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Invisible but consequential: root endophytic fungi have variable effects on belowground plant–insect interactions HUIJIE GAN,1, ALICE C. L. CHURCHILL,2 AND KYLE WICKINGS1 1

Department of Entomology, NY State Agricultural Experiment Station, Cornell University, Geneva, New York 14456 USA 2 Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, New York 14853 USA

Citation: Gan, H., A. C. L. Churchill, and K. Wickings. 2017. Invisible but consequential: root endophytic fungi have variable effects on belowground plant–insect interactions. Ecosphere 8(3):e01710. 10.1002/ecs2.1710

Abstract. Endophytic fungi are ubiquitous in nature and can play important roles in regulating plant– herbivore interactions. While some aboveground obligate symbionts are considered defensive mutualists of host plants, the importance of root endophytes in plant defense, especially against root-feeding insects, remains unclear. This study aimed to investigate the effects of root fungal endophytes on plant resistance against belowground herbivores and the recovery of host plants from damage. We grew the common grass Festuca arundinacea (tall fescue) semi-aeroponically in the laboratory and inoculated roots with one of five fungal endophytes isolated from field-collected tall fescue or meadow soil. Endophyte-inoculated and uninoculated control plants were subjected to feeding by larvae of the generalist root herbivore Rhizotrogus majalis (European chafer). Herbivory intensity was quantified after eight days, and regrowth of roots and shoots following root herbivory and mechanical shoot damage was measured thereafter for each treatment. Fungal identifications by DNA sequence analysis were conducted after completion of the herbivory experiments and revealed that the five endophytes included the decomposer fungi Trametes versicolor and Mortierella alpina, the entomopathogenic fungi Isaria fumosorosea and Beauveria bassiana, and a potential plant pathogen/entomopathogen Fusarium cf. equiseti. The effects of these root endophytes on plant defense against root-feeding insects were species-specific. While four endophytes had few effects on plant resistance, endophytic B. bassiana significantly reduced herbivore damage to roots. In comparison, plant tolerance to damage was impaired after colonization by all endophyte species except T. versicolor and M. alpina. The contrasting effects of endophytes on plant resistance and plant tolerance suggest that research solely evaluating plant resistance is likely to overestimate the benefits conferred by endophytes without accounting for potential negative effects on plant tolerance. We propose a conceptual framework to include both plant resistance and tolerance as two dimensions of a defensive strategy and show that plant associations with different root endophytes may shift the relative importance of resistance and tolerance for plant defense. Key words: Beauveria bassiana; entomopathogenic fungi; plant resistance; plant tolerance; plant–herbivore interaction; root fungal endophyte; root herbivory; tall fescue. Received 15 January 2017; accepted 20 January 2017. Corresponding Editor: Debra P. C. Peters. Copyright: © 2017 Gan et al. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. E-mail: [email protected]

INTRODUCTION

2013). As common root symbionts, mycorrhizal fungi are known to cause major changes in plant growth and chemical composition (Smith and Read 2008), as well as to modify relationships between hosts and other organisms, including herbivores (Gehring and Bennett 2009, Koricheva

Plant-associated microbes are ubiquitous in nature, and many of them are essential to the adaptation of their host plants to specific environments (Rodriguez et al. 2008, Rout and Southworth ❖ www.esajournals.org

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larvae, with both positive and negative effects observed depending on the fungal and insect species involved (Gange et al. 1994, Gange 2001, Currie et al. 2011, Borowicz 2013). The mechanisms underlying these effects are not well understood, although they are likely to involve modifications to plant nutrition (Bennett et al. 2006) and induction of plant chemical defense (Jung et al. 2012). In comparison, there appears to be only one recent empirical study to date that has explicitly addressed the roles of root endophytes on plant–herbivore interactions belowground (Cosme et al. 2016). In this study, the authors found that Piriformospora indica, a root endophytic fungus originally isolated from orchid plants, had no effect on the survival and growth of the root-feeding larvae of Lissorhoptrus oryzophilus (rice water weevil) when introduced to rice plants as root endophytes. However, this endophytic association interfered with gibberellin and jasmonate signaling in rice plants, thereby significantly improving tolerance of rice plants to root herbivory. While the root endophytic fungus P. indica has attracted increasing attention for its potential to enhance growth and performance of a wide range of crop plants (Weiß et al. 2016), the ecological significance of other endophyte species remains poorly characterized (Aguilar-Trigueros and Rillig 2016). Our study aimed to determine the effects of native root endophytic fungi on plant resistance and tolerance to a root-feeding insect. We chose the cool-season grass Festuca arundinacea Schreb (synonyms: Schedonorus arundinaceus and Lolium arundinaceum, i.e., tall fescue) as a host plant primarily because grass roots are commonly colonized by diverse and functionally distinct endophytes, including saprotrophic (Mandyam et al. 2010), entomopathogenic (Vega et al. 2008), or nematophagous fungi (Lopez-Llorca et al. 2006), as well as potential fungal pathogens (Malcolm et al. 2013). Native to Europe, tall fescue is cultivated worldwide as an important forage and turf grass (Gibson and Newman 2001). In the United States, more than 14 million hectares of tall fescue have been planted in pastures, lawns, and sports turf settings, providing important agronomic and economic value (Hoveland 2009). However, grass roots are frequently subject to intensive and destructive grazing by root-feeding soil invertebrates, such as scarab beetle larvae

