Fungal Ecology 30 (2017) 10e18

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Fungal Ecology

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Seed-associated fungi in the alpine tundra: Both mutualists and pathogens could impact recruitment

Terri Billingsley Tobias a, Emily C. Farrer b, Antonio a, Robert L. Sinsabaugh c, * Katharine N. Suding d, Andrea Porras-Alfaro a, a Department of Biological Sciences, Western Illinois University, Macomb, IL 61455, USA b Department of Ecology and Evolutionary Biology, Tulane University, New Orleans, LA 70118, USA c Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA d Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309, USA article info abstract

Article history: Seed-borne microbes are important pathogens and mutualists in agricultural crops but are understudied Received 26 October 2016 in natural systems. To understand the diversity and function of seed-borne fungi in alpine tundra, we Received in revised form cultured fungi from seeds of six dominant plant species prior to seed dispersal and evaluated their 2 July 2017 function using germination experiments in Zea mays. A total of 55 fungal cultures (9 species) were Accepted 4 August 2017 isolated with up to 4 genera per plant species. Dominant orders included Pleosporales and Hypocreales. Sixty-six percent of the isolates showed pathogenic effects. The most common genus was Alternaria Corresponding Editor: Nicole Hynson which had a negative effect on both seed germination and plant growth. Cladosporium was only isolated from the two dominant plant species and showed positive effects on germination and plant growth. The Keywords: high number of pathogenic fungi found coupled with the variation in seed endophytic communities Seed-borne fungi among plant species suggests that seed-associated fungi could affect community composition through Alpine tundra differential seedling recruitment. Mutualists © 2017 Elsevier Ltd and British Mycological Society. All rights reserved. Pathogens Cladosporium Alternaria

