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Expectations for habitat-adapted symbiosis in a winter annual grass
Fungal Ecology 29 (2017) 111e115
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Fungal Ecology journal homepage: www.elsevier.com/locate/funeco
Expectations for habitat-adapted symbiosis in a winter annual grass David L. Griffith a, c, *, Beau Larkin b, Andrew Kliskey c, Lilian Alessa c, George Newcombe d a
Environmental Science and Water Resources, University of Idaho, United States MPG Ranch, Florence, MT, United States c Center for Resilient Communities, University of Idaho, United States d Department of Forest, Rangeland, and Fire Sciences, University of Idaho, United States b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 April 2016 Received in revised form 30 May 2017 Accepted 18 July 2017 Available online 17 August 2017
The habitat-adapted symbiosis hypothesis predicts that the most positive effects of symbiosis are expected in the most stressful sites for a plant host. Stress varies with site characteristics but also during the life cycle of a plant, with winter annuals experiencing the most stress after fall emergence. For Bromus tectorum, fecundity can vary tremendously from a few to thousands of seeds per plant. We used endophytic Sordaria fimicola to test the hypothesis in three sites in western Montana. We hypothesized that the effects of S. fimicola inoculation would be most positive in the most stressful site after fall application. As predicted, the most positive effects on growth and fecundity were observed in the most stressful site after fall application of S. fimicola. However, the effects of treatments varied within and between sites considerably, and are best understood as an example of context-dependency in plantmicrobe interactions rather than habitat-adapted symbiosis. © 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
Corresponding Editor: Gareth W. Griffith Keywords: Coprophilous fungi Endophyte Habitat-adapted symbiosis Symbiosis Winter annual
Endophytic fungi have been isolated from above- and belowground tissues of all plants examined thus far (Arnold et al., 2000). Endophytes are phylogenetically diverse, but apart from well-studied examples, their ecological functions are largely hypothetical. One hypothesis regarding endophyte function is that of ‘habitat-adapted symbiosis’ (Rodriguez et al., 2009). According to this hypothesis, some non-clavicipitaceous, systemic endophytes help or enable plants to grow in unproductive, high-stress habitats. Habitat-adapted symbiosis is hypothesized to be critical or even obligate for plants in these habitats (Rodriguez et al., 2008). An outstanding example is that of the complex symbiosis in geothermally heated soil that involves a plant, a fungus, and a virus (Marquez et al., 2007). Another is the increased fitness of Oryza sativa in saline environments that has been attributed to infection by endophytic Fusarium culmorum (Rodriguez et al., 2004; Rodriguez and Redman, 2008; Redman et al., 2011). Similarly, drought is an abiotic stress that can be alleviated by
endophytes (Rodriguez et al., 2008). Disease represents a more complex biotic stress for plants, because the severity of a given disease can be modified by a large number of endophytes in either direction, and a single plant can simultaneously experience multiple diseases (Busby et al., 2015). However, for abiotic stresses such as heat and drought, a generalized form of the habitat-adapted symbiosis hypothesis of Rodriguez et al., (2009) can be stated as follows: the positive effects of endophyte-plant symbiosis are expected to be greatest in the most stressful environmental conditions a plant can tolerate. Stress for plants varies not only along abiotic, environmental gradients, but also phenologically during the life cycle of a plant. Winter annual grasses, such as Bromus japonicus and Bromus tectorum, experience greatest stress during the winter after fall germination (Baskin and Baskin, 1981; Mack and Pyke, 1984). This is likely to mean that the timing of endophyte infection matters, although most of the literature relating to this area is focused on order of assembly of members of the symbiont community rather than on life cycle of hosts. For example, the order of assembly of microbial communities can produce endophyte-pathogen in teractions that vary from inhibitory to facilitative (Adame-Alvarez
* Corresponding author. University of Idaho, 875 Perimeter Dr. MS 2481, Moscow, ID, 83844-2481, United States. E-mail address: griffi[email protected] (D.L. Griffith).
et al., 2014), or it can affect microbiome structure (Pan and May 2009; Cannon and Simmons, 2002). In our test of the habitat-adapted symbiosis hypothesis, we
1. Introduction
http://dx.doi.org/10.1016/j.funeco.2017.07.003 1754-5048/© 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
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simply predicted first that fall applications of a symbiont would affect B. tectorum more positively than spring applications within a given site. Secondly, we predicted that as site productivity increased for B. tectorum symbiont effects would become less positive. In other words, we predicted that the most positive overall effect would follow fall application in the least productive site. We employed a strain of Sordaria fimicola (CID 323) that was previously shown to increase growth and fecundity of B. tectorum (Newcombe et al., 2016), an invasive annual grass in western North America. Three sites with different biotic and abiotic stress characteristics, predicted to vary in B. tectorum productivity, were chosen at a location in the Bitterroot Valley, MT, and sites were divided into plots which were treated as uninoculated controls and others in which B. tectorum plants were inoculated in either fall or spring with S. fimicola spore suspension. 2. Materials and methods 2.1. Isolation and culturing of S. fimicola (CID 323) The Moscow strain of S. fimicola, designated as CID 323, was isolated from B. tectorum plants collected on Moscow Mountain (near W Twin Rd, 46 480 45.700 N 116 55011.000 W) at approximately 1,280 m elevation at a site with minimal slope, an aspect of ~300, on the roadside in a mixed conifer stand where B. tectorum was estimated at < 5% cover. Endophytic S. fimicola was isolated from surface-sterilized plant tissue incubated on potato dextrose agar (PDA) using methods previously described (Baynes et al., 2012a; Newcombe et al., 2016). Cultures were maintained by subculturing onto PDA from reference cultures stored at 4 C. Each plate of S. fimicola was examined microscopically (for appropriately formed and colored asci and ascospores) when producing perithecia to ensure that cultures were viable. Cultures with malformed or discolored perithecia, asci, or ascospores were discarded. 