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Seed-associated fungi in the alpine tundra: Both mutualists and pathogens could impact plant recruitment
Fungal Ecology 30 (2017) 10e18
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Fungal Ecology journal homepage: www.elsevier.com/locate/funeco
Seed-associated fungi in the alpine tundra: Both mutualists and pathogens could impact plant recruitment Terri Billingsley Tobias a, Emily C. Farrer b, Antonio Rosales 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 Department of Ecology and Evolutionary Biology, Tulane University, New Orleans, LA 70118, USA 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 b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 October 2016 Received in revised form 2 July 2017 Accepted 4 August 2017
Seed-borne microbes are important pathogens and mutualists in agricultural crops but are understudied in natural systems. To understand the diversity and function of seed-borne fungi in alpine tundra, we cultured fungi from seeds of six dominant plant species prior to seed dispersal and evaluated their function using germination experiments in Zea mays. A total of 55 fungal cultures (9 species) were 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 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 high number of pathogenic fungi found coupled with the variation in seed endophytic communities among plant species suggests that seed-associated fungi could affect community composition through differential seedling recruitment. © 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
Corresponding Editor: Nicole Hynson Keywords: Seed-borne fungi Alpine tundra Mutualists Pathogens Cladosporium Alternaria
1. Introduction Fungal communities are important drivers of plant diversity and community structure (Reynolds et al., 2003). Most studies on plantfungal interactions in natural systems in the last four decades have focused on root and leaf endophytes, rhizosphere and bulk soil communities, and soil-borne pathogens (Van Der Putten and Peters, 1997; Higgins et al., 2007; Hoffman and Arnold, 2008; Porras-Alfaro and Bayman, 2011); however, a potentially diverse and important group of microorganisms inhabit seeds. Seed-borne microbes are important pathogens and mutualists in agricultural crops (Newton et al., 2010), but they are understudied in natural systems (Clay, 1990; Ravel et al., 1997; Dalling et al., 2011). Most studies in natural systems have focused on fungi acquired in postdispersal events when the seeds come in contact with fungal soil
* Corresponding author. WIU Biology, Waggoner Hall 372, 1 University Circle, Macomb, IL 61455, USA. 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.
communities (Gallery et al., 2007; U'Ren et al., 2009). Seed-borne fungi colonize internal tissues (endophytes) and can show mutualistic or pathogenic activity (Baker and Smith, 1966; Stone et al., 2000; Rodriguez et al., 2009). They are highly ubiquitous and abundant: many cultivated crops and natural plants have some form of seed-borne fungi (e.g., Clay, 1990; Malone and Muskett, 1997; Dhingra et al., 2002; Ganley and Newcombe, 2006). Pathogenic seed-borne fungi have been extensively studied causing infectious diseases. Common species include Tilletia caries, Colletotrichum lindemuthianum, Botrytis cinerea, and several Fusarium species (Tillet, 1937; Noble et al., 1958; Malone and Muskett, 1997; Anderson et al., 2004). Epichlo€ e typhina in Lolium spp. improves embryo development (McLennan, 1920), but in adult plants, it also causes fescue toxicity as a way to control herbivory (Leuchtmann, 1992). There are also some saprotrophic fungal species 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 blight in oat seedlings (Tveit and Wood, 1955; Soytong et al., 2001). This study focuses on seed-borne fungi associated with
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dominant plants in the alpine tundra. The alpine tundra is characterized by harsh environmental conditions, including low temperatures, frost events, short growing season and high winds and solar radiation which lead to desiccation (Greenland, 1989). Diverse communities of fungi have been reported to be associated with plants in cold habitats (Dean et al., 2014; Tedersoo et al., 2014; Timling et al., 2014). In the alpine tundra, vertical transmission of fungi (via seeds) may be an important mechanism by which plants can pass on beneficial fungi to their offspring. Seed-borne fungi may also play an important role in the alpine environment by reducing dormancy or improving germination. Contrasting evidence is available about the role of microbial communities in breaking dormancy (Baskin and Baskin, 2000) but most of the studies have focused on external microbial fungi (e.g. SanchezDelgado et al., 2011) or seed-associated fungi acquired after dispersal (Baskin and Baskin, 2000; Zalamea et al., 2014). It is well known that alpine seeds can remain viable after many years in freezing conditions and have the ability to germinate and grow rapidly when the conditions become favorable (Billings and Mooney, 1968); seed-borne fungi may contribute to these processes. The main goal of this study was to describe the culturable fungal communities in alpine seeds and their potential contribution to plant growth. Fungi were isolated from surface sterilized seeds of six plant species and identified by sequencing the internal transcribed spacer (ITS) rRNA region. Potential roles of the fungal isolates were tested in germination and plant growth experiments using a model crop (maize; Zea mays) as well as one of the alpine tundra plants, Deschampsia cespitosa. All the isolates closely related to the genus Cladosporium showed positive results for seed germination as well as plant growth, so further phylogenetic analysis was performed on the genus to determine the placement of isolates with respect to known species. 2. Materials and methods
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palea were removed prior to surface sterilization. Sixty seeds of each species in small batches were surface sterilized in microcentrifuge tubes with 1 mL of 95% ethanol. Tubes were vortexed briefly to disperse seeds evenly and left to stand for 5 min. Ethanol was removed and 1 mL of 0.5% Clorox bleach (5.25% NaOCl; Clorox Company, Oakland, CA) was added for 5 min. This was followed by three rinses in 1 mL of distilled autoclaved water. Seeds were allowed to stand in sterile water for three additional minutes. To determine if the surface sterilization procedure was effective, seeds were tapped briefly on sterile malt extract agar (MEA) (Difco Laboratories, Detroit, MI) and incubated at room temperature. No fungi grew on these MEA plates. Five sterilized seeds were plated on MEA with streptomycin and tetracycline (50 mg mL1) to limit bacteria contamination. A total of 60 seeds per plant species were plated (5 seeds/12 plates). The seed lemma and palea were removed prior to sterilization but entire seeds were incubated for the isolation of putative endophytes. Plates were incubated at 25 C and evaluated daily for hyphal growth for 68 d. Fungal colonies were transferred to new MEA plates to obtain pure cultures. 2.2. Seed viability Alpine tundra seed viability was measured using tetrazolium (MP Biomedicals LLC. Solon, OH) following a protocol by Elias et al. (2006). Fifty seeds of each plant species (T. spicatum, A. scopulorum, E. simplex, G. rossii, P. bistortoides, D. cespitosa) were placed in microcentrifuge tubes with 1 mL of dH2O and allowed to soak overnight to hydrate. T. spicatum, P. bistortoides, D. cespitosa seeds were punctured with a needle and dissected longitudinally to allow exposure to the tetrazolium solution. Filter paper was soaked with a 1% (m/v) tetrazolium solution, seeds were placed on paper and incubated at 35 C for 3 h. After incubation, seeds were examined under a dissecting microscope to evaluate viability. Seeds stained 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 simplex, Polygonum bistortoides, Geum rossii and D. cespitosa were collected in 2011 and 2012. Both Deschampsia and Trisetum belong to the family Poaceae. Erigeron and Artemisia are members of the Asteraceae family while Polygonum belongs to Polygonaceae and Geum belongs to Rosaceae. Each of these seed families are reported to share common physiological dormancy (Baskin and Baskin, 2004). Preliminary germination trials suggest that all species, with the exception of P. bistortoides, exhibit germination rates between 15 and 52% without the use of any dormancy-breaking treatment (after a few months of dry, room-temperature storage) (Farrer, unpublished data). P. bistortoides requires 3e4 weeks of cold, moist stratification for germination (germination rate was 0% if not cold, moist stratified) (Farrer, unpublished data). All the seeds with the exception of A. scopulorum were kept refrigerated before the experiment; however our seeds were not stored moist in the refrigerator, which is likely why we observed 0% germination for P. bistortoides. The seeds were collected from plants in a moist meadow mainly composed of grasses and forbes in the alpine tundra at the Niwot Ridge Long Term Ecological Research (LTER) site located 35 km west of Boulder, Colorado. The site is located between 3297 and 3544 m above sea level, with a mean temperature for the winter of 8 C and the mean temperature for the summer of 13 C. In addition, the site remains under snow cover 9e10 months each year (Dean et al., 2014). Seeds were removed from infructescences and the lemma and
Fungal DNA was extracted from pure cultures grown on MEA using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) following the manufacturer's protocols. DNA was amplified using ITS rRNA fungal specific primers, ITS1F (50 CTTGGTCATTTAGAGGAAGTAA-30 ) and ITS4 (50 - TCCTCCGCTTATTGATATGC-30 ) (Gardes and Bruns, 1993; White et al., 1990). PCR was conducted in 25 mL reactions containing the following: 6.5 mL of nuclease free water, 1 mL of ITS1F (5 mM), 1 mL ITS4 (5 mM), 3 mL of 1% BSA (Sigma-Aldrich, St. Louis, MO), 12.5 mL of PCR Master Mix (Promega, Madison, WI), and 1 mL of the DNA sample. Reactions were amplified under the following conditions: 95 C for 5 min, followed by 30 cycles at 94 C for 30 s, then annealing at 50 C for 30 s with an extension at 72 C for 45 s, and a final extension of 72 C for 7 min. Gel electrophoresis with 1% agarose in Tris Acetate EDTA was used to verify the PCR reaction, PCR samples (3 mL DNA and 2 mL dye) were compared to a low DNA mass ladder (Invitrogen Corporation, Carlsbad CA) to determine fragment size and concentration for sequencing reactions. Prior to sequencing, PCR products were cleaned using ExoSAP-IT (Affymetrix, Cleveland, OH) following the manufacture's protocol. Sequences were prepared using BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) with ITS rRNA fungal specific primers for forward (ITS1F) and reverse sequences (ITS4). Each sequencing reaction contained the following: 5.5 mL of nuclease free water, 1.5 mL of BigDye sequencing buffer (Applied Biosystems, Foster City, CA.), 1 mL of ITS1F (3 mM), 1 mL of ITS4