1. Introduction communities (Gallery et al., 2007; U'Ren et al., 2009). Seed-borne fungi colonize internal tissues (endophytes) and can Fungal communities are important drivers of plant diversity and show mutualistic or pathogenic activity (Baker and Smith, 1966; community structure (Reynolds et al., 2003). Most studies on plant- Stone et al., 2000; Rodriguez et al., 2009). They are highly ubiqui- fungal interactions in natural systems in the last four decades have tous and abundant: many cultivated crops and natural have focused on root and leaf endophytes, rhizosphere and bulk soil some form of seed-borne fungi (e.g., Clay, 1990; Malone and communities, and soil-borne pathogens (Van Der Putten and Muskett, 1997; Dhingra et al., 2002; Ganley and Newcombe, Peters, 1997; Higgins et al., 2007; Hoffman and Arnold, 2008; 2006). Pathogenic seed-borne fungi have been extensively stud- Porras-Alfaro and Bayman, 2011); however, a potentially diverse ied causing infectious diseases. Common species include Tilletia and important group of microorganisms inhabit seeds. Seed-borne caries, Colletotrichum lindemuthianum, Botrytis cinerea, and several microbes are important pathogens and mutualists in agricultural Fusarium species (Tillet, 1937; Noble et al., 1958; Malone and crops (Newton et al., 2010), but they are understudied in natural Muskett, 1997; Anderson et al., 2004). Epichlo€e typhina in Lolium systems (Clay, 1990; Ravel et al., 1997; Dalling et al., 2011). Most spp. improves embryo development (McLennan, 1920), but in adult studies in natural systems have focused on fungi acquired in post- plants, it also causes fescue toxicity as a way to control herbivory dispersal events when the seeds come in contact with fungal soil (Leuchtmann, 1992). There are also some saprotrophic fungal spe- cies that produce toxic secondary metabolites that help control plant pathogens. For example, Chaetomium spp. have been reported as seed endophytes that can provide protection against Fusarium * Corresponding author. WIU Biology, Waggoner Hall 372, 1 University Circle, blight in oat seedlings (Tveit and Wood, 1955; Soytong et al., 2001). Macomb, IL 61455, USA. This study focuses on seed-borne fungi associated with E-mail address: [email protected] (A. Porras-Alfaro). http://dx.doi.org/10.1016/j.funeco.2017.08.001 1754-5048/© 2017 Elsevier Ltd and British Mycological Society. All rights reserved. T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18 11 dominant plants in the alpine tundra. The alpine tundra is char- palea were removed prior to surface sterilization. Sixty seeds of acterized by harsh environmental conditions, including low tem- each species in small batches were surface sterilized in micro- peratures, frost events, short growing season and high winds and centrifuge tubes with 1 mL of 95% ethanol. Tubes were vortexed solar radiation which lead to desiccation (Greenland, 1989). Diverse briefly to disperse seeds evenly and left to stand for 5 min. Ethanol communities of fungi have been reported to be associated with was removed and 1 mL of 0.5% Clorox bleach (5.25% NaOCl; Clorox plants in cold habitats (Dean et al., 2014; Tedersoo et al., 2014; Company, Oakland, CA) was added for 5 min. This was followed by Timling et al., 2014). In the alpine tundra, vertical transmission of three rinses in 1 mL of distilled autoclaved water. Seeds were fungi (via seeds) may be an important mechanism by which plants allowed to stand in sterile water for three additional minutes. To can pass on beneficial fungi to their offspring. Seed-borne fungi determine if the surface sterilization procedure was effective, seeds may also play an important role in the alpine environment by were tapped briefly on sterile malt extract agar (MEA) (Difco Lab- reducing dormancy or improving germination. Contrasting evi- oratories, Detroit, MI) and incubated at room temperature. No fungi dence is available about the role of microbial communities in grew on these MEA plates. breaking dormancy (Baskin and Baskin, 2000) but most of the Five sterilized seeds were plated on MEA with streptomycin and studies have focused on external microbial fungi (e.g. Sanchez- tetracycline (50 mg mL 1) to limit bacteria contamination. A total of Delgado et al., 2011) or seed-associated fungi acquired after 60 seeds per plant species were plated (5 seeds/12 plates). The seed dispersal (Baskin and Baskin, 2000; Zalamea et al., 2014). It is well lemma and palea were removed prior to sterilization but entire known that alpine seeds can remain viable after many years in seeds were incubated for the isolation of putative endophytes. freezing conditions and have the ability to germinate and grow Plates were incubated at 25 C and evaluated daily for hyphal rapidly when the conditions become favorable (Billings and growth for 68 d. Fungal colonies were transferred to new MEA Mooney, 1968); seed-borne fungi may contribute to these plates to obtain pure cultures. processes. The