2.2. Choice of field sites Field plots were established at three different sites (Table 1) on MPG Ranch land in the Bitterroot Valley, MT. The site judged to be the least stressful, or most productive, for B. tectorum was at the bottom of a wide draw with a 2% slope. The draw collects water from surrounding upland areas in the spring, and soils at this site are loamy in texture and contain little to no gravel to a depth of 20 cm. The site chosen as moderately stressful was more westerly (SSW) and mid-slope on the side of a wide draw, with more afternoon sun and gravelly sandy loam. The most stressful site was the steepest, with soils and aspect similar to the moderate site, but with more exposure to wind and sun than the other sites. It had the highest percentage of gravel and bare ground. To these site observations was added an assessment of soil resistance to root
penetration at each site with a soil hammer penetrometer. In the center of nine sub-plots at each site, we counted the number of hammer strikes needed to drive a 1.12 cm steel bar to a depth of 20 cm (Donaldson, 1986), in the expectation that soil resistance to root penetration would be inversely correlated with growth and fecundity of B. tectorum. In order to reach 20 cm, the least stressful site required an average of 12 strikes of the hammer; the moderately stressful site required an average of 19 strikes of the hammer; and the most stressful site required an average of 31 strikes of the hammer. 2.3. Inoculation of B. tectorum with S. fimicola in the field Inoculum was produced by incubating cultures on PDA at ambient laboratory conditions (20 C with a 10:14 light:dark photoperiod) for 28 d. Petri dishes with mature perithecia were scraped into sterile deionized water (SDW) and shaken to form a suspension of spores and mycelium. In total, 14 plates of mature S. fimicola were suspended in 5 L SDW. Final concentration of spores and mycelial fragments were estimated using a hemocytometer at ~1.05 million fungal units per mL of SDW. Each site was divided into 9 sub-plots of 3 5 m, with 1 m strips separating sub-plots, and treatments assigned to sub-plots so that each row and column contained each treatment level (control, spring application, and fall application). In early December 2012, after germination and emergence of B. tectorum was confirmed at the field sites, and there was sufficient precipitation (primarily light snow) to ensure constant leaf moisture, S. fimicola inoculum was applied to the three fall application sub-plots at each of the three sites at a rate of approximately 0.5 L per sub-plot with a backpack sprayer. In March of 2013, after snowmelt and during a period of continuous light rain, S. fimicola inoculum was applied to a further three sub-plots of each site at a rate of approximately 0.5 L per subplot with a backpack sprayer (with inoculum prepared in the same manner and quantities as described above). The final three subplots of each of the three sites were treated as uninoculated controls (treated with SDW rather than S. fimicola). Infection was confirmed by isolating S. fimicola from leaf samples from treatment plots using standard surface sterilization and plating on potato dextrose agar (c.f., Baynes et al., 2012a; Newcombe et al., 2016). 2.4. Observations and sampling Beginning in early summer, weekly observations of plots were made to monitor progress towards seed ripening and plant senescence in B. tectorum. When plants had dried sufficiently that little additional growth was expected, but before seed shatter, 5 cm 10 cm cotton bags were tied around at least 100 inflorescences per sub-plot to collect the seeds. We left the plants to
Table 1 Site descriptions. Site
Aspect ( ) Elevation (m) Slope (%) Plant Species (>5%)
Cover Estimates
Least Stressful
170
1113
2
Moderately Stressful 205
1200
11
bare ground (5%) annual grass monoculture litter (40%) bare ground (2%) shrubland litter (39%) moss/lichen (53%) gravel (3%)
Most Stressful
1177
15
204
Bromus tectorum (50%) Sisymbrium altissimum (5%) Poa bulbosa (18%) Hesperostipa comata (17%) Poa secunda (7%) Euphorbia esula (6%) Bromus tectorum (5%) Centaurea stoebe (5%) Bromus tectorum (41%) Hesperostipa comata (32%) Pseudoroegneria spicata (6%) Thinopyrum intermedium (6%)
Habitat-type
Average Penetrometer Strikes 12 19
bare ground (9%) non-irrigated grass plantation 31 litter (60%) moss/lichen (4%) gravel (12%)
D.L. Griffith et al. / Fungal Ecology 29 (2017) 111e115
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dry in the field, and then harvested all plots in the first week of August. With seed collection bags attached, plants were pulled from the ground and placed in paper envelopes. Despite the use of seed collection bags, for some plants seed shatter occurred earlier than expected, and all seeds were not collected. The harvested plants were dried for 72 h at 60 C. Seeds that remained attached to the plant were removed and roots were clipped just below the point of attachment of the lowest tiller. Aboveground biomass of plant tissue excluding seeds was recorded to 0.001 g, and these are the weights used in the analysis below. With plants that had not shattered prior to attachment of seed collection bags, the number of seeds (a measure of fecundity) were counted and seed mass was recorded to 0.001 g. When shatter had occurred prior to collection, the number of spikelets with glumes attached were counted and recorded as a proxy for seed number. We counted any seeds present and recorded seed mass to 0.001 g. Where estimates were not possible due to plant damage or other inability to discern seed counts, seed number was coded as missing (and not analyzed). 2.5. Statistical analysis Aboveground biomass (not including seed heads) and seed number per plant were each log-transformed (to remove heteroskedasticity). Total number of plants analyzed from the experiment was N ¼ 2,627, and the experiment was mildly unbalanced (due to damaged plants being discarded), with sub-plot average n ¼ 97 and a range from 92 to 106. A linear model [log(y) ¼ ax1 þ bx2 þ gx1x2 þ ε(x2:x1) þ ε; where y ¼ dependent variable, x1 ¼ site, and x2 ¼ treatment] was used to describe the experimental data, with treatment viewed as nested within site (as part of a strip-plot design). ANOVA was used to determine significance of site, treatment, and the interaction of site and treatment. Differences in treatment means within sites were analyzed using Tukey's HSD in order to reduce the chance of Type 1 error. All calculations were computed in R 2.14.0 (R Development Core Team, 2013).
