Mycologia, 107(1), 2015, pp. 1–11. DOI: 10.3852/14-106 # 2015 by The Mycological Society of America, Lawrence, KS 66044-8897 Issued 26 January 2015

Acid protease production in fungal root endophytes Michael S. Mayerhofer Erica Fraser Gavin Kernaghan1

root systems (Richard and Fortin 1974, Halmschlager and Kowalski 2004, Gru¨nig et al. 2008a) and they differ from mycorrhizal fungi in that they lack highly evolved absorptive structures (Brundrett 2006). The ecological roles of these fungi have been further obscured by conflicting results with respect to their influence on plant growth. Host plant growth response to inoculation with root endophytes in vitro is variable, depending on the fungal isolate and experimental conditions (Newsham 2011, Tellenbach et al. 2011, Mayerhofer et al. 2013). However, fungalroot endophytes might facilitate the mineralization of complex organic nitrogen sources, such as proteins, into forms available to the host plant (Mandyam and Jumpponen 2005, Upson et al. 2009, Newsham 2011), because host response to root endophytes is more often positive when organic nitrogen sources are added to the growing medium (Usuki and Narisawa 2007, Upson et al. 2009, Newsham 2011). Other authors have hypothesized that these fungi may be latent pathogens (Wilcox and Wang 1987, Schulz et al. 1999) or latent saprotrophs (Kernaghan and Patriquin 2011), in that they are typically capable of using a wide variety of organic compounds, including proteins and other plant cell-wall constituents (Caldwell et al. 2000, Mandyam et al. 2010). We expect proteolytic activity to be correlated with overall saprotrophic ability, in that protein is an important constituent of plant, fungal and bacterial cell walls (Ko¨gel-Knabner 2002) and the main source of nitrogen available in wood (Abraham 1995, Dunaevsky et al. 2006, Kudryavtseva et al. 2008). Fungi secrete extracellular proteases to hydrolyze proteins into shorter peptides or individual amino acids (Geisseler et al. 2010, Farrell et al. 2012). They produce aspartic, serine and metallo-proteases; classifications based on the molecular composition of the active site that cleaves the protein (Pavlukova et al. 1998, Monod et al. 2002, Kudryavtseva et al. 2008, Sˇimkovicˇ et al. 2008). Proteolytic activity is highly sensitive to environmental conditions, particularly pH. Consequently fungal protein utilization is often limited to a narrow range of pH values. For example, the ericoid mycorrhizal fungus Rhizoscyphus ericae, which is commonly associated with ericaceous plants in acidic heathlands, uses proteins at pH 2.0–4.0 while an endophyte of Rhodothamnus growing in a soil at pH 6.5 retains proteolytic activity up to pH 6.0 (Leake and Read 1990). The proteolytic characteristics of a given organism are also related to its preferred nutritional sources (St

Department of Biology, Mount Saint Vincent University, 166 Bedford Highway, Halifax, Nova Scotia, Canada, B3M 2J6

Abstract: 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. Rootassociated 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 wooddecay 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. Key words: aspartic protease, dark septate endophytes, ectomycorrhizal fungi, Helotiales, protein utilization, wood decay fungi INTRODUCTION Fungal-root endophytes are ubiquitous plant associates that colonize the root tissue of their host internally without causing any apparent harm (Saikkonen et al. 1998, Schulz and Boyle 2005). The association is difficult to classify because fungal root endophytes are found in both healthy and declining Submitted 28 Apr 2014; accepted for publication 1 Oct 2014. 1 Corresponding author. E-mail: [email protected]