main goal of this study was to describe the culturable fungal 2.2. Seed viability communities in alpine seeds and their potential contribution to plant growth. Fungi were isolated from surface sterilized seeds of Alpine tundra seed viability was measured using tetrazolium six plant species and identified by sequencing the internal tran- (MP Biomedicals LLC. Solon, OH) following a protocol by Elias et al. scribed spacer (ITS) rRNA region. Potential roles of the fungal iso- (2006). Fifty seeds of each plant species (T. spicatum, A. scopulorum, lates were tested in germination and plant growth experiments E. simplex, G. rossii, P. bistortoides, D. cespitosa) were placed in using a model crop (maize; Zea mays) as well as one of the alpine microcentrifuge tubes with 1 mL of dH2O and allowed to soak tundra plants, Deschampsia cespitosa. All the isolates closely related overnight to hydrate. T. spicatum, P. bistortoides, D. cespitosa seeds to the genus Cladosporium showed positive results for seed were punctured with a needle and dissected longitudinally to allow germination as well as plant growth, so further phylogenetic exposure to the tetrazolium solution. Filter paper was soaked with analysis was performed on the genus to determine the placement a 1% (m/v) tetrazolium solution, seeds were placed on paper and of isolates with respect to known species. incubated at 35 C for 3 h. After incubation, seeds were examined under a dissecting microscope to evaluate viability. Seeds stained 2. Materials and methods red were counted as viable and the colorless seeds were considered dead. 2.1. Seed collection and isolation of fungi 2.3. DNA extraction, PCR, and sequencing Seeds of Trisetum spicatum, Artemisia scopulorum, Erigeron sim- plex, Polygonum bistortoides, rossii and D. cespitosa were Fungal DNA was extracted from pure cultures grown on MEA collected in 2011 and 2012. Both Deschampsia and Trisetum belong using the Wizard Genomic DNA Purification Kit (Promega, Madi- to the family Poaceae. Erigeron and Artemisia are members of the son, WI) following the manufacturer's protocols. DNA was ampli- Asteraceae family while Polygonum belongs to Polygonaceae and fied using ITS rRNA fungal specific primers, ITS1F (50- Geum belongs to . Each of these seed families are reported CTTGGTCATTTAGAGGAAGTAA-30) and ITS4 (50- TCCTCCGCTTATT- to share common physiological dormancy (Baskin and Baskin, GATATGC-30)(Gardes and Bruns, 1993; White et al., 1990). PCR was 2004). Preliminary germination trials suggest that all species, conducted in 25 mL reactions containing the following: 6.5 mLof with the exception of P. bistortoides, exhibit germination rates be- nuclease free water, 1 mL of ITS1F (5 mM), 1 mL ITS4 (5 mM), 3 mLof1% tween 15 and 52% without the use of any dormancy-breaking BSA (Sigma-Aldrich, St. Louis, MO), 12.5 mL of PCR Master Mix treatment (after a few months of dry, room-temperature storage) (Promega, Madison, WI), and 1 mL of the DNA sample. Reactions (Farrer, unpublished data). P. bistortoides requires 3e4 weeks of were amplified under the following conditions: 95 C for 5 min, cold, moist stratification for germination (germination rate was 0% followed by 30 cycles at 94 C for 30 s, then annealing at 50 C for if not cold, moist stratified) (Farrer, unpublished data). All the seeds 30 s with an extension at 72 C for 45 s, and a final extension of with the exception of A. scopulorum were kept refrigerated before 72 C for 7 min. Gel electrophoresis with 1% agarose in Tris Acetate the experiment; however our seeds were not stored moist in the EDTA was used to verify the PCR reaction, PCR samples (3 mLDNA refrigerator, which is likely why we observed 0% germination for P. and 2 mL dye) were compared to a low DNA mass ladder (Invitrogen bistortoides. Corporation, Carlsbad CA) to determine fragment size and con- The seeds were collected from plants in a moist meadow mainly centration for sequencing reactions. composed of grasses and forbes in the alpine tundra at the Niwot Prior to sequencing, PCR products were cleaned using ExoSAP-IT Ridge Long Term Ecological Research (LTER) site located 35 km west (Affymetrix, Cleveland, OH) following the manufacture's protocol. of Boulder, Colorado. The site is located between 3297 and 3544 m Sequences were prepared using BigDye Terminator v1.1 Cycle above sea level, with a mean temperature for the winter of 8 C Sequencing Kit (Applied Biosystems, Foster City, CA) with ITS rRNA and the mean temperature for the summer of 13 C. In addition, the fungal specific primers for forward (ITS1F) and reverse sequences site remains under snow cover 9e10 months each year (Dean et al., (ITS4). Each sequencing reaction contained the following: 5.5 mLof 2014). nuclease free water, 1.5 mL of BigDye sequencing buffer (Applied Seeds were removed from infructescences and the lemma and Biosystems, Foster City, CA.), 1 mL of ITS1F (3 mM), 1 mL of ITS4 12 T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18

