first appearance of primordia), 65 days (sporocarp appearance on the substrate surface), and 4 months after seedlings were transplanted. The numbers of primordia and sporocarps were recorded during the experimental period. Total carbon (C) and N content were determined in seedlings at 4 months after transplantation, and in L. japonica sporocarps. Both mycelial growth and sporocarp production reached their maximum at an N application concentration of 50 mg/L, suggesting that the most suitable N concentration for ECM fungal sporocarp formation can easily be estimated in vitro during mycelial growth. This finding may help determine the most suitable N conditions for increasing edible ECM fungus sporocarp production in natural forests.
Community composition and seasonal variation of sporulation of arbuscular mycorrhizal fungi (AMF) have been studied in soils from many ecosystems including subtropical forest. Yet, AMF community composition has been surveyed only from the mineral soil but not from the litter layer and the root mat, and long-term variation in sporulation is not fully understood. We sampled a 75-m² plot from a subtropical forest to determine AMF community composition in the following habitats: the litter layer, the root mat, and the mineral soil. Moreover, samples were taken in fall, winter, spring, and summer over a 2-year period to follow the seasonal variation of AMF sporulation. We detected 47 AMF species belonging to six families and 14 genera, Glomeraceae and Acaulosporaceae being the most represented families. Sixteen species were common to all three habitats, five species were shared between two habitats, and 26 species were recovered exclusively from single habitats. While species richness was not significantly different among habitats, AMF total spore numbers were significantly higher in the litter and root mat compared to the soil. PERMANOVA did not detect a significant effect of habitats on community composition when species presence/absence was considered, but significant differences between litter versus soil and root mat versus soil were detected when spore abundance was considered. A seasonal pattern of spore abundance for species was not observed over the 2-year sampling period regardless of habitat. This study revealed that (i) different AMF species sporulate in the different habitats; thus, field surveys considering only the mineral soil might underestimate species richness and (ii) AMF species sporulate asynchronously in subtropical forest.
Dry weight of alfalfa and ryegrass shoots and roots (total per pot) with (M) and without (NM) arbuscular mycorrhizal inoculation. Root weights are shown as positive values below the abscissa. Bars (Means ± SD) topped by the same letter do not differ significantly using two-way ANOVA and Tukey (HSD) post hoc test (P < 0.05). Rank-transformed data were used for shoot weights
P concentration in alfalfa and ryegrass shoots and roots with (M) and without (NM) arbuscular mycorrhizal inoculation. P concentration in roots is shown as positive values below the abscissa. Bars (Means ± SD) topped by the same letter do not differ significantly using two-way ANOVA on rank-transformed data and Tukey (HSD) post hoc test (P < 0.05)
La, Ce, Sm and Yb concentrations in alfalfa and ryegrass a) shoots and b) roots with (M) and without (NM) arbuscular mycorrhizal inoculation. REE concentrations in roots are shown as positive values below the abscissa. Bars (Means ± SD) topped by the same letter do not differ significantly among treatments using two-way ANOVA (mycorrhization and plant species) on rank-transformed data and Tukey (HSD) post hoc test (P < 0.05). There was no significant difference among the REE concentrations in roots
Rare earth elements (REEs) are widely used in high-tech industries, and REE waste emissions have become a concern for ecosystems, food quality and human beings. Arbuscular mycorrhizal fungi (AMF) have repeatedly been reported to alleviate plant stress in metal-contaminated soils. To date, little information is available concerning the role of AMF in REE-contaminated soils. We recently showed that there was no transfer of Sm to alfalfa by Funneliformis mosseae, but only a single REE was examined, while light and heavy REEs are present in contaminated soils. To understand the role of AMF on the transfer of REEs to plants, we carried out an experiment using alfalfa (Medicago sativa) and ryegrass (Lolium perenne) in compartmented pots with separate bottom compartments that only were accessible by F. mosseae fungal hyphae. The bottom compartments contained a mixture of four REEs at equal concentrations (La, Ce, Sm and Yb). The concentration of REEs in plants was higher in roots than in shoots with higher REE soil–root than root–shoot transfer factors. Moreover, significantly higher light-REEs La and Ce were transferred to ryegrass shoots than Sm and the heavy-REE Yb, but this was not observed for alfalfa. Alfalfa dry weight was significantly increased by F. mosseae inoculation, but not ryegrass dry weight. For both plant species, there was significantly higher P uptake by the mycorrhizal plants than the nonmycorrhizal plants, but there was no significant transfer of La, Ce, Sm or Yb to alfalfa and ryegrass roots or shoots due to F. mosseae inoculation.
Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs, and the difficulty of growing them in asymbiotic or monoxenic (AMF + root) conditions limits research and their large-scale production as biofertilizer. We hypothesized that a combination of flavanols and strigolactones can mimic complex root signaling during the presymbiotic stages of AMF. We evaluated the germination, mycelial growth, branching, and auxiliary cell clusters formation by Gigaspora margarita during the presymbiotic stage in the presence (or absence) of transformed Cichorium intybus roots in basal culture medium enriched with glucose, a flavonol (quercetin or biochanin A) and a strigolactone analogue (1-Methyl-2-oxindole or indole propionic acid). With quercetin (5 µM), methyl oxindole (2.5 nM), and glucose (8.2 g/L) in the absence of roots, the presymbiotic mycelium of G. margarita grew without cytoplasmic retraction and produced auxiliary cells over 71 days similar to presymbiotic mycelium in the presence of roots but without glucose, strigolactones, and flavonols. Our results indicate that glucose and a specific combination of certain concentrations of a flavonol and a strigolactone might be used in asymbiotic or monoxenic liquid or semisolid cultures to stimulate AMF inoculant bioprocesses.
The main global determinants of taxonomic a, b and phylogenetic c, d soil AM fungal community composition. a and c distance-based redundancy analysis (dbRDA) ordination biplots. Arrows indicate the direction of maximum change in environmental variables. Ellipses show 1 standard deviation around the centroid of different biogeographic realms (using the same colors used to distinguish points on the plot); b and d variable importance in generalized dissimilarity models (GDM), measured as the change in model deviance caused by variable permutation. The GDM models do not include the categorical predictors of the dbRDA models but do include a spatial distance matrix that is not in dbRDA models. OrgC, % organic carbon; MAT, mean annual temperature; MAP, mean annual precipitation; SeaPrec, seasonal variation in precipitation
I-splines (partial ecological distance) from generalized dissimilarity modelling (GDM) of AM fungal a taxonomic and b phylogenetic community composition. The slope of the i-spline indicates the rate of compositional turnover and how it changes along the range of the variable (variable z-score). For instance, in a, community turnover is rapid at low values of pH and slower at high values, but the converse is true of turnover in relation to MAT. OrgC, % organic carbon; MAT, mean annual temperature; MAP, mean annual precipitation; SeaPrec, seasonal variation in precipitation. Note the different scales of the ordinate axes
Global interpolated maps of AM fungal communities using k-means clustering (k = 2) of a taxonomic and b phylogenetic distances. Greenland and the Sahara region were excluded from interpolations because of insufficient sampling and highly contrasted, different abiotic conditions
AM fungal family-level composition of a taxonomic and b phylogenetic community clusters. Stacked bars indicate the relative abundance of reads corresponding to different AM fungal families. Cluster labels follow the numbering in Fig. 3. Note that there is no direct correspondence between clusters in both classifications, with each bar only comprising read counts from those samples within it
Arbuscular mycorrhizal (AM) fungi are a ubiquitous group of plant symbionts, yet processes underlying their global assembly — in particular the roles of dispersal limitation and historical drivers — remain poorly understood. Because earlier studies have reported niche conservatism in AM fungi, we hypothesized that variation in taxonomic community composition (i.e., unweighted by taxon relatedness) should resemble variation in phylogenetic community composition (i.e., weighted by taxon relatedness) which reflects ancestral adaptations to historical habitat gradients. Because of the presumed strong dispersal ability of AM fungi, we also anticipated that the large-scale structure of AM fungal communities would track environmental conditions without regional discontinuity. We used recently published AM fungal sequence data (small‐subunit ribosomal RNA gene) from soil samples collected worldwide to reconstruct global patterns in taxonomic and phylogenetic community variation. The taxonomic structure of AM fungal communities was primarily driven by habitat conditions, with limited regional differentiation, and there were two well-supported clusters of communities — occurring in cold and warm conditions. Phylogenetic structure was driven by the same factors, though all relationships were markedly weaker. This suggests that niche conservatism with respect to habitat associations is weakly expressed in AM fungal communities. We conclude that the composition of AM fungal communities tracks major climatic and edaphic gradients, with the effects of dispersal limitation and historic factors considerably less apparent than those of climate and soil.
Map of Rhizophagus irregularis glomalin gene showing exon–intron structure and placement of primers listed in Table 2. Filled arrowheads indicate new primers used in this publication, empty arrowheads are from (Magurno et al. 2019)
Phylogenetic tree based on partial glomalin gene sequence alignments from diverse species of AM fungi, consisting of 1314 positions. Units of the number of base substitutions per site. Bootstrap values are shown. Phylogenetic analyses were conducted in MEGA X (Kumar et al. 2018)
Percent root colonization (± SE) in corn farming practices based on the root-intersect method. BP is black plastic. P value is from one-way ANOVA. Bars topped by the same letter do not differ at p ≤ 0.05 according to Tukey–Kramer HSD post hoc analysis
Currently, root colonization measurements of arbuscular mycorrhizal fungi (AMF) require staining and microscopy, and species-level identification of the fungi by such observations is not possible. Here, we present novel multiplex real-time PCR assays targeting the glomalin genes of 11 different species of AMF commonly found in temperate agricultural soils, which independently detect and measure the abundance of these fungi using DNA extracts from soil and or root tissue. The availability of these tools will not only increase throughput in determining levels of root colonization but can provide species-specific levels of root colonization from a single sample. This will help to establish which AMF species, or combinations of different species, provide the most benefits to crops, and will aid in the development of AMF for use as biofertilizers.