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(3 mM), 1 mL of BigDye Terminator (Applied Biosystems, Foster City, CA), and 1 mL of PCR product. Sequencing reactions were completed under the following conditions: 96 C for 10 s, 50 C for 5 s, 60 C for 4 min during 30 cycles. Sequencing reactions were precipitated using 1 mL of 125 mM EDTA, 1 mL 3 M sodium acetate and 25 mL of 100% ethanol following Johnson et al. (2013). Samples were sequenced in both directions at the Molecular Biology Facility at the University of New Mexico, Albuquerque, NM. Forward and reverse sequences were grouped in contigs and edited in Sequencher 4.9 (Gene Codes Corporation, Ann Arbor, MI). Contigs were assembled into operational taxonomic units (OTUs) at 97% similarity. Sequences were deposited in GenBank under accession numbers KX839269-KX839323. Preliminary identification of the sequences at the genus level was done using RDP naïve Bayesian rRNA classifier version 2.1 (Wang et al., 2007) with a bootstrap cutoff of 80% and compared with Basic Local Alignment Search Tools (BLAST) in NCBI (http://www.ncbi.nlm.nih.gov) (Altschul et al., 1990). Fungi with a bootstrap support higher of 80% in RDP and with a score higher than 700 and 95% identity similarity in NCBI were reported as the closest relative for each taxon. Principle component analysis was done using XLSTAT. Dissimilarity between plant seeds and seed-borne fungal isolates was calculated and graphed on a distance biplot. For phylogenetic analysis the ITS rRNA sequences were aligned using MUSCLE (multiple comparison by log expectations) (Edgar, 2004). They were trimmed and edited by hand using MEGA 6 (Tamura et al., 2013). Maximum Likelihood analysis was used to infer the evolutionary history. Taxa from Crous et al. (2009), Zalar et al. (2007) and Genbank were used for comparison. 2.4. Germination experiments To determine potential functional roles of the fungal isolates, germination experiments were conducted on two isolates for each representative OTU, defined at 97% similarity. Germination and plant growth was evaluated in commercial Z. mays plants (commercial non-GMO hybrid Z. mays seeds, Arrow Seed). Z. mays was used as a model because of its rapid growth and high germination rates. In addition, this seed was chosen because it is endophyte-free (treated with captan and trifloxystrobin, a metalaxyl fungicide, and Poncho™ 600, an insecticide containing clothianidin) allowing an initial testing without the influence of seed-borne fungi present in the host plants. The lack of seed-borne fungi in Z. mays was confirmed by plating surface sterilized seeds on MEA agar for 1 week. Since Z. mays was not the primary host for fungi that showed positive plant growth results we conducted additional germination experiments on one alpine tundra plant (Deschampsia). With the exception of Deschampsia the majority of the alpine tundra seeds in this study had very low germination rates (0e48%) under the conditions tested in our experiments. Fungal isolates were plated and grown for 14 d on MEA with antibiotics until the fungus covered the plate. Ten plates were made for each fungal isolate with the same number of control plates (no fungus). Five surface sterilized seeds were plated in each plate (50 seeds per fungus). Prior to surface sterilization, Z. mays seeds were washed three times in sterile water for five minutes to remove traces of fungicide and insecticide. The Deschampsia seeds were not commercially treated with fungicide and likely contained seedborne fungal endophytes. Plates were incubated in the dark at 25 C for 7 d. Seeds were checked daily and germination rates were recorded. After 7 d, plates were placed under grow lights with a photoperiod of 12 h of light and 12 h of darkness. Plants were left to grow up to 15 d in vitro. The number of roots, number of leaves, length of longest
root, and length of stem (measured from the tip of the plant to the seed) were recorded. A t-test was performed with StatPlus to determine statistical differences between treatments to compare the number of roots, and root and stem length. An ANOVA with post hoc testing was used to compare the percentage of germination for the different fungal treatments with respect to the controls. 2.5. Root necrosis index and plant vigor index Root necrosis was measured using a dissecting microscope. The percentage of root necrosis was calculated following Hauser (2007); surface of necrotic cortical tissue/total surface of cortical tissue x 100%. In addition, a modified plant vigor index (PVI) was also calculated as (mean longest root length þ mean shoot length) x (% germination) (Raj et al., 2003). 2.6. Root colonization Root colonization was examined in plants exposed to isolates that showed positive growth effects. Z. mays roots were stained following a modified protocol from Vielerheilig et al. (1998). Roots were cut into 3 cm segments and placed in microcentrifuge tubes. A 10% KOH solution (1 mL) was added and tubes were placed in a water bath for 15 min at 60 C. The KOH was then removed and 1 mL 5% Parker Ink solution (2.5 mL blue ink, 5% acetic acid, 50 mL dH2O) solution was added to the roots. Roots were heated again in a water bath for 15 min at 60 C. Roots were washed 3 with water and stored in 20% glycerol until use. A total of 100 intersections were evaluated from ten plants per treatment using the gridline intersection method (McGonigle et al., 1990). Root sections were ranked for colonization per field of view: 0 ¼ no DSE hyphae present, 1 ¼ 1 or 2 hyphae present, and 2 ¼ more than 2 hyphae present (Mandyam et al., 2013). 3. Results 3.1. Isolation and identification of fungi from seeds A total of 55 putative fungal endophytes were isolated and sequenced using the ITS rRNA region from the 360 seeds plated (60 seeds per plant species) (Table S1). All fungal isolates belong to the phylum Ascomycota representing five orders, 8 unique genera, and 9 OTUs defined at 97% similarity (Table 1 and Table S1). The most abundant order was Pleosporales (26% of the cultures), followed by Hypocreales (23%), Capnodiales (18%), Eurotiales (17%), and Helotiales (7%). Surface sterilized alpine seeds contained few putative endophytes per plant species. We recovered 2e4 fungal species per plant species using culture-based methods and one type of media, and plant species differed in the putative endophyte communities their seeds contained (Fig. 1). No fungal growth was observed on the negative control plates. Across plant species, 5e27% of seeds were colonized by endophytic fungi. T. spicatum and P. bistortoides had the highest colonization rates with 27% and 22%, respectively. Little to no colonization was observed in A. scopulorum (5%) and E. simplex (8%) (Table 1). Twenty-two percent of the seeds plated contained fungal isolates closely related to three common plant pathogenic genera: Alternaria, Fusarium, and Aspergillus (Table 1). The most abundant genus was closely related to Alternaria accounting for 25% of the pure isolates (Table S1). Alternaria was found in four of the six plant species but the majority of isolates (64%) were found in association with P. bistortoides (Table 1, Fig. 1). Other common taxa included Aspergillus isolated from A. scopulorum, T. spicatum, and E. simplex and Fusarium isolated from D. cespitosa and P. bistortoides (Fig. 1).
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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 bistortoides Geum rossii Deschampsia cespitosa Trisetum spicatum Artemisia scopulorum Erigeron simplex
Alternaria, Fusarium
30%
0%
22%
Alternaria, Botrytis, Cladosporium, Lewia Alternaria, Cladosporium, Fusarium Epichlo€ e, Alternaria, Aspergillus, Botrytis Aspergillus Aspergillus, Engyodontium
60% 28% 44% 24% 36%
48% 38% 18% 5% 32%
13% 13% 27% 8% 5%
Cladosporium was also abundant, accounting for 19% of the fungi isolated from alpine seeds. This genus was only cultured from G. rossii (37% of the isolates) and D. cespitosa (77% of the isolates) (Table 1, Fig. 1).
3.2. Effect of fungal isolates on germination and plant growth A total of 11 representative fungi were selected for germination experiments based on identification using the ITS rRNA. Six of the 11 fungi tested on Z. mays showed some level of pathogenicity ranging from low germination rates to high levels of root necrosis compared to the controls (Figs. 2 and 3). Isolates in the genus Fusarium (Bb12 and Bb20) and Alternaria (Bb17) showed the greatest negative effects in early germination stages with only 32e58% of the Z. mays seeds germinated after seven days as compared to the controls (98% germination) (Fig. 2). Botrytis (Gr5) isolated from G. rossii along with Aspergillus (As1, As5) isolated from
A. scopulorum also showed significant negative effects on early germination in Z. mays (Fig. 2). The fungal taxa that negatively affected germination also 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 (Fig. 3E). Fusarium (Fig. 3A, B), Aspergillus (Fig. 3C, D) and Botrytis (Fig. 3F) also significantly decreased root and stem length compared to the controls (p < 0.05). Fusarium, Aspergillus, and Botrytis (Fig. 3G) significantly reduced the number of roots (Fig. 3AeD, G) (p < 0.05). Fusarium isolates (Bb20 and Bb12) showed the greatest level of root necrosis (60e70% root necrosis), followed by Aspergillus (As1, As5, 47e52%) and Botrytis (Gr5, 52%). plant vigor index was lower for isolates of Fusarium (45 PVI), Aspergillus (49 PVI), Botrytis (98 PVI), and Alternaria (14 PVI) as compared to the control (229 PVI) suggesting a general reduction of plant health.
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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.
were obtained. After 15 d, a significant percentage of D. cespitosa seeds exposed to Cladosporium isolates germinated with rates of 28e36% with respect to 0% for the controls. The highest germination rate was observed for isolate Dc7 with 36% as compared to 0% for the control seeds. The three isolates did not differ in their effect on the number of roots and plant length (p > 0.5). Phylogenetic analysis for the Cladosporium isolates showed two major clades. The Cladosporium oxysporum/tenuissimum/cladosporioides clade contained the majority of the isolates (7 isolates in total) (Fig. S1) and it is in agreement with previously published phylogenies that show little to no resolution at the species level (Crous et al., 2009). Fungi in this clade were closely related to a fungal endophyte in Pinus ponderosa and Quercus litter from Mexico. The second clade contained isolates Dc8 and Dc9 and groups with other fungal endophytes from coastal dune grasses and Pinus taeda and are closely related to Cladosporium velox. Of the isolates tested in vitro, Dc6, Dc7, Gr4 were all closely related to C. oxysporum and all show similar PVI ranging from 1151 to 888. Dc9 was closely related to C. velox showed a lower PVI of 532, lower germination rates (Fig. 2), and lower growth rates for D. cespitosa, but a significantly greater effect on root growth for Z. mays (Fig. 3K).
In addition to negative effects on Z. mays, one isolate had neutral effects (no major differences with the controls) and four isolates positively affected Z. mays performance. Engyodontium had mostly neutral impact on Z. mays growth; it increased the number of roots but negatively affected root length, and had no impact on any of the other metrics. The four isolates that improved Z. mays growth all belong to the genus Cladosporium. Three isolates (Dc6, Dc7 and Gr4) showed significantly faster germination rates after three days ranging from 60% to 88% (Gr4) compared to 26% for the controls (Fig. 2). After 7 d both the controls and experimental seeds had germination rates higher than 92%. Significant positive effects on root and stem length were observed for all Cladosporium isolates with Dc9 and Dc7 showing the highest increase in root length (Fig. 3HeK) (p < 0.005). For example, the average length of the longest roots for Z. mays exposed to isolate Dc9 after 7 d was nearly three times that of the control (13.8 ± 0.78 vs. 4.7 ± 0.32 cm) (p < 0.005) and the length of the stem after 7 d was almost two times higher than the control (5.2 ± 0.40 cm vs. 2.7 ± 0.21 cm) (p < 0.005) (Fig. 3K). This could be a result of the faster germination rates observed for those plants exposed to Cladosporium with respect to the other fungi and the controls. An increase in the number of roots was also observed for all plants exposed to Cladosporium. Isolates Dc6, Dc7, Dc9, and Gr4 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). None of the Cladosporium isolates showed signs of necrosis and fungal colonization was observed in the roots of the three Cladosporium isolates tested (Gr4 culture lost viability and could not be tested). Isolate Dc7 had the highest rate of colonization (96% of the roots) compared to the controls (0%) followed by isolates Dc9 (62%) and Dc6 (60%). Cladosporium isolates Dc6 (1118 PVI), Dc9 (532 PVI), Gr4 (1151 PVI), and Dc7 (888 PVI) all had significantly higher plant vigor index as compared to the controls (229 PVI).
