Environmental heterogeneity structures root-associated fungal communities in Daphne arbuscula (Thymelaeaceae), a shrub adapted to extreme rocky habitats.

Rocky habitats, globally distributed ecosystems, harbour diverse biota, including numerous endemic and endangered species. Vascular plants thriving in these environments face challenging abiotic conditions, requiring diverse morphological and physiological adaptations. Their engagement with the surrounding microbiomes is, however, equally vital for their adaptation, fitness, and long-term survival. Nevertheless, there remains a lack of understanding surrounding this complex interplay within this fascinating biotic ecosystem. Using microscopic observations and metabarcoding analyses, we examined the fungal abundance and diversity in the root system of the rock-dwelling West Carpathian endemic shrub, Daphne arbuscula (Thymelaeaceae). We explored the diversification of root-associated fungal communities in relation to microclimatic variations across the studied sites. We revealed extensive colonization of the Daphne roots by diverse taxonomic fungal groups attributed to different ecological guilds, predominantly plant pathogens, dark septate endophytes (DSE), and arbuscular mycorrhizal fungi (AMF). Notably, differences in taxonomic composition and ecological guilds emerged between colder and warmer microenvironments. Apart from omnipresent AMF, warmer sites exhibited a prevalence of plant pathogens, while colder sites were characterized by a dominance of DSE. This mycobiome diversification, most likely triggered by the environment, suggests that D. arbuscula populations in warmer areas may be more vulnerable to fungal diseases, particularly in the context of global climate change.

©Relationship between endophytic fungal diversity and colonization and soil factors of cultured blueberry roots in Guizhou Province, Southwest China.

Dark septate endophytes (DSEs) inhabit plant roots and soil in ecosystems and host plants worldwide. DSE colonization is influenced by cultivars, soil factors, and specific habitat conditions. The regular diversity of DSEs in blueberries in Guizhou, China, is still unclear. In this study, four cultivars (Gardenblue, Powderblue, O'Neal, and Legacy) in three areas (Gaopo, Majiang, and Fenggang) in Guizhou were used to identify DSEs by morphological and molecular biological methods and to clarify the relationship between DSE diversity and DSE colonization and soil factors of cultivated blueberries in Guizhou. The DSEs isolated from cultivated blueberry roots in 3 areas in Guizhou Province were different, belonging to 17 genera, and the dominant genera were Penicillium, Phialocephala, and Thozetella. DSEs isolated from Majiang belonged to 12 genera and 16 species, those from Gaopo belonged to 7 genera and 15 species, and those from Fenggang belonged to 5 genera and 7 species. Among the different blueberry varieties, 11 genera were isolated from O'Neal, 12 genera were isolated from Powderblue, 11 genera were isolated from Legacy and 13 genera were isolated from Gardenblue. Coniochaeta is endemic to O'Neal, Chaetomium and Curvularia are endemic to Powderblue, and Thielavia is endemic to Legacy. Correlation analysis showed that DSE diversity was significantly correlated with DSE colonization and soil factors.

Are dark septate endophytes an ancestral ecological state in the evolutionary history of the order Chaetothyriales?

Dark septate endophytes (DSE) are pigmented fungi that colonize plant roots. They represent a morpho-functional status composed of many species belonging to the phylum Ascomycota, distributed in different orders. The order Chaetothyriales has representatives with diverse lifestyles, among which the rock-inhabiting one has been proposed to be the ancestral ecological character state. However, all taxa have the phenotypic characteristic of being highly melanized. This trait has been considered relevant in most Chaetothyriales because it allows them to tolerate extreme or toxic environmental conditions. In the present study, aiming to reconstruct the evolutionary history of this order, we analyzed the contribution of the DSE habit to the diversification of the Chaetothyriales. We also report the distribution of the DSE habit among the main families and/or clades within the order. Our results suggest that DSE had a key position in the evolution of the order Chaetothyriales, both as an ancestral ecological character and as a character from which other specialized forms such as Domatium probably derived.

Endorhizal fungal symbiosis in lycophytes and metal(loid)-accumulating ferns growing naturally in mine wastes in Mexico.