et al. 2009). Diverse endophytic fungi also inhabit roots. They are typically transmitted horizontally and appear to form unspecialized and variable associations in roots without causing apparent disease symptoms (Rodriguez et al. 2009). In spite of the fact that endophytic fungi are just as common in roots as mycorrhizae (Li et al. 2005, Mandyam and Jumpponen 2008), the ecological functions of many root fungal endophytes are not well understood (Rodriguez et al. 2009). There is evidence that root fungal endophytes can facilitate plant nitrogen uptake (Upson et al. 2009) and increase plant stress tolerance through elevated antioxidant capacity (Waller et al. 2005, Kumar et al. 2009). Additionally, some root endophytic fungi have been shown to up-regulate gene expression of defense pathways and increase production of plant defensive compounds (Mandyam and Jumpponen 2014, Pieterse et al. 2014). As such, it has been suggested that root endophytic fungi may play important roles in plant defense against herbivores (Mandyam and Jumpponen 2005). However, empirical studies to address the ecological significance of root endophytes in regulating plant–herbivore interactions remain scarce. Studies on root endophytic fungi have provided evidence that they can either suppress (Jallow et al. 2004, Zhou et al. 2016) or enhance (Barazani et al. 2005) the performance of foliar-feeding insects. However, few studies have quantified the impacts of root endophytes on plant–herbivore interactions in belowground tissues. One may speculate that root endophytes could have a stronger effect against belowground herbivores than against aboveground herbivores because the former directly consume the endophytes and, thus, may be impacted by both direct and indirect effects of endophyte colonization. Given the fact that root herbivores are diverse and widespread in many ecosystems and cause considerable plant damage that rivals aboveground herbivory, it is critical that we improve our understanding of the impacts that root endophytes have on belowground plant defense (Rasmann and Agrawal 2008, van Dam 2009). Our current understanding of plant–fungi– herbivore interactions in the rhizosphere comes primarily from the study of mycorrhizae. Several studies have shown that mycorrhizae can affect plant resistance and tolerance to root-feeding ❖ www.esajournals.org

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(Coleoptera: Scarabaeidae) (Potter and Braman 1991). Some tall fescue cultivars are known to harbor vertically transmitted endophytes of the genus Epichlo€e (asexual form = Neotyphodium), which are well documented for their ability to produce alkaloids that are toxic to both vertebrate and invertebrate herbivores (Schardl et al. 2004, Saikkonen et al. 2013). However, the growth of these endophytes is largely restricted to aboveground tissues with limited effects on root-feeding insects (Hinton and Bacon 1985, Potter et al. 1992, Moy et al. 2000). In contrast to the intensive research on Epichlo€e endophytes, root endophytic fungi in grasses have received only limited attention (Hartley and Gange 2009, S anchez Marquez et al. 2012). It remains unknown whether root endophytes can promote plant defense against herbivores in a way comparable to aboveground obligate symbionts, like Epichlo€e. In this study, we isolated naturally occurring fungal endophytes from field-collected tall fescue and meadow soils and established five diverse morphotypes as endophytes in the roots of lab-grown tall fescue. We investigated their effects singly on plant resistance to feeding by larvae of the generalist root herbivore Rhizotrogus majalis Razoumowsky (European chafer), as well as their impacts on the ability of tall fescue to recover from damage. We hypothesized that root endophytic fungi can alter both plant resistance and tolerance to root herbivory. In addition, based on the insight that mycorrhizal effects on root defense vary with mycorrhizal species, we also hypothesized that the magnitude of root endophyte effects on plant resistance and tolerance will vary with endophyte species.

Fig. 1. (a) Semi-aeroponic system for growing Festuca arundinacea consisting of a plastic planting basket secured within a 50-mL tube. (b) The planting basket is removed and transferred to a specimen cup to create a feeding arena where roots are exposed to a single Rhizotrogus majalis larva.