The rRNA gene region with commonly used AMF primers. Primers used in studies from which database sequences were extracted. The scale represents basepair number. The SSU is represented by 18S, while the LSU is represented by 28S. ITS1 and ITS2 are represented by the grey regions either side of 5.8S, with ITS1 to the left and ITS2 to the right. Full sequences can be found in Table S1
The new reference tree for placement of AMF via LSU amplicon sequences. This expanded tree includes 174 AMF across 11 families (indicated by shades of blue) and sequences representing the major neighboring clades of the Basidiomycota, Ascomycota, Mucoromycota, Mortierellomycota, and Chytridomycota as well as two animal and two plant outgroups (indicated by shades of grey)
An example of non-homologous sequences corrected by the new pipeline. The same 15 OTUs were placed in the tree without an initial BLAST screening (A; shaded in purple), but including this step removed these non-homologous sequences, with only OTU 10 retained (B)
Arbuscular mycorrhizal fungi (AMF; Glomeromycota) are difficult to culture; therefore, establishing a robust amplicon-based approach to taxa identification is imperative to describe AMF diversity. Further, due to low and biased sampling of AMF taxa, molecular databases do not represent the breadth of AMF diversity, making database matching approaches suboptimal. Therefore, a full description of AMF diversity requires a tool to determine sequence-based placement in the Glomeromycota clade. Nonetheless, commonly used gene regions, including the SSU and ITS, do not enable reliable phylogenetic placement. Here, we present an improved database and pipeline for the phylogenetic determination of AMF using amplicons from the large subunit (LSU) rRNA gene. We improve our database and backbone tree by including additional outgroup sequences. We also improve an existing bioinformatics pipeline by aligning forward and reverse reads separately, using a universal alignment for all tree building, and implementing a BLAST screening prior to tree building to remove non-homologous sequences. Finally, we present a script to extract AMF belonging to 11 major families as well as an amplicon sequencing variant (ASV) version of our pipeline. We test the utility of the pipeline by testing the placement of known AMF, known non-AMF, and Acaulospora sp. spore sequences. This work represents the most comprehensive database and pipeline for phylogenetic placement of AMF LSU amplicon sequences within the Glomeromycota clade.
Mycorrhizosphere bacteria composition during seasons a based on microscopy and biochemical phenotype characterization of 417 isolated strains and b based on plant growth-promoting traits in the 68 different OTUs for auxin production (IAA), phosphate solubilization (P solubilization), siderophore production, ACC deaminase production (ACCD), or PGRP trait not detected (none)
Non-metric multidimensional scaling ordination of isolated bacterial communities associated with desert truffle plants in different seasons. Filled circles denote samples, open circles denote bacterial OTUs. Ellipses denote 95% confidence intervals. Permanova results regard season showed significant differences (F = 2.7061, p = 0.001, R² = 0.474)
RLQ joined ordination showing the relationship between PGPR traits and seasons from bacterial isolates. Direction and length of vectors indicate correlation with other variables and contribution to the ordination, respectively (model #2, p = 0.0045; model #4, p = 0.0004)
Community weighted means (CWMs) analysis of the PGPR activities in bacterial colonies across seasons. Different letters indicate significant differences between groups (p < 0.05)
Desert truffle is becoming a new crop in semiarid areas. Climatic parameters and the presence of microorganisms influence the host plant physiology and alter desert truffle production. Desert truffle plants present a typical summer deciduous plant phenology divided into four stages: summer dormancy, autumn bud break, winter photosynthetic activity, and spring fruiting. We hypothesize that the bacterial community associated with desert truffle plants will show a seasonal trend linked to their plant growth–promoting rhizobacteria (PGPR) traits. This information will provide us with a better understanding about its potential role in this symbiosis and possible management implementations. Bacteria were isolated from root-adhering soil at the four described seasons. A total of 417 isolated bacteria were phenotypically and biochemically characterized and gathered by molecular analysis into 68 operational taxonomic units (OTUs). They were further characterized for PGPR traits such as indole acetic acid production, siderophore production, calcium phosphate solubilization, and ACCD (1-amino-cyclopropane-1-carboxilatedeaminase) activity. These PGPR traits were used to infer functional PGPR diversity and cultivable bacterial OTU composition at different phenological moments. The different seasons induced shifts in the OTU composition linked to their PGPR traits. Summer was the phenological stage with the lowest microbial diversity and PGPR functions, whereas spring was the most active one. Among the PGPR traits analyzed, P-solubilizing rhizobacteria were harbored in the mycorrhizosphere during desert truffle fruiting in spring.
Ectomycorrhizal (EM) and arbuscular mycorrhizal (AM) fungi are often studied independently, and thus little is known regarding differences in vertical distribution of these two groups in forests where they co-occur. We sampled roots at two soil depths in two northern hardwood stands in Bartlett, New Hampshire, co-dominated by tree species that associate with AM or EM fungi. Root length of both groups declined with depth. More importantly, root length of EM plant species exceeded that of AM plants at 0-10-cm depth, while AM exceeded EM root length at 30-50-cm depth. Colonization rates were similar between mineral and organic portions of the shallow (0-10 cm) samples for EM and AM fungi and declined dramatically with depth (30-50 cm). The ratio of EM to AM fungal colonization declined with depth, but not as much as the decline in root length with depth, resulting in greater dominance by EM fungi near the surface and by AM fungi at depth. The depth distribution of EM and AM roots may have implications for soil carbon accumulation as well as for the success of the associated tree species.
Schematic representation of nurse plant seedling inoculation systems A and corresponding recipient plant mycorrhization parameters B. Seedling inoculation systems with the nurse plant planted upwards (A1) and downwards (A2). Mycorrhizal colonization (B1), arbuscule colonization (B2), and hyphal density (B3) of recipient plants cultivated in M-NU and M-ND conditions at 45, 60, and 75 days after plant-
Plant mycorrhization can be achieved by transplanting new seedlings with mycorrhizal nurse plants; however, this method inevitably induces plant interactions. Transplanting nurse plants downwards may prevent light competition among new seedlings and nurse plants in the same pot. We hypothesized that seedling mycorrhization via mycorrhizal provision from plants planted downwards would be a feasible and efficient strategy. We used seedlings cultivated for 6 months after inoculation with arbuscular mycorrhizal fungi (AMF) as nurse plants, and seedlings cultivated for 1 month without AMF as recipient plants, transplanting one nurse plant and three recipient plants together in one pot. We compared two approaches for cultivating mycorrhizal Broussonetia papyrifera seedlings: planting mycorrhizal nurse plants upwards (M-NU) and downwards (M-ND). We also planted non-mycorrhizal nurse plants upwards (NM-NU) and downwards (NM-ND) as controls. We analyzed growth parameters and the mycorrhizal colonization status of recipient plants at 45, 60, and 75 days after planting (DAP). As expected, the plant growth, gas exchange, and root morphological parameters of recipient plants with mycorrhizal nurse plants were higher than those of recipient plants with non-mycorrhizal nurse plants at 60 and 75 DAP. Furthermore, the AMF colonization status and physiological growth status of M-ND recipient plants were improved compared with M-NU recipient plants. Our results demonstrate that inducing seedling mycorrhization by planting mycorrhizal nurse plants downwards is a feasible strategy for achieving AMF symbiosis while mitigating negative interactions among plants.
Relationship between site variables and water table depth among plots along drained Picea mariana — dominated peatland gradients near Meadowlands, MN, USA. Peat tissue was harvested from the top 20 cm. Canopy density represents the first axis of a PCA (accounting for 95% of variation) using basal area and canopy openness
Canonical analysis of principal coordinates of fungal communities on root tips of P. mariana seedlings collected from drained peatland gradients near Meadowlands, MN, USA. Ordinations depict (a) all fungal genera (n = 34, only top 5% of taxa with best fit are shown, all others transparent), (b) fungal genera classified as mycorrhizal (n = 34, only top 20% of taxa with best fit are shown, all others transparent), (c) fungal genera classified as saprotrophic (n = 36, only top 5% of taxa with best fit are shown, all others transparent), and (d) ectomycorrhizal (ECM) exploration types (n = 35) derived from FUNGuild (Nguyen et al. 2016), Agerer (2001), Clemmensen et al. (2015), and Hagenbo et al. (2018)
Results of path analyses for ECM community composition (represented by the first PCA axis of mycorrhizal genera, accounting for 61.3% of variance), medium-distance fringe exploration type, short-distance exploration type, relative abundance of class II peroxidase gene copies (calculated by multiplying the relative abundance of a given genus with the average number of published gene copies for that genus; see Supplementary Data), Cortinarius-relative abundance, Cenococcum geophilum–relative abundance, and Piloderma sphaerosporum relative abundance, collected from fine roots of P. mariana seedlings along drained peatland gradients near Meadowlands, MN, USA (n = 36). Only significant (p < 0.05) paths were retained. Values indicate standardized path coefficients, and arrow width is proportional to the standardized path coefficient; black arrows indicate positive coefficients while red arrows indicate negative coefficients
The relative abundance (residuals of multiple regression) of (a) medium-distance fringe and short-distance ectomycorrhizal exploration types as a function of seedling stem radial growth; (b) medium-distance fringe exploration types as a function of pH; (c) medium-distance fringe and short-distance exploration types as a function of peat % N; (d) class II peroxidase gene copies in the ectomycorrhizal community (calculated by multiplying the relative abundance of a given genus with the average number of published gene copies found for that genus; see Supplementary Data) as a function of seedling stem radial growth; (e) class II peroxidase gene copies as a function of pH; (f) class II peroxidase gene copies as a function of peat % N. Picea mariana seedlings were collected from drained peatlands near Meadowlands, MN, USA (n = 36 samples). Exploration types were classified using FUNGuild (Nguyen et al. 2016), Agerer (2001), Clemmensen et al. (2015), and Hagenbo et al. (2018)
(a) The relative abundance (residuals of multiple regression) of Cortinarius spp. as a function of host seedling stem radial growth; (b) the relative abundance (proportion) of Cenococcum geophilum and Piloderma sphaerosporum as a function of host seedling stem radial growth; (c) the relative abundance (residuals of multiple regression) of Cortinarius spp. as a function of pH; and (d) the relative abundance (residuals of multiple regression) as a function of peat % N, on fine roots of P. mariana seedlings from drained peatland gradients near Meadowlands, MN, USA (n = 36)
Many trees depend on symbiotic ectomycorrhizal fungi for nutrients in exchange for photosynthetically derived carbohydrates. Trees growing in peatlands, which cover 3% of the earth’s terrestrial surface area yet hold approximately one-third of organic soil carbon stocks, may benefit from ectomycorrhizal fungi that can efficiently forage for nutrients and degrade organic matter using oxidative enzymes such as class II peroxidases. However, such traits may place a higher carbon cost on both the fungi and host tree. To investigate these trade-offs that might structure peatland ectomycorrhizal fungal communities, we sampled black spruce (Picea mariana (Mill.)) seedlings along 100-year-old peatland drainage gradients in Minnesota, USA, that had resulted in higher soil nitrogen and canopy density. Structural equation models revealed that the relative abundance of the dominant ectomycorrhizal fungal genus, Cortinarius, which is known for relatively high fungal biomass coupled with elevated class II peroxidase potential, was negatively linked to site fertility but more positively affected by recent host stem radial growth, suggesting carbon limitation. In contrast, Cenococcum, known for comparatively lower fungal biomass and less class II peroxidase potential, was negatively linked to host stem radial growth and unrelated to site fertility. Like Cortinarius, the estimated relative abundance of class II peroxidase genes in the ectomycorrhizal community was more related to host stem radial growth than site fertility. Our findings indicate a trade-off between symbiont foraging traits and associated carbon costs that consequently structure seedling ectomycorrhizal fungal communities in peatlands.