Ferns and lycophytes are pioneer plants that can be useful for revegetation. Their natural distribution and interaction with soil fungal endophytes can increase plant fitness but have received little attention. This study aimed to identify these plant species in mine wastes, and determine colonization by arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE). The pseudo-total and diethylenetriamine pentaacetic acid (DTPA)-extractable rhizosphere concentrations of As, Cu, Cd, Pb, and Zn, bioavailability index (BI), and bioconcentration factor (BCF) were analyzed. Six ferns and one lycophyte were identified. Arsenic and metal concentrations were high, which were plant and site-dependent. All species showed hyperaccumulation of As in fronds, especially Argyrochosma formosa (2,883) and Notholaena affinis (2,160) had the highest concentrations (mg kg-1). All plants were colonized by AMF (3%-24%) and DSE (2%-33%). Astrolepis sinuata and Myriopteris notholaenoides had the maximum colonization by AMF and A. formosa by DSE. This study identifies for the first time five ferns and one lycophyte species on mine wastes, their As hyperaccumulation capacity and the simultaneous fungal colonization by AMF and DSE. These are relevant plant traits for phytoremediation. However, fungal identification and the role colonization by AMF and DSE requires full analysis.

Arsenic accumulator ferns and a lycophytes species naturally established on three polluted sites were found. Astrolepis integerrima, A. sinuata, Myriopteris notholaenoides, Notholaena affinis, N. sulphurea, and the lycophyte Selaginella lepidophylla are for the first time reported in these sites. This is the first evidence of DSE colonization in these plants growing on mine wastes in interaction with AMF-colonization. These plants may be useful in developing phytoremediation alternatives.

The relationship between chlorophyllous spores and mycorrhizal associations in ferns: evidence from an evolutionary approach.

PREMISE: Approximately 14% of all fern species have physiologically active chlorophyllous spores that are much more short-lived than the more common and dormant achlorophyllous spores. Most chlorophyllous-spored species (70%) are epiphytes and account for almost 37% of all epiphytic ferns. Chlorophyllous-spored ferns are also overrepresented among fern species in habitats with waterlogged soils, of which nearly 60% have chlorophyllous spores. Ferns in these disparate habitat types also have a low incidence of mycorrhizal associations. We therefore hypothesized that autotrophic chlorophyllous spores represent an adaptation of ferns to habitats with scarce mycorrhizal associations. METHODS: We evaluated the coevolution of chlorophyllous spores and mycorrhizal associations in ferns and their relation to habitat type using phylogenetic comparative methods. RESULTS: Although we did not find support for the coevolution of spore type and mycorrhizal associations, we did find that chlorophyllous spores and the absence of mycorrhizal associations have coevolved with epiphytic and waterlogged habitats. Transition rates to epiphytic and waterlogged habitats were significantly higher in species with chlorophyllous spores compared to achlorophyllous lineages. CONCLUSIONS: Spore type and mycorrhizal associations appear to play important roles in the radiation of ferns into different habitat types. Future work should focus on clarifying the functional significance of these associations.

Micro-scale Experimental System Coupled with Fluorescence-based Estimation of Fungal Biomass to Study Utilisation of Plant Substrates.

The degradation capacity and utilisation of complex plant substrates are crucial for the functioning of saprobic fungi and different plant symbionts with fundamental functions in ecosystems. Measuring the growth capacity and biomass of fungi on such systems is a challenging task. We established a new micro-scale experimental setup using substrates made of different plant species and organs as media for fungal growth. We adopted and tested a reliable and simple titration-based method for the estimation of total fungal biomass within the substrates using fluorescence-labelled lectin. We found that the relationship between fluorescence intensity and fungal dry weight was strong and linear but differed among fungi. The effect of the plant organ (i.e. root vs. shoot) used as substrate on fungal growth differed among plant species and between root endophytic fungal species. The novel microscale experimental system is useful for screening the utilisation of different substrates, which can provide insight into the ecological roles and functions of fungi. Furthermore, our fungal biomass estimation method has applications in various fields. As the estimation is based on the fungal cell wall, it measures the total cumulative biomass produced in a certain environment.

Dark septate endophytes (DSE): potential bioremedial promoters of oil derivatives.

Oil spills are a global environmental problem. One of the management tools used to solve this problem is phytoremediation, a process that uses the capacity of plants and microorganisms to metabolize the components of the oil. The aims of the present study were to isolate, identify and characterize the fungi obtained from plants growing in an oil-contaminated area and evaluate their growth response and emulsifying and degrading capacity in two petroleum derivatives (kerosene and lube oil). Four dark septate endophytes (DSE) strains were isolated and identified: Exserohilum pedicellatum, Ophiosphaerella sp., and two Alternaria alternata strains. E. pedicellatum was found in an oil-contaminated environment for the first time. All strains were grown in kerosene, although some showed inhibition, whereas in lube oil, all showed growth induction. Ophiosphaerella sp. showed "drops" in kerosene, but the four strains showed surfactant capacity in lube oil. Ophiosphaerella sp. showed the highest emulsifying activity index but both A. alternata strains presented the highest lube oil degradation, which was directly related to the weight of the fungal biomass. There was not relationship between emulsifying capacity and oil degradation. However, these fungi show technological potential for application in phytoremediation processes.