(3 mM), 1 mL of BigDye Terminator (Applied Biosystems, Foster City, root, and length of stem (measured from the tip of the plant to the CA), and 1 mL of PCR product. Sequencing reactions were completed seed) were recorded. A t-test was performed with StatPlus to under the following conditions: 96 C for 10 s, 50 C for 5 s, 60 C for determine statistical differences between treatments to compare 4 min during 30 cycles. the number of roots, and root and stem length. An ANOVA with post Sequencing reactions were precipitated using 1 mL of 125 mM hoc testing was used to compare the percentage of germination for EDTA, 1 mL 3 M sodium acetate and 25 mL of 100% ethanol following the different fungal treatments with respect to the controls. Johnson et al. (2013). Samples were sequenced in both directions at the Molecular Biology Facility at the University of New Mexico, 2.5. Root necrosis index and plant vigor index Albuquerque, NM. Forward and reverse sequences were grouped in contigs and Root necrosis was measured using a dissecting microscope. The edited in Sequencher 4.9 (Gene Codes Corporation, Ann Arbor, MI). percentage of root necrosis was calculated following Hauser Contigs were assembled into operational taxonomic units (OTUs) at (2007); surface of necrotic cortical tissue/total surface of cortical 97% similarity. Sequences were deposited in GenBank under tissue x 100%. In addition, a modified plant vigor index (PVI) was accession numbers KX839269-KX839323. Preliminary identifica- also calculated as (mean longest root length þ mean shoot length) x tion of the sequences at the genus level was done using RDP naïve (% germination) (Raj et al., 2003). Bayesian rRNA classifier version 2.1 (Wang et al., 2007) with a bootstrap cutoff of 80% and compared with Basic Local Alignment 2.6. Root colonization Search Tools (BLAST) in NCBI (http://www.ncbi.nlm.nih.gov) (Altschul et al., 1990). Fungi with a bootstrap support higher of 80% Root colonization was examined in plants exposed to isolates in RDP and with a score higher than 700 and 95% identity similarity that showed positive growth effects. Z. mays roots were stained in NCBI were reported as the closest relative for each taxon. Prin- following a modified protocol from Vielerheilig et al. (1998). Roots ciple component analysis was done using XLSTAT. Dissimilarity were cut into 3 cm segments and placed in microcentrifuge tubes. A between plant seeds and seed-borne fungal isolates was calculated 10% KOH solution (1 mL) was added and tubes were placed in a and graphed on a distance biplot. water bath for 15 min at 60 C. The KOH was then removed and For phylogenetic analysis the ITS rRNA sequences were aligned 1 mL 5% Parker Ink solution (2.5 mL blue ink, 5% acetic acid, 50 mL using MUSCLE (multiple comparison by log expectations) (Edgar, dH2O) solution was added to the roots. Roots were heated again in a 2004). They were trimmed and edited by hand using MEGA 6 water bath for 15 min at 60 C. Roots were washed 3 with water (Tamura et al., 2013). Maximum Likelihood analysis was used to and stored in 20% glycerol until use. A total of 100 intersections infer the evolutionary history. Taxa from Crous et al. (2009), Zalar were evaluated from ten plants per treatment using the gridline et al. (2007) and Genbank were used for comparison. intersection method (McGonigle et al., 1990). Root sections were ranked for colonization per field of view: 0 ¼ no DSE hyphae pre- 2.4. Germination experiments sent, 1 ¼ 1 or 2 hyphae present, and 2 ¼ more than 2 hyphae pre- sent (Mandyam et al., 2013). To determine potential functional roles of the fungal isolates, germination experiments were conducted on two isolates for each 3. Results representative OTU, defined at 97% similarity. Germination and plant growth was evaluated in commercial Z. mays plants (com- 3.1. Isolation and identification of fungi from seeds mercial non-GMO hybrid Z. mays seeds, Arrow Seed). Z. mays was used as a model because of its rapid growth and high germination A total of 55 putative fungal endophytes were isolated and rates. In addition, this seed was chosen because it is endophyte-free sequenced using the ITS rRNA region from the 360 seeds plated (60 (treated with captan and trifloxystrobin, a metalaxyl fungicide, and seeds per plant species) (Table S1). All fungal isolates belong to the Poncho™ 600, an insecticide containing clothianidin) allowing an phylum Ascomycota representing five orders, 8 unique genera, and initial testing without the influence of seed-borne fungi present in 9 OTUs defined at 97% similarity (Table 1 and Table S1). The most the host plants. The lack of seed-borne fungi in Z. mays was abundant order was Pleosporales (26% of the cultures), followed by confirmed by plating surface sterilized seeds on MEA agar for 1 Hypocreales (23%), Capnodiales (18%), Eurotiales (17%), and Helot- week. Since Z. mays was not the primary host for fungi that showed iales (7%). positive plant growth results we conducted additional germination Surface sterilized alpine seeds contained few putative endo- experiments on one alpine tundra plant (Deschampsia). With the phytes per plant species. We recovered 2e4 fungal species per exception of Deschampsia the majority of the alpine tundra seeds in plant species using culture-based methods and one type of media, this study had very low germination rates (0e48%) under the and plant species differed in the putative endophyte communities conditions tested in our experiments. their seeds contained (Fig. 1). No fungal growth was observed on Fungal isolates were plated and grown for 14 d on MEA with the negative control plates. antibiotics until the fungus covered the plate. Ten plates were made Across plant species, 5e27% of seeds were colonized by endo- for each fungal isolate with the same number of control plates (no phytic fungi. T. spicatum and P. bistortoides had the highest coloni- fungus). Five surface sterilized seeds were plated in each plate (50 zation rates with 27% and 22%, respectively. Little to no colonization seeds per fungus). Prior to surface sterilization, Z. mays seeds were was observed in A. scopulorum (5%) and E. simplex (8%) (Table 1). washed three times in sterile water for five minutes to remove Twenty-two percent of the seeds plated contained fungal iso- traces of fungicide and insecticide. The Deschampsia seeds were not lates closely related to three common plant pathogenic genera: commercially treated with fungicide and likely contained seed- Alternaria, Fusarium, and Aspergillus (Table 1). The most abundant borne fungal endophytes. genus was closely related to Alternaria accounting for 25% of the Plates were incubated in the dark at 25 C for 7 d. Seeds were pure isolates (Table S1). Alternaria was found in four of the six plant checked daily and germination rates were recorded. After 7 d, species but the majority of isolates (64%) were found in association plates were placed under grow lights with a photoperiod of 12 h of with P. bistortoides (Table 1, Fig. 1). Other common taxa included light and 12 h of darkness. Plants were left to grow up to 15 d Aspergillus isolated from A. scopulorum, T. spicatum, and E. simplex in vitro. The number of roots, number of leaves, length of longest and Fusarium isolated from D. cespitosa and P. bistortoides (Fig. 1). T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18 13