This study shows alpine tundra seeds collected from the plants are colonized by fungi that have been mainly described as pathogens or saprotrophs (Alternaria, Fusarium, Aspergillus, Botrytis). These fungi showed some level of pathogenicity on germination experiments conducted on Z. mays as a model plant. We also report Cladosporium as one of the seed-associated fungi that showed growth-promoting activity in Z. mays and one of the hosts, D. cespitosa. This is one of the first studies describing diversity and 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 D. cespitosa, one of the original hosts from which these isolates
Most of the isolated taxa in this study were associated with common plant pathogenic fungi. Alternaria and Fusarium, common
4. Discussion
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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 fungi isolated from cereal crops (Bain, 1950; Clear et al., 2000; Abdulsalaam and Shenge, 2011). Fungi in the Alternaria complex include saprotrophic to parasitic species and can be easily detected in seeds, as colonies will begin to grow on MEA after 5 d (Malone and Muskett, 1997). Pathogenic isolates such as Fusarium and Alternaria were both cultured from plants with low to 0% germination rates (Table 1). The percentage of viability within the seeds containing these fungal isolates ranged from 28 to 44% suggesting seed dormancy as one of the potential causes of low germination rates (Table 1). 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 observed in Fusarium isolates (Bb20, 71% and Bb12, 58%) followed by Botrytis (Gr5, 52%) and Aspergillus (As5, 52%). Fusarium species are ubiquitous and have been found in most ecosystems including alpine grasslands (Burgess and Bryden, 2012). Klironomos (2002) reported Fusarium, Verticillium, and Cylindrocarpon as the most commonly isolated fungi in Canadian meadows and grasslands. When tested, these isolates all caused systemic infections in the roots of native plants. In our study, a
significant number of the fungi cultured (63%) were also common plant pathogens associated with seeds. In bioassays, these isolates responded in a similar way, causing root necrosis and poor germination rates. Plant pathogens can range from highly specific to generalist. In this study, we used an alternative host (Z. mays) to evaluate pathogenicity due to the low germination rates of the native seeds and the presence of endophytic fungi in all the plants evaluated. Additional testing in the greenhouse and field conditions is necessary to determine the impact of these putative pathogens in native ecosystems.
4.2. Diversity and growth promoting properties of seed-borne fungi It is possible that other non-culturable fungi were associated with the alpine seeds since this study was cultured-based and we used a single medium. However, our study is in agreement with previous studies showing that surface sterilized seeds before dispersal contained either none or only one endophyte per seed (e.g. Dhingra et al., 2002; Ganley and Newcombe, 2006). Two to four fungal species were isolated per plant species with most being pathogenic and a few showing growth-promoting properties.
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Dhingra et al. (2002) showed similar results in which one fungus per seed was isolated in a high percentage of the seeds and many of the isolates showed pathogenicity in Anadenanthera macrocarpa in Brazil. U'Ren et al. (2009) and Gallery et al. (2007) showed that seedassociated fungal diversity was low before dispersal in the lowland forests of Panama and Costa Rica but high colonization rates were found after dispersal (i.e., the seeds have been in contact with soil communities after a period of time). The few fungi identified in pre-dispersal seeds using molecular methods were highly similar to isolated plant endophytes in other studies. Gallery et al. (2007) cultured 220 fungal isolates with 73 unique genotypes from four Cecropia species and only 5 genotypes were sequenced from predispersal seeds. Of the 73 unique genotypes, 64.3% were recovered only once, indicating high diversity of fungal taxa within the seeds once they have been in contact with the soil. These suggest that the majority of seed-associated fungi are acquired after dispersal. We isolated four fungi related to the genus Cladosporium that significantly increased plant vigor. Z. mays and D. cespitosa seeds exposed to these isolates germinated and grew significantly faster than control seeds (Figs. 2 and 3). Cladosporium have been previously reported as growth promoting endophytes in Glycine max and Cucumis sativus influencing both seed germination and plant growth (Hamayun et al., 2009, 2010). Hamayun et al. (2009) reported a novel species Cladosporium sphaerospermum, with growth promoting effects. Both Hamayun et al. (2009, 2010) determined that the production of gibberellins by Cladosporium isolates was responsible for the increase in plant growth. In addition, recent reports also indicate that endohyphal bacteria can affect host plants by the production of plant growth hormones (Hoffman et al., 2013).
4.3. Potential mechanisms of transmission of fungi in alpine tundra Vertically transmitted fungi often form mutualistic relationships with their plant hosts (Clay, 1989, 1990; Leuchtmann, 1992). These fungi spend their entire lifecycle within the plant tissue and are passed from parent to offspring via the host's seeds (Saikkonen et al., 2010). Vertically transmitted endophytes like Epichlo€ e can remain symptomless and have direct benefits on the plant host (Clay, 1989; Saikkonen et al., 2004). However, some Epichlo€ e species such as E. typhina can also become pathogenic to the host plant (Vazquez-de-Aldana et al., 2013). A single isolate of Epichlo€ e sp., a vertically transmitted fungus in grass species, was recovered in this study. Epichlo€ e spp. in grasses are very well studied and play important ecological roles in plant communities (Rudgers et al., 2004) and it has been reported in Trisetum flavescens (White and Baldwin, 1992). In our study, this genus was specific to T. spicatum, accounting for 15% of the cultures isolated from Trisetum seeds. The isolate did not survive well in culture and could not be tested in germination experiments. The fungi in this study were isolated before dispersal but the transmission mechanisms of seed endophytes can be complicated to determine; while certain groups are vertically transmitted others may be horizontally transmitted via spores in the air or soil (Sanchez-Marquez et al., 2012). A recent study by Hodgson et al. (2014) suggested that vertical transmission can occur in different plant groups in addition to grasses. Fungal endophytes can be vertically transmitted in forbs through both pollen and seeds. The same genera found in this study, Alternaria, Cladosporium, Aspergillus, and Fusarium were reported as vertically transmitted fungi in forbs by Hodgson et al. (2014), but additional studies will be necessary to demonstrate that these fungi could potentially be vertically transmitted in alpine tundra plants.
4.4. Potential role in seed recruitment and survival in alpine ecosystems Reproduction by seeds is thought to be a rare occurrence among alpine plants (Bell and Bliss, 1980; Weppler et al., 2006). Many of the plant species in this study are clonal (e.g. Trisetum, Erigeron, Geum and Deschampsia) and the tradeoffs of investment between sexual and vegetative reproduction (Barrett, 2015) explain in part the low germination rates. The alpine tundra is dominated by longlived perennials (Billings and Mooney, 1968). Adaptive strategies, such as clonality, provide alpine plant populations with stability during harsh environmental conditions that are not favorable for seed germination and seedling establishment (Bierzychudek, 1985). However, in a 4-year demographic study at Niwot, Forbis (2003) found both seedling recruitment and mortality rates to be similar to published rates from perennial plant species from other environments including tropical and temperate ecosystems, suggesting that reproduction by seeds may be more important than previously thought in the alpine. In our study, germination rates and seed viability varied greatly among seeds (Table 1). This is likely due to the interaction of multiple factors including seed dormancy, viability, and the high presence of putative pathogens that could impact germination success. For example, P. bistortoides seeds had 0% germination rates, contained the highest percentage of fungal isolates all closely related to common plant pathogens such as Alternaria and Fusarium. When tested in vitro these fungal isolates significantly decreased germination rates in Z. mays (Fig. 2) and showed high percentages of root necrosis. In addition to plant pathogens, mutualistic fungi can also be associated with germination success and seedling survival. In this study, four Cladosporium isolates significantly increased germination rates and root growth in both Z. mays and D. cespitosa (Figs. 2 and 3). Both D. cespitosa and G. rossii are dominant plants, accounting for 60% of the cover at Niwot (Farrer et al., 2015). All the Cladosporium isolates in our study were cultured from only G. rossii and D. cespitosa accounting for 70% of the fungi cultured from D. cespitosa and 37.5% of the fungi cultured from G. rossii (Table S1). Having a beneficial relationship with growth promoting fungi could be advantageous for alpine plants given the harsh environmental conditions and a very short growing season. Faster germination rates with production of roots could have a bigger impact on quick establishment of seedlings and associations with Cladosporium in seeds and could contribute to the dominance of these two plant species. Acknowledgments This project was funded by the National Science Foundation (APA, KS, RS NSF-DEB 919510; KS, NSF-DEB 1637686) and a Western Illinois University Graduate and Professional Research Grant. In addition, we would like to thank three high school students Abraham Matlack, Ali Kerr, and Dexter Redenius who dedicated a summer helping with this project (APA, NSF-DEB 1314459). Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.funeco.2017.08.001. References Abdulsalaam, S., Shenge, K.C., 2011. Seed borne pathogens on farmer-saved sorghum (Sorghum bicolor L.) seeds. J. Stored Prod. Postharvest Res. 2, 24e28. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403e410. Anderson, P.K., Cunningham, A.A., Patel, N.G., Morales, F.J., Epstein, P.R., Daszak, P.,