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MYCOLOGIA

Leger et al. 1997, Dubovenko et al. 2010). Ubiquitous, opportunistic fungi such as Aspergillus and Trichoderma spp. secrete a wide variety of proteases to utilize multiple substrates over a broad pH spectrum (St Leger et al. 1997), while parasites and pathogens, such as mycoparasites and nematophagous fungi, typically secrete extracellular serine proteases (Kredics et al. 2009). Dermatophytes, such as Trichophyton, produce large quantities of keratinase to specifically target the elastic proteins of skin and hair (Asahi et al. 1985). Because ecological functions are often reflected in enzymatic profiles, information on specific proteolytic abilities may shed light on the ecology of the root endophytes. With the exception of Hedlund et al. (1991) who detected extracellular serine and metalloproteases from Umbelopsis isabellina, protease assays from root endophytes have been restricted to measuring the clearing of gelatin (Caldwell et al. 2000, Mandyam et al. 2010) or soy protein media (Gru¨nig et al. 2008b). In the present study we sought to determine the patterns of protein utilization and extracellular protease activities, and characterize the protease types, in a selection of common fungal root endophytes of boreal trees in comparison with saprophytic and ectomycorrhizal (ECM) fungi. MATERIALS AND METHODS Fungal isolates.—We maintained one isolate of each of the ECM fungi Hebeloma incarnatulum (syn. H. longicaudum) and Laccaria bicolor, one isolate of the wood decay fungus Irpex lacteus and multiple isolates of the common root endophytes Meliniomyces variabilis (three), Umbelopsis isabellina (two) and Phialocephala fortinii sensu lato (six) (TABLE I) on 1% w/v water agar media (25 mL/plate). We used six isolates of P. fortinii s.l. to capture potential intraspecific variation within the Phialocephala fortinii s.l.Acephala applanata species complex (Gru¨nig et al. 2008a, Kernaghan and Patriquin 2011). The fungal-root endophytes were originally isolated from surface-sterilized boreal tree roots and identified by ITS sequencing. ECM fungi were isolated from sporocarps on malt-yeast media containing antibiotics and 5 ppm benomyl to select for basidiomycetes (Danielson 1984). I. lacteus was isolated from forest soil onto media containing 1% bovine serum albumin (BSA) as the sole carbon and nitrogen source. We confirmed the identity of the ECM and I. lacteus cultures by ITS sequencing and comparison with GenBank and UNITE (Abarenkov et al. 2010) sequence data bases. Utilization of BSA.—We assessed protein utilization of the 14 isolates over 21 d on liquid BSA media: 0.3 g BSA (96% lyophilized powder, Sigma), 0.03 g Yeast Extract (BBL), 1 g MgSO4?7H2O, 2 g KH2PO4, 0.132 mg MnSO4?H2O, 0.1 mg H3BO3, 0.03 mg ZnSO4?7H2O, 0.000 1 mg CoCl2?6H2O and 0.0001 mg CuSO4?5H2O per liter. The media was titrated to range from pH 2 to 9 (in one-unit increments) with HCl or

NaOH before autoclaving. The BSA and yeast extract were added by filter sterilization (0.22 m) and the pH confirmed. Mycelial plugs (5 mm) of each fungal isolate were transferred from water agar into culture tubes containing 10 mL BSA media at each of the eight pH levels. All treatments and controls were incubated at 22 C in triplicate. Beginning 24 h after inoculation, we quantified the concentration of protein remaining in the media with the Bradford assay (Bradford 1976), in which 6 mL media was removed from each culture tube under sterile conditions (non-destructive sampling) and combined with 56 mL Coomassie protein assay reagent (Thermo-Scientific) in a clear, flat-bottom, 96-well microplate. We then measured absorbance at 580 nm with a Biotek Synergy HT microplate reader after a 15 min incubation period. Each assay was conducted in triplicate for a total of nine assays per species times pH combination. We calculated the amount of protein remaining in the media based on a BSA standard curve. Finally we determined fungal biomass by filtering, drying and weighing mycelia either after 21 d or after protein no longer could be detected in the media. Utilization of soy protein.—To determine the utilization patterns of plant protein, we assayed the utilization of soy protein media at pH 3 (the single best pH for endophytes), using the endophyte isolates with the highest protein utilization rates on BSA; P. fortinii ARSL180907.2, M. variabilis ARSL060907.24 and U. isabellina ARSL230507.15. We also included L. bicolor, H. incarnatulum and I. lacteus for comparison. Soy media was made by adding 100 g Swiss Natur Soya Pro protein isolate, 1 g MgSO4, 1 g KH2PO4 and 0.7 g Na2HPO4 to 1 L H2O, titrated to pH 3 with 5M H2SO4. This solution was left overnight at 4 C and the supernatant decanted and filter-sterilized with Millipore Stericup Express PLUS with a 0.22 mm PES filter. The same isolates were also inoculated onto BSA media (prepared as above) to directly compare utilization of the two protein sources. For both the soy and BSA media, we inoculated 21 tubes (8 mL media each) per fungal isolate. Every 2 d over a 14 d period, we filtered and weighed the fungal hyphae from three tubes per isolate (destructive sampling) and used the supernatant to conduct the Bradford assay as described above. Protease activity and inhibition.—Protease activity of 14 d cultures on BSA media (grown at the optimum pH for protein utilization by each species) was quantified with a fluorescently labeled casein assay (EnzChek protease assay kit, Invitrogen). We combined 50 mL culture or control media with 50 mL of fluorescently labeled casein in citratecitric acid buffer in black 96-well microplates. The casein buffer was adjusted to give a pH range from 2.0–6.0 in 0.5 unit increments. We loaded each replicate in triplicate wells and incubated the plates in the dark for 18 h and read the fluorescence at an excitation of 590 nm and emission of 645 nm on a Biotek Synergy HT. We calculated the fluorescence for each sample by subtracting un-inoculated control values from sample values after averaging triplicate wells. We also tested the culture media for the inhibition of aspartic, serine, metallo- and cysteine proteases by the