Fig. 1. Principal component analysis (PCA) of plant seeds and seed associated fungi. Green triangles and black text represent alpine plant species and red dots and grey text represent seed-associated fungi cultured from those plant species. Blue lines with blue squares represent fungal species positive for plant growth and germination. All other fungi cultured are represented by red lines.

Table 1 Evaluation of seed viability, germination, and fungal colonization in alpine seeds.

Plant species Fungal genera recovered from plant seeds Percentage of viable seeds after 2 years Percentage of seed germination Percentage of seeds with fungi

Polygonum Alternaria, Fusarium 30% 0% 22% bistortoides Geum rossii Alternaria, Botrytis, Cladosporium, Lewia 60% 48% 13% Deschampsia cespitosa Alternaria, Cladosporium, Fusarium 28% 38% 13% Trisetum spicatum Epichlo€e, Alternaria, Aspergillus, Botrytis 44% 18% 27% Artemisia scopulorum Aspergillus 24% 5% 8% Erigeron simplex Aspergillus, Engyodontium 36% 32% 5%

Cladosporium was also abundant, accounting for 19% of the fungi A. scopulorum also showed significant negative effects on early isolated from alpine seeds. This genus was only cultured from germination in Z. mays (Fig. 2). G. rossii (37% of the isolates) and D. cespitosa (77% of the isolates) The fungal taxa that negatively affected germination also (Table 1, Fig. 1). showed consistent negative effects on root and stem length and root number in Z. mays (Fig. 3). Alternaria showed the greatest decrease in root and stem length with respect to the controls 3.2. Effect of fungal isolates on germination and plant growth (Fig. 3E). Fusarium (Fig. 3A, B), Aspergillus (Fig. 3C, D) and Botrytis (Fig. 3F) also significantly decreased root and stem length A total of 11 representative fungi were selected for germination compared to the controls (p < 0.05). Fusarium, Aspergillus, and experiments based on identification using the ITS rRNA. Six of the Botrytis (Fig. 3G) significantly reduced the number of roots 11 fungi tested on Z. mays showed some level of pathogenicity (Fig. 3AeD, G) (p < 0.05). Fusarium isolates (Bb20 and Bb12) ranging from low germination rates to high levels of root necrosis showed the greatest level of root necrosis (60e70% root necrosis), compared to the controls (Figs. 2 and 3). Isolates in the genus followed by Aspergillus (As1, As5, 47e52%) and Botrytis (Gr5, 52%). Fusarium (Bb12 and Bb20) and Alternaria (Bb17) showed the plant vigor index was lower for isolates of Fusarium (45 PVI), greatest negative effects in early germination stages with only Aspergillus (49 PVI), Botrytis (98 PVI), and Alternaria (14 PVI) as 32e58% of the Z. mays seeds germinated after seven days as compared to the control (229 PVI) suggesting a general reduction of compared to the controls (98% germination) (Fig. 2). Botrytis (Gr5) plant health. isolated from G. rossii along with Aspergillus (As1, As5) isolated from 14 T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18

Fig. 2. Effect of fungal isolates on seed germination of Z. mays after 3 d and 7 d of exposure. Vertical bars represent means with standard errors for 50 seeds (ten replicates x five seeds per replicate). Bars with asterisks are significantly different (p < 0.05) from controls.