T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18 2004. Emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers. Trends Ecol. Evol. 19, 535e544. Bain, D.C., 1950. Fungi recovered from seed of Sorghum vulgare Pers. Phytopathology 40, 521e522. Baker, K.F., Smith, S.H., 1966. Dynamics of seed transmission of plant pathogens. Annu. Rev. Phytopathol. 4, 311e332. Barrett, S.C., 2015. Influences of clonality on plant sexual reproduction. P. Natl. A. Sci. U. S. A. 112, 8859e8866. Baskin, J.M., Baskin, C.C., 2004. A classification system for seed dormancy. Seed Sci. Res. 14, 1e16. Baskin, J.M., Baskin, C.C., 2000. Evolutionary considerations of claims for physical dormancy-break by microbial action and abrasion by soil particles. Seed Sci. Res. 10, 409e414. Bell, K.L., Bliss, L.C., 1980. Plant reproduction in a high arctic environment. Arct. Alp. Res. 12, 1e10. Bierzychudek, P., 1985. Patterns in parthenogenesis. Experientia 41, 1255e1264. Billings, W.D., Mooney, H.A., 1968. The ecology of arctic and alpine plants. Biol. Rev. 43, 481e529. Burgess, L.W., Bryden, W.L., 2012. Fusarium: a ubiquitous fungus of global significance. Microbiol. Aust. 33.1, 22e25. Clay, K., 1989. Clavicipitaceous endophytes of grasses: their potential as biocontrol agents. Mycol. Res. 92, 1e12. Clay, K., 1990. The impact of parasitic and mutualistic fungi on competitive interactions among plants. In: Grace, J.B., Tilman, D. (Eds.), Perspectives on Plant Competition. Academic Press Inc, San Diego, pp. 391e407. Clear, R.M., Patrick, S.K., Gaba, D., 2000. Prevalence of fungi and fusariotoxins on barley seed from western Canada. Can. J. Plant Pathol 22, 44e50. Crous, P.W., Schoch, C.L., Hyde, K.D., Wood, A.R., Gueidan, C., De Hoog, G.S., Groenewald, J.Z., 2009. Phylogenetic lineages in the Capnodiales. Stud. Mycol 64, 17e47. Dalling, J.W., Davis, A.S., Schutte, B.J., Arnold, A.E., 2011. Seed survival in soil: interacting effects of predation, dormancy, and soil microbial community. J. Ecol. 99, 89e95. Dean, S.L., Farrer, E.C., Taylor, D.L., Porras-Alfaro, A., Suding, K.N., Sinsabaugh, R.L., 2014. Nitrogen deposition alters plant-fungal relationships: linking belowground dynamics to aboveground vegetation change. Mol. Ecol. 23, 1364e1378. Dhingra, O.D., Maia, C.B., Lustosa, D.C., Mesquita, J.B., 2002. Seedborne pathogenic fungi that affect seedling quality of red angico (Anadenanthera macrocarpa) trees in Brazil. J. Phytopathol. 150, 451e455. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792e1797. Elias, S., Garay, A., Schweitzer, L., Hanning, S., 2006. Seed quality testing of native species. Native Plants J. 7, 15e19. Farrer, E.C., Ashton, I.W., Spasojevic, M.J., Fu, S., Gonzalez, D.J.X., Suding, K.N., 2015. Indirect effects of global climate change accumulate to alter plant diversity but not ecosystem function in alpine tundra. 2015. J. Ecol. 103, 351e360. Forbis, T.A., 2003. Seedling demography in an alpine ecosystem. Am. J. Bot. 90, 1197e1206. Gallery, R.E., Dalling, J.W., Arnold, A.E., 2007. Diversity, host affinity, and distribution of seed-infecting fungi: a case study with Cecropia. Ecology 88, 582e588. Ganley, R.J., Newcombe, G., 2006. Fungal endophytes in seeds and needles of Pinus monticola. Mycol. Res. 110, 318e327. Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomyceteseapplication to the identification of mycorrhizae and rusts. Mol. Ecol. 2, 113e118. Greenland, D., 1989. The climate of NIWOT Ridge, Front Range, Colorado, USA. Arct. Alp. Res. 21, 380e391. Hamayun, M., Khan, S.A., Ahmad, N., Tang, D.S., Kang, S.M., Na, C.I., Sohn, E.Y., Hwang, Y.H., Shin, D.H., Lee, B.H., Kim, J.G., Lee, I.J., 2009. Cladosporium sphaerospermum as a new plant growth-promoting endophyte from the roots of Glycine max (L.) Merr. World J. Microbiol. Biotechnol. 25, 627e632. Hamayun, M., Khan, S.A., Khan, A.L., Rehman, G., Kim, Y.H., Iqbal, I., Hussain, J., Sohn, E.Y., Lee, I.J., 2010. Gibberellin production and plant growth promotion from pure cultures of Cladosporium sp MH-6 isolated from cucumber (Cucumis sativus L.). Mycologia 102, 989e995. Hauser, S., 2007. Plantain (Musa spp. AAB) bunch yield and root health responses to combinations of physical and chemical sucker sanitation measures. Afr. Plant Prot. 13, 1e15. Higgins, K.L., Arnold, A.E., Miadlikowska, J., Sarvate, S.D., Lutzoni, F., 2007. Phylogenetic relationships, host affinity, and geographic structure of boreal and arctic endophytes from three major plant lineages. Mol. Phylogenet. Evol. 42, 543e555. Hodgson, S., De Cates, C., Hodgson, J., Morley, N.J., Sutton, B.C., Gange, A.C., 2014. Vertical transmission of fungal endophytes is widespread in forbs. Ecol. Evol. 4, 1199e1208. Hoffman, M.T., Arnold, A.E., 2008. Geographic locality and host identity shape fungal endophyte communities in cupressaceous trees. Mycol. Res. 112, 331e344. Hoffman, M.T., Gunatilaka, M.K., Wijeratne, K., Gunatilaka, L., Arnold, A.E., 2013. Endohyphal bacterium enhances production of indole-3-acetic acid by a foliar fungal endophyte. PLoS One 8, e73132. Johnson, L.J., Miller, A.N., McCleery, R.A., McClanahan, R., Kath, J.A., Lueschow, S., Porras-Alfaro, A., 2013. Psychrophilic and psychrotolerant fungi on bats and the presence of Geomyces spp. on bat wings prior to the arrival of white nose syndrome. Appl. Environ. Microbiol. 79, 5465e5471.
17
Klironomos, J.N., 2002. Feedback with soil biota contributes to plant rarity and invasiveness in communities. Nature 417, 67e70. Leuchtmann, A., 1992. Systematics, distribution, and host specificity of grass endophytes. Nat. Toxins 1, 150e162. Malone, J.P., Muskett, A.E., 1997. Seed-borne Fungi, Description of 77 Fungus Species. The International Seed Testing Association, Zurich. Mandyam, K.G., Roe, J., Jumpponen, A., 2013. Arabidopsis thaliana model system reveals a continuum of responses to root endophyte colonization. Fungal Biol. 117, 250e260. McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L., Swan, J.A., 1990. A new method which gives an objective measure of colonization of roots by vesiculararbuscular mycorrhizal fungi. New Phytol. 115, 495e501. McLennan, E.T., 1920. Endophytic fungus of Lolium: part 1. Proc. R. Soc. Vic. 32, 252e301. Newton, A.C., Fitt, B.D.L., Atkins, S.D., Walters, D.R., Daniell, T.J., 2010. Pathogenesis, parasitism and mutualism in the trophic space of microbe-plant interactions. TIM 18, 365e373. Noble, M.J., De Tempe, J., Neergaard, P., 1958. An Annotated List of Seed-borne Diseases. Surrey, England, CMI, Kew. Porras-Alfaro, A., Bayman, P., 2011. Hidden fungi, emergent properties: endophytes and microbiomes. Annu. Rev. Phytopathol. 49, 291e315. Raj, S.N., Deepak, S.A., Basavaraju, P., Shetty, S.H., Reddy, M.S., Kloepper, J.W., 2003. Comparative performance of formulations of plant growth promoting rhizobacteria in growth promotion and suppression of downy mildew in pearl millet. Crop Prot. 22, 579e588. Ravel, C., Michalakis, Y., Charmet, G., 1997. The effect of imperfect transmission on the frequency of mutualistic seed-borne endophytes in natural populations of grasses. Oikos 80, 18e24. Reynolds, H.L., Packer, A., Bever, J.D., Clay, K., 2003. Grassroots ecology: plantmicrobe-soil interactions as drivers of plant community structure and dynamics. Ecology 84, 2281e2291. Rodriguez, R.J., White, J.F., Arnold, A.E., Redman, R.S., 2009. Fungal endophytes: diversity and functional roles. New Phytol. 182, 314e330. Rudgers, J.A., Koslow, J.M., Clay, K., 2004. Endophytic fungi alter relationships between diversity and ecosystem properties. Ecol. Lett. 7, 42e51. Saikkonen, K., Saari, S., Helander, M., 2010. Defensive mutualism between plants and endophytic fungi. Fungal Divers 41, 101e113. €li, P., Helander, M., Faeth, S.H., 2004. Evolution of endophyteeSaikkonen, K., Wa plant symbioses. Trends Plant Sci. 9, 275e280. Sanchez-Delgado, P., Ortega-Amaro, M., Jimenez-Bremont, J., Flores, J., 2011. Are fungi important for breaking seed dormancy in desert species? Experimental evidence in Opuntia streptacantha (Cactaceae). Plant Biol. 13, 154e159. Sanchez-Marquez, S., Bills, G.F., Herrero, N., Zabalgogeazcoa, I., 2012. Non-systemic fungal endophytes of grasses. Fungal Ecol. 5, 289e297. Soytong, K., Kanokmedhakul, S., Kukkongviriyapa, V., Isobe, M., 2001. Application of Chaetomium species (Ketomium(R)) as a new broad spectrum biological fungicide for plant disease control: a review article. Fungal Divers 7, 1e15. Stone, J.K., Bacon, C.W., White, J.F., 2000. An overview of endophytic microbes: endophytism defined. In: Bacon, C.W., White, J.F. (Eds.), Microbial Endophytes. Marcel Dekker Inc., New York, pp. 3e30. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis. Mol. Biol. Evol. 30, 2725e2729, version 6.0. . ~ lme, S., Ko ~ljalg, U., Yorou, N.S., Wijesundera, R., Tedersoo, L., Bahram, M., Po Ruiz, L.V., Vasco-Palacios, A.M., Thu, P.Q., Suija, A., Smith, M.E., 2014. Global diversity and geography of soil fungi. Science 346, 1256688. Tillet, M., 1937. Dissertation on the cause of the corruption and smutting of the kernels of wheat in the head and the means of preventing these untoward circumstances. Phytopathol. Class. 5, 1e150. Timling, I., Walker, D.A., Nusbaum, C., Lennon, N.J., Taylor, D.L., 2014. Rich and cold: diversity, distribution, and drivers of fungal communities in patterned-ground ecosystems of the North American Arctic. Mol. Ecol. 23, 3258e3272. Tveit, M., Wood, R.K.S., 1955. The control of Fusarium blight in oat seedlings with antagonistic species of Chaetomium. Ann. Appl. Biol. 43, 538e552. U'Ren, J.M., Dalling, J.W., Gallery, R.E., Maddison, D.R., Davis, E.C., Gibson, C.M., Arnold, A.E., 2009. Diversity and evolutionary orgins of fungi associated with seeds of a neotropical pioneer tree: a case study for analyzing fungal environmental samples. Mycol. Res. 113, 432e449. Van Der Putten, W.H., Peters, B.A.M., 1997. How soil-borne pathogens may affect plant competition. Ecology 78, 1785e1795. Vazquez-de-Aldana, B.R., Garcia-Criado, B., Vicente-Tavera, S., Zabalgogeazcoa, I., 2013. Fungal endophyte (Epichlo€ e festucae) alters the nutrient content of Festuca rubra regardless of water availability. PLoS One 8, e84539. Vielerheilig, H., Coughlan, A.P., Wyss, U., Piche, Y., 1998. Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Appl. Environ. Microbiol. 64, 5004e5007. Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261e5267. €cklin, J., 2006. The relative importance of sexual and clonal Weppler, T., Stoll, P., Sto reproduction for population growth in the long-lived alpine plant Geum reptans. J Ecol 94, 869e879. White, J.F., Baldwin, N.A., 1992. A preliminary enumeration of grass endophytes in west central England. Sydowia 44, 78e84. White, T.J., Bruns, T.D., Lee, S.B., Taylor, J.W., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A.,
18
T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18
Gelfand, D.H., Sninsky, J.J., White, T.J., protocols, P.C.R. (Eds.), A Guide to Methods and Applications. Academic Press, San Diego, pp. 315e322. Zalamea, P.C., Sarmiento, C., Arnold, A.E., Davis, A.S., Dalling, J.W., 2014. Do soil microbes and abrasion by soil particles influence persistence and loss of physical dormancy in seeds of tropical pioneers? Front. Plant Sci. 5, 799.
Zalar, P., De Hoog, G.S., Schroers, H.J., Groenewald, J.Z., Gunde-Cimerman, N., 2007. Phylogeny and ecology of the ubiquitous saprobe Cladosporium spaerospermum with descriptions of seven new species from hypersaline environments. Stud. Mycol. 58, 157e183.