MAYERHOFER ET AL.: FUNGAL ROOT ENDOPHYTE PROTEASES TABLE I.

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Species, ecological group, origin and accession numbers of fungal isolates Accession Nos. Species

Ecology

ARSLa

GenBank

Origin

Phialocephala fortinii s.l.

Root endophyte

Meliniomyces variabilis

Root endophyte

Umbelopsis isabellina

Root endophyte

Laccaria bicolor Hebeloma incarnatulum Irpex lacteus

ECM ECM Saprotroph

190907.6 070907.26 180907.2 250507.1 170507.44 180507.18 060907.24 190907.72 220507.11 230507.15b 170507.25II 261109.1 051012.1 150911.11

HQ157899 HQ157940 HQ157945 HQ157854 KM460828 HQ157956 HQ157845 HQ157888 HQ157931 HQ157862 HQ157911 KF639964 KF639965 KF639961

Fir root, Cape Breton, NS Spruce root, Cape Breton, NS Spruce root, Cape Breton, NS Birch root, Abitibi-Te´miscamingue, Qc Fir root, Abitibi-Te´miscamingue, Qc Spruce root, Abitibi-Te´miscamingue, Qc Birch root, Cape Breton, NS Fir root, Cape Breton, NS Spruce root, Abitibi-Te´miscamingue, Qc Birch root, Abitibi-Te´miscamingue, Qc Fir root, Abitibi-Te´miscamingue, Qc Fruiting Body, Cape Breton, NS Fruiting Body, Purcell’s Cove, NS Soil, Halifax, NS

a b

Atlantic Root Symbiosis Laboratory accessions. Also deposited in the University of Alberta Microfungus Collection and Herbarium as UAMH 11125.

addition of 1 mM Pepstatin A, 20 mM E64, 5 mM EDTA or 0.1 mM PMSF respectively (ten Have et al. 2004) to the protease activity assay. We used casein buffered at pH 4.0 for the endophytic fungi and I. lacteus and pH 6.0 for the ECM fungi. We incubated the plates for 22 h to increase overall fluorescence and detection of inhibition. Because we observed a small amount of variation between wells of the same sample including controls, we considered fluorescence as detectable only when the mean, minus the standard error of a sample, was larger than 10 fluorescence units. Zymography.— We used gelatin zymography to characterize proteases produced by our isolates. Before zymography, we grew all cultures at their optimal pH for protease production based on the previous Bradford assay results. Both gels and buffer systems were below pH 7.0 to prevent permanent denaturation of acid proteases (Hachmann and Amshey 2005). After growth on BSA media, we centrifuged 7.5 mL of liquid media for 7.5 min at 18 400 g and concentrated 5 mL of the resulting supernatant using MicrosepTM or AmiconH centrifugal filters with 10 KDa cutoff at 4 C and 7600 g for 20 min. Concentrated supernatants were diluted 43 in loading buffer (125 mM Tris HCl pH 6.8, 8% SDS, 50% glycerol and 0.02% Bromophenol blue). We loaded the samples into an ice-cooled SDS-page system with a resolving gel composed of 1.15 mL H2O, 2.85 mL 1.25 mM bis tris buffer pH 6.7, 4 mL 30% acrylamide:bis- acrylamide (29 : 1), 0.1% SDS, 20 mL TMED, 7.5 mg ammonium persulfate and 1% gelatin. Stacking gels were composed of 0.75 mL 30% acrylamide : bis acrylamide 29 : 1, 40 mL 0.1% SDS, 25 mL TMED, 7.5 mg ammonium persulfate and 2.5 mL H2O in 1.42 mL of 1.25 mM bis tris buffer pH 6.7. The running buffer consisted of 250 mM MOPS, 250 mM Tris and 1% SDS. We used a 10–250 kDa protein standard ladder (Precision Plus, Bio-Rad) to determine molecular mass.