1% sodium hypochlorite for 2 min, followed by several rinses in sterile water (Wearn et al. 2012). Approximately 20 tall fescue seeds were sown in PVC (polyvinyl chloride) tubes (2 cm inner diameter, 2 cm height) with cheesecloth secured to the bottom (Fig. 1a, referred to as a “planting basket” hereafter). Prior to use, all planting baskets were surface-sterilized as described above, dried, and pre-weighed (M_basket) for later determination of net plant biomass. Once seeded, each planting basket was inserted into a 50-mL sterile centrifuge tube and secured in place with wire to control the depth of the planting basket within the tube (Fig. 1a). This allowed us to raise or lower each planting basket to accommodate progressive root growth. At the start of the experiment, an aliquot (5 mL) of sterile deionized water was introduced into each centrifuge tube, and the planting basket was positioned 1 cm above the water surface. All plants received 5 mL of ¼-strength Hoagland’s solution (MP Biomedicals, Solon, Ohio, USA)

MATERIALS AND METHODS Plant establishment

We selected an Epichlo€e-free variety of turf-type tall fescue, F. arundinacea (“Fawn,” Ernst Conservation Seeds, Meadville, Pennsylvania, USA), to avoid potential confounding effects from foliar endophytes. Plants were grown from surfacesterilized seeds in semi-aeroponic systems in the laboratory (Fig. 1a), which allowed us to effectively manipulate root endophytes and quantify root feeding by larvae. Seeds were surface-sterilized by soaking sequentially in 95% ethanol for 30 s, sterile water (deionized and autoclaved) for 30 s, ❖ www.esajournals.org

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endophytic fungi in Appendix S1). All root inoculation and insect-feeding studies described herein were conducted without prior knowledge of the taxonomic affiliations of HG1, HG9, HG30, HG35, and HG44, thereby insuring non-biased evaluations of the impact of specific endophytes on plant health and response to larval feeding. Thereafter, we identified the five morphotypes to species level by DNA sequencing of the internal transcribed spacer region of the nuclear ribosomal DNA (ITS) or the translation elongation factor 1-alpha (EF1-a) gene. See Fungal identifications in Appendix S1 for DNA isolation and gene sequencing methods.

twice per week from day 8 to day 46 until they were transferred to feeding arenas (Fig. 1b) for the next phase of the experiment. The water level and depth of each planting basket was adjusted as needed to ensure that root tips (~1/3 of the root system) were submerged in water. All seedlings within the same planting basket (~20 plants) were treated identically, with each planting basket representing an independent sampling unit. The semi-aeroponic systems were placed into growth chambers with a 16-h light (21°C) and 8-h dark (18°C) cycle. To maintain a dark environment for the roots, the portions of the tubes below the planting baskets were wrapped with aluminum foil. The growth chambers were set to 75% relative humidity to maintain sufficient moisture for plant growth.

Fungal inoculation of plants

The five selected fungal morphotypes were cultured on potato dextrose agar (PDA) plates at room temperature before being inoculated onto three-week-old tall fescue roots. Because fungal morphotypes HG1, HG9, and HG35 did not sporulate sufficiently on PDA plates, we used mycelia for inoculation. Briefly, for each planting basket, mycelia were scraped from an approximately 3 cm 9 3 cm agar block from each fungal culture (1 month old) using a sterile spatula and spread evenly over the full length of the roots. For the presumptive entomopathogenic fungi isolated from insect cadavers (HG30 and HG44), conidia were scraped from the surface of three 1-monthold agar plates into 800 mL of sterile deionized water. The conidia solutions were agitated on a magnetic stir plate for ~10 min with five drops of surfactant (Tween-80) to minimize surface tension. An aliquot of each conidia suspension was enumerated by hemocytometer to determine a final concentration of ~5 9 105 conidia/mL. Tall fescue planting baskets were removed from centrifuge tubes and submerged in aliquots of conidia suspensions (six to seven planting baskets per 200 mL conidia suspension) in a surface-sterilized plastic container at room temperature for 1 h to ensure adequate contact between conidia and roots. After inoculation, all planting baskets were then placed back into their respective 50-mL centrifuge tubes in the growth chambers, again with approximately 1/3 of the root length submerged in nutrient solution. A total of 156 planting baskets were prepared with 26 baskets per treatment, including five fungal treatments and the uninoculated control. Two planting baskets from each fungal treatment and the untreated control were harvested

Fungal isolation and identification Fungi used in the study were originally isolated from tall fescue roots and soils from central New York State, United States (see Isolation of root endophytic fungi in Appendix S1). A preliminary trial was conducted to assess the ability of fungi collected from the field to establish endophytically in tall fescue growing in the semi-aeroponic systems described above (see Preliminary in planta fungal endophyte evaluation in Appendix S1). Fungal colonization within root tissues was assessed 18 d after inoculation via compound microscopy after roots were cleared and stained using a vinegarink method (Vierheilig et al. 1998). Fungal colonization was quantified as the percentage of observations (~150 observations per slide for each planting basket) in which fungal hyphae were detected inside the roots (McGonigle et al. 1990). The trial revealed five fungal isolates (HG1, HG9, HG30, HG35, and HG44) that consistently colonized tall fescue roots in vitro in the semi-aeroponic system, with a root infection rate of 25.3% for HG1, 20.6% for HG9, 14.1% for HG30, 10% for HG35, and 15.1% for HG44. None of the fungi caused detectable disease symptoms in treated F. arundinacea plants and were, therefore, selected for further study. Among these five morphotypes, HG30 and HG44 were isolated from soils using a sentinel insect bait method and presumed to be entomopathogenic fungi, whereas HG1, HG9, and HG35 were isolated as endophytes from fieldcollected tall fescue roots (see Isolation of root ❖ www.esajournals.org