Combined inoculation with dark septate endophytes and arbuscular mycorrhizal fungi: synergistic or competitive growth effects on maize?

BACKGROUND: Effects on maize were assessed of dual inoculation with arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) isolated from other plant species. METHODS: Suspensions of DSE isolated from Stipa krylovii were prepared at different densities (2, 4, and 8 × 105 CFU mL- 1) and inoculated separately (AMF or DSE) or together (AMF + DSE), to explore their effects on maize growth. RESULTS: Inoculation with AMF or medium and high densities of DSE and combined inoculation (AMF + DSE) increased plant above-ground growth and altered root morphology. Differences in plant growth were attributable to differences in DSE density, with negative DSE inoculation responsiveness at low density. AMF promoted plant above-ground growth more than DSE and the high density of DSE promoted root development more than AMF. Combined inoculation might lead to synergistic growth effects on maize at low density of DSE and competitive effects at medium and high DSE densities. CONCLUSIONS: AMF and DSE co-colonized maize roots and they had positive effects on the host plants depending on DSE density. These findings indicate the optimum maize growth-promoting combination of AMF and DSE density and provide a foundation for further exploration of potentially synergistic mechanisms between AMF and DSE in physiological and ecological effects on host plants.

Melatonin confers heavy metal-induced tolerance by alleviating oxidative stress and reducing the heavy metal accumulation in Exophiala pisciphila, a dark septate endophyte (DSE).

BACKGROUND: Melatonin (MT), ubiquitous in almost all organisms, functions as a free radical scavenger. Despite several reports on its role as an antioxidant in animals, plants, and some microorganisms, extensive studies in filamentous fungi are limited. Based upon the role of melatonin as an antioxidant, we investigated its role in heavy metal-induced stress tolerance in Exophiala pisciphila, a dark septate endophyte (DSE), by studying the underlying mechanisms in alleviating oxidative stress and reducing heavy metal accumulation. RESULTS: A significant decrease in malondialdehyde (MDA) and oxygen free radical (OFR) in E. pisciphila was recorded under Cd, Zn, and Pb stresses as compared to the control. Pretreatment of E. pisciphila with 200.0 µM exogenous melatonin significantly increased the activity of superoxide dismutase (SOD) under Zn and Pb stresses. Pretreatment with 200.0 µM melatonin also lowered Cd, Zn, and Pb concentrations significantly. Melatonin production was enhanced by Cd, Cu, and Zn after 2 d, and melatonin biosynthetic enzyme genes, E. pisciphila tryptophan decarboxylase (EpTDC1) and serotonin N-acetyltransferase (EpSNAT1), were transcriptionally upregulated. The overexpression of EpTDC1 and N-acetylserotonin O-methyltransferase (EpASMT1) in Escherichia coli and Arabidopsis thaliana enhanced its heavy metal-induced stress tolerance. The overexpression of EpTDC1 and EpASMT1 reduced the Cd accumulation in the whole A. thaliana plants, especially in the roots. CONCLUSIONS: Melatonin conferred heavy metal-induced stress tolerance by alleviating oxidative stress, activating antioxidant enzyme SOD, and reducing heavy metal accumulation in E. pisciphila. Melatonin biosynthetic enzyme genes of E. pisciphila also played key roles in limiting excessive heavy metal accumulation in A. thaliana. These findings can be extended to understand the role of melatonin in other DSEs associated with economically important plants and help develop new strategies in sustainable agriculture practice where plants can grow in soils contaminated with heavy metals.

Plant performance of enhancing licorice with dual inoculating dark septate endophytes and Trichoderma viride mediated via effects on root development.