In addition to negative effects on Z. mays, one isolate had neutral were obtained. After 15 d, a significant percentage of D. cespitosa effects (no major differences with the controls) and four isolates seeds exposed to Cladosporium isolates germinated with rates of positively affected Z. mays performance. Engyodontium had mostly 28e36% with respect to 0% for the controls. The highest germina- neutral impact on Z. mays growth; it increased the number of roots tion rate was observed for isolate Dc7 with 36% as compared to 0% but negatively affected root length, and had no impact on any of the for the control seeds. The three isolates did not differ in their effect other metrics. The four isolates that improved Z. mays growth all on the number of roots and plant length (p > 0.5). belong to the genus Cladosporium. Three isolates (Dc6, Dc7 and Gr4) Phylogenetic analysis for the Cladosporium isolates showed two showed significantly faster germination rates after three days major clades. The Cladosporium oxysporum/tenuissimum/clado- ranging from 60% to 88% (Gr4) compared to 26% for the controls sporioides clade contained the majority of the isolates (7 isolates in (Fig. 2). After 7 d both the controls and experimental seeds had total) (Fig. S1) and it is in agreement with previously published germination rates higher than 92%. Significant positive effects on phylogenies that show little to no resolution at the species level root and stem length were observed for all Cladosporium isolates (Crous et al., 2009). Fungi in this clade were closely related to a with Dc9 and Dc7 showing the highest increase in root length fungal endophyte in Pinus ponderosa and Quercus litter from (Fig. 3HeK) (p < 0.005). For example, the average length of the Mexico. The second clade contained isolates Dc8 and Dc9 and longest roots for Z. mays exposed to isolate Dc9 after 7 d was nearly groups with other fungal endophytes from coastal dune grasses and three times that of the control (13.8 ± 0.78 vs. 4.7 ± 0.32 cm) Pinus taeda and are closely related to Cladosporium velox. Of the (p < 0.005) and the length of the stem after 7 d was almost two isolates tested in vitro, Dc6, Dc7, Gr4 were all closely related to times higher than the control (5.2 ± 0.40 cm vs. 2.7 ± 0.21 cm) C. oxysporum and all show similar PVI ranging from 1151 to 888. Dc9 (p < 0.005) (Fig. 3K). This could be a result of the faster germination was closely related to C. velox showed a lower PVI of 532, lower rates observed for those plants exposed to Cladosporium with germination rates (Fig. 2), and lower growth rates for D. cespitosa, respect to the other fungi and the controls. but a significantly greater effect on root growth for Z. mays (Fig. 3K). An increase in the number of roots was also observed for all plants exposed to Cladosporium. Isolates Dc6, Dc7, Dc9, and Gr4 4. Discussion produced an average of 5.3 ± 0.29 roots per plant after seven days with respect to the controls with 4.5 ± 0.28 (p < 0.005) (Fig. 3HeK). This study shows alpine tundra seeds collected from the plants None of the Cladosporium isolates showed signs of necrosis and are colonized by fungi that have been mainly described as patho- fungal colonization was observed in the roots of the three Clado- gens or saprotrophs (Alternaria, Fusarium, Aspergillus, Botrytis). sporium isolates tested (Gr4 culture lost viability and could not be These fungi showed some level of pathogenicity on germination tested). Isolate Dc7 had the highest rate of colonization (96% of the experiments conducted on Z. mays as a model plant. We also report roots) compared to the controls (0%) followed by isolates Dc9 (62%) Cladosporium as one of the seed-associated fungi that showed and Dc6 (60%). Cladosporium isolates Dc6 (1118 PVI), Dc9 (532 PVI), growth-promoting activity in Z. mays and one of the hosts, Gr4 (1151 PVI), and Dc7 (888 PVI) all had significantly higher plant D. cespitosa. This is one of the first studies describing diversity and vigor index as compared to the controls (229 PVI). function of alpine tundra seed-borne fungi in pre-dispersal stages.