After electrophoresis gels were washed three times in 2.5% Triton-X 100 and twice in development solution (200 mM NaCl, 5 mM CaCl2, 0.02% Brij L23 solution) and incubated overnight in the same solution at 37 C. We initially characterized all isolates with a pH 4.0 development solution (buffered with 50 mM sodium citrate-citric acid) to assess both inter- and intraspecific differences. We then characterized the most proteolytically active isolates of each endophyte species (P. fortinii ARSL180907.2, M. variabilis ARSL190907.72, U. isabellina ARSL230507.15) as well as the two ECM fungi and I. lacteus at pH 2, 3, 4, 5 and 6 by buffering Triton-X 100 and development solutions with 50 mM sodium citrate-citric acid. Gels were stained with an amido black staining solution (45% methanol, 10% glacial acetic acid, 45% H2O, 0.1% w/v amido black) and destained with 50% methanol, 2% glacial acetic acid and 48% distilled H2O, until clear bands were observed. Gels were scanned at 600 DPI on a flatbed scanner and image analysis conducted with AlphaView software (Protein Simple). Statistics.—We tested for differences in protein utilization and protease activity patterns among fungal isolates across a pH gradient using hierarchical cluster analysis. Protein utilization was estimated as a rate; the linear decrease in protein concentration over time between the start and end of the experiment (or when protein could no longer be detected in the media). Before cluster analyses, the data all were made positive by adding the lowest negative value plus one and then log10 transformed. We ran the analyses using correlation values as distance and Ward’s method for clustering, identifying significant groups with the package pvclust (Suzuki and Shimodaira 2006). We also used linear ANCOVA with time as the covariate to assess betweenspecies differences in the destructive soy and BSA protein utilization experiment and ANOVA to assess the effects of the different protease inhibitors on the hydrolysis of fluorescently labeled casein. All analyses were conducted with R (R Core Team 2013).

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MYCOLOGIA reached 61 mg protein per L per day, while the two ECM fungi achieved only 5 and 9 mg per day. The protein utilization rates in the endophytic fungi were intermediate between these extremes (FIG. 1). Species rankings based on protein utilization rates were generally similar for growth on BSA and soy protein, although statistically significant groupings (Tukey’s HSD) differed between the two (TABLE II, SUPPLEMENTARY TABLE II). On BSA, I. lacteus and the root endophytes had significantly higher utilization rates than the ECM fungi, while on soy, I. lacteus and U. isabellina exhibited significantly higher utilization rates than all the other fungi. In general, the fungi hydrolyzed BSA faster than the more complex soy protein, although growth on soy tended to be greater than on BSA.

FIG. 1. Protein utilization rates between pH 2–9 for root endophytes (solid lines), the saprotrophic wood decay fungus I. lacteus (dotted line), and ECM fungi (dashed lines). Data points represent an average of three replicates per isolate, with six isolates for P. fortinii (n 5 18), three isolates for M. variabilis (n 5 9), two for U. isabellina (n 5 6) and one isolate for each of the ECM and wood decay fungi (n 5 3). Error bars represent the standard error of the mean.

RESULTS Protein utilization rates.—The fungi exhibited three distinct patterns with respect to pH optima and pH range for protein utilization (FIG. 1, SUPPLEMENTARY TABLE I). M. variabilis had a pH optimum between pH 2 and 3, and an overall pH range between 2 and 4. P. fortinii, U. isabellina, and I. lacteus all displayed pH optima at 3 and ranged between 2 and 6 (7 for I. lacteus) and the ECM fungi H. incarnatulum and L.bicolor showed optima between pH 4 and 6 and ranged from 3 to 7. The fungi also varied in their maximum protein utilization rates. For example, on BSA, I. lacteus TABLE II.