BACKGROUND: This study aimed to assess whether licorice (Glycyrrhiza uralensis) can benefit from dual inoculation by Trichoderma viride and dark septate endophytes (DSE) isolated from other medicinal plants. METHODS: First, we isolated and identified three DSE (Paraboeremia putaminum, Scytalidium lignicola, and Phoma herbarum) and Trichoderma viride from medicinal plants growing in farmland of China. Second, we investigated the influences of these three DSE on the performance of licorice at different T. viride densities (1 × 106, 1 × 107, and 1 × 108 CFU/mL) under sterilised condition in a growth chamber. RESULTS: Three DSE strains could colonize the roots of licorice, and they established a positive symbiosis with host plants depending on DSE species and T. viride densities. Inoculation of P. putaminum increased the root biomass, length, surface area, and root:shoot ratio. S. lignicola increased the root length, diameter and surface area and decreased the root:shoot ratio. P. herbarum increased the root biomass and surface area. T. viride increased the root biomass, length, and surface area. Structural equation model (SEM) analysis showed that DSE associated with T. viride augmented plant biomass and height, shoot branching, and root surface area. Variations in root morphology and biomass were attributed to differences in DSE species and T. viride density among treatments. P. putaminum or P. herbarum with low- or medium T. viride density and S. lignicola with low- or high T. viride density improved licorice root morphology and biomass. CONCLUSIONS: DSE isolated from other medicinal plants enhanced the root growth of licorice plants under different densities T. viride conditions and may also be used to promote the cultivation of medicinal plants.

Angiosperm symbioses with non-mycorrhizal fungal partners enhance N acquisition from ancient organic matter in a warming maritime Antarctic.

In contrast to the situation in plants inhabiting most of the world's ecosystems, mycorrhizal fungi are usually absent from roots of the only two native vascular plant species of maritime Antarctica, Deschampsia antarctica and Colobanthus quitensis. Instead, a range of ascomycete fungi, termed dark septate endophytes (DSEs), frequently colonise the roots of these plant species. We demonstrate that colonisation of Antarctic vascular plants by DSEs facilitates not only the acquisition of organic nitrogen as early protein breakdown products, but also as non-proteinaceous d-amino acids and their short peptides, accumulated in slowly-decomposing organic matter, such as moss peat. Our findings suggest that, in a warming maritime Antarctic, this symbiosis has a key role in accelerating the replacement of formerly dominant moss communities by vascular plants, and in increasing the rate at which ancient carbon stores laid down as moss peat over centuries or millennia are returned to the atmosphere as CO2 .

Plant performance response to eight different types of symbiosis.

Almost all plant species interact with one or more symbioses somewhere within their distribution range. Bringing together plant trait data and growth responses to symbioses spanning 552 plant species, we provide for the first time on a large scale (597 studies) a quantitative synthesis on plant performance differences between eight major types of symbiosis, including mycorrhizas, N-fixing bacteria, fungal endophytes and ant-plant interactions. Frequency distributions of plant growth responses varied considerably between different types of symbiosis, in terms of both mean effect and 'risk', defined here as percentage of experiments reporting a negative effect of symbiosis on plants. Contrary to expectation, plant traits were poor predictors of growth response across and within all eight symbiotic associations. Our analysis showed no systematic additive effect when a host plant engaged in two functionally different symbioses. This synthesis suggests that plant species' ecological strategies have little effect in determining the influence of a symbiosis on host plant growth. Reliable quantification of differences in plant performance across symbioses will prove valuable for developing general hypotheses on how species become engaged in mutualisms without a guarantee of net returns.

Subterranean Desert Rodents (Genus Ctenomys) Create Soil Patches Enriched in Root Endophytic Fungal Propagules.

Subterranean rodents are considered major soil engineers, as they can locally modify soil properties by their burrowing activities. In this study, the effect of a subterranean rodent of the genus Ctenomys on soil properties and root endophytic fungal propagules in a shrub desert of northwest Argentina was examined. Our main goal was to include among root endophytic fungi not only arbuscular mycorrhiza but also the dark septate endophytes. We compared the abundance of fungal propagules as well as several microbiological and physicochemical parameters between soils from burrows and those from the surrounding landscape. Our results show that food haulage, the deposition of excretions, and soil mixing by rodents' burrowing promote soil patchiness by (1) the enrichment in both types of root endophytic fungal propagules; (2) the increase in organic matter and nutrients; and (3) changes in soil edaphic properties including moisture, field capacity, and texture. These patches may play a critical role as a source of soil heterogeneity in desert ecosystems, where burrows constructed in interpatches of bare soil can act, once abandoned, as "islands of fertility," promoting the establishment of plants in an otherwise hostile environment.

Pinewood nematode infection alters root mycoflora of Pinus tabulaeformis Carr.