Arbuscular mycorrhizal (AM) fungi and rhizobium are likely important drivers of plant coexistence and grassland productivity due to complementary roles in supplying limiting nutrients. However, the interactive effects of mycorrhizal and rhizobial associations on plant community productivity and competitive dynamics remain unclear. To address this, we conducted a greenhouse experiment to determine the influences of these key microbial functional groups on communities comprising three plant species by comparing plant communities grown with or without each symbiont. We also utilized N-fertilization and clipping treatments to explore potential shifts in mycorrhizal and rhizobial benefits across abiotic and biotic conditions. Our research suggests AM fungi and rhizobium co-inoculation was strongly facilitative for plant community productivity and legume (Medicago sativa) growth and nodulation. Plant competitiveness shifted in the presence of AM fungi and rhizobium, favoring M. sativa over a neighboring C4 grass (Andropogon gerardii) and C3 forb (Ratibida pinnata). This may be due to rhizobial symbiosis as well as the relatively greater mycorrhizal growth response of M. sativa, compared to the other model plants. Clipping and N-fertilization altered relative costs and benefits of both symbioses, presumably by altering host-plant nitrogen and carbon dynamics, leading to a relative decrease in mycorrhizal responsiveness and proportional biomass of M. sativa relative to the total biomass of the entire plant community, with a concomitant relative increase in A. gerardii and R. pinnata proportional biomass. Our results demonstrate a strong influence of both microbial symbioses on host-plant competitiveness and community dynamics across clipping and N-fertilization treatments, suggesting the symbiotic rhizosphere community is critical for legume establishment in grasslands.
Diversity in arbuscular mycorrhizal fungi (AMF) contributes to biodiversity and resilience in natural environments and healthy agricultural systems. Functional complementarity exists among species of AMF in symbiosis with their plant hosts, but the molecular basis of this is not known. We hypothesise this is in part due to the difficulties that current sequence assembly methodologies have assembling sequences for intrinsically disordered proteins (IDPs) due to their low sequence complexity. IDPs are potential candidates for functional complementarity because they often exist as extended (non-globular) proteins providing additional amino acids for molecular interactions. Rhizophagus irregularis arabinogalactan-protein-like proteins (AGLs) are small secreted IDPs with no known orthologues in AMF or other fungi. We developed a targeted bioinformatics approach to identify highly variable AGLs/IDPs in RNA-sequence datasets. The approach includes a modified multiple k -mer assembly approach (Oases) to identify candidate sequences, followed by targeted sequence capture and assembly (mirabait-mira). All AMF species analysed, including the ancestral family Paraglomeraceae, have small families of proteins rich in disorder promoting amino acids such as proline and glycine, or glycine and asparagine. Glycine- and asparagine-rich proteins also were found in Geosiphon pyriformis (an obligate symbiont of a cyanobacterium), from the same subphylum (Glomeromycotina) as AMF. The sequence diversity of AGLs likely translates to functional diversity, based on predicted physical properties of tandem repeats (elastic, amyloid, or interchangeable) and their broad pI ranges. We envisage that AGLs/IDPs could contribute to functional complementarity in AMF through processes such as self-recognition, retention of nutrients, soil stability, and water movement.
Historically, Hyaloscypha s. lat. (Hyaloscyphaceae, Helotiales) included various saprobes with small apothecia formed on decaying plant matter, usually wood, that were defined by chemical and (ultra)structural aspects. However, recent molecular phylogenetic and resynthesis studies have narrowed the concept of the genus and shown that it contains several widely distributed species with unknown sexual morphs that form ectomycorrhizae, ericoid mycorrhizae, and mycothalli and also grow endophytically in plant roots and hypogeous ectomycorrhizal (EcM) fruitbodies (i.e., the historical Hymenoscyphus ericae aggregate). Hence, some of the sexually reproducing saprobic Hyaloscypha s. lat. and the symbionts belong to the monophyletic Hyaloscypha s. str. Here, we introduce two new root-symbiotic Hyaloscypha s. str. species, i.e., H. gabretae and H. gryndleri spp. nov. While the former was isolated only from ericaceous hosts (Vaccinium myrtillus from Southern Bohemia, Czechia and Calluna vulgaris from England, UK), the latter was obtained from a basidiomycetous EcM root tip of Picea abies (Pinaceae), roots of Pseudorchis albida (Orchidaceae), and hair roots of V. myrtillus from Southern Bohemia and C. vulgaris from England. Hyaloscypha gryndleri comprises two closely related lineages, suggesting ongoing speciation, possibly connected with the root-symbiotic life-style. Fungal isolates from ericaceous roots with sequences similar to H. gabretae and H. gryndleri have been obtained in Japan and in Canada and Norway, respectively, suggesting a wide and scattered distribution across the Northern Hemisphere. In a series of in vitro experiments, both new species failed to form orchid mycorrhizal structures in roots of P. albida and H. gryndleri repeatedly formed what morphologically corresponds to the ericoid mycorrhizal (ErM) symbiosis in hair roots of V. myrtillus, whereas the ErM potential of H. gabretae remained unresolved. Our results highlight the symbiotic plasticity of root-associated hyaloscyphoid mycobionts as well as our limited knowledge of their diversity and distribution, warranting further ecophysiological and taxonomic research of these important and widespread fungi.
(a) Study sites and schematic diagram of study plots. Map of Kii Peninsula on Honshu Island, Japan, with locations of Pseudotsuga japonica forests studied. (b) Each site contained three subplots, one for each forest type: black, P. japonica forest; gray, transition zone; white, Cryptomeria japonica and/or Chamaecyparis obtusa artificial plantation. See soil sampling section and Fig. S1 of Online Resource 1 for more details. Abbreviations: OM, Ohmata; KW, Kawamatakannon; SN, Sannokogawa
Rank frequency plot of ectomycorrhizal fungal operational taxonomic units (OTUs) detected in soil from Pseudotsuga japonica forests, with fungal frequency data pooled by forest type. Symbol colors indicate forest type: black, P. japonica forest; gray, transition zone; white, Cryptomeria japonica and/or Chamaecyparis obtusa artificial plantation
Non-metric multidimensional scaling plot for ectomycorrhizal (EcM) fungal communities (excluding Cenococcum geophilum) in soil from Pseudotsuga japonica forests, using Bray–Curtis distance. Community composition was based on frequency data for EcM fungal operational taxonomic units detected by bioassay in each subplot. Circles, triangles, and squares represent the Ohmata (OM), Kawamatakannon (KW), and Sannokogawa (SN) sites, respectively. Symbol colors show subplots based on forest type: black, P. japonica forest; gray, transition zone; white, Cryptomeria japonica and/or Chamaecyparis obtusa artificial plantation. Stress value = 0.053. Community structure differed significantly among locations (PERMANOVA, F1,7 = 3.909, R² = 0.358, p = 0.035)
Ectomycorrhizal (EcM) fungal spores play an important role in seedling establishment and forest regeneration, especially in areas where compatible host tree species are absent. However, compared to other Pinaceae trees with a wide distribution, limited information is available for the interaction between the endangered Pseudotsuga trees and EcM fungi, especially the spore bank. The aim of this study was to investigate EcM fungal spore bank communities in soil in remnant patches of Japanese Douglas-fir (Pseudotsuga japonica) forest. We conducted a bioassay of 178 soil samples collected from three P. japonica forests and their neighboring arbuscular mycorrhizal artificial plantations, using the more readily available North American Douglas-fir (Pseudotsuga menziesii) as bait seedlings. EcM fungal species were identified by a combination of morphotyping and DNA sequencing of the ITS region. We found that EcM fungal spore banks were present not only in P. japonica forests but also in neighboring plantations. Among the 13 EcM fungal species detected, Rhizopogon togasawarius had the second highest frequency and was found in all plots, regardless of forest type. Species richness estimators differed significantly among forest types. The community structure of EcM fungal spore banks differed significantly between study sites but not between forest types. These results indicate that EcM fungal spore banks are not restricted to EcM forests and extend to surrounding forest dominated by arbuscular mycorrhizal trees, likely owing to the durability of EcM fungal spores in soils.
An Aphelenchoides sp. feeding on hyphae of four ectomycorrhizal fungi. (a) Cenococcum geophilum, (b) Pisolithus tinctorius, (c) Rhizopogon roseolus and (d) Suillus granulatus. Bars indicate 50 μm
Population growth of Aphelenchoides sp. on four ectomycorrhizal fungi. An initial number of 15 nematode individuals were inoculated per plate. Data are means (n = 5) with standard errors. Different small letters indicate significant differences in nematode numbers among different fungi at each week (Steel–Dwass test, p < 0.05)
Distribution of body-length classes of nematodes in four ectomycorrhizal fungi, (a) Cenococcum geophilum, (b) Pisolithus tinctorius, (c) Rhizopogon roseolus and (d) Suillus granulatus. First 150 individuals including both juveniles and adults encountered in each fungal media were measured. k indicates the minimum number of modes when the null hypothesis of the Silverman test is not rejected (p > 0.05)
Food attraction of Aphelenchoides sp. to four ectomycorrhizal fungi. Cg, Cenococcum geophilum; Pt, Pisolithus tinctorius; Rr, Rhizopogon roseolus; Sg, Suillus granulatus; and Cont, no fungus (i.e., control). Initial number of nematodes including both juveniles and adults was 242 ± 9 (n = 10, mean ± SE) individuals. Nematodes were extracted 24 h after inoculation. Attraction (%) = (number of nematodes in a certain fungal disc) / (number of nematodes in the medium) × 100. Median (horizontal line), first and third quartiles (bottom and top of the box) and minimum and maximum concentrations (whiskers) are shown. Different small letters indicate significant differences in attraction among different fungi (Steel–Dwass test, p < 0.05)
Fungivorous nematodes can use ectomycorrhizal (ECM) fungi as food resources in forest soils, and they may establish close predator–prey relationships in forest ecosystems. However, the effect of ECM fungal species on the growth of fungivorous nematodes is poorly studied. To identify fungivorous nematode propagation and preference for ECM fungi, we investigated the in vitro population growth and food attraction of the fungivorous nematode Aphelenchoides sp. on media with four ECM fungal species: Cenococcum geophilum, Pisolithus tinctorius, Rhizopogon roseolus and Suillus granulatus. Individual nematodes were fed on hyphae of all four ECM fungal species grown on modified Melin-Norkrans agar media. Nematode numbers were significantly lower on P. tinctorius than on all other fungal species. The other three species produced similar population growth rates, with S. granulatus producing the greatest number of nematodes at 2, 3 and 4 weeks and C. geophilum and R. roseolus producing the largest number after 8 weeks. In the histogram for nematode length classes, a unimodal pattern was fitted for P. tinctorius and R. roseolus, but a bimodal pattern was fitted for C. geophilum and S. granulatus by the Silverman test. The attraction of nematodes to S. granulatus was significantly higher than that to other ECM fungi. Our findings suggest that the propagation and body size of nematodes are ECM fungal species dependent. Predator–prey relationships between fungivorous nematodes and ECM fungi may accelerate nutrient cycles in forest ecosystems.