Proteolytic activity.—Media from U. isabellina and I. lacteus cultures were more proteolytically active than from any of the other fungi (FIG. 2, SUPPLEMENTARY TABLE III), which correlates with the data on protein utilization. Protease activity was generally comparable among the other fungi when only absolute fluorescence is considered. However, when expressed as activity per mg dry fungal weight, the ECM fungi displayed somewhat higher activities than the ascomycetous root endophytes, P. fortinii and M. variabilis. As with protein utilization, pH had variable effects on proteolytic activity. M. variabilis protease exhibited activity from pH 2.0 to 3.0, while the ECM fungi were active only between pH 4.0 and 6.0. P. fortinii, U. isabellina and I. lacteus all displayed much broader pH ranges for proteolytic activity. Hierarchical cluster analysis.—Hierarchical clustering analysis synthesizes data on both protein utilization and activity (FIG. 3). The fungi form three statistically significant clusters. I. lacteus, all isolates of U. isabellina and P. fortinii form one cluster, all isolates of M. variabilis form a second cluster, and the ECM fungi form a third. These main groupings likely

Protein utilization rates and final weights of selected isolates on BSA and soy-protein isolate media BSA media

Species

Isolate

Ecology

L. bicolor H. incarnatulum M. variabilis P. fortinii I. lacteus U. isabellina

261109.1 051012.1 060907.24 180907.2 150911.11 230507.15

Mycorrhizal Mycorrhizal Endophyte Endophyte Saprotroph Endophyte

Soy-protein isolate media

Utilization rate Tukey’s Final weight Utilization rate HSD (mg) (mg/L?day21) (mg/L?day21) 0.3 18.8 27.8 30.1 42.8 47.8

a b c c c c

0.1 0.4 2.3 0.8 0.3 1.7

6 6 6 6 6 6

0.1 0.1 0.1 0.1 0.1 0.2

0.3 2.2 2.3 4.8 11.9 10.8

Tukey’s Final weight HSD (mg) a a a a b b

1.6 6 0.8 0.9 6 0.2 2.4 6 0.1 3.1 2.4 6 0.7 9.7 6 0.7

Values represent averages of three replicates per sampling time (n 5 21 per isolate) 6 standard error, except P. fortinii 180907.2 grown on soy media, which had no replicates at the last sampling time (n 5 19).

MAYERHOFER ET AL.: FUNGAL ROOT ENDOPHYTE PROTEASES

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FIG. 2. Protease activity between pH 2.0–6.0 in fungi grown in liquid BSA media at their optimum pH for protein utilization. Black bars represent absolute fluorescence units and gray bars are fluorescence units per mg dry weight. The average final dry weights (mg 6 standard error) are given with the species names. The number of isolates and replicates measured are as in FIG. 1. Error bars represent the standard error of the mean.

reflect differences in effective pH ranges between species. Two sub-clusters also occur; one including I. lacteus and U. isabellina and the other including two of the three isolates of M. variabilis. These subclusters reflect overall differences in maximum activity and utilization rates, as I. lacteus and U. isabellina were the most active fungi overall, and M. variabilis ARSL220507.11 was the least active M. variabilis isolate. Specific protease inhibitors.— Of the four protease inhibitors tested, only pepstatin and EDTA caused significant reductions in protease activity (FIG. 4, SUPPLEMENATRY TABLE IV). The addition of pepstatin resulted in 93–100% inhibition in protease activity in the root endophytes and 83% inhibition in I. lacteus, indicating that these fungi produce aspartic proteases. Furthermore, it is unlikely that these fungi produce other protease types, as none of the other inhibitors elicited significant reduction in activity

compared to the control. In the ECM fungi, only EDTA significantly reduced activity (86% in L. bicolor and 56% in H. incarnatulum), indicating the production of metallo-proteases. Zymography.— Bands of proteolytic activity varied from 20 kDa to 125 kDa (FIG. 5). We observed one distinct band in I. lacteus (42 kDa) and U. isabelina (70 kDa), and two distinct bands in H. incarnatulum (125 and 84 kDa), L. bicolor (86 and 72 kDa), M. variabilis (60 and 20 kDa) and P. fortinii (63 and 26 kDa). In some cases, bands of much higher molecular mass were observed, but these were likely protein aggregates (Lantz and Ciborowski 1994). Intraspecific variation was low, with similar band sizes for all isolates within a given species. The influence of pH on band presence/absence varied among the fungal species tested, with the ECM fungi L. bicolor and H. incarnatulum lacking activity at the lowest pH values and the root endophytes P. fortinii and M.