AIMS: This study investigates pinewood nematode's impacts on root mycoflora of Pinus tabulaeformis. METHODS AND RESULTS: The biomass, colonization rate, community structure and diversity of root-associated fungi were investigated in pinewood nematode-infected and nematode-noninfected P. tabulaeformis. The results indicated that the roots of P. tabulaeformis were colonized highly by root-associated fungi, mainly ectomycorrhizal fungi (ECMF) and dark septate endophytes. Infection of pinewood nematode was associated with a significant (P < 0·05) decrease in root colonization rates by ECMF, dark septate endophytes and total hyphae, as well as in fungal biomass in the roots. Illumina MiSeq sequences of tagged amplicons of 18S rDNA region revealed Basidiomycota (65·70%) and Ascomycota (34·14%) as the dominant root-associated fungi in roots of P. tabulaeformis. Among the detected operational taxonomic units (OTUs), ECMF and dark septate endophytes exhibited a higher relative abundance in trees infected by pinewood nematode compared with noninfected ones. CONCLUSIONS: The infection of pinewood nematode altered the composition and OTU abundance of root-associated fungi community in P. tabulaeformis roots with a decrease in the biomass, species richness and diversity of root-associated fungi, as well as in the colonization rates and abundance of ECMF and dark septate endophytes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is an important contribution for better understanding the interaction between pine wilt disease and root-associated fungi.

Rapid temporal changes in root colonization by arbuscular mycorrhizal fungi and fine root endophytes, not dark septate endophytes, track plant activity and environment in an alpine ecosystem.

Fungal root endophytes play an important role in plant nutrition, helping plants acquire nutrients in exchange for photosynthates. We sought to characterize the progression of root colonization by arbuscular mycorrhizal fungi (AMF), dark septate endophytes (DSE), and fine root endophytes (FRE) over an alpine growing season, and to understand the role of the host plant and environment in driving colonization levels. We sampled four forbs on a regular schedule from June 26th-September 11th from a moist meadow (3535 m a.s.l) on Niwot Ridge, Rocky Mountain Front Range, CO, USA. We quantified the degree of root colonization by storage structures, exchange structures, and hyphae of all three groups of fungi. AMF and FRE percent colonization fluctuated significantly over time, while DSE did not. All AMF structures changed over time, and the degree of change in vesicles differed by plant species. FRE hyphae, AMF arbuscules and AMF vesicles peaked late in the season as plants produced seeds. AMF hyphae levels started high, decreased, and then increased within 20 days, highlighting the dynamic nature of plant-fungal interactions. Overall, our results show that AMF and FRE, not DSE, root colonization rapidly changes over the course of a growing season and these changes are driven by plant phenology and seasonal changes in the environment.

A shift from arbuscular mycorrhizal to dark septate endophytic colonization in Deschampsia flexuosa roots occurs along primary successional gradient.

Soil fungal community and dominant mycorrhizal types are known to shift along with plant community changes during primary succession. However, it is not well understood how and why root fungal symbionts and colonization types vary within the plant host when the host species is able to thrive both at young and at old successional stages with different light and nutrient resource availability. We asked (i) how root fungal colonization of Deschampsia flexuosa (Poaceae) by arbuscular mycorrhizal (AM) fungi and dark septate endophytes (DSE) changes along a postglacial primary successional land uplift gradient. As neighboring vegetation may play a role in root fungal colonization, we also asked (ii) whether removal of the dominant neighbor, Empetrum nigrum ssp. hermaphroditum (Ericaceae), affects root fungal colonization of Deschampsia. We also studied whether (iii) foliar carbon (C) and nitrogen (N) concentration of Deschampsia is related to successional changes along a land uplift gradient. AM colonization decreased (-50 %), DSE colonization increased (+200 %), and foliar C declined in Deschampsia along with increasing successional age, whereas foliar N was not affected. Empetrum removal did not affect AM colonization but increased DSE sclerotial colonization especially at older successional stages. The observed decrease in foliar C coincides with an increase in canopy closure along with increasing successional age. We suggest that the shift from an AM-dominated to a DSE-dominated root fungal community in Deschampsia along a land uplift successional gradient may be related to different nutritional benefits gained through these root fungal groups.

Communities of Cultivable Root Mycobionts of the Seagrass Posidonia oceanica in the Northwest Mediterranean Sea Are Dominated by a Hitherto Undescribed Pleosporalean Dark Septate Endophyte.