Contents lists available at ScienceDirect
Fungal Ecology journal homepage: www.elsevier.com/locate/funeco
Seed-associated fungi in the alpine tundra: Both mutualists and pathogens could impact plant recruitment Terri Billingsley Tobias a, Emily C. Farrer b, Antonio Rosales 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 Department of Ecology and Evolutionary Biology, Tulane University, New Orleans, LA 70118, USA 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 b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 October 2016 Received in revised form 2 July 2017 Accepted 4 August 2017
Seed-borne microbes are important pathogens and mutualists in agricultural crops but are understudied in natural systems. To understand the diversity and function of seed-borne fungi in alpine tundra, we cultured fungi from seeds of six dominant plant species prior to seed dispersal and evaluated their function using germination experiments in Zea mays. A total of 55 fungal cultures (9 species) were 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 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 high number of pathogenic fungi found coupled with the variation in seed endophytic communities among plant species suggests that seed-associated fungi could affect community composition through differential seedling recruitment. © 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
Corresponding Editor: Nicole Hynson Keywords: Seed-borne fungi Alpine tundra Mutualists Pathogens Cladosporium Alternaria
1. Introduction Fungal communities are important drivers of plant diversity and community structure (Reynolds et al., 2003). Most studies on plantfungal interactions in natural systems in the last four decades have focused on root and leaf endophytes, rhizosphere and bulk soil communities, and soil-borne pathogens (Van Der Putten and Peters, 1997; Higgins et al., 2007; Hoffman and Arnold, 2008; Porras-Alfaro and Bayman, 2011); however, a potentially diverse and important group of microorganisms inhabit seeds. Seed-borne microbes are important pathogens and mutualists in agricultural crops (Newton et al., 2010), but they are understudied in natural systems (Clay, 1990; Ravel et al., 1997; Dalling et al., 2011). Most studies in natural systems have focused on fungi acquired in postdispersal events when the seeds come in contact with fungal soil
* Corresponding author. WIU Biology, Waggoner Hall 372, 1 University Circle, Macomb, IL 61455, USA. 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.
communities (Gallery et al., 2007; U'Ren et al., 2009). Seed-borne fungi colonize internal tissues (endophytes) and can show mutualistic or pathogenic activity (Baker and Smith, 1966; Stone et al., 2000; Rodriguez et al., 2009). They are highly ubiquitous and abundant: many cultivated crops and natural plants have some form of seed-borne fungi (e.g., Clay, 1990; Malone and Muskett, 1997; Dhingra et al., 2002; Ganley and Newcombe, 2006). Pathogenic seed-borne fungi have been extensively studied causing infectious diseases. Common species include Tilletia caries, Colletotrichum lindemuthianum, Botrytis cinerea, and several Fusarium species (Tillet, 1937; Noble et al., 1958; Malone and Muskett, 1997; Anderson et al., 2004). Epichlo€ e typhina in Lolium spp. improves embryo development (McLennan, 1920), but in adult plants, it also causes fescue toxicity as a way to control herbivory (Leuchtmann, 1992). There are also some saprotrophic fungal species 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 blight in oat seedlings (Tveit and Wood, 1955; Soytong et al., 2001). This study focuses on seed-borne fungi associated with
T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18
dominant plants in the alpine tundra. The alpine tundra is characterized by harsh environmental conditions, including low temperatures, frost events, short growing season and high winds and solar radiation which lead to desiccation (Greenland, 1989). Diverse communities of fungi have been reported to be associated with plants in cold habitats (Dean et al., 2014; Tedersoo et al., 2014; Timling et al., 2014). In the alpine tundra, vertical transmission of fungi (via seeds) may be an important mechanism by which plants can pass on beneficial fungi to their offspring. Seed-borne fungi may also play an important role in the alpine environment by reducing dormancy or improving germination. Contrasting evidence is available about the role of microbial communities in breaking dormancy (Baskin and Baskin, 2000) but most of the studies have focused on external microbial fungi (e.g. SanchezDelgado et al., 2011) or seed-associated fungi acquired after dispersal (Baskin and Baskin, 2000; Zalamea et al., 2014). It is well known that alpine seeds can remain viable after many years in freezing conditions and have the ability to germinate and grow rapidly when the conditions become favorable (Billings and Mooney, 1968); seed-borne fungi may contribute to these processes. The main goal of this study was to describe the culturable fungal communities in alpine seeds and their potential contribution to plant growth. Fungi were isolated from surface sterilized seeds of six plant species and identified by sequencing the internal transcribed spacer (ITS) rRNA region. Potential roles of the fungal isolates were tested in germination and plant growth experiments using a model crop (maize; Zea mays) as well as one of the alpine tundra plants, Deschampsia cespitosa. All the isolates closely related to the genus Cladosporium showed positive results for seed germination as well as plant growth, so further phylogenetic analysis was performed on the genus to determine the placement of isolates with respect to known species. 2. Materials and methods
11
palea were removed prior to surface sterilization. Sixty seeds of each species in small batches were surface sterilized in microcentrifuge tubes with 1 mL of 95% ethanol. Tubes were vortexed briefly to disperse seeds evenly and left to stand for 5 min. Ethanol was removed and 1 mL of 0.5% Clorox bleach (5.25% NaOCl; Clorox Company, Oakland, CA) was added for 5 min. This was followed by three rinses in 1 mL of distilled autoclaved water. Seeds were allowed to stand in sterile water for three additional minutes. To determine if the surface sterilization procedure was effective, seeds were tapped briefly on sterile malt extract agar (MEA) (Difco Laboratories, Detroit, MI) and incubated at room temperature. No fungi grew on these MEA plates. Five sterilized seeds were plated on MEA with streptomycin and tetracycline (50 mg mL1) to limit bacteria contamination. A total of 60 seeds per plant species were plated (5 seeds/12 plates). The seed lemma and palea were removed prior to sterilization but entire seeds were incubated for the isolation of putative endophytes. Plates were incubated at 25 C and evaluated daily for hyphal growth for 68 d. Fungal colonies were transferred to new MEA plates to obtain pure cultures. 2.2. Seed viability Alpine tundra seed viability was measured using tetrazolium (MP Biomedicals LLC. Solon, OH) following a protocol by Elias et al. (2006). Fifty seeds of each plant species (T. spicatum, A. scopulorum, E. simplex, G. rossii, P. bistortoides, D. cespitosa) were placed in microcentrifuge tubes with 1 mL of dH2O and allowed to soak overnight to hydrate. T. spicatum, P. bistortoides, D. cespitosa seeds were punctured with a needle and dissected longitudinally to allow exposure to the tetrazolium solution. Filter paper was soaked with a 1% (m/v) tetrazolium solution, seeds were placed on paper and incubated at 35 C for 3 h. After incubation, seeds were examined under a dissecting microscope to evaluate viability. Seeds stained 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 simplex, Polygonum bistortoides, Geum rossii and D. cespitosa were collected in 2011 and 2012. Both Deschampsia and Trisetum belong to the family Poaceae. Erigeron and Artemisia are members of the Asteraceae family while Polygonum belongs to Polygonaceae and Geum belongs to Rosaceae. Each of these seed families are reported to share common physiological dormancy (Baskin and Baskin, 2004). Preliminary germination trials suggest that all species, with the exception of P. bistortoides, exhibit germination rates between 15 and 52% without the use of any dormancy-breaking treatment (after a few months of dry, room-temperature storage) (Farrer, unpublished data). P. bistortoides requires 3e4 weeks of cold, moist stratification for germination (germination rate was 0% if not cold, moist stratified) (Farrer, unpublished data). All the seeds with the exception of A. scopulorum were kept refrigerated before the experiment; however our seeds were not stored moist in the refrigerator, which is likely why we observed 0% germination for P. bistortoides. The seeds were collected from plants in a moist meadow mainly composed of grasses and forbes in the alpine tundra at the Niwot Ridge Long Term Ecological Research (LTER) site located 35 km west of Boulder, Colorado. The site is located between 3297 and 3544 m above sea level, with a mean temperature for the winter of 8 C and the mean temperature for the summer of 13 C. In addition, the site remains under snow cover 9e10 months each year (Dean et al., 2014). Seeds were removed from infructescences and the lemma and
Fungal DNA was extracted from pure cultures grown on MEA using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) following the manufacturer's protocols. DNA was amplified using ITS rRNA fungal specific primers, ITS1F (50 CTTGGTCATTTAGAGGAAGTAA-30 ) and ITS4 (50 - TCCTCCGCTTATTGATATGC-30 ) (Gardes and Bruns, 1993; White et al., 1990). PCR was conducted in 25 mL reactions containing the following: 6.5 mL of nuclease free water, 1 mL of ITS1F (5 mM), 1 mL ITS4 (5 mM), 3 mL of 1% BSA (Sigma-Aldrich, St. Louis, MO), 12.5 mL of PCR Master Mix (Promega, Madison, WI), and 1 mL of the DNA sample. Reactions were amplified under the following conditions: 95 C for 5 min, followed by 30 cycles at 94 C for 30 s, then annealing at 50 C for 30 s with an extension at 72 C for 45 s, and a final extension of 72 C for 7 min. Gel electrophoresis with 1% agarose in Tris Acetate EDTA was used to verify the PCR reaction, PCR samples (3 mL DNA and 2 mL dye) were compared to a low DNA mass ladder (Invitrogen Corporation, Carlsbad CA) to determine fragment size and concentration for sequencing reactions. Prior to sequencing, PCR products were cleaned using ExoSAP-IT (Affymetrix, Cleveland, OH) following the manufacture's protocol. Sequences were prepared using BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) with ITS rRNA fungal specific primers for forward (ITS1F) and reverse sequences (ITS4). Each sequencing reaction contained the following: 5.5 mL of nuclease free water, 1.5 mL of BigDye sequencing buffer (Applied Biosystems, Foster City, CA.), 1 mL of ITS1F (3 mM), 1 mL of ITS4