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were subjected to root feeding by larvae of R. majalis. Third-instar larvae were collected from fairways of a public golf course in central New York State and placed singly into cells of ice cube trays for 1 d at 20°C in the dark to void their gut contents prior to the start of the feeding experiment. Larval feeding arenas were constructed using 125-mL specimen cups (Fig. 1b). The outside of each specimen cup was painted black to minimize light penetration, thereby simulating belowground light conditions. A 2-cm diameter hole was cut in the center of each cup lid to receive a single planting basket. A single larva was placed into each feeding arena and allowed to feed on the roots for 8 d. The plant roots were dipped into deionized water for 2 min and then blotted with paper towels to remove excess nutrient solution on the root surface before being transferred to the feeding arenas. The planting baskets were positioned so that larvae had direct access to root tips, and baskets were lowered further into the arenas as feeding progressed to ensure that roots were always accessible to the larvae. Herbivore-free planting baskets were also placed inside feeding arenas and did not receive larvae. The feeding arenas were maintained in growth chambers with a 12-h light (21°C) and 12-h dark cycle (18°C) and 90% humidity to minimize abiotic plant stress during larval feeding. All larvae were weighed prior to and after the feeding experiment to examine the potential impact of endophytes on larval performance. Daily production of fecal pellets was used as a proxy for root consumption, assuming assimilation rates were similar among individual larvae (Potter et al. 1992). Fecal pellets produced by larvae were counted and collected daily. All fecal pellets collected from each planting basket were air-dried at room temperature for 1 week and weighed. The maximal root length in each planting basket was also measured prior to and at the end of the feeding experiment to calculate the amount of root loss due to herbivory (i.e., the reduction in root length due to herbivory).

18 d after inoculation. Colonization by endophytes was confirmed by placing segments of surfacesterilized roots onto PDA plates with streptomycin (100 mg/L) and neomycin (26 mg/L) added to suppress bacterial growth. The PDA plates were checked over a three-week period for growth from cut root surfaces of the same morphotype of fungus used as inoculum. While all seeds used in our study were surface-sterilized and grown in a controlled, soil-free environment, resident bacteria and fungi (potential seed symbionts) could not be completely eliminated from seeds. Plating of surface-sterilized seeds and roots on PDA plates confirmed that resident bacteria and fungi were observed uniformly, but in very low numbers across all plant treatments (i.e., control and endophyte-inoculated). These microbes were distinguishable from the inoculated endophytes and did not prevent establishment of the latter.

Plant resistance to root herbivory Plant resistance is generally assessed by quantifying herbivore damage (Fineblum and Rausher 1995). We evaluated tall fescue resistance to belowground herbivory by measuring reductions in root length after an eight-day feeding period as described below. We also quantified root carbon (C) and nitrogen (N) concentrations as an integrated measure of endophyte effects on plant root chemistry. We chose this approach since the array of secondary metabolites potentially produced in planta by the endophytic fungi used in our study is essentially unknown, and there is a concurrent lack of information on plant defensive compounds effective against herbivore feeding in tall fescue roots (Rasmann and Agrawal 2008). Six planting baskets from each group were destructively harvested three weeks after fungal inoculation, and C and N concentrations (percentage of dry mass) in the proximal 20-mm root tip sections were measured using the dry combustion method (Nelson and Sommers 1982) on an elemental analyzer (Costech Analytical Technologies, Valencia, California, USA). To assess potential changes in plant moisture during the feeding period, two to three planting baskets from each endophyte treatment and the uninoculated control (16 in total) were set aside and did not receive root herbivores. Plants in the remaining baskets (15–16 replicates per endophyte treatment and the uninoculated control) ❖ www.esajournals.org

Plant tolerance to larval and mechanical damage Plant tolerance to herbivory refers to the ability to regrow and reproduce after herbivore damage (Strauss and Agrawal 1999). We focused on plant regrowth as one aspect of plant tolerance, which was measured as follows. Because all plants received varying degrees of root damage from 5