Effects of different AM inoculation (I) and O3 treatments on shoot, root, and total biomass and root-shoot ratio of alfalfa plants. EM, IM, and −M represent inoculation with exogenous AM fungi, inoculation with indigenous AM fungi, and non-inoculated controls, respectively. AA and EO represent ambient air and elevated O3, respectively. Values presented are mean ± SE (n = 3). Bars topped by the same letter do not differ significantly by Tukey’s HSD test (P < 0.05)
Effects of different AM inoculation (I) and O3 treatments on shoot and root malondialdehyde (MDA) concentrations of alfalfa plants. EM, IM, and −M represent inoculation with exogenous AM fungi, inoculation with indigenous AM fungi, and non-inoculated controls, respectively. AA and EO represent ambient air and elevated O3, respectively. Values presented are mean ± SE (n = 3). Bars topped by the same letter do not differ significantly by Tukey’s HSD test (P < 0.05)
Effects of AM inoculation (I) and O3 treatments on net photosynthetic rate and stomatal conductance of alfalfa plants 46 (A, C) and 64 days (B, D) after sowing. EM, IM, and −M represent inoculation with exogenous AM fungi, inoculation with indigenous AM fungi, and non-inoculated controls, respectively. AA and EO represent ambient air and elevated O3, respectively. Values presented are mean ± SE (n = 3). Means followed by the same letter do not differ significantly by Tukey’s HSD test (P < 0.05)
Effects of different AM inoculation (I) and O3 treatments on shoot and root C, N, and P concentrations. EM, IM, and −M represent inoculation with exogenous AM fungi, inoculation with indigenous AM fungi, and non-inoculated controls, respectively. AA and EO represent ambient air and elevated O3, respectively. Values presented are mean ± SE (n = 3). Bars topped by the same letter do not differ significantly by Tukey’s HSD test (P < 0.05)
Enriched surface ozone (O3) can impose harmful effects on plants. Conversely, arbuscular mycorrhizal (AM) symbiosis can enhance plant tolerance to various environmental stresses and facilitate plant growth. The interaction of AM fungi and O3 on plant performance, however, seldom has been investigated. In this study, alfalfa (Medicago sativa L.) was used as a test plant to study the effects of O3 and AM symbiosis on plant physiology and growth under two O3 levels (ambient air and elevated O3 with 60 nmol·mol⁻¹ O3 enrichment) and three AM inoculation treatments (inoculation with exogenous or indigenous AM fungi and non-inoculation control). The results showed that elevated O3 decreased plant net photosynthetic rate and biomass, and increased malondialdehyde concentration, while AM inoculation (with both exogenous and indigenous AM fungi) could promote plant nutrient acquisition and growth irrespective of O3 levels. The positive effects of AM symbiosis on plant nutrient acquisition and antioxidant enzyme (superoxide dismutase and peroxidase) activities were most likely offset by increased stomatal conductance and O3 intake. As a result, AM inoculation and O3 generally showed no significant interactions on plant performance: although elevated O3 did not diminish the beneficial effects of AM symbiosis on alfalfa plants, AM symbiosis also did not alleviate the harmful effects of O3 on plants.
Relative abundances of AMF in the roots of cassava in three agroecology zones in Nigeria. (a) Relative read abundance (%) of AMF families present in cassava root samples. (b) Relative read abundance (%) of AMF genera present in cassava root samples. (c) Relative read abundance (%) of AMF genera present in cassava root samples excluding the genus Glomus
The difference in diversity indices in 60 cassava fields across three agroecology zones in Nigeria. (a) AMF richness, (b) Log (Exp H’). Bars represent estimated marginal means. Bars topped by the same letter do not differ significantly by Tukey HSD post hoc tests on GLMM and LMM models with fields as random factors
The means of AMF diversity indices in 60 cassava fields across six states in Nigeria based on some agricultural practices. (A) AMF richness as affected by fallow periods. (B) Exp H’ as affected by fallow periods. (C) AMF richness as affected by tillage. (D) Exp H’ as affected by tillage. Boxplots topped by the same letter do not differ significantly by Tukey HSD post hoc tests. The horizontal line in the middle of the box is the median value of the scores and the lower and upper boundaries indicate the 25th and 75th percentiles, respectively. The whisker presents the smallest/largest value greater/less than the lower /upper quantile minus/plus times the interquartile range where outliers fall beyond whiskers
Ordination plot of the redundancy analysis (RDA) of AMF communities in the root of cassava in 60 sampling locations in three agroecology zones in Nigeria showing the effects of soil chemical properties and soil management practices on AMF composition. Arrows indicate environmental variables explaining a significant proportion of the AMF communities (as determined with forward selection, Table 2). Ellipses represent confidence regions based on SD from the centroid for management practice: green for hoe tillage, black for no-tillage and red for tractor tillage
Venn diagrams depicting variance partitioning of three groups of explanatory variables, geographical distance (Distance), soil chemical properties (SCP) and soil management practices (SMPs). The overlap represents shared variation among explanatory matrices. Numbers indicate adjusted R² values
Cassava, forming starch-rich, tuberous roots, is an important staple crop in smallholder farming systems in sub-Saharan Africa. Its relatively good tolerance to drought and nutrient-poor soils may be partly attributed to the crop’s association with arbuscular mycorrhiza fungi (AMF). Yet insights into AMF-community composition and richness of cassava, and knowledge of its environmental drivers are still limited. Here, we sampled 60 cassava fields across three major cassava-growing agro-ecological zones in Nigeria and used a DNA meta-barcoding approach to quantify large-scale spatial variation and evaluate the effects of soil characteristics and common agricultural practices on AMF community composition, richness and Shannon diversity. We identified 515 AMF operational taxonomic units (OTUs), dominated by Glomus, with large variation across agro-ecological zones, and with soil pH explaining most of the variation in AMF community composition. High levels of soil available phosphorus reduced OTU richness without affecting Shannon diversity. Long fallow periods (> 5 years) reduced AMF richness compared with short fallows, whereas both zero tillage and tractor tillage reduced AMF diversity compared with hoe tillage. This study reveals that the symbiotic relationship between cassava and AMF is strongly influenced by soil characteristics and agricultural management and that it is possible to adjust cassava cultivation practices to modify AMF diversity and community structure. Graphical abstract
Tropical montane forests are threatened by uncontrolled fire events because of agricultural expansion. Consequently, deforested areas frequently are dominated by the bracken fern, Pteridium spp., for long periods, and forest regeneration is limited. Despite considerable research on bracken-dominated ecosystems, little is known about the relationship between bracken mycorrhizal fungi and tree seedlings. Arbuscular mycorrhizal fungi (AMF) form symbiotic relationships with terrestrial plants, providing nutrients and protection against pathogens and promoting seedling growth and establishment. Therefore, AMF inoculum have high potential for forest restoration programs. Here, we compare the species diversity of AMF spores, root colonization, and seedling growth of Clusia trochiformis 1 year after the addition of different liquefied root inocula: forest conspecific, forest heterospecific, and from Pteridium rhizomes. Thirteen morphospecies of arbuscular mycorrhizal fungi were identified on the roots of C. trochiformis, and Glomus spp. were the most abundant in all treatments. No differences were observed in spore species richness and diversity among treatments, but spore density was the highest subsequent to the Pteridium inoculum. There was no significant difference in mycorrhizal root colonization and seedling growth of C. trochiformis among inoculated treatments. We found a positive relation between root colonization and total biomass. This study shows that the AMF communities in bracken areas and forests present similar characteristics and that the bracken fern does not limit AMF inoculum potential, favouring seedling growth of Clusia.