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(3 mM), 1 mL of BigDye Terminator (Applied Biosystems, Foster City, CA), and 1 mL of PCR product. Sequencing reactions were completed under the following conditions: 96 C for 10 s, 50 C for 5 s, 60 C for 4 min during 30 cycles. Sequencing reactions were precipitated using 1 mL of 125 mM EDTA, 1 mL 3 M sodium acetate and 25 mL of 100% ethanol following Johnson et al. (2013). Samples were sequenced in both directions at the Molecular Biology Facility at the University of New Mexico, Albuquerque, NM. Forward and reverse sequences were grouped in contigs and edited in Sequencher 4.9 (Gene Codes Corporation, Ann Arbor, MI). Contigs were assembled into operational taxonomic units (OTUs) at 97% similarity. Sequences were deposited in GenBank under accession numbers KX839269-KX839323. Preliminary identification of the sequences at the genus level was done using RDP naïve Bayesian rRNA classifier version 2.1 (Wang et al., 2007) with a bootstrap cutoff of 80% and compared with Basic Local Alignment Search Tools (BLAST) in NCBI (http://www.ncbi.nlm.nih.gov) (Altschul et al., 1990). Fungi with a bootstrap support higher of 80% in RDP and with a score higher than 700 and 95% identity similarity in NCBI were reported as the closest relative for each taxon. Principle component analysis was done using XLSTAT. Dissimilarity between plant seeds and seed-borne fungal isolates was calculated and graphed on a distance biplot. For phylogenetic analysis the ITS rRNA sequences were aligned using MUSCLE (multiple comparison by log expectations) (Edgar, 2004). They were trimmed and edited by hand using MEGA 6 (Tamura et al., 2013). Maximum Likelihood analysis was used to infer the evolutionary history. Taxa from Crous et al. (2009), Zalar et al. (2007) and Genbank were used for comparison. 2.4. Germination experiments To determine potential functional roles of the fungal isolates, germination experiments were conducted on two isolates for each representative OTU, defined at 97% similarity. Germination and plant growth was evaluated in commercial Z. mays plants (commercial non-GMO hybrid Z. mays seeds, Arrow Seed). Z. mays was used as a model because of its rapid growth and high germination rates. In addition, this seed was chosen because it is endophyte-free (treated with captan and trifloxystrobin, a metalaxyl fungicide, and Poncho™ 600, an insecticide containing clothianidin) allowing an initial testing without the influence of seed-borne fungi present in the host plants. The lack of seed-borne fungi in Z. mays was confirmed by plating surface sterilized seeds on MEA agar for 1 week. Since Z. mays was not the primary host for fungi that showed positive plant growth results we conducted additional germination experiments on one alpine tundra plant (Deschampsia). With the exception of Deschampsia the majority of the alpine tundra seeds in this study had very low germination rates (0e48%) under the conditions tested in our experiments. Fungal isolates were plated and grown for 14 d on MEA with antibiotics until the fungus covered the plate. Ten plates were made for each fungal isolate with the same number of control plates (no fungus). Five surface sterilized seeds were plated in each plate (50 seeds per fungus). Prior to surface sterilization, Z. mays seeds were washed three times in sterile water for five minutes to remove traces of fungicide and insecticide. The Deschampsia seeds were not commercially treated with fungicide and likely contained seedborne fungal endophytes. Plates were incubated in the dark at 25 C for 7 d. Seeds were checked daily and germination rates were recorded. After 7 d, plates were placed under grow lights with a photoperiod of 12 h of light and 12 h of darkness. Plants were left to grow up to 15 d in vitro. The number of roots, number of leaves, length of longest
root, and length of stem (measured from the tip of the plant to the seed) were recorded. A t-test was performed with StatPlus to determine statistical differences between treatments to compare the number of roots, and root and stem length. An ANOVA with post hoc testing was used to compare the percentage of germination for the different fungal treatments with respect to the controls. 2.5. Root necrosis index and plant vigor index Root necrosis was measured using a dissecting microscope. The percentage of root necrosis was calculated following Hauser (2007); surface of necrotic cortical tissue/total surface of cortical tissue x 100%. In addition, a modified plant vigor index (PVI) was also calculated as (mean longest root length þ mean shoot length) x (% germination) (Raj et al., 2003). 2.6. Root colonization Root colonization was examined in plants exposed to isolates that showed positive growth effects. Z. mays roots were stained following a modified protocol from Vielerheilig et al. (1998). Roots were cut into 3 cm segments and placed in microcentrifuge tubes. A 10% KOH solution (1 mL) was added and tubes were placed in a water bath for 15 min at 60 C. The KOH was then removed and 1 mL 5% Parker Ink solution (2.5 mL blue ink, 5% acetic acid, 50 mL dH2O) solution was added to the roots. Roots were heated again in a water bath for 15 min at 60 C. Roots were washed 3 with water and stored in 20% glycerol until use. A total of 100 intersections were evaluated from ten plants per treatment using the gridline intersection method (McGonigle et al., 1990). Root sections were ranked for colonization per field of view: 0 ¼ no DSE hyphae present, 1 ¼ 1 or 2 hyphae present, and 2 ¼ more than 2 hyphae present (Mandyam et al., 2013). 3. Results 3.1. Isolation and identification of fungi from seeds A total of 55 putative fungal endophytes were isolated and sequenced using the ITS rRNA region from the 360 seeds plated (60 seeds per plant species) (Table S1). All fungal isolates belong to the phylum Ascomycota representing five orders, 8 unique genera, and 9 OTUs defined at 97% similarity (Table 1 and Table S1). The most abundant order was Pleosporales (26% of the cultures), followed by Hypocreales (23%), Capnodiales (18%), Eurotiales (17%), and Helotiales (7%). Surface sterilized alpine seeds contained few putative endophytes per plant species. We recovered 2e4 fungal species per plant species using culture-based methods and one type of media, and plant species differed in the putative endophyte communities their seeds contained (Fig. 1). No fungal growth was observed on the negative control plates. Across plant species, 5e27% of seeds were colonized by endophytic fungi. T. spicatum and P. bistortoides had the highest colonization rates with 27% and 22%, respectively. Little to no colonization was observed in A. scopulorum (5%) and E. simplex (8%) (Table 1). Twenty-two percent of the seeds plated contained fungal isolates closely related to three common plant pathogenic genera: Alternaria, Fusarium, and Aspergillus (Table 1). The most abundant genus was closely related to Alternaria accounting for 25% of the pure isolates (Table S1). Alternaria was found in four of the six plant species but the majority of isolates (64%) were found in association with P. bistortoides (Table 1, Fig. 1). Other common taxa included Aspergillus isolated from A. scopulorum, T. spicatum, and E. simplex and Fusarium isolated from D. cespitosa and P. bistortoides (Fig. 1).
T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18
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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 bistortoides Geum rossii Deschampsia cespitosa Trisetum spicatum Artemisia scopulorum Erigeron simplex
Alternaria, Fusarium
30%
0%
22%
Alternaria, Botrytis, Cladosporium, Lewia Alternaria, Cladosporium, Fusarium Epichlo€ e, Alternaria, Aspergillus, Botrytis Aspergillus Aspergillus, Engyodontium
60% 28% 44% 24% 36%
48% 38% 18% 5% 32%
13% 13% 27% 8% 5%
Cladosporium was also abundant, accounting for 19% of the fungi isolated from alpine seeds. This genus was only cultured from G. rossii (37% of the isolates) and D. cespitosa (77% of the isolates) (Table 1, Fig. 1).
3.2. Effect of fungal isolates on germination and plant growth A total of 11 representative fungi were selected for germination experiments based on identification using the ITS rRNA. Six of the 11 fungi tested on Z. mays showed some level of pathogenicity ranging from low germination rates to high levels of root necrosis compared to the controls (Figs. 2 and 3). Isolates in the genus Fusarium (Bb12 and Bb20) and Alternaria (Bb17) showed the greatest negative effects in early germination stages with only 32e58% of the Z. mays seeds germinated after seven days as compared to the controls (98% germination) (Fig. 2). Botrytis (Gr5) isolated from G. rossii along with Aspergillus (As1, As5) isolated from
A. scopulorum also showed significant negative effects on early germination in Z. mays (Fig. 2). The fungal taxa that negatively affected germination also 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 (Fig. 3E). Fusarium (Fig. 3A, B), Aspergillus (Fig. 3C, D) and Botrytis (Fig. 3F) also significantly decreased root and stem length compared to the controls (p < 0.05). Fusarium, Aspergillus, and Botrytis (Fig. 3G) significantly reduced the number of roots (Fig. 3AeD, G) (p < 0.05). Fusarium isolates (Bb20 and Bb12) showed the greatest level of root necrosis (60e70% root necrosis), followed by Aspergillus (As1, As5, 47e52%) and Botrytis (Gr5, 52%). plant vigor index was lower for isolates of Fusarium (45 PVI), Aspergillus (49 PVI), Botrytis (98 PVI), and Alternaria (14 PVI) as compared to the control (229 PVI) suggesting a general reduction of plant health.
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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.
were obtained. After 15 d, a significant percentage of D. cespitosa seeds exposed to Cladosporium isolates germinated with rates of 28e36% with respect to 0% for the controls. The highest germination rate was observed for isolate Dc7 with 36% as compared to 0% for the control seeds. The three isolates did not differ in their effect on the number of roots and plant length (p > 0.5). Phylogenetic analysis for the Cladosporium isolates showed two major clades. The Cladosporium oxysporum/tenuissimum/cladosporioides clade contained the majority of the isolates (7 isolates in total) (Fig. S1) and it is in agreement with previously published phylogenies that show little to no resolution at the species level (Crous et al., 2009). Fungi in this clade were closely related to a fungal endophyte in Pinus ponderosa and Quercus litter from Mexico. The second clade contained isolates Dc8 and Dc9 and groups with other fungal endophytes from coastal dune grasses and Pinus taeda and are closely related to Cladosporium velox. Of the isolates tested in vitro, Dc6, Dc7, Gr4 were all closely related to C. oxysporum and all show similar PVI ranging from 1151 to 888. Dc9 was closely related to C. velox showed a lower PVI of 532, lower germination rates (Fig. 2), and lower growth rates for D. cespitosa, but a significantly greater effect on root growth for Z. mays (Fig. 3K).
In addition to negative effects on Z. mays, one isolate had neutral effects (no major differences with the controls) and four isolates positively affected Z. mays performance. Engyodontium had mostly neutral impact on Z. mays growth; it increased the number of roots but negatively affected root length, and had no impact on any of the other metrics. The four isolates that improved Z. mays growth all belong to the genus Cladosporium. Three isolates (Dc6, Dc7 and Gr4) showed significantly faster germination rates after three days ranging from 60% to 88% (Gr4) compared to 26% for the controls (Fig. 2). After 7 d both the controls and experimental seeds had germination rates higher than 92%. Significant positive effects on root and stem length were observed for all Cladosporium isolates with Dc9 and Dc7 showing the highest increase in root length (Fig. 3HeK) (p < 0.005). For example, the average length of the longest roots for Z. mays exposed to isolate Dc9 after 7 d was nearly three times that of the control (13.8 ± 0.78 vs. 4.7 ± 0.32 cm) (p < 0.005) and the length of the stem after 7 d was almost two times higher than the control (5.2 ± 0.40 cm vs. 2.7 ± 0.21 cm) (p < 0.005) (Fig. 3K). This could be a result of the faster germination rates observed for those plants exposed to Cladosporium with respect to the other fungi and the controls. An increase in the number of roots was also observed for all plants exposed to Cladosporium. Isolates Dc6, Dc7, Dc9, and Gr4 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). None of the Cladosporium isolates showed signs of necrosis and fungal colonization was observed in the roots of the three Cladosporium isolates tested (Gr4 culture lost viability and could not be tested). Isolate Dc7 had the highest rate of colonization (96% of the roots) compared to the controls (0%) followed by isolates Dc9 (62%) and Dc6 (60%). Cladosporium isolates Dc6 (1118 PVI), Dc9 (532 PVI), Gr4 (1151 PVI), and Dc7 (888 PVI) all had significantly higher plant vigor index as compared to the controls (229 PVI).