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FIG. 3. Hierarchical cluster analysis of combined data on protein utilization rates and protease activities from isolates of fungal root endophyte species (black), ECM fungi and wood decay fungi (gray). Horizontal lines and associated P-values (P , 0.05) indicate statistically significant clusters (black) and sub-clusters (gray).

variabilis lacking activity at the highest pH values (TABLE III). Within individual species, different bands responded similarly to pH changes, with the exception of the 60 kDa M. variabilis band, which was

absent at pH 5. The influence of pH on zymogram banding patterns was in greater accordance with protein utilization than protease activity, indicating that zymograms may be more sensitive to pH

FIG. 4. Protease activity after the addition of specific inhibitors (inhibited protease type in brackets). Significant inhibition relative to controls (P , 0.05) is denoted by an asterisk. The number of isolates and replicates measured are as in FIG. 1. Error bars represent the standard error of the mean.

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TABLE III. Presence (*) of activity bands on gelatin zymograms across a pH gradient

Species

Ecology

60 20 P. fortinii Endophyte 65 26 U. isabellina Endophyte 70 I. lacteus Saprotroph 42 H. incarnatulum Mycorrhizal 125 84 L. bicolor Mycorrhizal 86 72 M. variabilis

FIG. 5. Gelatin zymogram activity patterns for representative fungal isolates. A protein ladder with band sizes ranging from 10 to 250 kDa is present on each image. The zone of activity (above 200 kDa) in L. bicolor is likely a protein aggregate.

dependent proteolytic activity than the fluorescent casein assay. DISCUSSION We have characterized the proteolytic abilities of a selection of fungal root endophytes as well as fungi that are clearly saprotrophic (wood decay) and mutualistic (ECM). All fungi tested could hydrolize BSA, soy protein isolate, casein and gelatin but differed in protein utilization efficiency, protease activity, type of protease and pH optima and range. Conversely, intraspecific variation was relatively low, even among the six isolates of Phialocephala fortinii, part of the Phialocephala fortinii s.l.-Acephala applanata complex thought to represent many cryptic species (Gru¨nig et al. 2008b). The saprotrophic wood-decay fungus, I. lacteus, was the most proteolytically active, secreting a 42 kDa aspartic protease, likely the Irpex lacteus aspartic protease (ILAP) (Fujimoto et al. 2004). The efficient utilization of protein by I. lacteus might be related to the fact that protein is the main source of nitrogen in wood (Abraham 1995, Dunaevsky et al. 2006, Kudryavtseva et al. 2008). Wood-decay fungi are also thought to produce extracellular proteases that

Endophyte

Band (kDa)

pH 2

3

4

5

6

* * * * * *

* * * * * * * *

* * * * * * * * * *

* * * * * * * * *

* * * * * *

autolyze older hyphae, an adaptation to life in a nitrogen deficient environment (Levi and Cowling 1969, Watkinson et al. 2001). Hyphal autolysis might partially explain why I. lacteus exhibited a high protein utilization rate but relatively low hyphal weight on both BSA and soy media. Umbelopsis isabellina also displayed high proteolytic activity (although significantly less than I. lacteus) and grew well on both BSA and soy protein media. We detected one 70 kDa aspartic protease from our isolates of U. isabellina, but Hedlund et al. (1991) reported serine and metallo-proteases from the same species. This discrepancy may be due to the fact that the isolates in the previous study were grown on malt media, whereas we used BSA and soy to induce fungal protease production. It is possible that a different choice of substrates might have resulted in the production of protease types other than the aspartic proteases detected. While Umbelopsis spp. commonly occur as endophytes of healthy roots (Hoff et al. 2004, Kernaghan and Patriquin 2011), they are also common in soils (Domsch et al. 1980, Summerbell 2005, Baldrian et al. 2011) and decaying and dead roots (Halmschlager and Kowalski 2004, Giordano et al. 2009), where they might be involved in root decomposition (Fisk et al. 2011). Given their saprotrophic use of a range of substrates, together with their proteolytic activity, the occurrence of Umbelopsis spp. within healthy roots might be of an opportunistic nature. Conversely, the common root endophyte P. fortinii appears to be more obligate with respect to its endophytic living strategy. Unlike Umbelopsis spp., P. fortinii is not commonly isolated from soil and seems restricted mainly to root tissue (Gru¨nig et al. 2008a), even though it has been found in decaying roots and stumps (Menkis et al. 2004, 2006) and is capable of using a wide variety of carbon and nitrogen compounds (Caldwell et al. 2000).