3.3. Cladosporium effect on germination and phylogenetic analysis 4.1. Plant pathogenic fungi as seed-borne fungi

Three of the Cladosporium isolates were tested in vitro with Most of the isolated taxa in this study were associated with D. cespitosa, one of the original hosts from which these isolates common plant pathogenic fungi. Alternaria and Fusarium, common T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18 15

Fig. 3. Effect of fungal isolates on Z. mays growth. Bars represent means with standard error for ten replicates, five seeds per replicate (50 seeds). Asterisks indicate significant differences between fungi and control (t-test, p < 0.05). isolates in this study, are also among the most common seed-borne significant number of the fungi cultured (63%) were also common fungi isolated from cereal crops (Bain, 1950; Clear et al., 2000; plant pathogens associated with seeds. In bioassays, these isolates Abdulsalaam and Shenge, 2011). Fungi in the Alternaria complex responded in a similar way, causing root necrosis and poor include saprotrophic to parasitic species and can be easily detected germination rates. in seeds, as colonies will begin to grow on MEA after 5 d (Malone Plant pathogens can range from highly specific to generalist. In and Muskett, 1997). Pathogenic isolates such as Fusarium and this study, we used an alternative host (Z. mays) to evaluate path- Alternaria were both cultured from plants with low to 0% germi- ogenicity due to the low germination rates of the native seeds and nation rates (Table 1). The percentage of viability within the seeds the presence of endophytic fungi in all the plants evaluated. containing these fungal isolates ranged from 28 to 44% suggesting Additional testing in the greenhouse and field conditions is seed dormancy as one of the potential causes of low germination necessary to determine the impact of these putative pathogens in rates (Table 1). native ecosystems. The pathogenicity of fungi from alpine tundra seeds ranged from poor germination rates, to slow or stunted growth, to high root necrosis in Z. mays. The highest percentage of root necrosis was 4.2. Diversity and growth promoting properties of seed-borne fungi observed in Fusarium isolates (Bb20, 71% and Bb12, 58%) followed by Botrytis (Gr5, 52%) and Aspergillus (As5, 52%). Fusarium species It is possible that other non-culturable fungi were associated are ubiquitous and have been found in most ecosystems including with the alpine seeds since this study was cultured-based and we alpine grasslands (Burgess and Bryden, 2012). used a single medium. However, our study is in agreement with Klironomos (2002) reported Fusarium, Verticillium, and Cylin- previous studies showing that surface sterilized seeds before drocarpon as the most commonly isolated fungi in Canadian dispersal contained either none or only one endophyte per seed meadows and grasslands. When tested, these isolates all caused (e.g. Dhingra et al., 2002; Ganley and Newcombe, 2006). Two to systemic infections in the roots of native plants. In our study, a four fungal species were isolated per plant species with most being pathogenic and a few showing growth-promoting properties. 16 T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18