This study shows alpine tundra seeds collected from the plants are colonized by fungi that have been mainly described as pathogens or saprotrophs (Alternaria, Fusarium, Aspergillus, Botrytis). These fungi showed some level of pathogenicity on germination experiments conducted on Z. mays as a model plant. We also report Cladosporium as one of the seed-associated fungi that showed growth-promoting activity in Z. mays and one of the hosts, D. cespitosa. This is one of the first studies describing diversity and 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 D. cespitosa, one of the original hosts from which these isolates
Most of the isolated taxa in this study were associated with common plant pathogenic fungi. Alternaria and Fusarium, common
4. Discussion
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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 fungi isolated from cereal crops (Bain, 1950; Clear et al., 2000; Abdulsalaam and Shenge, 2011). Fungi in the Alternaria complex include saprotrophic to parasitic species and can be easily detected in seeds, as colonies will begin to grow on MEA after 5 d (Malone and Muskett, 1997). Pathogenic isolates such as Fusarium and Alternaria were both cultured from plants with low to 0% germination rates (Table 1). The percentage of viability within the seeds containing these fungal isolates ranged from 28 to 44% suggesting seed dormancy as one of the potential causes of low germination rates (Table 1). 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 observed in Fusarium isolates (Bb20, 71% and Bb12, 58%) followed by Botrytis (Gr5, 52%) and Aspergillus (As5, 52%). Fusarium species are ubiquitous and have been found in most ecosystems including alpine grasslands (Burgess and Bryden, 2012). Klironomos (2002) reported Fusarium, Verticillium, and Cylindrocarpon as the most commonly isolated fungi in Canadian meadows and grasslands. When tested, these isolates all caused systemic infections in the roots of native plants. In our study, a
significant number of the fungi cultured (63%) were also common plant pathogens associated with seeds. In bioassays, these isolates responded in a similar way, causing root necrosis and poor germination rates. Plant pathogens can range from highly specific to generalist. In this study, we used an alternative host (Z. mays) to evaluate pathogenicity due to the low germination rates of the native seeds and the presence of endophytic fungi in all the plants evaluated. Additional testing in the greenhouse and field conditions is necessary to determine the impact of these putative pathogens in native ecosystems.
4.2. Diversity and growth promoting properties of seed-borne fungi It is possible that other non-culturable fungi were associated with the alpine seeds since this study was cultured-based and we used a single medium. However, our study is in agreement with previous studies showing that surface sterilized seeds before dispersal contained either none or only one endophyte per seed (e.g. Dhingra et al., 2002; Ganley and Newcombe, 2006). Two to four fungal species were isolated per plant species with most being pathogenic and a few showing growth-promoting properties.
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Dhingra et al. (2002) showed similar results in which one fungus per seed was isolated in a high percentage of the seeds and many of the isolates showed pathogenicity in Anadenanthera macrocarpa in Brazil. U'Ren et al. (2009) and Gallery et al. (2007) showed that seedassociated fungal diversity was low before dispersal in the lowland forests of Panama and Costa Rica but high colonization rates were found after dispersal (i.e., the seeds have been in contact with soil communities after a period of time). The few fungi identified in pre-dispersal seeds using molecular methods were highly similar to isolated plant endophytes in other studies. Gallery et al. (2007) cultured 220 fungal isolates with 73 unique genotypes from four Cecropia species and only 5 genotypes were sequenced from predispersal seeds. Of the 73 unique genotypes, 64.3% were recovered only once, indicating high diversity of fungal taxa within the seeds once they have been in contact with the soil. These suggest that the majority of seed-associated fungi are acquired after dispersal. We isolated four fungi related to the genus Cladosporium that significantly increased plant vigor. Z. mays and D. cespitosa seeds exposed to these isolates germinated and grew significantly faster than control seeds (Figs. 2 and 3). Cladosporium have been previously reported as growth promoting endophytes in Glycine max and Cucumis sativus influencing both seed germination and plant growth (Hamayun et al., 2009, 2010). Hamayun et al. (2009) reported a novel species Cladosporium sphaerospermum, with growth promoting effects. Both Hamayun et al. (2009, 2010) determined that the production of gibberellins by Cladosporium isolates was responsible for the increase in plant growth. In addition, recent reports also indicate that endohyphal bacteria can affect host plants by the production of plant growth hormones (Hoffman et al., 2013).
4.3. Potential mechanisms of transmission of fungi in alpine tundra Vertically transmitted fungi often form mutualistic relationships with their plant hosts (Clay, 1989, 1990; Leuchtmann, 1992). These fungi spend their entire lifecycle within the plant tissue and are passed from parent to offspring via the host's seeds (Saikkonen et al., 2010). Vertically transmitted endophytes like Epichlo€ e can remain symptomless and have direct benefits on the plant host (Clay, 1989; Saikkonen et al., 2004). However, some Epichlo€ e species such as E. typhina can also become pathogenic to the host plant (Vazquez-de-Aldana et al., 2013). A single isolate of Epichlo€ e sp., a vertically transmitted fungus in grass species, was recovered in this study. Epichlo€ e spp. in grasses are very well studied and play important ecological roles in plant communities (Rudgers et al., 2004) and it has been reported in Trisetum flavescens (White and Baldwin, 1992). In our study, this genus was specific to T. spicatum, accounting for 15% of the cultures isolated from Trisetum seeds. The isolate did not survive well in culture and could not be tested in germination experiments. The fungi in this study were isolated before dispersal but the transmission mechanisms of seed endophytes can be complicated to determine; while certain groups are vertically transmitted others may be horizontally transmitted via spores in the air or soil (Sanchez-Marquez et al., 2012). A recent study by Hodgson et al. (2014) suggested that vertical transmission can occur in different plant groups in addition to grasses. Fungal endophytes can be vertically transmitted in forbs through both pollen and seeds. The same genera found in this study, Alternaria, Cladosporium, Aspergillus, and Fusarium were reported as vertically transmitted fungi in forbs by Hodgson et al. (2014), but additional studies will be necessary to demonstrate that these fungi could potentially be vertically transmitted in alpine tundra plants.
4.4. Potential role in seed recruitment and survival in alpine ecosystems Reproduction by seeds is thought to be a rare occurrence among alpine plants (Bell and Bliss, 1980; Weppler et al., 2006). Many of the plant species in this study are clonal (e.g. Trisetum, Erigeron, Geum and Deschampsia) and the tradeoffs of investment between sexual and vegetative reproduction (Barrett, 2015) explain in part the low germination rates. The alpine tundra is dominated by longlived perennials (Billings and Mooney, 1968). Adaptive strategies, such as clonality, provide alpine plant populations with stability during harsh environmental conditions that are not favorable for seed germination and seedling establishment (Bierzychudek, 1985). However, in a 4-year demographic study at Niwot, Forbis (2003) found both seedling recruitment and mortality rates to be similar to published rates from perennial plant species from other environments including tropical and temperate ecosystems, suggesting that reproduction by seeds may be more important than previously thought in the alpine. In our study, germination rates and seed viability varied greatly among seeds (Table 1). This is likely due to the interaction of multiple factors including seed dormancy, viability, and the high presence of putative pathogens that could impact germination success. For example, P. bistortoides seeds had 0% germination rates, contained the highest percentage of fungal isolates all closely related to common plant pathogens such as Alternaria and Fusarium. When tested in vitro these fungal isolates significantly decreased germination rates in Z. mays (Fig. 2) and showed high percentages of root necrosis. In addition to plant pathogens, mutualistic fungi can also be associated with germination success and seedling survival. In this study, four Cladosporium isolates significantly increased germination rates and root growth in both Z. mays and D. cespitosa (Figs. 2 and 3). Both D. cespitosa and G. rossii are dominant plants, accounting for 60% of the cover at Niwot (Farrer et al., 2015). All the Cladosporium isolates in our study were cultured from only G. rossii and D. cespitosa accounting for 70% of the fungi cultured from D. cespitosa and 37.5% of the fungi cultured from G. rossii (Table S1). Having a beneficial relationship with growth promoting fungi could be advantageous for alpine plants given the harsh environmental conditions and a very short growing season. Faster germination rates with production of roots could have a bigger impact on quick establishment of seedlings and associations with Cladosporium in seeds and could contribute to the dominance of these two plant species. Acknowledgments This project was funded by the National Science Foundation (APA, KS, RS NSF-DEB 919510; KS, NSF-DEB 1637686) and a Western Illinois University Graduate and Professional Research Grant. In addition, we would like to thank three high school students Abraham Matlack, Ali Kerr, and Dexter Redenius who dedicated a summer helping with this project (APA, NSF-DEB 1314459). Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.funeco.2017.08.001. References Abdulsalaam, S., Shenge, K.C., 2011. Seed borne pathogens on farmer-saved sorghum (Sorghum bicolor L.) seeds. J. Stored Prod. Postharvest Res. 2, 24e28. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403e410. Anderson, P.K., Cunningham, A.A., Patel, N.G., Morales, F.J., Epstein, P.R., Daszak, P.,