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MYCOLOGIA

Phialocephala fortinii is part of the Phialocephala fortinii s.l.-Acephala applanata complex (Gru ¨ nig et al. 2008a), a group of dark septate endophytes (DSE) common in coniferous forests (Jumpponen and Trappe 1998a, Gru¨nig et al. 2008b, Tellenbach et al. 2011). The ubiquitous nature of these fungi within plant roots has led to theories proposing mutualistic relationships with the host plant (Jumpponen 2001). These are supported by reports of increased plant growth after inoculation with DSE (Jumpponen and Trappe 1998b, Ruotsalainen and Kytoviita 2004), although other studies show more variable results (Mayerhofer et al. 2013). One proposed mutualism involves the DSE mineralizing organic nitrogen in the rhizosphere into a form more available to the host (Upson et al. 2009). Although P. fortinii displayed intermediate levels of proteolytic activity and growth on protein, this still would seem to be a plausible scenario. Meliniomyces variabilis, part of the Rhizoscyphus ericae aggregate (Hambleton and Sigler 2005), exists as a root endophyte of trees (Kernaghan and Patriquin 2011) and also forms ericoid mycorrhizae with ericaceous plants (Grelet et al. 2010, Vohnı´k et al. 2013). Although it has been isolated from the fruiting body of the hypogeous fungus Hydnotrya (Vohnı´k et al. 2007), it appears mainly restricted to plant roots (Hambleton and Sigler 2005). We found the protein utilization patterns in M. variablis to be similar to those of the closely related ericoid mycorrhizal fungus Rhizoscyphus ericae, in that they both have very low pH optima for proteolytic activity (pH 2–4), which would appear to be an adaption to the acidic soils in which they associate with ericaceous plants (Leake and Read 1991). The overall protein utilization rates of the two ECM species were lower than any of the other fungi tested, although this seems partly due to delayed protease production in the ECM fungi relative to the other fungi tested (data not shown). Also, the proteolytic activity of the ECM fungi on fluorescent casein (tested at 14 d) was relatively high when expressed as fluorescence per milligram of fungal biomass. Although ECM fungi generally possess lower proteolytic potentials than soil saprophytes (Lundeberg 1970, Colpaert and van Laere 1996, Baldrian 2009, Nagendran et al. 2009), the genomes of both Laccaria bicolor and Tuber melanosporum contain many genes with the potential to produce a variety of extracellular proteases (Martin et al. 2008, 2010). The ECM fungi tested secreted metallo-proteases and exhibited pH optima for both protein utilization and activity pH 4–6, while all other fungi tested produced aspartic proteases with pH optima at 3 (or lower for M. variabilis). A pH optimum at 4–6 appears typical for ECM fungi (Abuzinadah and Read

1986, Colpaert and van Laere 1996, Chalot and Brun 1998), although Nehls et al. (2001) reported aspartic proteases from Amanita muscaria with pH optima as low as 3.5. However, protease type has not often been studied in the ECM fungi, and to our knowledge, the production of extracellular metallo-proteases has not previously been confirmed in this group. The goal of this study was to gain insight into the ecophysiological characteristics of fungal root endophytes through characterization of their proteolytic profiles in comparison with fungi with well defined ecological roles. Extrapolating from our data on proteolytic ability, it would seem that the endophyte U. isabellina (Mucoromycotina) is relatively saprotrophic in nature while the ascomycetous endophytes P. fortinii s.l. and M. variablilis are somewhat more limited saprotrophically. Although not all are strongly saprotrophic, the fungal root endophytes may still play an important role in the decay of fine roots, in that they are already within the root tissue upon senescence (Kernaghan 2013). Furthermore, some of the nitrogen liberated by fungal root endophytes might become available to the host plant indirectly via ECM fungi or perhaps directly through plant amino acid uptake (Na¨sholm et al. 2009). Finally intermediate proteolytic activity in the two common fungal root endophytes, P. fortinii and M. variablilis seems appropriate for inhabitants of healthy roots, as we would not expect fungi with this strategy to be overly aggressive. ACKNOWLEDGMENTS Financial support was made possible by the Natural Sciences and Engineering Research Council of Canada (NSERC). We thank Russell Easy for his advice on gelatin zymography and Zhongmin Dong for his helpful input throughout the project.

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