Fig. 1. Effects of S. fimicola (CID323) on B. tectorum biomass (bars indicate means and whiskers indicate SEM; n~300 per treatment) at different sites (ranked from most to least stressful) and time of inoculation (Fall ¼ more stressful; Spring ¼ less stressful). Treatment means that were significantly different from control are marked with an * over the bar. All collections of biomass and measurements were made in August following inoculation (8 mo following Fall inoculation; 5 mo following Spring inoculations).
3. Results Growth and fecundity of B. tectorum, as represented by the aboveground biomass and number of seeds produced by individuals, were lowest in the most stressful site, higher in the moderately stressful site, and highest in the least stressful sites, respectively (Figs. 1 and 2). Uninoculated, control plants in the least stressful site had more than nine times the biomass and produced ~7.5 times as many seeds, on average, as those in the control plants in the most stressful site (see ‘Mean Weight’ in Table S1). As expected, there were statistically significant differences in biomass (F ¼ 54.557, df ¼ 2, p < 0.0001) and number of seeds (F ¼ 66.123, df ¼ 2, p < 0.0001) per plant at the site level, but the interaction of site and treatment was not significant for either measure across the entire experiment. There were differences between treatment means within sites, sometimes of great magnitude (e.g., the difference between Fall and Control biomass was þ48.96% in the most stressful site) (Figs. 1 and 2; Table S1). Tukey's honest significant difference test, which is conservative in respect to Type 1 error, revealed significant differences in treatment means in 8 of 12 treatment groups when compared to controls from the same sites (p values are given in Table S1). 4. Discussion Results for this rough test of the habitat-adapted symbiosis hypothesis were mixed. On the one hand, we confirmed the
Fig. 2. Effect of S. fimicola (CID323) on B. tectorum fecundity (number of seeds per plant, where bars indicate means and whiskers indicate SEM; n~300 per treatment) at different sites (ranked from most to least stressful) and time of inoculation (Fall ¼ more stressful; Spring ¼ less stressful). Treatment means that were significantly different from control are marked with an * over the bar. All collections of biomass and measurements were made in August following inoculation (8 mo following Fall inoculation; 5 mo following Spring inoculations).
hypothesis in that the most positive effect overall followed fall application of S. fimicola in the most stressful site: 49% and 39% greater growth and fecundity, respectively, than uninoculated controls (Table S1). Fall application in the moderately stressful site also was followed by a more positive response in fecundity than the response to spring application, as predicted. However, in the least stressful site, the growth and fecundity responses to fall application were negative whereas they were positive after spring application of S. fimicola. In other words, these results were the opposite of
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those predicted by the habitat-adapted symbiosis hypothesis unless we were to modify our understanding of the hypothesis in such a way that it only applies to sites like our two most stressful ones. The problem with that arbitrary inference is that all three of these sites were likely marginal for B. tectorum. Range-wide, individual plants are reported to produce between 25 and 5000 seeds, with average plants in good conditions producing 300e400 seeds (Zouhar, 2003). This suggests that with 30 seeds per uninoculated plant (Fig. 2) even our least stressful site was at the lower end of a regional productivity gradient for B. tectorum. B. tectorum has been an invasive species in western North America for more than a century (Piemeisel, 1938; Mack, 2011) and is thus quite well studied for a winter annual grass in the range. Research into its endophytes is newer, but novel, mutualistic relationships have been found that confer greater thermotolerance and increased growth and fecundity to the plant (Baynes et al., 2012a). We have also shown that a fungivorous nematode can ‘farm’ an endophytic fungus within B. tectorum without affecting the plant (Baynes et al., 2012b). More recently, we have shown that S. fimicola and two other dung fungi can have negative effects on B. tectorum biomass and fecundity in greenhouse experiments that are likely to be relatively ‘unstressful’ (Newcombe et al., 2016). Despite its status as a model genetic organism, the functions of endophytic S. fimicola are poorly studied. It now has a cosmopolitan distribution (Haberle et al., 2015), although its range prior to the age of biotic homogenization is unknown (Newcombe and Dugan, 2010). In prior experiments, we demonstrated that S. fimicola can have multiple lifestyles (endophytic, saprophytic, and coprophilous). This latter inference is supported by other studies. Isolated as an endophyte from maize, S. fimicola can act as a weak pathogen (Chambers and De Wet, 1987), and it has been shown to increase biomass and reduce disease effects in wheat and rye-grass (Dewan et al. 1994). Our isolate of S. fimicola fits the description of a ‘Class-2 endophyte’ (Rodriguez et al., 2009), that is expected to be involved in habitat-adapted symbioses with hosts because it can infect both roots and shoots and become systemic (Newcombe et al., 2016). Whereas our results appear mixed with regard to the habitatadapted symbiosis hypothesis, they are in line with studies demonstrating the context-dependency of endophytic and mycorrhizal fungi, and plant symbionts generally. Clavicipitaceous, grass endophytes are usually described as being defense mutualists, but they have been shown to have other positive effects on host fitness, including increasing host growth and competitive ability, under certain environmental conditions (Marks et al., 1991). For example, Neotyphodium coenophialum has been implicated in helping an exotic grass (Lolium arundinaceum) establish in existing plant communities in a novel range (Rudgers et al., 2005). On the other hand, grass endophytes can decrease host fitness in some hosts (e.g., Festuca arizonica e Faeth and Sullivan, 2003) or under different herbivory or soil moisture levels (Saikkonen et al., 1999; Koh and Hik, 2007; Miranda et al., 2011). Environmental conditions and complex biotic interactions can cause a range of outcomes, from mutualistic to parasitic (Saikkonen et al., 2004, 2013; Schardl et al., 2004). Context-dependency may have to be analyzed on a case-by-case basis. For example, it has been shown that Epichlo€ e festucae can have either positive or negative effects on Festuca rubra in Sweden; in dry, nutrient-rich conditions the endophyte increased plant productivity, while in wetter or poorer soils infected plants fared poorly compared to uninfected plants (Saona et al., 2010). Schulz and Boyle (2005) demonstrated that other non-clavicipitaceous endophytes cause disease symptoms when hosts are placed in conditions of extreme physical or environmental stress. These, and many more, examples of context-dependency are consistent with findings from plant-microbe studies generally (Johnson et al., 1997;
Kiss, 2003; Wardle et al., 2004; Heath and Tiffin, 2007; Hoeksema et al., 2010). Our results are consistent with other findings that context-dependency in plant-fungal interactions is best seen as the rule, rather than the exception. Acknowledgments Much of the work represented in this paper was funded by the United States Forest Service (Agreement 10-CR-11221632-182). Additional support was provided by MPG Ranch, the Environmental Sciences and Water Resources Program at the University of Idaho, the Center for Resilient Communities at the University of Idaho, and the Mountain Social Ecological Observatory Network (MtnSEON; Award No. DEB 1231233). We'd like to thank Frederick Olmos and Heather Baltes for assisting with processing samples in the lab and field. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.funeco.2017.07.003. References Adame-Alvarez, R.-A., Mendiola-Soto, J., Heil, M., 2014. Order of arrival shifts endophyte- pathogen interactions in bean from resistance induction to disease facilitation. FEMS Microbiol. Lett. 355, 100e107. Arnold, A.E., Maynard, Z., Gilbert, G., Coley, P.D., Kursar, T.A., 2000. Are tropical fungal endophytes hyperdiverse? Ecol. Lett. 3, 267e274. Baskin, J.M., Baskin, C.C., 1981. Ecology of germination and flowering in the weedy winter annual grass Bromus japonicus. J. Range Manag. 34, 369e372. Baynes, M., Newcombe, G., Dixon, L., Castlebury, L., O'Donnell, K., 2012a. A novel plant- fungal mutualism associated with fire. Fungal Biol. 116, 133e144. Baynes, M., Russell, D.M., Newcombe, G., Carta, L.K., Rossman, A.Y., Ismaiel, A., 2012b. A mutualistic interaction between a fungivorous nematode and a fungus within the endophytic community of Bromus tectorum. Fungal Ecol. 5, 610e623. Busby, P.E., Ridout, M., Newcombe, G., 2015. Fungal endophytes: modifiers of plant disease. Plant Mol. Biol. 1e11. Cannon, P.F., Simmons, C.M., 2002. Diversity and host preference of leaf endophytic fungi in the Iwokrama Forest Reserve, Guyana. Mycologia 94, 210e220. Chambers, K.R., De Wet, D.C., 1987. Isolation of Sordaria fimicola from maize stalks. J. Phytopathol. 120, 369e371. Dewan, M., Ghisalbertib, E., Rowland, C., Sivasithamparam, K., 1994. Reduction of symptoms of take-all of wheat and rye-grass seedlings by the soil-borne fungus Sordaria fimicola. Appl. Soil Ecol. 1, 45e51. Donaldson, C.H., 1986. Simple technique for estimating soil compaction. Karoo Agric. 3, 55e58. Faeth, S.H., Sullivan, T.J., 2003. Mutualistic asexual endophytes in a native grass are usually parasitic. Am. Nat. 161, 310e325. Haberle, S., Turney, C., Kershaw, P., Brook, B., 2015. Using dung fungi to interpret the decline and extinction of megaherbivores: problems and solutions. Quat. Sci. Rev. 110, 107e113. Heath, K.D., Tiffin, P., 2007. Context dependence in the coevolution of plant and rhizobial mutualists. Proc. R. Soc. Lond. B Biol. Sci. 274, 1905e1912. Hoeksema, J.D., Chaudhary, V.B., Gehring, C.A., Johnson, N.C., Karst, J., Koide, R.T., Pringle, A., Zabinski, C., Bever, J.D., Moore, J.C., Wilson, G.W., 2010. A metaanalysis of context-dependency in plant response to inoculation with mycorrhizal fungi. Ecol. Lett. 13, 394e407. Johnson, N.C., Graham, J.H., Smith, F.A., 1997. Functioning of mycorrhizal associations along the mutualismeparasitism continuum. New Phytol. 135, 575e585. Kiss, L., 2003. A review of fungal antagonists of powdery mildews and their potential as biocontrol agents. Pest Manag. Sci. 59, 475e483. Koh, S., Hik, D.S., 2007. Herbivory mediates grass-endophyte relationships. Ecology 88, 2752e2757. Mack, R.N., 2011. Fifty years of ‘waging war on cheatgrass’: research advances, while meaningful control languishes. In: Richardson, D.M. (Ed.), Fifty Years of Invasion Ecology: the Legacy of Charles Elton. Blackwell Publishing Ltd, Hoboken, NJ, pp. 253e265. Mack, R.N., Pyke, D.A., 1984. The demography of Bromus tectorum: the role of microclimate, grazing and disease. J. Ecol. 72, 731e748. Marks, S., Clay, K., Cheplick, G.P., 1991. Effects of fungal endophytes on interspecific and intraspecific competition in the grasses Festuca arundinacea and Lolium perenne. J. Appl. Ecol. 28, 194e204. Marquez, L.M., Redman, R.S., Rodriguez, R.J., Roossinck, M.J., 2007. A virus in a fungus in a plant: three-way symbiosis required for thermal tolerance. Science 315, 513e515. Miranda, M.I., Omacini, M., Chaneton, E.J., 2011. Environmental context of
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Contents lists available at ScienceDirect
Fungal Ecology journal homepage: www.elsevier.com/locate/funeco
Expectations for habitat-adapted symbiosis in a winter annual grass David L. Griffith a, c, *, Beau Larkin b, Andrew Kliskey c, Lilian Alessa c, George Newcombe d a
Environmental Science and Water Resources, University of Idaho, United States MPG Ranch, Florence, MT, United States c Center for Resilient Communities, University of Idaho, United States d Department of Forest, Rangeland, and Fire Sciences, University of Idaho, United States b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 April 2016 Received in revised form 30 May 2017 Accepted 18 July 2017 Available online 17 August 2017