T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18 2004. Emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers. Trends Ecol. Evol. 19, 535e544. Bain, D.C., 1950. Fungi recovered from seed of Sorghum vulgare Pers. Phytopathology 40, 521e522. Baker, K.F., Smith, S.H., 1966. Dynamics of seed transmission of plant pathogens. Annu. Rev. Phytopathol. 4, 311e332. Barrett, S.C., 2015. Influences of clonality on plant sexual reproduction. P. Natl. A. Sci. U. S. A. 112, 8859e8866. Baskin, J.M., Baskin, C.C., 2004. A classification system for seed dormancy. Seed Sci. Res. 14, 1e16. Baskin, J.M., Baskin, C.C., 2000. Evolutionary considerations of claims for physical dormancy-break by microbial action and abrasion by soil particles. Seed Sci. Res. 10, 409e414. Bell, K.L., Bliss, L.C., 1980. Plant reproduction in a high arctic environment. Arct. Alp. Res. 12, 1e10. Bierzychudek, P., 1985. Patterns in parthenogenesis. Experientia 41, 1255e1264. Billings, W.D., Mooney, H.A., 1968. The ecology of arctic and alpine plants. Biol. Rev. 43, 481e529. Burgess, L.W., Bryden, W.L., 2012. Fusarium: a ubiquitous fungus of global significance. Microbiol. Aust. 33.1, 22e25. Clay, K., 1989. Clavicipitaceous endophytes of grasses: their potential as biocontrol agents. Mycol. Res. 92, 1e12. Clay, K., 1990. The impact of parasitic and mutualistic fungi on competitive interactions among plants. In: Grace, J.B., Tilman, D. (Eds.), Perspectives on Plant Competition. Academic Press Inc, San Diego, pp. 391e407. Clear, R.M., Patrick, S.K., Gaba, D., 2000. Prevalence of fungi and fusariotoxins on barley seed from western Canada. Can. J. Plant Pathol 22, 44e50. Crous, P.W., Schoch, C.L., Hyde, K.D., Wood, A.R., Gueidan, C., De Hoog, G.S., Groenewald, J.Z., 2009. Phylogenetic lineages in the Capnodiales. Stud. Mycol 64, 17e47. Dalling, J.W., Davis, A.S., Schutte, B.J., Arnold, A.E., 2011. Seed survival in soil: interacting effects of predation, dormancy, and soil microbial community. J. Ecol. 99, 89e95. Dean, S.L., Farrer, E.C., Taylor, D.L., Porras-Alfaro, A., Suding, K.N., Sinsabaugh, R.L., 2014. Nitrogen deposition alters plant-fungal relationships: linking belowground dynamics to aboveground vegetation change. Mol. Ecol. 23, 1364e1378. Dhingra, O.D., Maia, C.B., Lustosa, D.C., Mesquita, J.B., 2002. Seedborne pathogenic fungi that affect seedling quality of red angico (Anadenanthera macrocarpa) trees in Brazil. J. Phytopathol. 150, 451e455. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792e1797. Elias, S., Garay, A., Schweitzer, L., Hanning, S., 2006. Seed quality testing of native species. Native Plants J. 7, 15e19. Farrer, E.C., Ashton, I.W., Spasojevic, M.J., Fu, S., Gonzalez, D.J.X., Suding, K.N., 2015. Indirect effects of global climate change accumulate to alter plant diversity but not ecosystem function in alpine tundra. 2015. J. Ecol. 103, 351e360. Forbis, T.A., 2003. Seedling demography in an alpine ecosystem. Am. J. Bot. 90, 1197e1206. Gallery, R.E., Dalling, J.W., Arnold, A.E., 2007. Diversity, host affinity, and distribution of seed-infecting fungi: a case study with Cecropia. Ecology 88, 582e588. Ganley, R.J., Newcombe, G., 2006. Fungal endophytes in seeds and needles of Pinus monticola. Mycol. Res. 110, 318e327. Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomyceteseapplication to the identification of mycorrhizae and rusts. Mol. Ecol. 2, 113e118. Greenland, D., 1989. The climate of NIWOT Ridge, Front Range, Colorado, USA. Arct. Alp. Res. 21, 380e391. Hamayun, M., Khan, S.A., Ahmad, N., Tang, D.S., Kang, S.M., Na, C.I., Sohn, E.Y., Hwang, Y.H., Shin, D.H., Lee, B.H., Kim, J.G., Lee, I.J., 2009. Cladosporium sphaerospermum as a new plant growth-promoting endophyte from the roots of Glycine max (L.) Merr. World J. Microbiol. Biotechnol. 25, 627e632. Hamayun, M., Khan, S.A., Khan, A.L., Rehman, G., Kim, Y.H., Iqbal, I., Hussain, J., Sohn, E.Y., Lee, I.J., 2010. Gibberellin production and plant growth promotion from pure cultures of Cladosporium sp MH-6 isolated from cucumber (Cucumis sativus L.). Mycologia 102, 989e995. Hauser, S., 2007. Plantain (Musa spp. AAB) bunch yield and root health responses to combinations of physical and chemical sucker sanitation measures. Afr. Plant Prot. 13, 1e15. Higgins, K.L., Arnold, A.E., Miadlikowska, J., Sarvate, S.D., Lutzoni, F., 2007. Phylogenetic relationships, host affinity, and geographic structure of boreal and arctic endophytes from three major plant lineages. Mol. Phylogenet. Evol. 42, 543e555. Hodgson, S., De Cates, C., Hodgson, J., Morley, N.J., Sutton, B.C., Gange, A.C., 2014. Vertical transmission of fungal endophytes is widespread in forbs. Ecol. Evol. 4, 1199e1208. Hoffman, M.T., Arnold, A.E., 2008. Geographic locality and host identity shape fungal endophyte communities in cupressaceous trees. Mycol. Res. 112, 331e344. Hoffman, M.T., Gunatilaka, M.K., Wijeratne, K., Gunatilaka, L., Arnold, A.E., 2013. Endohyphal bacterium enhances production of indole-3-acetic acid by a foliar fungal endophyte. PLoS One 8, e73132. Johnson, L.J., Miller, A.N., McCleery, R.A., McClanahan, R., Kath, J.A., Lueschow, S., Porras-Alfaro, A., 2013. Psychrophilic and psychrotolerant fungi on bats and the presence of Geomyces spp. on bat wings prior to the arrival of white nose syndrome. Appl. Environ. Microbiol. 79, 5465e5471.
17
Klironomos, J.N., 2002. Feedback with soil biota contributes to plant rarity and invasiveness in communities. Nature 417, 67e70. Leuchtmann, A., 1992. Systematics, distribution, and host specificity of grass endophytes. Nat. Toxins 1, 150e162. Malone, J.P., Muskett, A.E., 1997. Seed-borne Fungi, Description of 77 Fungus Species. The International Seed Testing Association, Zurich. Mandyam, K.G., Roe, J., Jumpponen, A., 2013. Arabidopsis thaliana model system reveals a continuum of responses to root endophyte colonization. Fungal Biol. 117, 250e260. McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L., Swan, J.A., 1990. A new method which gives an objective measure of colonization of roots by vesiculararbuscular mycorrhizal fungi. New Phytol. 115, 495e501. McLennan, E.T., 1920. Endophytic fungus of Lolium: part 1. Proc. R. Soc. Vic. 32, 252e301. Newton, A.C., Fitt, B.D.L., Atkins, S.D., Walters, D.R., Daniell, T.J., 2010. Pathogenesis, parasitism and mutualism in the trophic space of microbe-plant interactions. TIM 18, 365e373. Noble, M.J., De Tempe, J., Neergaard, P., 1958. An Annotated List of Seed-borne Diseases. Surrey, England, CMI, Kew. Porras-Alfaro, A., Bayman, P., 2011. Hidden fungi, emergent properties: endophytes and microbiomes. Annu. Rev. Phytopathol. 49, 291e315. Raj, S.N., Deepak, S.A., Basavaraju, P., Shetty, S.H., Reddy, M.S., Kloepper, J.W., 2003. Comparative performance of formulations of plant growth promoting rhizobacteria in growth promotion and suppression of downy mildew in pearl millet. Crop Prot. 22, 579e588. Ravel, C., Michalakis, Y., Charmet, G., 1997. The effect of imperfect transmission on the frequency of mutualistic seed-borne endophytes in natural populations of grasses. Oikos 80, 18e24. Reynolds, H.L., Packer, A., Bever, J.D., Clay, K., 2003. Grassroots ecology: plantmicrobe-soil interactions as drivers of plant community structure and dynamics. Ecology 84, 2281e2291. Rodriguez, R.J., White, J.F., Arnold, A.E., Redman, R.S., 2009. Fungal endophytes: diversity and functional roles. New Phytol. 182, 314e330. Rudgers, J.A., Koslow, J.M., Clay, K., 2004. Endophytic fungi alter relationships between diversity and ecosystem properties. Ecol. Lett. 7, 42e51. Saikkonen, K., Saari, S., Helander, M., 2010. Defensive mutualism between plants and endophytic fungi. Fungal Divers 41, 101e113. €li, P., Helander, M., Faeth, S.H., 2004. Evolution of endophyteeSaikkonen, K., Wa plant symbioses. Trends Plant Sci. 9, 275e280. Sanchez-Delgado, P., Ortega-Amaro, M., Jimenez-Bremont, J., Flores, J., 2011. Are fungi important for breaking seed dormancy in desert species? Experimental evidence in Opuntia streptacantha (Cactaceae). Plant Biol. 13, 154e159. Sanchez-Marquez, S., Bills, G.F., Herrero, N., Zabalgogeazcoa, I., 2012. Non-systemic fungal endophytes of grasses. Fungal Ecol. 5, 289e297. Soytong, K., Kanokmedhakul, S., Kukkongviriyapa, V., Isobe, M., 2001. Application of Chaetomium species (Ketomium(R)) as a new broad spectrum biological fungicide for plant disease control: a review article. Fungal Divers 7, 1e15. Stone, J.K., Bacon, C.W., White, J.F., 2000. An overview of endophytic microbes: endophytism defined. In: Bacon, C.W., White, J.F. (Eds.), Microbial Endophytes. Marcel Dekker Inc., New York, pp. 3e30. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis. Mol. Biol. Evol. 30, 2725e2729, version 6.0. . ~ lme, S., Ko ~ljalg, U., Yorou, N.S., Wijesundera, R., Tedersoo, L., Bahram, M., Po Ruiz, L.V., Vasco-Palacios, A.M., Thu, P.Q., Suija, A., Smith, M.E., 2014. Global diversity and geography of soil fungi. Science 346, 1256688. Tillet, M., 1937. Dissertation on the cause of the corruption and smutting of the kernels of wheat in the head and the means of preventing these untoward circumstances. Phytopathol. Class. 5, 1e150. Timling, I., Walker, D.A., Nusbaum, C., Lennon, N.J., Taylor, D.L., 2014. Rich and cold: diversity, distribution, and drivers of fungal communities in patterned-ground ecosystems of the North American Arctic. Mol. Ecol. 23, 3258e3272. Tveit, M., Wood, R.K.S., 1955. The control of Fusarium blight in oat seedlings with antagonistic species of Chaetomium. Ann. Appl. Biol. 43, 538e552. U'Ren, J.M., Dalling, J.W., Gallery, R.E., Maddison, D.R., Davis, E.C., Gibson, C.M., Arnold, A.E., 2009. Diversity and evolutionary orgins of fungi associated with seeds of a neotropical pioneer tree: a case study for analyzing fungal environmental samples. Mycol. Res. 113, 432e449. Van Der Putten, W.H., Peters, B.A.M., 1997. How soil-borne pathogens may affect plant competition. Ecology 78, 1785e1795. Vazquez-de-Aldana, B.R., Garcia-Criado, B., Vicente-Tavera, S., Zabalgogeazcoa, I., 2013. Fungal endophyte (Epichlo€ e festucae) alters the nutrient content of Festuca rubra regardless of water availability. PLoS One 8, e84539. Vielerheilig, H., Coughlan, A.P., Wyss, U., Piche, Y., 1998. Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Appl. Environ. Microbiol. 64, 5004e5007. Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261e5267. €cklin, J., 2006. The relative importance of sexual and clonal Weppler, T., Stoll, P., Sto reproduction for population growth in the long-lived alpine plant Geum reptans. J Ecol 94, 869e879. White, J.F., Baldwin, N.A., 1992. A preliminary enumeration of grass endophytes in west central England. Sydowia 44, 78e84. White, T.J., Bruns, T.D., Lee, S.B., Taylor, J.W., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A.,
18
T. Billingsley Tobias et al. / Fungal Ecology 30 (2017) 10e18
Gelfand, D.H., Sninsky, J.J., White, T.J., protocols, P.C.R. (Eds.), A Guide to Methods and Applications. Academic Press, San Diego, pp. 315e322. Zalamea, P.C., Sarmiento, C., Arnold, A.E., Davis, A.S., Dalling, J.W., 2014. Do soil microbes and abrasion by soil particles influence persistence and loss of physical dormancy in seeds of tropical pioneers? Front. Plant Sci. 5, 799.
Zalar, P., De Hoog, G.S., Schroers, H.J., Groenewald, J.Z., Gunde-Cimerman, N., 2007. Phylogeny and ecology of the ubiquitous saprobe Cladosporium spaerospermum with descriptions of seven new species from hypersaline environments. Stud. Mycol. 58, 157e183.