Effect of silicon (Si) and two arbuscular mycorrhizal (AM) species-M1 (Claroideoglomus etunicatum) and M2 (Rhizoglomus intraradices) on a root colonization (%) and b root dry weight (g plant⁻¹) of pigeonpea genotype Pusa 2001 under different levels of AsV (AsV20- 20 mg kg⁻¹ and AsV40- 40 mg kg⁻¹) and AsIII (AsIII4- 4 mg kg⁻¹ and AsIII8- 8 mg kg⁻¹) stress. Values represented are means of six replicates ± standard error (SE). Means followed by same letter do not differ significantly by Tukey’s HSD tests at p ≤ 0.05. Si–AM– = Si and AM absent; Si+  = Si present; M1+  = M1 present; M2+  = M2 present; Si+M1+  = Si and M1 present; Si+M2+  = Si and M2 present
Effect of silicon (Si) and two arbuscular mycorrhizal (AM) species-M1 (Claroideoglomus etunicatum) and M2 (Rhizoglomus intraradices) on a starch in leaves (mg g⁻¹ FW) and b total soluble sugars (mg g⁻¹ FW) in leaves of pigeonpea genotype Pusa 2001 under different levels of AsV (AsV20- 20 mg kg⁻¹ and AsV40- 40 mg kg⁻¹) and AsIII (AsIII4- 4 mg kg⁻¹ and AsIII8- 8 mg kg⁻¹) stress. Values represented are means of six replicates ± standard error (SE). Means followed by same letter do not differ significantly by Tukey’s HSD tests at p ≤ 0.05. Si–AM– = Si and AM absent; Si+  = Si present; M1+  = M1 present; M2+  = M2 present; Si+M1+  = Si and M1 present; Si+M2+  = Si and M2 present
Effect of silicon (Si) and two arbuscular mycorrhizal (AM) species-M1 (Claroideoglomus etunicatum) and M2 (Rhizoglomus intraradices) on a proline in leaves (mg g⁻¹ DW) and b proline dehydrogenase activity (ProDH; nkat mg⁻¹ protein) in leaves of pigeonpea genotype Pusa 2001 under different levels of AsV (AsV20- 20 mg kg⁻¹ and AsV40- 40 mg kg⁻¹) and AsIII (AsIII4- 4 mg kg⁻¹ and AsIII8- 8 mg kg⁻¹) stress. Values represented are means of six replicates ± standard error (SE). Means followed by same letter do not differ significantly by Tukey’s HSD tests at p ≤ 0.05. Si–AM– = Si and AM absent; Si+  = Si present; M1+  = M1 present; M2+  = M2 present; Si+M1+  = Si and M1 present; Si+M2+  = Si and M2 present
Effect of silicon (Si) and two arbuscular mycorrhizal (AM) species-M1 (Claroideoglomus etunicatum) and M2 (Rhizoglomus intraradices) on a total glomalin related soil proteins (mg g⁻¹ soil) and b soil acid phosphatase (µg pNP g⁻¹ soil h⁻¹) activity of pigeonpea genotype Pusa 2001 under different levels of AsV (AsV20- 20 mg kg⁻¹ and AsV40- 40 mg kg⁻¹) and AsIII (AsIII4- 4 mg kg⁻¹ and AsIII8- 8 mg kg⁻¹) stress. Values represented are means of six replicates ± standard error (SE). Means followed by same letter do not differ significantly by Tukey’s HSD tests at p ≤ 0.05. Si–AM– = Si and AM absent; Si+  = Si present; M1+  = M1 present; M2+  = M2 present; Si+M1+  = Si and M1 present; Si+M2+  = Si and M2 present
Effect of silicon (Si) and two arbuscular mycorrhizal (AM) species-M1 (Claroideoglomus etunicatum) and M2 (Rhizoglomus intraradices) on a arsenic content in leaves (µg g⁻¹ DW) and b Si content in leaves (mg g⁻¹ DW) of pigeonpea genotype Pusa 2001 under different levels of AsV (AsV20- 20 mg kg⁻¹ and AsV40- 40 mg kg⁻¹) and AsIII (AsIII4- 4 mg kg⁻¹ and AsIII8- 8 mg kg⁻¹) stress. Values represented are means of six replicates ± standard error (SE). Means followed by same letter do not differ significantly by Tukey’s HSD tests at p ≤ 0.05. Si–AM– = Si and AM absent; Si+  = Si present; M1+  = M1 present; M2+  = M2 present; Si+M1+  = Si and M1 present; Si+M2+  = Si and M2 present
Arsenic (As) pollution of soil reduces the growth and reproductive potential of plants. Silicon (Si) and arbuscular mycorrhizal (AM) fungi play significant roles in alleviating adverse effects of As stress. However, studies are scant regarding alleviative effects of Si in pigeonpea (Cajanus cajan L. Millsp.) because legumes are considered low Si-accumulators. We investigated the individual as well as synergistic potential of Si with two AM species (M1-Claroideoglomus etunicatum and M2-Rhizoglomus intraradices) in modulating soil properties, thereby improving growth and productivity of pigeonpea genotype Pusa 2001 grown in AsV and AsIII challenged soils. Both As species hampered the establishment of AM symbiosis, thus, reducing nutrient uptake, growth and yield, with AsIII more toxic than AsV. Exogenously applied Si and AM species enhanced soil glomalin and phosphatases activity, hence decreased metal bioavailability in soil, increased plant nutrient acquisition, biomass and chlorophylls; with maximum benefits provided by M2, closely followed by Si and least by M1. These amendments boosted the activities of starch hydrolytic enzymes (α-, β-amylase, starch phosphorylase) in plants, along with a simultaneous increase in total soluble sugars (TSS). This enhanced sugar accumulation directly led to improved reproductive attributes, more efficiently by M2 and Si than by M1. Moreover, there was a substantial increase in proline biosynthesis due to significantly enhanced activities of its biosynthetic enzymes. Additionally, combined applications of Si and AM, especially +Si+M2, complemented each other where AM enhanced Si uptake, while Si induced mycorrhization, suggesting their mutual and beneficial roles in ameliorating metal(loid) toxicity and achieving sustainability in pigeonpea production under As stress.
Colonization (a hyphal; b total colonization) of AM fungi in roots of Carex capillacea and Poa annua along the elevational gradient. All the data are presented as mean ± SE, n = 3. Significant differences between the two plant species in AM colonization at same altitude by paired Student t tests are shown with asterisks. *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001
Comparison of AM fungus family relative abundances between sample types (rhizosphere soil versus and Carex capillacea roots, or Carex capillacea versus Poa annua roots). Significant differences are marked with an asterisk (Wilcoxon rank-sum test: FDR < 0.05)
Non-metric multidimensional scaling (NMDS) plot of AM fungi community composition (based on Bray–Curtis distances) and the vectors of significant environmental variables (p < 0.05) across sites. Arrows indicate the correlation of the two NMDS axes with the ratio of Poa annua biomass to total plant biomass (PAB), ratio of Poa annua number to total plant number (PAN) and elevation
Little is known about Arbuscular mycorrhizal (AM) fungal colonization and community composition in non-mycorrhizal (NM) plants, especially along elevational gradients. This study explores this question using a NM plant, Carex capillacea, at Mount Segrila, Tibet. Here, C. capillacea, its rhizosphere soil, and the neighboring mycotrophic plant Poa annua were sampled at four elevations to evaluate and compare their AM fungi colonization and communities. The results showed that AM fungal colonization density of C. capillacea was negatively correlated with elevation and biomass of total NM plants per quadrat. AM fungal diversity and community composition between C. capillacea and P. annua showed a similar pattern. In addition, elevation and soil did not significantly influence the AM community in C. capillacea, while they were important abiotic factors for assemblages in rhizosphere soil and P. annua. These findings support that a broad array of AM fungi colonize the root of C. capillacea, and a mycelial network from a co-occurring host plant might shape the AM fungal communities in C. capillacea along the elevation gradient. The co-occurrence patterns of AM fungi associated with non-mycotrophic species and adjacent mycotrophic species have important implications for understanding AM fungal distribution patterns and plant–AM interactions.
Frank showed for the first time the empirical evidence of the nutritional role of mycorrhizal symbiosis (Franck 1885, 1894). Photograph of Franck’s first assay on mycorrhization. Franck conducted a 3-year experiment on spruce grown in pots containing either Spruce forest natural soil (Unsterilisirt) and the same heat-sterilized soil without (Sterilisirt) or with developing ectomycorrhizas (Sterilisirt, spontan inficirt). Spruce trees grown in unsterilized soil had many needle ramifications (they were “with strong health and wild” and bright green), whereas those grown in sterilized soil were small, unhealthy, and yellowish. According to Frank, spruces in the leftmost pots were recently colonized by mycorrhizal fungi (presence of a mantle only on the young roots) probably originated from the unsterilized pots
Gallaud’s (1904) drawings of different endomycorrhiza types and recent corresponding photomicrographs (2021). (1) The Arum maculatum type, produced by most land plants, was defined as the most ordered degree of fungal root colonization. Fungal hyphae penetrate roots through the epidermis and alongside cortical cells in the apoplast pathways to eventually enter a cell and form a unique arbuscular structure. Drawings of (A) a longitudinal section of Arum maculatum root and (B) a young arbuscule; (C) Photomicrograph of a longitudinal section of a Medicago truncatula root and (D) a single arbuscule. (2) The Paris Quadrifolia type is mainly observed in trees and forest herbs. In this specific type, mycorrhizal hyphae pass from cell to cell (cortical layer) while forming structures such as arbuscules in the traversed cells. A Drawings of a transversal section of Paris quadrifolia; B photomicrograph of a longitudinal section of a Paris quadrifolia root and C a single arbuscule. (3) The Hepatic type is highly similar to the Paris-type mycorrhiza, but without a layered organization. Nowadays, it is no longer accepted as distinct (Dickson et al. 2007) A Drawings of transversal section of a Pellia epiphylla thallus; B photomicrograph of a transversal section of a Pellia epiphylla thallus. Noy, nucleus root hair; ap, pillar seat; as, corky seat; c, passage cell; end, endoderm; epi, epiderm; r, rhizoid; d, collapsed arbuscule
Chronology, with key dates of the history of arbuscular mycorrhizal symbiosis
Arbuscular mycorrhiza, one of the oldest interactions on earth (~ 450 million years old) and a first-class partner for plants to colonize emerged land, is considered one of the most pervasive ecological relationships on the globe. Despite how important and old this interaction is, its discovery was very recent compared to the long story of land plant evolution. The story of the arbuscular mycorrhiza cannot be addressed apart from the history, controversies, and speculations about mycorrhiza in its broad sense. The chronicle of mycorrhizal research is marked by multiple key milestones such as the initial description of a “persistent epiderm and pellicular wall structure” by Hartig; the introduction of the “Symbiotismus” and “Mycorrhiza” concepts by Frank; the description of diverse root-fungal morphologies; the first description of arbuscules by Gallaud; Mosse’s pivotal statement of the beneficial nature of the arbuscular mycorrhizal symbiosis; the impact of molecular tools on the taxonomy of mycorrhizal fungi as well as the development of in vitro root organ cultures for producing axenic arbuscular mycorrhizal fungi (AMF). An appreciation of the story – full of twists and turns – of the arbuscular mycorrhiza, going from the roots of mycorrhiza history, along with the discovery of different mycorrhiza types such as ectomycorrhiza, can improve research to help face our days’ challenge of developing sustainable agriculture that integrates the arbuscular mycorrhiza and its ecosystem services.
Diagram summarising the different possible impacts of mycorrhization on quality traits in crop production. Mycorrhization impacts the production of compounds involved in the nutritional value of foodstuffs (i.e. seeds, leaves, vegetables, fruits, roots) such as fatty acids, amino acids or vitamins. Mycorrhization of crops impacts the concentrations of sugars and acids in fruit and thus modifies the sweetness/acidity ratio that may influence the perception of the taste and palatability of food products. Finally, mycorrhization also increases the production of secondary metabolites in different plant tissues. This increased production of bioactive compounds is of great interest for the agri-food and pharmaceutical industries. The production of secondary metabolites in the form of aromatic compounds also is relevant for the fragrance industry. This diagram was created using the Biorender tool (
Modern agriculture is currently undergoing rapid changes in the face of the continuing growth of world population and many ensuing environmental challenges. Crop quality is becoming as important as crop yield and can be characterised by several parameters. For fruits and vegetables, quality descriptors can concern production cycle (e.g. conventional or organic farming), organoleptic qualities (e.g. sweet taste, sugar content, acidity) and nutritional qualities (e.g. mineral content, vitamins). For other crops, however, the presence of secondary metabolites such as anthocyanins or certain terpenes in the targeted tissues is of interest as well, especially for their human health properties. All plants are constantly interacting with microorganisms. These microorganisms include arbuscular mycorrhizal fungi as well as certain soil bacteria that provide ecosystem services related to plant growth, nutrition and quality parameters. This review is an update of current research on the single and combined (co-inoculation) use of arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria in crop production, with a focus on their positive impacts on crop quality traits (e.g. nutritional value, organoleptic properties). We also highlight the need to dissect mechanisms regulating plant-symbionts and symbiont-symbiont interactions, to develop farming practices and to study a broad range of interactions to optimize the symbiotic potential of root-associated microorganisms.