The habitat-adapted symbiosis hypothesis predicts that the most positive effects of symbiosis are expected in the most stressful sites for a plant host. Stress varies with site characteristics but also during the life cycle of a plant, with winter annuals experiencing the most stress after fall emergence. For Bromus tectorum, fecundity can vary tremendously from a few to thousands of seeds per plant. We used endophytic Sordaria fimicola to test the hypothesis in three sites in western Montana. We hypothesized that the effects of S. fimicola inoculation would be most positive in the most stressful site after fall application. As predicted, the most positive effects on growth and fecundity were observed in the most stressful site after fall application of S. fimicola. However, the effects of treatments varied within and between sites considerably, and are best understood as an example of context-dependency in plantmicrobe interactions rather than habitat-adapted symbiosis. © 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
Corresponding Editor: Gareth W. Griffith Keywords: Coprophilous fungi Endophyte Habitat-adapted symbiosis Symbiosis Winter annual
Endophytic fungi have been isolated from above- and belowground tissues of all plants examined thus far (Arnold et al., 2000). Endophytes are phylogenetically diverse, but apart from well-studied examples, their ecological functions are largely hypothetical. One hypothesis regarding endophyte function is that of ‘habitat-adapted symbiosis’ (Rodriguez et al., 2009). According to this hypothesis, some non-clavicipitaceous, systemic endophytes help or enable plants to grow in unproductive, high-stress habitats. Habitat-adapted symbiosis is hypothesized to be critical or even obligate for plants in these habitats (Rodriguez et al., 2008). An outstanding example is that of the complex symbiosis in geothermally heated soil that involves a plant, a fungus, and a virus (Marquez et al., 2007). Another is the increased fitness of Oryza sativa in saline environments that has been attributed to infection by endophytic Fusarium culmorum (Rodriguez et al., 2004; Rodriguez and Redman, 2008; Redman et al., 2011). Similarly, drought is an abiotic stress that can be alleviated by
endophytes (Rodriguez et al., 2008). Disease represents a more complex biotic stress for plants, because the severity of a given disease can be modified by a large number of endophytes in either direction, and a single plant can simultaneously experience multiple diseases (Busby et al., 2015). However, for abiotic stresses such as heat and drought, a generalized form of the habitat-adapted symbiosis hypothesis of Rodriguez et al., (2009) can be stated as follows: the positive effects of endophyte-plant symbiosis are expected to be greatest in the most stressful environmental conditions a plant can tolerate. Stress for plants varies not only along abiotic, environmental gradients, but also phenologically during the life cycle of a plant. Winter annual grasses, such as Bromus japonicus and Bromus tectorum, experience greatest stress during the winter after fall germination (Baskin and Baskin, 1981; Mack and Pyke, 1984). This is likely to mean that the timing of endophyte infection matters, although most of the literature relating to this area is focused on order of assembly of members of the symbiont community rather than on life cycle of hosts. For example, the order of assembly of microbial communities can produce endophyte-pathogen in teractions that vary from inhibitory to facilitative (Adame-Alvarez
* Corresponding author. University of Idaho, 875 Perimeter Dr. MS 2481, Moscow, ID, 83844-2481, United States. E-mail address: griffi[email protected] (D.L. Griffith).
et al., 2014), or it can affect microbiome structure (Pan and May 2009; Cannon and Simmons, 2002). In our test of the habitat-adapted symbiosis hypothesis, we
1. Introduction
http://dx.doi.org/10.1016/j.funeco.2017.07.003 1754-5048/© 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
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simply predicted first that fall applications of a symbiont would affect B. tectorum more positively than spring applications within a given site. Secondly, we predicted that as site productivity increased for B. tectorum symbiont effects would become less positive. In other words, we predicted that the most positive overall effect would follow fall application in the least productive site. We employed a strain of Sordaria fimicola (CID 323) that was previously shown to increase growth and fecundity of B. tectorum (Newcombe et al., 2016), an invasive annual grass in western North America. Three sites with different biotic and abiotic stress characteristics, predicted to vary in B. tectorum productivity, were chosen at a location in the Bitterroot Valley, MT, and sites were divided into plots which were treated as uninoculated controls and others in which B. tectorum plants were inoculated in either fall or spring with S. fimicola spore suspension. 2. Materials and methods 2.1. Isolation and culturing of S. fimicola (CID 323) The Moscow strain of S. fimicola, designated as CID 323, was isolated from B. tectorum plants collected on Moscow Mountain (near W Twin Rd, 46 480 45.700 N 116 55011.000 W) at approximately 1,280 m elevation at a site with minimal slope, an aspect of ~300, on the roadside in a mixed conifer stand where B. tectorum was estimated at < 5% cover. Endophytic S. fimicola was isolated from surface-sterilized plant tissue incubated on potato dextrose agar (PDA) using methods previously described (Baynes et al., 2012a; Newcombe et al., 2016). Cultures were maintained by subculturing onto PDA from reference cultures stored at 4 C. Each plate of S. fimicola was examined microscopically (for appropriately formed and colored asci and ascospores) when producing perithecia to ensure that cultures were viable. Cultures with malformed or discolored perithecia, asci, or ascospores were discarded. 2.2. Choice of field sites Field plots were established at three different sites (Table 1) on MPG Ranch land in the Bitterroot Valley, MT. The site judged to be the least stressful, or most productive, for B. tectorum was at the bottom of a wide draw with a 2% slope. The draw collects water from surrounding upland areas in the spring, and soils at this site are loamy in texture and contain little to no gravel to a depth of 20 cm. The site chosen as moderately stressful was more westerly (SSW) and mid-slope on the side of a wide draw, with more afternoon sun and gravelly sandy loam. The most stressful site was the steepest, with soils and aspect similar to the moderate site, but with more exposure to wind and sun than the other sites. It had the highest percentage of gravel and bare ground. To these site observations was added an assessment of soil resistance to root