Seagrasses, a small group of submerged marine macrophytes, were reported to lack mycorrhizae, i.e., the root-fungus symbioses most terrestrial plants use for nutrient uptake. On the other hand, several authors detected fungal endophytes in seagrass leaves, shoots, rhizomes, and roots, and an anatomically and morphologically unique dark septate endophytic (DSE) association has been recently described in the roots of the Mediterranean seagrass Posidonia oceanica. Nevertheless, the global diversity of seagrass mycobionts is not well understood, and it remains unclear what fungus forms the DSE association in P. oceanica roots. We isolated and determined P. oceanica root mycobionts from 11 localities in the northwest Mediterranean Sea with documented presence of the DSE association and compared our results with recent literature. The mycobiont communities were low in diversity (only three species), were dominated by a single yet unreported marine fungal species (ca. 90 % of the total 177 isolates), and lacked common terrestrial and freshwater root mycobionts. Our phylogenetic analysis suggests that the dominating species represents a new monotypic lineage within the recently described Aigialaceae family (Pleosporales, Ascomycota), probably representing a new genus. Most of its examined colonies developed from intracellular microsclerotia occupying host hypodermis and resembling microsclerotia of terrestrial DSE fungi. Biological significance of this hitherto overlooked seagrass root mycobiont remains obscure, but its presence across the NW Mediterranean Sea and apparent root intracellular lifestyle indicate an intriguing symbiotic relationship with the dominant Mediterranean seagrass. Our microscopic observations suggest that it may form the DSE association recently described in P. oceanica roots.

Aboveground Epichloë coenophiala-Grass Associations Do Not Affect Belowground Fungal Symbionts or Associated Plant, Soil Parameters.

Cool season grasses host multiple fungal symbionts, such as aboveground Epichloë endophytes and belowground arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSEs). Asexual Epichloë endophytes can influence root colonization by AMF, but the type of interaction-whether antagonistic or beneficial-varies. In Schedonorus arundinaceus (tall fescue), Epichloë coenophiala can negatively affect AMF, which may impact soil properties and ecosystem function. Within field plots of S. arundinaceus that were either E. coenophiala-free (E-), infected with the common, mammal-toxic E. coenophiala strain (CTE+), or infected with one of two novel, non-toxic strains (AR542 NTE+ and AR584 NTE+), we hypothesized that (1) CTE+ would decrease AMF and DSE colonization rates and reduce soil extraradical AMF hyphae compared to E- or NTE+, and (2) this would lead to E- and NTE+ plots having greater water stable soil aggregates and C than CTE+. E. coenophiala presence and strain did not significantly alter AMF or DSE colonization, nor did it affect extraradical AMF hypha length, soil aggregates, or aggregate-associated C and N. Soil extraradical AMF hypha length negatively correlated with root AMF colonization. Our results contrast with previous demonstrations that E. coenophiala symbiosis inhibits belowground AMF communities. In our mesic, relatively nutrient-rich grassland, E. coenophiala symbiosis did not antagonize belowground symbionts, regardless of strain. Manipulating E. coenophiala strains within S. arundinaceus may not significantly alter AMF communities and nutrient cycling, yet we must further explore these relationships under different soils and environmental conditions given that symbiont interactions can be important in determining ecosystem response to global change.

Acid protease production in fungal root endophytes.

Fungal endophytes are ubiquitous in healthy root tissue, but little is known about their ecosystem functions, including their ability to utilize organic nutrient sources such as proteins. Root-associated fungi may secrete proteases to access the carbon and mineral nutrients within proteins in the soil or in the cells of their plant host. We compared the protein utilization patterns of multiple isolates of the root endophytes Phialocephala fortinii s.l., Meliniomyces variabilis and Umbelopsis isabellina with those of two ectomycorrhizal (ECM) fungi, Hebeloma incarnatulum and Laccaria bicolor, and the wood-decay fungus Irpex lacteus at pH values of 2-9 on liquid BSA media. We also assessed protease activity using a fluorescently labeled casein assay and gelatin zymography and characterized proteases using specific protease inhibitors. I. lacteus and U. isabellina utilized protein efficiently, while the ECM fungi exhibited poor protein utilization. ECM fungi secreted metallo-proteases and had pH optima above 4, while other fungi produced aspartic proteases with lower pH optima. The ascomycetous root endophytes M. variabilis and P. fortinii exhibited intermediate levels of protein utilization and M. variabilis exhibited a very low pH optimum. Comparing proteolytic profiles between fungal root endophytes and fungi with well defined ecological roles provides insight into the ecology of these cryptic root associates.