(a) Relative abundance of arbuscular mycorrhizal (AM) fungal genera, presented for the entire dataset and (b) AM fungal richness at each location in Qatar. The 19 locations are categorized as one of four habitat types: (1) rawdha: calcareous soil depressions with relatively good soil quality and water accessibility due to influxes of water and minerals from surrounding higher ground; (2) mangrove: mass of shrubs growing in coastal saline water; (3) saltmarsh: wet muddy soil with open access to coastal saltwater; and (4) sabkha: salt pans (either with or without vegetation) that tend to accumulate minerals due to calcareous surface evaporation
Principal component analysis (PCA) plot showing the associations between chemical element concentrations, pH, and salinity among locations (shown in blue) in Qatar. The significance of influence of the chemical parameters (shown in red) is indicated by the distance of each point from the origin
Non-metric multidimensional scaling (NMDS) plots of arbuscular mycorrhizal (AM) fungal communities versus (a)chemical parameters and (b) AM fungal genera at 19 sites in Qatar, representing four type of habitat: mangrove, rawdha, sabkha, and saltmarsh. The circles represent correlation of 1 of vectors to variables
Qatar is largely characterized by a hyper-arid climate and low soil fertility which create a stressful soil environment for arbuscular mycorrhizal (AM) fungi. In a study of AM fungal communities and their relationship with soil chemical characteristics, we used a high-throughput sequencing technique to explore AM fungal diversity and community composition in different habitats across Qatar. We identified a total of 79 AM fungal taxa, over 77% of which were species from the Glomeraceae family. The lowest AM fungal diversity was observed in saltmarsh and in one rawdha site, while the highest richness, effective number of species, and diversity were observed in rawdha and sabkha communities. NMDS and multiple regression analyses showed that AM fungi were negatively correlated with soil pH and TC, but positively correlated with K and NO3−. AM fungi also were positively correlated with Cd, with the latter suggesting that very low levels of heavy metals may not always be harmful to AM fungi. These findings provide baseline information on AM fungal assemblages and the chemical drivers of diversity across communities in Qatar. This work partly compensates for the current lack of broad-scale studies in the Arabian Peninsula by providing understanding of overall patterns of AM fungi and their drivers in the region.
Comparisons of error rates for the mycorrhizal databases listed in Table 2 (AM = arbuscular mycorrhizal, NM = nonmycorrhizal; EM = ectomycorrhizal). Errors were identified by a rigorous auditing process using methodology in Box 1 (incorrect reports for species = xNM, xAM or xEM). Error rates are for raw data, but FungalRoot (Soudzilovskaia et al. 2020) also provides corrected data
Accumulation of errors in databases resulting from data transfer between them (see text). Errors were tracked back to original sources by reference citations (incorrect reports for species = xNM, xAM or xEM; x% = minimum estimate of total errors for database)
The decadal frequency of ectomycorrhizal association designation errors included in Harley and Harley (1987). These records are listed in Table S1
a The relationship between total records per genus and the number of cases where mycorrhizal diagnosis is highly likely to be incorrect. Records are for the 200 most sampled vascular plant genera in the FungalRoot database (Soudzilovskaia et al. 2020). Plants are separated by growth form (16,533 records for 4,737 species), but two with fewer samples were omitted (sedges, ferns). b. The relationship between sample numbers and errors totalled for plant families for the same data (80 families)
Frequency histogram of mycorrhizal traits allocated to the 500 most sampled plant genera (23,488 records for 7,259 species) in the FungalRoot database (Soudzilovskaia et al. 2020). a All records for species reported to be nonmycorrhizal (NM). b. All records for species reported to be arbuscular mycorrhizal (AM). Plants are separated into categories by their actual mycorrhizal status (AM = arbuscular mycorrhizal, NM = nonmycorrhizal, NM-AM = variable AM) and mycorrhizal records by error state (xNM = incorrect report of NM, xAM = incorrect AM)
Nearly 150 years of research has accumulated large amounts of data on mycorrhizal association types in plants. However, this important resource includes unreliable allocated traits for some species. An audit of six commonly used data sources revealed a high degree of consistency in the mycorrhizal status of most species, genera and families of vascular plants, but there were some records that contradict the majority of other data (~ 10% of data overall). Careful analysis of contradictory records using rigorous definitions of association types revealed that the majority were diagnosis errors, which often stem from references predating modern knowledge of mycorrhiza types. Other errors are linked to inadequate microscopic examinations of roots or plants with complex root anatomy, such as phi thickenings or beaded roots. Errors consistently occurred at much lower frequencies than correct records but have accumulated in uncorrected databases. This results in less accurate knowledge about dominant plants in some ecosystems because they were sampled more often. Errors have also propagated from one database to another over decades when data were amalgamated without checking their suitability. Due to these errors, it is often incorrect to designate plants reported to have inconsistent mycorrhizas as “facultatively mycorrhizal”. Updated protocols for resolving conflicting mycorrhizal data are provided here. These are based on standard morphological definitions of association types, which are the foundations of mycorrhizal science. This analysis also identifies the need for adequate training and mentoring of researchers to maintain the quality of mycorrhizal research.
Frequency distribution of mean root colonization by Rhizophagus intraradices for 288 lines from the mapping panel, 6 weeks after planting. Least-squares means are reported to account for environmental gradients in the greenhouse. Mean colonization for lines ranged from 0.0 to 70.1%
Manhattan plots for A mean root colonization, B maximum root colonization, and C range of root colonization. The genomic position of each SNP is represented on the x-axis, colored by chromosome, and the negative logarithm of the association p-value for each SNP is presented on each y-axis. Red lines represent the significance threshold (p = 2.43 × 10⁻⁶) using a Gao correction with the effective number of tests (n = 20,562). Blue lines represent the threshold for suggestive SNPs, which were the top 0.01% of all SNPs
Plant symbiosis with arbuscular mycorrhizal (AM) fungi provides many benefits, including increased nutrient uptake, drought tolerance, and belowground pathogen resistance. To develop a better understanding of the genetic architecture of mycorrhizal symbiosis, we conducted a genome-wide association study (GWAS) of this plant-fungal interaction in cultivated sunflower. A diversity panel of cultivated sunflower (Helianthus annuus L.) was phenotyped for root colonization under inoculation with the AM fungus Rhizophagus intraradices. Using a mixed linear model approach with a high-density genetic map, we identified genomic regions that are likely associated with R. intraradices colonization in sunflower. Additionally, we used a set of twelve diverse lines to assess the effect that inoculation with R. intraradices has on dried shoot biomass and macronutrient uptake. Colonization among lines in the mapping panel ranged from 0–70% and was not correlated with mycorrhizal growth response, shoot phosphorus response, or shoot potassium response among the Core 12 lines. Association mapping yielded three single-nucleotide polymorphisms (SNPs) that were significantly associated with R. intraradices colonization. This is the first study to use GWAS to identify genomic regions associated with AM colonization in an Asterid eudicot species. Three genes of interest identified from the regions containing these SNPs are likely related to plant defense.
Importance value indices (IVI) of host plants and concentrations of environmental variables in their rhizospheres at Aghanashini (AG) and Gangavali (GN). Values represent the average of three replicates. Error bars indicate standard error of the mean. Statistical comparisons between the five plant species present at both Aghanashini and Gangavali are presented in Table 1
Neighbour-joining (NJ) tree representing phylogenetic relationships of the representative AMF sequences from Aghanashini and Gangavali and their closest matched sequences from GenBank. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. Sequences marked with asterisks (clones IKNM 1.1 to IKNM25) are from the present study and clone names followed by same numbers (e.g., IKNM 1.1 and 1.2) belong to the same OTU. The bootstrap values (1000 replicates) are indicated at each node. Bootstrap support of only 70% and above is shown in the tree. GloP1–GloP13, PgloP1, AcauloP1–AcauloP3, and EntroP1 represent AMF phylotypes found at Aghanashini and Gangavali
Non-metric multidimensional scaling (NMDS) ordinations of (A) AMF community composition based on Bray and Curtis dissimilarities with regard to relative abundance of AMF phylotypes, (B) rhizospheric concentrations of PO4− P, % soil organic matter, salinity, and pH based on Euclidean distances at both estuaries. Ellipses are drawn around each group’s centroid
We investigated the role of plant host and soil variables in determining arbuscular mycorrhizal fungi (AMF) community composition in plant roots of two spatially separated mangrove estuaries on the rivers Aghanashini (14° 30′ 30″ N–74° 22′ 44″ E) and Gangavali (14° 35′ 26″ N–74° 17′ 51″ E) on the west coast of India. Both mangrove estuaries had similar plant species composition but differed in soil chemistries. We amplified a 550-bp portion of 18S small subunit (SSU) rDNA from mangrove plant roots and analysed it by restriction fragment length polymorphism (RFLP). Clones representing unique RFLP patterns were sequenced. A total of 736 clones were obtained from roots of seven and five plant species sampled at Aghanashini and Gangavali, respectively. AMF phylotype numbers in plant roots at Aghanashini (12) were higher than at Gangavali (9) indicating quantitative differences in the AMF community composition in plant roots at the two mangrove estuaries. Because both estuaries had similar plant species composition, the quantitative difference in AMF communities between the estuaries could be an attribute of the differences in rhizospheric chemistry between the two sites. Non-metric multidimensional scaling (NMDS) revealed overlap in the AMF communities of the two sites. Three and two AMF phylotypes had significant indicator value indices with specific hosts at Aghanashini and Gangavali, respectively. Environmental vector fitting to NMDS ordination did not reveal a significant effect of any soil variable on AMF composition at the two sites. However, significant effects of both plant hosts and sites were observed on rhizospheric P. Our results indicate that root AMF community composition may be an outcome of plant response to rhizospheric variables. This suggests that plant identity may have a primary role in shaping AMF communities in mangroves.