penetration at each site with a soil hammer penetrometer. In the center of nine sub-plots at each site, we counted the number of hammer strikes needed to drive a 1.12 cm steel bar to a depth of 20 cm (Donaldson, 1986), in the expectation that soil resistance to root penetration would be inversely correlated with growth and fecundity of B. tectorum. In order to reach 20 cm, the least stressful site required an average of 12 strikes of the hammer; the moderately stressful site required an average of 19 strikes of the hammer; and the most stressful site required an average of 31 strikes of the hammer. 2.3. Inoculation of B. tectorum with S. fimicola in the field Inoculum was produced by incubating cultures on PDA at ambient laboratory conditions (20 C with a 10:14 light:dark photoperiod) for 28 d. Petri dishes with mature perithecia were scraped into sterile deionized water (SDW) and shaken to form a suspension of spores and mycelium. In total, 14 plates of mature S. fimicola were suspended in 5 L SDW. Final concentration of spores and mycelial fragments were estimated using a hemocytometer at ~1.05 million fungal units per mL of SDW. Each site was divided into 9 sub-plots of 3 5 m, with 1 m strips separating sub-plots, and treatments assigned to sub-plots so that each row and column contained each treatment level (control, spring application, and fall application). In early December 2012, after germination and emergence of B. tectorum was confirmed at the field sites, and there was sufficient precipitation (primarily light snow) to ensure constant leaf moisture, S. fimicola inoculum was applied to the three fall application sub-plots at each of the three sites at a rate of approximately 0.5 L per sub-plot with a backpack sprayer. In March of 2013, after snowmelt and during a period of continuous light rain, S. fimicola inoculum was applied to a further three sub-plots of each site at a rate of approximately 0.5 L per subplot with a backpack sprayer (with inoculum prepared in the same manner and quantities as described above). The final three subplots of each of the three sites were treated as uninoculated controls (treated with SDW rather than S. fimicola). Infection was confirmed by isolating S. fimicola from leaf samples from treatment plots using standard surface sterilization and plating on potato dextrose agar (c.f., Baynes et al., 2012a; Newcombe et al., 2016). 2.4. Observations and sampling Beginning in early summer, weekly observations of plots were made to monitor progress towards seed ripening and plant senescence in B. tectorum. When plants had dried sufficiently that little additional growth was expected, but before seed shatter, 5 cm 10 cm cotton bags were tied around at least 100 inflorescences per sub-plot to collect the seeds. We left the plants to
Table 1 Site descriptions. Site
Aspect ( ) Elevation (m) Slope (%) Plant Species (>5%)
Cover Estimates
Least Stressful
170
1113
2
Moderately Stressful 205
1200
11
bare ground (5%) annual grass monoculture litter (40%) bare ground (2%) shrubland litter (39%) moss/lichen (53%) gravel (3%)
Most Stressful
1177
15
204
Bromus tectorum (50%) Sisymbrium altissimum (5%) Poa bulbosa (18%) Hesperostipa comata (17%) Poa secunda (7%) Euphorbia esula (6%) Bromus tectorum (5%) Centaurea stoebe (5%) Bromus tectorum (41%) Hesperostipa comata (32%) Pseudoroegneria spicata (6%) Thinopyrum intermedium (6%)
Habitat-type
Average Penetrometer Strikes 12 19
bare ground (9%) non-irrigated grass plantation 31 litter (60%) moss/lichen (4%) gravel (12%)
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dry in the field, and then harvested all plots in the first week of August. With seed collection bags attached, plants were pulled from the ground and placed in paper envelopes. Despite the use of seed collection bags, for some plants seed shatter occurred earlier than expected, and all seeds were not collected. The harvested plants were dried for 72 h at 60 C. Seeds that remained attached to the plant were removed and roots were clipped just below the point of attachment of the lowest tiller. Aboveground biomass of plant tissue excluding seeds was recorded to 0.001 g, and these are the weights used in the analysis below. With plants that had not shattered prior to attachment of seed collection bags, the number of seeds (a measure of fecundity) were counted and seed mass was recorded to 0.001 g. When shatter had occurred prior to collection, the number of spikelets with glumes attached were counted and recorded as a proxy for seed number. We counted any seeds present and recorded seed mass to 0.001 g. Where estimates were not possible due to plant damage or other inability to discern seed counts, seed number was coded as missing (and not analyzed). 2.5. Statistical analysis Aboveground biomass (not including seed heads) and seed number per plant were each log-transformed (to remove heteroskedasticity). Total number of plants analyzed from the experiment was N ¼ 2,627, and the experiment was mildly unbalanced (due to damaged plants being discarded), with sub-plot average n ¼ 97 and a range from 92 to 106. A linear model [log(y) ¼ ax1 þ bx2 þ gx1x2 þ ε(x2:x1) þ ε; where y ¼ dependent variable, x1 ¼ site, and x2 ¼ treatment] was used to describe the experimental data, with treatment viewed as nested within site (as part of a strip-plot design). ANOVA was used to determine significance of site, treatment, and the interaction of site and treatment. Differences in treatment means within sites were analyzed using Tukey's HSD in order to reduce the chance of Type 1 error. All calculations were computed in R 2.14.0 (R Development Core Team, 2013).