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Data analyses and statistics

larval feeding, plant roots were deliberately clipped to a length of 30 mm to standardize root length and to ensure an equivalent amount of root access to nutrient solution at the start of the regrowth experiment. Plant shoots were also clipped to a height of 30 mm prior to initiating the regrowth experiment. This was done to standardize starting root and shoot length for the plants but is also representative of the mechanical damage that managed turf grasses experience during mowing. The plants in each planting basket, therefore, experienced both belowground herbivore feeding and mechanical damage. The clipped plants within each planting basket were weighed (M_t0_wet) before being placed back into their semi-aeroponic system to grow for an additional 3 weeks to quantify the effects of endophytes on the ability of F. arundinacea to regrow after damage. Herbivore-free planting baskets were destructively harvested before the regrowth experiment, and the wet and dry biomasses (dried at 60°C for 48 h) of roots and shoots were determined independently for the purpose of estimating moisture content (%H2O) of the plants before the regrowth experiment. About 1/3 of the roots were submerged in 15 mL of ¼-strength Hoagland’s solution at the beginning of the regrowth experiment to facilitate new plant growth. Another 5 mL aliquot of ¼-strength Hoagland’s solution was added at day 7. No nutrients were provided to the plants for the final 10 d of regrowth based on our observation that the plants appeared healthy and showed no signs of nutrient stress. At the termination of the regrowth experiment, plants were destructively harvested. For each planting basket, roots (M_t1_ root_dried) and shoots (M_t1_shoot_dried) were separated and dried at 60°C for 48 h and weighed separately, which allowed a determination of above- and belowground resource allocation at the end of the regrowth experiment in response to endophyte colonization. We calculated relative plant regrowth as an assessment of plant tolerance to damage using the following formula: (final biomass initial biomass)/initial biomass 9 100% (Bultman et al. 2004), wherein initial dry biomass was converted from initial wet weight as follows: (M_t0_wet M_basket) 9 (1 %H2O), and final biomass was the sum of the final root and shoot dry weight: M_t1_root_dried + M_t1_shoot_dried. ❖ www.esajournals.org

To test the effects of endophyte colonization on plant resistance and plant tolerance to herbivore damage, one-way ANOVA was performed for the following data metrics: C and N concentrations (percentage of dry mass), root damage (the reduction in root length), dry mass of fecal pellets, larval weight, plant regrowth, and shoot-to-root ratio at the end of the regrowth experiment. Dunnett’s post hoc multiple comparisons were then made for any ANOVA tests showing significance at P < 0.05 to compare each endophyte treatment with the uninoculated control. A paired t test was used to compare the larval weights before and after the feeding experiment. Repeated-measures ANOVA was used to analyze consecutive fecal pellet production by fitting a mixed linear model, with fungal treatment as a fixed, between-subject factor and day as a within-subject factor. Normality of the distribution and homogeneity of the variance were tested using Shapiro and Levene’s tests, respectively. To meet the assumptions of ANOVA, the number of fecal pellets was logtransformed, and fecal pellet dry mass, relative plant regrowth, and shoot-to-root ratio were square-root-transformed. To better visualize the overall effect of endophyte treatments on plant defense, we also calculated the net effects of fungal colonization on root damage (i.e., assessment of plant resistance) and plant regrowth (i.e., assessment of plant tolerance) in comparison with control plants using the following formula: (fungal treatment control)/control 9 100%. All statistical analyses were conducted using the R software version 3.31 (R Development Core Team 2016). In particular, the following packages were used: nlme (Pinheiro et al. 2007) for repeatedmeasures ANOVA; multcomp (Hothorn et al. 2008) for subsequent Dunnett’s multiple comparisons.

RESULTS Identification of endophytic fungal morphotypes Gene sequencing analyses revealed that the five isolates used in our study belong to five genera from three fungal phyla: Fusarium cf. equiseti (HG1; Ascomycete), Trametes versicolor (HG9; Basidiomycete), Isaria fumosorosea (HG30; Ascomycete), Mortierella alpina (HG35; Zygomycete), and Beauveria bassiana (HG44; Ascomycete). See Fungal Identifications in Appendix S1 for specific 6

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levels of identity of the EF1-a and ITS gene sequences used to justify taxonomic assignments and for summaries of known biological characteristics of each species. DNA sequences have been deposited in GenBank under Accession Numbers KU500171 (HG1), KU517157 (HG9), KU523252 (HG30), KU523253 (HG35), and KU523254 (HG44).

The effect of endophyte associations on root chemistry and herbivory Endophyte treatments had no significant impact on N concentrations in the root tips (Table 1, F5,23 = 0.67, P = 0.65). However, plants inoculated with B. bassiana had a significantly higher C concentration (42.59% 0.19; mean SE) in the root tips compared to control plants receiving no fungal treatment (41.46% 0.15) (P = 0.021). ANOVA also revealed varying effects of the endophyte treatments on root herbivory (Fig. 2, F5,83 = 3.62, P = 0.005). While four of five endophytes (i.e., F. cf. equiseti, T. versicolor, M. alpina, and I. fumosorosea) did not significantly alter herbivore damage to roots (Fig. 2, Dunnett’s test, P > 0.60), plants colonized by B. bassiana suffered slightly less (27%) root damage than the uninoculated plants (Fig. 2, P = 0.067). Concurrently, the total number of fecal pellets produced by R. majalis larvae after feeding on B. bassiana-colonized roots was significantly lower than for larvae feeding on uninoculated control plants (Fig. 3, P = 0.049). In contrast, other endophyte treatments had no significant effect on the numbers of fecal pellets produced by R. majalis larvae (Fig. 3, P > 0.43). Endophyte infection had a