Ectomycorrhizal fungi contribute to the nutrition of many woody plants, including those in the Pinaceae family. Loblolly pine (Pinus taeda L.), a native species of the Southeastern USA, can be colonized by multiple species of ectomycorrhizal fungi. The role of these symbionts in P. taeda potassium (K+) nutrition has not been previously investigated. Here, we assessed the contribution of four ectomycorrhizal fungi, Hebeloma cylindrosporum, Paxillus ammoniavirescens, Laccaria bicolor, and Suillus cothurnatus, in P. taeda K+ acquisition under different external K+ availabilities. Using a custom-made two-compartment system, P. taeda seedlings were inoculated with one of the four fungi, or kept non-colonized, and grown under K+-limited or -sufficient conditions for 8 weeks. Only the fungi had access to separate compartments in which rubidium, an analog tracer for K+, was supplied before harvest. Resulting effects of the fungi were recorded, including root colonization, biomass, and nutrient concentrations. We also analyzed the fungal performance in axenic conditions under varying supply of K+ and sodium. Our study revealed that these four ectomycorrhizal fungi are differentially affected by external K+ and sodium variations, that they are not able to provide similar benefits to the host P. taeda in our growing conditions, and that rubidium may be used with some limitations to estimate K+ transport from ectomycorrhizal fungi to colonized plants.
Main effect of mycorrhizal on polyphenol oxidase activity (a), interactive effect of nematode inoculation and evaluation time on polyphenol oxidase activity (b), and interactive effect of nematode inoculation and mycorrhizal on peroxidase activity (c) in Cymbopogon citratus roots at 4, 8, and 12 days after inoculation (DANI) of Pratylenchus brachyurus. Plants were inoculated with one of two arbuscular mycorrhizal fungi, Claroideoglomus etunicatum, or Rhizophagus clarus. The same letter above bars indicates no significant difference based on Tukey’s test, p < 0.05. Data are expressed as mean (columns) ± standard error (error bars)
Cymbopogon citratus (lemongrass) is an important medicinal and aromatic plant containing citral-rich essential oil, of which the quality and quantity may be affected by nematode infection. Research has shown that arbuscular mycorrhizal fungi (AMF) may act as nematode biocontrol agents and improve the chemical composition of plants. Three experiments were conducted to assess the effects of AMF inoculation on vegetative growth, essential oil composition, induction of defense-related proteins, and control of Pratylenchus brachyurus in C. citratus. Seedlings were transplanted into pots inoculated with one of two AMF species (Rhizophagus clarus or Claroideoglomus etunicatum). At 30 days after AMF inoculation, plants were inoculated with P. brachyurus. Evaluations were performed at 75 days after nematode inoculation. Although both AMF treatments led to effective root colonization (> 84%), fungus inoculation was not effective in reducing P. brachyurus population density. Nevertheless, C. etunicatum promoted an increase in shoot weight, and AMF treatments contributed to preserving essential oil composition in nematode-infected plants. In addition, both AMF treatments enhanced polyphenol oxidase activity and R. clarus increased peroxidase activity after nematode inoculation.
Workflow of our statistical analyses. To demonstrate our approach, we simulated data so that plant mycorrhizal status was negatively linked to plant-available P and N (i.e. the prediction tested in this study). On the other hand, we expected plant mycorrhizal status to be positively linked to soil Ca and Mg. We used six datasets including coastal, semi-natural, mesic and broad-leaved semi-dry grasslands from Scotland, Latvia and the Czech Republic. Statistical inference was based on tests focusing on both community (upper part of the figure) and species (bottom part of the figure) levels. (1a) Community mycorrhization (Moora 2014) was computed using both weighted (CWM) and unweighted (presence/absence, CM) community data. We accounted for mycorrhizal status uncertainty by comparing two databases: empirical (Bueno et al. 2019b) and taxonomic (Soudzilovskaia et al. 2020) and applied statistical weights in both (1b) community mycorrhization (CWM and CM) and (1d) species niche centroids (SNC). (1c) Phylogenetic signal in SNC values was examined. (2a, b) We performed a max test (ter Braak et al. 2018), which combines both community and species data to test the central tendency of the link between mycorrhizal status and soil nutrients. This analysis was done for each dataset separately. (3a, b) We estimated quantile slopes using quantile regression in each dataset separately. (3c, d) Quantile slopes (i.e. effect sizes for quantiles Q0.90, 0.75, 0.50, 0.25 and 0.10) were then pooled using the random-effects meta-analysis (here, only the quantile 0.90 is shown)
Pooled effect sizes (quantile slopes and their 95% CI) from random-effect model meta-analyses testing the response of abundance-weighted community mycorrhization (CWM of mycorrhizal status) to soil nutrient availability in all datasets. a) P, b) Ca and c) Mg. Effect sizes were pooled for quantiles of the response variable (Q0.1, 0.25, 0.50, 0.75 and 0.90)
Pooled effect sizes (quantile slopes and their 95% CI) from random-effect model meta-analyses testing the response of species niche centroids (SNC) to plant mycorrhizal status. a) P, b) Ca and c) Mg. Effect sizes were pooled for quantiles of the response variable (Q0.1, 0.25, 0.50, 0.75 and 0.90)
Plant mycorrhizal status (a trait indicating the ability to form mycorrhizas) can be a useful plant trait for predicting changes in vegetation influenced by increased fertility. Mycorrhizal fungi enhance nutrient uptake and are expected to provide a competitive advantage for plants growing in nutrient-poor soils; while in nutrient-rich soils, mycorrhizal symbiosis may be disadvantageous. Some studies in natural systems have shown that mycorrhizal plants can be more frequent in P and N-poor soils (low nutrient availability) or Ca and Mg-high (high pH) soils, but empirical support is still not clear. Using vegetation and soil data from Scottish coastal habitats, and Latvian and Czech grasslands, we examined whether there is a link between plant mycorrhizal status and plant-available P, N, Ca and Mg. We performed the max test analysis (to examine the central tendency) and a combination of quantile regression and meta-analysis (to examine tendencies in different quantiles) on both community and plant species data combined with plant phylogenies. We consistently found no changes in mycorrhizal status at the community and species levels along the gradients of plant-available P, N, Ca and Mg in the central tendency and in almost all quantiles across all datasets. Thus, we found no support for the hypotheses that herbaceous species which are able to form mycorrhizas are more frequent in nutrient-poor and high pH environments. Obligatory, facultatively and non-mycorrhizal herbaceous species appear to assemble randomly along the gradients of nutrient availability in several European herbaceous habitats, suggesting that all these strategies perform similarly under non-extreme soil nutrient conditions.
Arbuscular mycorrhizal fungi (AMF) are known to improve plant growth and nutrition and therefore are likely to affect the competitive relationships between crops and weeds. In this study, we evaluated whether AMF (Funneliformis mosseae, Rhizoglomus fasciculatum, Rhizoglomus intraradices) change plant competition between Phaseolus vulgaris and the weeds Solanum nigrum L., Digitaria sanguinalis L., and Ipomoea purpurea L. Mycorrhizal colonization, aggressivity index, photosynthetic rates, and yield parameters were measured. While the presence of AMF reduced the total biomass of D. sanguinalis and S. nigrum when grown in competition with P. vulgaris, it increased the total biomass of I. purpurea when grown with P. vulgaris. Significantly, elevated mycorrhizal growth responses (38–44%) improved the competitive ability of I. purpurea. In contrast, the competitive ability of S. nigrum was increased only when plants colonized by R. intraradices. The total protein content of P. vulgaris pods when in competition was negatively affected by AMF, thus leading to low nutritional quality. The results suggest that AMF have the potential to affect the outcome of weed—P. vulgaris competition. We demonstrate that not only colonization with AMF but also AMF species can affect the competitive relationships between crops and weeds, and thus, AMF represent key soil organisms to be taken into account in sustainable weed management strategies.
Principal component analyses of bacteria A and fungi B, from soils sampled 20 days after soil microbiome manipulation. Treatment abbreviations are as follows: natural soil (NS); natural soil heated at 50 ºC for 1 h (NH50), natural soil heated at 80 ºC for 1 h (NH80); natural soil heated at 100 ºC for 1 h (NH100); sterilized soil by autoclaving twice (121 ºC, 103 kPa, 1 h) (AS) followed by re-inoculation with different dilutions from the native soil (10% wv⁻¹ of natural soil) (AS + 10⁻¹); (AS + 10⁻³); (AS + 10⁻⁶); and without dilution inoculation (AS)
Soil acid phosphatase activity 20 days after soil microbiome manipulation. Treatment abbreviations are as follows: natural soil (NS); soil heated at 50 ºC for 1 h (NH50), soil heated at 80 ºC for 1 h (NH80); soil heated at 100 ºC for 1 h (NH100); sterilized soil by autoclaving twice (121 ºC, 103 kPa, 1 h) (AS) followed by re-inoculation with different dilutions from the native soil (10% wv⁻¹ of natural soil) (AS + 10⁻¹); (AS + 10⁻³); (AS + 10⁻⁶); and without dilution inoculation (AS). Bars topped by the same letter do not differ significantly between soil manipulation treatments by LSD test (p > 0.05). Error bars represent standard error of the mean
Mycorrhizal colonization of Brachiaria (A–D) and Crotalaria (E–H) cultivated in a soil which had been subjected to microbiome manipulation. Plants were cultivated without AMF inoculation (A and E) or inoculated with Acaulospora colombiana (B and F); Rhizophagus clarus (C and G); or Dentiscutata heterogama (D and H). Treatment abbreviations are as follows: natural soil (NS); soil heated at 50 ºC for 1 h (NH50), soil heated at 80 ºC for 1 h (NH80); soil heated at 100 ºC for 1 h (NH100); sterilized soil by autoclaving twice (121 ºC, 103 kPa, 1 h) (AS) followed by re-inoculation with different dilutions from the native soil (10% wv⁻¹ of natural soil) (AS + 10⁻¹); (AS + 10⁻³); (AS + 10⁻⁶); and without dilution inoculation. Bars topped by the same letters do not differ significantly among soil manipulation treatments within each AMF treatment by LSD test (p > 0.05). ns, not significant (p > 0.05). *missing data. Error bars represent standard error of the mea
Change in acid phosphatase activity in soils cultivated with Brachiaria (A–D) and Crotalaria (E–H) after microbiome manipulations. Plants were cultivated without AMF inoculation (A and E) or inoculated with Acaulospora colombiana (B and F); Rhizophagus clarus (C and G); or Dentiscutata heterogama (D and H). Treatment abbreviations are as follows: natural soil (NS); soil heated at 50 ºC for 1 h (NH50), soil heated at 80 ºC for 1 h (NH80); soil heated at 100 ºC for 1 h (NH100); sterilized soil by autoclaving twice (121 ºC, 103 kPa, 1 h) (AS) followed by re-inoculation with different dilutions from the native soil (10% wv⁻¹ of natural soil) (AS + 10⁻¹); (AS + 10⁻³); (AS + 10⁻⁶); and without dilution inoculation. Positive values on the ordinate axis show increasing APASE; negative values on the ordinate axis y show decreasing APASE. *means significant difference of soil acid phosphatase activity after cultivation by Dunnett test (p ≤ 0.05). Error bars represent standard error of the mean
Arbuscular mycorrhizal fungi (AMF) are important symbionts of many plant species, facilitating the acquisition of soil nutrients by roots. We hypothesized that AMF root colonization is strongly influenced by the composition of the soil microbiome. Here, we evaluated mycorrhizal colonization of two plants, the grass Urochloa brizantha (Brachiaria) and the legume Crotalaria juncea (Crotalaria). These were cultivated in the same soil but hosting eight distinct microbiomes: natural soil (i); soil exposed to heat treatments for 1 h at 50 ºC (ii), 80 ºC (iii), or 100 ºC (iv); sterilized soil by autoclaving (AS) followed by re-inoculation of dilutions of the natural soil community at 10−1 (v), 10−3 (vi), and 10−6 (vii); and AS without re-inoculation (viii). Microbial diversity (bacteria and fungi) was assessed through 16S rDNA and ITS1 metabarcoding, respectively, and the soil acid phosphatase activity (APASE) was measured. Sequencing results showed the formation of distinct microbial communities according to the soil manipulations, which also correlated with the decline of APASE. Subsequently, seedlings of Brachiaria and Crotalaria were grown in those soils inoculated separately with three AMF (Acaulospora colombiana, Rhizophagus clarus, and Dentiscutata heterogama) which were compared to an AMF-free control treatment. Brachiaria showed higher colonization in natural soil when compared to the microbial community manipulations, regardless of the AMF species inoculated. In contrast, two mycorrhiza species were able to colonize Crotalaria under modified microbial communities at similar rates to natural soil. Furthermore, Brachiaria showed a possible inverse relationship between APASE and mycorrhization, but this trend was absent for Crotalaria. We conclude that mycorrhizal root colonization and soil acid phosphatase activity were associated with the structure of the soil microbiome, depending on the plant species evaluated.