Fig. 1. Effects of S. fimicola (CID323) on B. tectorum biomass (bars indicate means and whiskers indicate SEM; n~300 per treatment) at different sites (ranked from most to least stressful) and time of inoculation (Fall ¼ more stressful; Spring ¼ less stressful). Treatment means that were significantly different from control are marked with an * over the bar. All collections of biomass and measurements were made in August following inoculation (8 mo following Fall inoculation; 5 mo following Spring inoculations).
3. Results Growth and fecundity of B. tectorum, as represented by the aboveground biomass and number of seeds produced by individuals, were lowest in the most stressful site, higher in the moderately stressful site, and highest in the least stressful sites, respectively (Figs. 1 and 2). Uninoculated, control plants in the least stressful site had more than nine times the biomass and produced ~7.5 times as many seeds, on average, as those in the control plants in the most stressful site (see ‘Mean Weight’ in Table S1). As expected, there were statistically significant differences in biomass (F ¼ 54.557, df ¼ 2, p < 0.0001) and number of seeds (F ¼ 66.123, df ¼ 2, p < 0.0001) per plant at the site level, but the interaction of site and treatment was not significant for either measure across the entire experiment. There were differences between treatment means within sites, sometimes of great magnitude (e.g., the difference between Fall and Control biomass was þ48.96% in the most stressful site) (Figs. 1 and 2; Table S1). Tukey's honest significant difference test, which is conservative in respect to Type 1 error, revealed significant differences in treatment means in 8 of 12 treatment groups when compared to controls from the same sites (p values are given in Table S1). 4. Discussion Results for this rough test of the habitat-adapted symbiosis hypothesis were mixed. On the one hand, we confirmed the
Fig. 2. Effect of S. fimicola (CID323) on B. tectorum fecundity (number of seeds per plant, where bars indicate means and whiskers indicate SEM; n~300 per treatment) at different sites (ranked from most to least stressful) and time of inoculation (Fall ¼ more stressful; Spring ¼ less stressful). Treatment means that were significantly different from control are marked with an * over the bar. All collections of biomass and measurements were made in August following inoculation (8 mo following Fall inoculation; 5 mo following Spring inoculations).
hypothesis in that the most positive effect overall followed fall application of S. fimicola in the most stressful site: 49% and 39% greater growth and fecundity, respectively, than uninoculated controls (Table S1). Fall application in the moderately stressful site also was followed by a more positive response in fecundity than the response to spring application, as predicted. However, in the least stressful site, the growth and fecundity responses to fall application were negative whereas they were positive after spring application of S. fimicola. In other words, these results were the opposite of
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those predicted by the habitat-adapted symbiosis hypothesis unless we were to modify our understanding of the hypothesis in such a way that it only applies to sites like our two most stressful ones. The problem with that arbitrary inference is that all three of these sites were likely marginal for B. tectorum. Range-wide, individual plants are reported to produce between 25 and 5000 seeds, with average plants in good conditions producing 300e400 seeds (Zouhar, 2003). This suggests that with 30 seeds per uninoculated plant (Fig. 2) even our least stressful site was at the lower end of a regional productivity gradient for B. tectorum. B. tectorum has been an invasive species in western North America for more than a century (Piemeisel, 1938; Mack, 2011) and is thus quite well studied for a winter annual grass in the range. Research into its endophytes is newer, but novel, mutualistic relationships have been found that confer greater thermotolerance and increased growth and fecundity to the plant (Baynes et al., 2012a). We have also shown that a fungivorous nematode can ‘farm’ an endophytic fungus within B. tectorum without affecting the plant (Baynes et al., 2012b). More recently, we have shown that S. fimicola and two other dung fungi can have negative effects on B. tectorum biomass and fecundity in greenhouse experiments that are likely to be relatively ‘unstressful’ (Newcombe et al., 2016). Despite its status as a model genetic organism, the functions of endophytic S. fimicola are poorly studied. It now has a cosmopolitan distribution (Haberle et al., 2015), although its range prior to the age of biotic homogenization is unknown (Newcombe and Dugan, 2010). In prior experiments, we demonstrated that S. fimicola can have multiple lifestyles (endophytic, saprophytic, and coprophilous). This latter inference is supported by other studies. Isolated as an endophyte from maize, S. fimicola can act as a weak pathogen (Chambers and De Wet, 1987), and it has been shown to increase biomass and reduce disease effects in wheat and rye-grass (Dewan et al. 1994). Our isolate of S. fimicola fits the description of a ‘Class-2 endophyte’ (Rodriguez et al., 2009), that is expected to be involved in habitat-adapted symbioses with hosts because it can infect both roots and shoots and become systemic (Newcombe et al., 2016). Whereas our results appear mixed with regard to the habitatadapted symbiosis hypothesis, they are in line with studies demonstrating the context-dependency of endophytic and mycorrhizal fungi, and plant symbionts generally. Clavicipitaceous, grass endophytes are usually described as being defense mutualists, but they have been shown to have other positive effects on host fitness, including increasing