Fig. 2. Herbivore damage to tall fescue roots colonized by different endophytes. Root damage was measured as the reduction in root length (mm) after root feeding. Values are mean SE; n = 15–17. Dashed line represents the mean value of the control plants, with the shaded area representing SE. Denotes marginally difference between Beauveria bassiana treatment and controls from Dunnett’s multiple comparisons at P = 0.067.

similar effect on total fecal pellet dry mass, with a 47% decrease in frass mass from larvae feeding on B. bassiana-infected plants compared to uninoculated plants; however, the effect was only marginally significant (P = 0.090; data not shown). While we observed continuous consumption of root materials, larvae lost an average of 3% of their original body weight (wet weight) by the end of the eight-day feeding period (paired t test, P < 0.001; data not shown). However, the loss of larval weight did not differ among the larvae feeding on endophyte-treated plants and the uninoculated control (F5,82 = 0.21, P = 0.96). In addition, over 95% of larvae survived the eightday feeding experiment. The few larvae that died exhibited no detectable symptoms of entomopathogen infection (e.g., fungal growth from the insect cuticle).

Table 1. C and N concentrations (percentage of dry mass) and carbon-to-nitrogen ratio of Festuca arundinacea root tips three weeks after colonization by different species of endophytic fungi. Endophyte species Fusarium cf. equiseti Trametes versicolor Mortierella alpina Isaria fumosorosea Beauveria bassiana Control

C

N

C: N

41.59 (0.21) 41.31 (0.26) 41.31 (0.34) 41.64 (0.21) 42.59 (0.19)* 41.46 (0.15)

1.55 (0.05) 1.69 (0.1) 1.48 (0.07) 1.54 (0.12) 1.54 (0.08) 1.58 (0.12)

26.94 (0.89) 24.80 (1.41) 28.35 (1.57) 27.43 (1.94) 27.96 (1.43) 26.72 (1.90)

Plant tolerance to larval and mechanical damage Endophyte associations in roots also significantly affected plant regrowth after damage (Fig. 4, F5,82 = 6.90, P < 0.001). Notably, plants inoculated with F. cf. equiseti, I. fumosorosea, or B. bassiana exhibited significantly lower regrowth of total plant biomass compared to plants

Note: Values are mean with standard error in brackets (n = 4–6). * Significant difference between endophyte treatment and control plants from Dunnett’s multiple comparisons at P < 0.05.

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known as a wood-decay and decomposer fungus (Aguiar et al. 2014). Several studies have demonstrated that Trametes spp., including T. versicolor, can grow endophytically in the sapwood, needles, fruits, and twigs of woody plants (Martin et al. 2015, and references within), as well as in the leaves of oil palm (Pinruan et al. 2010), and the aerial tissues and roots of wheat (Comby et al. 2016) and other grasses (Sanchez Marquez et al. 2007, 2010). M. alpina is commonly found as a soil-dwelling saprophyte but also has been described as an endophyte of leaves in Antarctic mosses (Melo et al. 2014), in roots of wheat (Comby et al. 2016), and in leaves and roots of other grasses (Sanchez Marquez et al. 2007, 2010); only recently, M. alpina was described as an entomopathogen (Edgington et al. 2014). The ascomycetous endophytes characterized in our study are well described as entomopathogens (B. bassiana and I. fumosorosea) (Zimmermann 2008, Boomsma et al. 2014) or, in the case of F. cf. equiseti, as a cereal pathogen (Villani et al. 2016) or entomopathogen (Wenda-Piesik et al. 2009). All three Ascomycetes have been commonly reported as endophytes of grasses (Sanchez Marquez et al. 2007, Behie et al. 2015, Mantzoukas et al. 2015, Martin and Dombrowski 2015).

Fig. 3. Cumulative number of fecal pellets produced by Rhizotrogus majalis after feeding on tall fescue roots colonized by individual endophyte species over the course of 8 d. Values are mean SE; n = 15–17. Dashed line represents the mean value for control plants, with the shaded area representing SE. Denotes significant difference between individual endophyte treatment and controls from Dunnett’s multiple comparisons at P < 0.05.

receiving no endophyte treatment (Fig. 4, P < 0.05). Regrowth of M. alpina- or T. versicolor-colonized plants did not differ from the uninoculated control (Fig. 4, P = 0.15 and 0.91, respectively). Resource allocation between shoots and roots, measured at the end of the regrowth experiment, also varied with endophyte treatments (Fig. 5, F5,82 = 2.68, P = 0.027), with a significantly lower shoot-to-root ratio in plants colonized by endophytic M. alpina compared to plants receiving no endophytes (Fig. 5, P = 0.004). There was a similar, but statistically insignificant, trend of lower shoot-to-root ratio in I. fumosorosea-colonized plants (Fig. 5, P = 0.086) that was not observed in the other endophytetreated plants.