Almost all land plants form symbiotic associations with arbuscular mycorrhizal fungi (AMF). Individual plants usually are colonized by a wide range of phylogenetically diverse AMF species. The impact that different AMF taxa have on plant growth is only partly understood. We screened 44 AMF isolates for their effect on growth promotion and nutrient uptake of leek plants ( Allium porrum ), including isolates that have not been tested previously. In particular, we aimed to test weather AMF lineages with an ancient evolutionary age differ from relatively recent lineages in their effects on leek plants. The AMF isolates that were tested covered 18 species from all five AMF orders, eight families, and 13 genera. The experiment was conducted in a greenhouse. A soil–sand mixture was used as substrate for the leek plants. Plant growth response to inoculation with AMF varied from − 19 to 232% and depended on isolate, species, and family identity. Species from the ancient families Archaeosporaceae and Paraglomeraceae tended to be less beneficial, in terms of stimulation plant growth and nutrient uptake, than species of Glomeraceae, Entrophosporaceae, and Diversisporaceae, which are considered phylogenetically more recent than those ancient families. Root colonization levels also depended on AMF family. This study indicates that plant benefit in the symbiosis between plants and AMF is linked to fungal identity and phylogeny and it shows that there are large differences in effectiveness of different AMF.
Some epiphytic orchids in the tribe Vandeae are characterized by extremely vestigial leaves (even leafless). Thus, their leaves provide only a small proportion of carbon required for their growth and development, while a large portion of carbon may need to be supplied by their roots and mycorrhizal fungi (MF). The MF richness and composition of leafless epiphytic orchids, which belong to numerous genera with diverse ecophysiologies and wide geographical ranges, remain poorly understood. In this study, we identified the MF communities of seven leafless epiphytic species from three orchid genera from up to 17 sites in China using high-throughput sequencing. Our analyses revealed that the leafless epiphytic orchids have a highly specialized association with Ceratobasidiaceae. Several fungal OTUs were found in three different orchid genera and have promoted germinations of Chiloschista and Phalaenopsis, which may have been caused by convergent evolution of leafless epiphytic orchids. Furthermore, the MF composition of Taeniophyllum glandulosum was significantly affected by collection site and host tree. Our study provides new insights into mycorrhizal associations of epiphytic orchids.
Arbuscular mycorrhizal fungi (AMF) represent an important group of root symbionts, given the key role they play in the enhancement of plant nutrition, health, and product quality. The services provided by AMF often are facilitated by large and diverse beneficial bacterial communities, closely associated with spores, sporocarps, and extraradical mycelium, showing different functional activities, such as N2 fixation, nutrient mobilization, and plant hormone, antibiotic, and siderophore production and also mycorrhizal establishment promotion, leading to the enhancement of host plant performance. The potential functional complementarity of AMF and associated microbiota poses a key question as to whether members of AMF-associated bacterial communities can colonize the root system after establishment of mycorrhizas, thereby becoming endophytic. Root endophytic bacterial communities are currently studied for the benefits provided to host plants in the form of growth promotion, stress reduction, inhibition of plant pathogens, and plant hormone release. Their quantitative and qualitative composition is influenced by many factors, such as geographical location, soil type, host genotype, and cultivation practices. Recent data suggest that an additional factor affecting bacterial endophyte recruitment could be AMF and their associated bacteria, even though the mechanisms allowing members of AMF-associated bacterial communities to actually establish in the root system, becoming endophytic, remain to be determined. Given the diverse plant growth–promoting properties shown by AMF-associated bacteria, further studies are needed to understand whether AMF may represent suitable tools to introduce beneficial root endophytes in sustainable and organic agriculture where the functioning of such multipartite association may be crucial for crop production.
A Venn diagram depicting the distribution of OTUs across (i) AM high (> 15% woody AM-associating plant coverage) and AM low plots (< 15% woody AM-associating plant coverage) and (ii) the two plant hosts. Fifteen out of the thirty-two OTUs were observed in all
Standardized effect sizes of observed checkerboard scores which were compared against a null model generated with the sim4 algorithm (y-axis). We plotted these values against total OTU richness of the respective subsets of the dataset to capture a factor that may influence them. The two discontinuous lines highlight confidence intervals within which the community matrix can be considered random. The green point represents samples from AM high plots (> 15% woody AM-associating plant coverage), the brown from AM low plots (< 15% woody AM-associating plant coverage), and the orange points from combinations of the two. A pink border was used for spring and black for autumn; we used no border where we pooled samples from spring and autumn. We used white “x” symbols to highlight the location in the panel of samples taken over the first year. The square represents samples on Euonymus whereas circles those on Hedera. The diamond shows the complete data set. Differences in standardized effect sizes above 1.96 and below −1.96 are significant at a 0.05% confidence level
a Principal component analysis of Hellinger-transformed AMF community data (we plotted respective diagrams with axis one, explaining 13.5% of variability in Figs. S4 and S5). Green symbols represent samples from AM high (> 15% woody AM-associating plant coverage), whereas brown represent AM low plots (< 15% woody AM-associating plant coverage). A pink border was used for spring and a black for autumn. We used white “x” symbols to highlight the location in the panel of samples taken over the first year. Triangles describe samples on Euonymus, whereas circles those on Hedera. b Distributions of pairwise community distances (Bray–Curtis distances) for a range of pairwise combinations (dark green: within plots sampled at the same time; orange: same plot differing in sampling time; purple: same harvest but different plot; pink: same plot in the 4th harvest but different host plant; light green: same harvest but different plot grouped based on the relative coverage of AMF-associating woody plants). Larger values signify more dissimilar samples, meaning that the responsible factor induced a stronger AMF community shift than in the case of smaller values. As an example, the pairs belonging on the same plot which are presented in the four first histograms (in dark green and orange) consistently showed smaller values than those across different plots (two purples histograms) suggesting that space played a role in shaping AMF communities. Note that Bray–Curtis community distances between Hedera and Euonymus (in pink; same plot) were smaller than respective distances between individuals of Hedera (dark green). c Mean relative abundances of the seven AMF families (Acaulosporaceae, Archaeosporaceae, Claroideoglomeraceae, Diversisporaceae, Gigasporaceae, Glomeraceae, Paraglomaceae) grouped based on (top) the time of sampling, plant host, and (bottom) our classification into high and low plots
A Venn diagram depicting the distribution of OTUs across (i) AM high (> 15% woody AM-associating plant coverage) and AM low plots (< 15% woody AM-associating plant coverage) and (ii) the two plant hosts. Fifteen out of the thirty-two OTUs were observed in all four types of habitats. B Frequency of occurrence of the fifteen most abundant OTUs across ten groups of samples describing plant host, plot quality in relation to AMF abundance, and season of sampling
Partitioning of variance explained by spatial, temporal, host specific, and AM plant cover related parameters across AMF communities in our forest site. Spatial parameters comprised three PCNM axes, and temporal parameters comprised the effects of season (i.e. explaining zero variance; insert at the bottom left) and year. The estimates are biased and are presented only for comparative purposes: for example, the impact of host effects on AMF community structure should have been considerably higher than shown, but because we harvested Euonymus only once the parameter explained a relatively small part of the total variance. The variance partitioning additionally unrealistically assumes a completely balanced design with an equal representation of samples on all plots and invariable sampling effort across the four harvests. By including parameters that explained no variance such as season (insert at the bottom left), we further biased estimates. Finally, the analysis also does not capture that some plots have been assayed more than once and thus are not independent samples
Many woody and herbaceous plants in temperate forests cannot establish and survive in the absence of mycorrhizal associations. Most temperate forests are dominated by ectomycorrhizal woody plant species, which implies that the carrying capacity of the habitat for arbuscular mycorrhizal fungi (AMF) is relatively low and AMF could in some cases experience a limitation of propagules. Here we address how the AMF community composition varied in a small temperate forest site in Germany in relation to time, space, two plant host species, and also with regard to the degree to which plots were covered with AMF-associating woody species. The AMF communities in our study were non-random. We observed that space had a greater impact on fungal community composition than either time, mycorrhizal state of the close-by woody species, or the identity of the host plant. The identity of the host plant was the only parameter that modified AMF richness in the roots. The set of parameters which we addressed has rarely been studied together, and the resulting ranking could ease prioritizing some of them to be included in future surveys. AMF are crucial for the establishment of understory plants in temperate forests, making it desirable to further explore how they vary in time and space.
Top-cited authors
Dirk Redecker
  • University of Burgundy
Leho Tedersoo
  • University of Tartu
Daniel Wipf
  • University of Burgundy
Tom W. May
  • Royal Botanic Gardens Victoria
Sidney Luiz Stürmer
  • Universidade Regional de Blumenau