Dhingra et al. (2002) showed similar results in which one fungus 4.4. Potential role in seed recruitment and survival in alpine per seed was isolated in a high percentage of the seeds and many of ecosystems the isolates showed pathogenicity in Anadenanthera macrocarpa in Brazil. Reproduction by seeds is thought to be a rare occurrence among U'Ren et al. (2009) and Gallery et al. (2007) showed that seed- alpine plants (Bell and Bliss, 1980; Weppler et al., 2006). Many of associated fungal diversity was low before dispersal in the low- the plant species in this study are clonal (e.g. Trisetum, Erigeron, land forests of Panama and Costa Rica but high colonization rates Geum and Deschampsia) and the tradeoffs of investment between were found after dispersal (i.e., the seeds have been in contact with sexual and vegetative reproduction (Barrett, 2015) explain in part soil communities after a period of time). The few fungi identified in the low germination rates. The alpine tundra is dominated by long- pre-dispersal seeds using molecular methods were highly similar lived perennials (Billings and Mooney, 1968). Adaptive strategies, to isolated plant endophytes in other studies. Gallery et al. (2007) such as clonality, provide alpine plant populations with stability cultured 220 fungal isolates with 73 unique genotypes from four during harsh environmental conditions that are not favorable for Cecropia species and only 5 genotypes were sequenced from pre- seed germination and seedling establishment (Bierzychudek,1985). dispersal seeds. Of the 73 unique genotypes, 64.3% were recov- However, in a 4-year demographic study at Niwot, Forbis (2003) ered only once, indicating high diversity of fungal taxa within the found both seedling recruitment and mortality rates to be similar seeds once they have been in contact with the soil. These suggest to published rates from perennial plant species from other envi- that the majority of seed-associated fungi are acquired after ronments including tropical and temperate ecosystems, suggesting dispersal. that reproduction by seeds may be more important than previously We isolated four fungi related to the genus Cladosporium that thought in the alpine. In our study, germination rates and seed significantly increased plant vigor. Z. mays and D. cespitosa seeds viability varied greatly among seeds (Table 1). This is likely due to exposed to these isolates germinated and grew significantly faster the interaction of multiple factors including seed dormancy, than control seeds (Figs. 2 and 3). Cladosporium have been previ- viability, and the high presence of putative pathogens that could ously reported as growth promoting endophytes in Glycine max and impact germination success. For example, P. bistortoides seeds had Cucumis sativus influencing both seed germination and plant 0% germination rates, contained the highest percentage of fungal growth (Hamayun et al., 2009, 2010). Hamayun et al. (2009) re- isolates all closely related to common plant pathogens such as ported a novel species Cladosporium sphaerospermum, with growth Alternaria and Fusarium. When tested in vitro these fungal isolates promoting effects. Both Hamayun et al. (2009, 2010) determined significantly decreased germination rates in Z. mays (Fig. 2) and that the production of gibberellins by Cladosporium isolates was showed high percentages of root necrosis. responsible for the increase in plant growth. In addition, recent In addition to plant pathogens, mutualistic fungi can also be reports also indicate that endohyphal bacteria can affect host plants associated with germination success and seedling survival. In this by the production of plant growth hormones (Hoffman et al., 2013). study, four Cladosporium isolates significantly increased germina- tion rates and root growth in both Z. mays and D. cespitosa (Figs. 2 and 3). Both D. cespitosa and G. rossii are dominant plants, ac- 4.3. Potential mechanisms of transmission of fungi in alpine tundra counting for 60% of the cover at Niwot (Farrer et al., 2015). All the Cladosporium isolates in our study were cultured from only G. rossii Vertically transmitted fungi often form mutualistic relationships and D. cespitosa accounting for 70% of the fungi cultured from with their plant hosts (Clay, 1989, 1990; Leuchtmann, 1992). These D. cespitosa and 37.5% of the fungi cultured from G. rossii (Table S1). fungi spend their entire lifecycle within the plant tissue and are Having a beneficial relationship with growth promoting fungi could passed from parent to offspring via the host's seeds (Saikkonen be advantageous for alpine plants given the harsh environmental et al., 2010). Vertically transmitted endophytes like Epichlo€e can conditions and a very short growing season. Faster germination remain symptomless and have direct benefits on the plant host rates with production of roots could have a bigger impact on quick (Clay, 1989; Saikkonen et al., 2004). However, some Epichlo€e species establishment of seedlings and associations with Cladosporium in such as E. typhina can also become pathogenic to the host plant seeds and could contribute to the dominance of these two plant (Vazquez-de-Aldana et al., 2013). species. A single isolate of Epichlo€e sp., a vertically transmitted fungus in grass species, was recovered in this study. Epichlo€e spp. in grasses Acknowledgments are very well studied and play important ecological roles in plant communities (Rudgers et al., 2004) and it has been reported in This project was funded by the National Science Foundation Trisetum flavescens (White and Baldwin, 1992). In our study, this (APA, KS, RS NSF-DEB 919510; KS, NSF-DEB 1637686) and a Western genus was specifictoT. spicatum, accounting for 15% of the cultures Illinois University Graduate and Professional Research Grant. In isolated from Trisetum seeds. The isolate did not survive well in addition, we would like to thank three high school students culture and could not be tested in germination experiments. Abraham Matlack, Ali Kerr, and Dexter Redenius who dedicated a The fungi in this study were isolated before dispersal but the summer helping with this project (APA, NSF-DEB 1314459). transmission mechanisms of seed endophytes can be complicated to determine; while certain groups are vertically transmitted others Supplementary data may be horizontally transmitted via spores in the air or soil (Sanchez-Marquez et al., 2012). A recent study by Hodgson et al. Supplementary data related to this article can be found at http:// (2014) suggested that vertical transmission can occur in different dx.doi.org/10.1016/j.funeco.2017.08.001. plant groups in addition to grasses. Fungal endophytes can be vertically transmitted in forbs through both pollen and seeds. The References same genera found in this study, Alternaria, Cladosporium, Asper- gillus, and Fusarium were reported as vertically transmitted fungi in Abdulsalaam, S., Shenge, K.C., 2011. 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