DISCUSSION

Fig. 4. Effects of endophyte colonization on plant regrowth after mechanical and feeding damage. Plant regrowth was calculated as the percent increase in total plant biomass relative to initial plant biomass over a three-week regrowth period. Values are mean SE; n = 15–17. Dashed line represents the mean value of the control plants, with the shaded area representing SE. Denotes significant difference between individual endophyte treatment and controls from Dunnett’s multiple comparisons at P < 0.05.

The predominant fungal endophytes most commonly reported from grass roots reside in the Ascomycota (Mandyam and Jumpponen 2008, S anchez M arquez et al. 2010). To our knowledge, our study is among the first to reveal fungal species from the Basidiomycota (T. versicolor) and Zygomycota (M. alpina) as endophytic colonizers of F. arundinacea roots. T. versicolor is primarily ❖ www.esajournals.org

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an Epichlo€e fungus comes at the cost of reducing plant tolerance. In contrast, colonization of F. arundinacea by F. cf. equiseti tended to lower both host plant tolerance and resistance (Fig. 6, Quadrant IV). By comparison, the endophytic fungus P. indica in rice plants increased plant tolerance with little effect on plant resistance (Cosme et al. 2016). Other root-associated fungi, such as mycorrhizae, can enhance both plant defense and plant tolerance (Koricheva et al. 2009), likely by enhancing nitrogen and phosphorus uptake in roots (Tao et al. 2016), as well as by priming the n-Aguilar plant defense system (Pozo and Azco 2007). However, some mycorrhizal species actually reduce plant resistance and tolerance (see review in Borowicz 2013). Overall, our findings highlight the importance of species identity in the influence of root-associated fungi on plant defense against herbivores. However, since plant roots are consistently exposed to and colonized by diverse fungal species (Wearn et al. 2012), it will be critical that future studies investigate whether different combinations of endophytic and mycorrhizal fungi interact synergistically or antagonistically in plant tissues and how their overall effects on plant defense change with environmental conditions (Partida-Martınez and Heil 2011, AguilarTrigueros and Rillig 2016). The species-specific effects of indigenous root endophytes on plant defense may relate to functional aspects of the fungi during their life stages outside of a plant host. As a ubiquitous entomopathogen, B. bassiana has been established endophytically in many economically important crops, including banana, corn, cotton, fava bean, poppy, sorghum, tomato, and wheat (Gurulingappa et al. 2010, Mantzoukas et al. 2015, also see references reviewed by Vidal and Jaber 2015). The majority of these studies revealed that endophytic B. bassiana reduces herbivore damage by a wide range of sucking, chewing, and tunneling insects in leaves and stems (Vidal and Jaber 2015). Our study is the first to demonstrate that endophytic B. bassiana in roots can confer suppression of belowground herbivory by rootchewing insects. In comparison, colonization by another well-known entomopathogenic fungus, I. fumosorosea, had little measurable effect on plant resistance in our study. In contrast, endophytic I. fumosorosea in sweet sorghum was found to greatly increase larval mortality of the

Fig. 5. The ratio between shoot and root biomass at the end of the regrowth experiments. Values are mean SE; n = 15–17. Dashed line represents the mean value of the control plants, with the shaded area representing SE. Denotes significant difference between individual endophyte treatment and controls from Dunnett’s multiple comparisons at P < 0.05.

Our study demonstrates that these naturally occurring fungal endophytes in F. arundinacea roots can alter both plant resistance and tolerance against root-feeding insects, and the effects vary with endophyte species. While T. versicolor and M. alpina had few effects on plant defense against root herbivores, all three of the ascomycete fungi reduced plant regrowth after damage. In addition, B. bassiana also increased plant resistance to root-feeding larvae. The contrasting effects we observed for B. bassiana on plant resistance and plant tolerance suggest that research solely evaluating plant resistance is likely to overestimate the benefits conferred by ascomycete endophytes without accounting for other concurrent effects on plant defense. Because plant tolerance and plant resistance are often expressed jointly as alternative defense strategies, we propose a conceptual framework to include both plant resistance and tolerance as two dimensions of plant defense (Fig. 6) and posit that root endophytes may determine the relative importance of these two plant defensive strategies. For example, endophytic B. bassiana shifts the defense strategy of F. arundinacea to a position of higher resistance but lower tolerance (Fig. 6, Quadrant III) compared to uninoculated plants in our study. Similarly, Bultman et al. (2004) reported that the increase in plant resistance conferred by ❖ www.esajournals.org

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Fig. 6. Conceptual illustration of plant defense strategies including both plant resistance (x-axis, herbivore damage to roots) and plant tolerance (y-axis, plant regrowth of shoots and roots after damage), relative to the control. Associations with different root endophytes alter the relative importance of plant resistance vs. tolerance in host plant defensive strategies. Values are mean SE; n = 15–17.