host growth and competitive ability, under certain environmental conditions (Marks et al., 1991). For example, Neotyphodium coenophialum has been implicated in helping an exotic grass (Lolium arundinaceum) establish in existing plant communities in a novel range (Rudgers et al., 2005). On the other hand, grass endophytes can decrease host fitness in some hosts (e.g., Festuca arizonica e Faeth and Sullivan, 2003) or under different herbivory or soil moisture levels (Saikkonen et al., 1999; Koh and Hik, 2007; Miranda et al., 2011). Environmental conditions and complex biotic interactions can cause a range of outcomes, from mutualistic to parasitic (Saikkonen et al., 2004, 2013; Schardl et al., 2004). Context-dependency may have to be analyzed on a case-by-case basis. For example, it has been shown that Epichlo€ e festucae can have either positive or negative effects on Festuca rubra in Sweden; in dry, nutrient-rich conditions the endophyte increased plant productivity, while in wetter or poorer soils infected plants fared poorly compared to uninfected plants (Saona et al., 2010). Schulz and Boyle (2005) demonstrated that other non-clavicipitaceous endophytes cause disease symptoms when hosts are placed in conditions of extreme physical or environmental stress. These, and many more, examples of context-dependency are consistent with findings from plant-microbe studies generally (Johnson et al., 1997;
Kiss, 2003; Wardle et al., 2004; Heath and Tiffin, 2007; Hoeksema et al., 2010). Our results are consistent with other findings that context-dependency in plant-fungal interactions is best seen as the rule, rather than the exception. Acknowledgments Much of the work represented in this paper was funded by the United States Forest Service (Agreement 10-CR-11221632-182). Additional support was provided by MPG Ranch, the Environmental Sciences and Water Resources Program at the University of Idaho, the Center for Resilient Communities at the University of Idaho, and the Mountain Social Ecological Observatory Network (MtnSEON; Award No. DEB 1231233). We'd like to thank Frederick Olmos and Heather Baltes for assisting with processing samples in the lab and field. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.funeco.2017.07.003. References Adame-Alvarez, R.-A., Mendiola-Soto, J., Heil, M., 2014. Order of arrival shifts endophyte- pathogen interactions in bean from resistance induction to disease facilitation. FEMS Microbiol. Lett. 355, 100e107. Arnold, A.E., Maynard, Z., Gilbert, G., Coley, P.D., Kursar, T.A., 2000. Are tropical fungal endophytes hyperdiverse? Ecol. Lett. 3, 267e274. Baskin, J.M., Baskin, C.C., 1981. Ecology of germination and flowering in the weedy winter annual grass Bromus japonicus. J. Range Manag. 34, 369e372. Baynes, M., Newcombe, G., Dixon, L., Castlebury, L., O'Donnell, K., 2012a. A novel plant- fungal mutualism associated with fire. Fungal Biol. 116, 133e144. Baynes, M., Russell, D.M., Newcombe, G., Carta, L.K., Rossman, A.Y., Ismaiel, A., 2012b. A mutualistic interaction between a fungivorous nematode and a fungus within the endophytic community of Bromus tectorum. Fungal Ecol. 5, 610e623. Busby, P.E., Ridout, M., Newcombe, G., 2015. Fungal endophytes: modifiers of plant disease. Plant Mol. Biol. 1e11. Cannon, P.F., Simmons, C.M., 2002. Diversity and host preference of leaf endophytic fungi in the Iwokrama Forest Reserve, Guyana. Mycologia 94, 210e220. Chambers, K.R., De Wet, D.C., 1987. Isolation of Sordaria fimicola from maize stalks. J. Phytopathol. 120, 369e371. Dewan, M., Ghisalbertib, E., Rowland, C., Sivasithamparam, K., 1994. Reduction of symptoms of take-all of wheat and rye-grass seedlings by the soil-borne fungus Sordaria fimicola. Appl. Soil Ecol. 1, 45e51. Donaldson, C.H., 1986. Simple technique for estimating soil compaction. Karoo Agric. 3, 55e58. Faeth, S.H., Sullivan, T.J., 2003. Mutualistic asexual endophytes in a native grass are usually parasitic. Am. Nat. 161, 310e325. Haberle, S., Turney, C., Kershaw, P., Brook, B., 2015. Using dung fungi to interpret the decline and extinction of megaherbivores: problems and solutions. Quat. Sci. Rev. 110, 107e113. Heath, K.D., Tiffin, P., 2007. Context dependence in the coevolution of plant and rhizobial mutualists. Proc. R. Soc. Lond. B Biol. Sci. 274, 1905e1912. Hoeksema, J.D., Chaudhary, V.B., Gehring, C.A., Johnson, N.C., Karst, J., Koide, R.T., Pringle, A., Zabinski, C., Bever, J.D., Moore, J.C., Wilson, G.W., 2010. A metaanalysis of context-dependency in plant response to inoculation with mycorrhizal fungi. Ecol. Lett. 13, 394e407. Johnson, N.C., Graham, J.H., Smith, F.A., 1997. Functioning of mycorrhizal associations along the mutualismeparasitism continuum. New Phytol. 135, 575e585. Kiss, L., 2003. A review of fungal antagonists of powdery mildews and their potential as biocontrol agents. Pest Manag. Sci. 59, 475e483. Koh, S., Hik, D.S., 2007. Herbivory mediates grass-endophyte relationships. Ecology 88, 2752e2757. Mack, R.N., 2011. Fifty years of ‘waging war on cheatgrass’: research advances, while meaningful control languishes. In: Richardson, D.M. (Ed.), Fifty Years of Invasion Ecology: the Legacy of Charles Elton. Blackwell Publishing Ltd, Hoboken, NJ, pp. 253e265. Mack, R.N., Pyke, D.A., 1984. The demography of Bromus tectorum: the role of microclimate, grazing and disease. J. Ecol. 72, 731e748. Marks, S., Clay, K., Cheplick, G.P., 1991. Effects of fungal endophytes on interspecific and intraspecific competition in the grasses Festuca arundinacea and Lolium perenne. J. Appl. Ecol. 28, 194e204. Marquez, L.M., Redman, R.S., Rodriguez, R.J., Roossinck, M.J., 2007. A virus in a fungus in a plant: three-way symbiosis required for thermal tolerance. Science 315, 513e515. Miranda, M.I., Omacini, M., Chaneton, E.J., 2011. Environmental context of
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