secondary metabolites (i.e., destruxins) in plant tissues and cause mycosis in insects after exposure in planta for 2 weeks (Golo et al. 2014, Keyser et al. 2014). We did not observe mycosis in R. majalis larvae after feeding on living roots colonized by endophytic fungi and acknowledge that the short-term duration of the feeding experiment may have prevented us from doing so. Further research examining plant root defense compounds and in planta production of insecticidal metabolites of fungal origin will be needed to elucidate the direct and indirect effects of endophytic entomopathogens on root herbivores. We also observed that multiple species of root endophyte can alter plant tolerance and resource allocation following damage, including a reduction in plant regrowth and a decrease in shoot-to-root ratio upon recovery. The specific mechanisms by which endophytes altered plant tolerance and resource allocation in our study are unclear. Although endophytes are defined as conferring no apparent harm to their host, such associations could have hidden metabolic costs for plants, as evidenced by their suppression of plant growth, especially under plant resource limitation (Faeth and Hamilton 2006, Cheplick 2007, Mayerhofer et al. 2013). Cosme et al. (2016) recently found that colonization of rice plant

stem borer Sesamia nonagrioides and reduce stemtunneling activities by 60–87% (Mantzoukas et al. 2015). Although M. alpina and F. cf. equiseti have been described as entomopathogens (Wenda-Piesik et al. 2009, Edgington et al. 2014) with capacities to produce insecticidal and insectdeterrent compounds in some plants (Melo et al. 2014, Villani et al. 2016), we were unable to measure any effects of these two endophytes on root feeding of R. majalis larvae, further highlighting the context dependency of plant–endophyte– insect interactions. While we did not examine plant secondary metabolite production in our study, the slightly higher C concentration observed in B. bassianainfected roots might suggest an increased resource allocation to C-based defensive compounds that could deter herbivore feeding (Coviella et al. 2002, Jia et al. 2016). Indeed, a recent study reports that co-infection of endophytic B. bassiana and mycorrhizae in tomato plants can significantly increase terpenoid content in leaves leading to a reduction in foliar feeding by Spodoptera exigua (Shrivastava et al. 2015). In addition, it is yet unknown whether B. bassiana produces insecticidal metabolites in planta (Vega et al. 2008). However, the endophytic entomopathogenic fungus Metarhizium robertsii has been shown to produce ❖ www.esajournals.org

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of our laboratory study to field conditions and other plant–insect systems. However, our results suggest that fungi that engage in robust antagonistic insect interactions, such as B. bassiana, are more likely to enhance plant resistance to insect feeding when they become endophytic compared to other fungi. Evaluating both plant resistance and tolerance in the same study and manipulating endophyte consortia under various field conditions will be rewarding for future studies to advance our understanding of the role of root endophytes in the adaptation of plants to their surrounding soils and their consequences for belowground plant–insect interactions.

roots by the fungus P. indica elicited gibberellic acid biosynthesis and increased the tolerance of plants to belowground herbivory. Another recent study revealed that the presence of horizontally transmitted endophytes (presumably both aboveand belowground) suppresses plant regrowth after clipping, supporting the argument that some endophytes may confer metabolic costs to their hosts (Santangelo and Kotanen 2016). In our study, plants were exposed to significant damage (both root feeding and mechanical damage from clipping) and did not receive nutrients for the final 10 d of the regrowth experiment. Consequently, the reduction in plant tolerance following herbivore damage may be due to elevated resource competition between the host plant and some endophyte species (Wise and Abrahamson 2007). It will be valuable for future studies to investigate whether endophyte effects on plant tolerance vary under different nutrient conditions (Aguilar-Trigueros and Rillig 2016). It should be noted that the plants in our study were grown initially in simple semi-aeroponic systems with readily available mineral nutrients. Root endophytes in the field may confer other benefits to their hosts, which we were unable to identify in our study. For example, the well-studied endophytic entomopathogenic fungus M. robertsii has been shown to directly transfer nutrients from dead infected insects to plants (Behie et al. 2012). Also, some root endophytes, such as T. versicolor, are known to produce extracellular enzymes to break down organic matter, which may increase nutrient availability for host plants (Mandyam and Jumpponen 2005, Aguiar et al. 2014). Indeed, Cosme et al. (2016) recently reported an increase in plant tolerance in conjunction with a slightly higher shoot phosphorus concentration in endophyte-treated plants grown in soil. In such cases, the nutritional benefit provided by endophytic fungi may mitigate their metabolic costs and increase plant tolerance to damage rather than decrease it, as seen for F. cf. equiseti, I. fumosorosea, and B. bassiana in our study. To conclude, our study revealed that only one of the five horizontally transferred root endophytic fungi we evaluated altered plant resistance to root-feeding insects, but multiple species reduced the ability of plants to recover from damage under our experimental conditions. We remain cautious about extrapolating the results ❖ www.esajournals.org

ACKNOWLEDGMENTS A special thanks to Pengfei Wu for his assistance with the herbivore feeding experiments. We would also like to thank Katelyn Berry for maintenance of the tall fescue.

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SUPPORTING INFORMATION Additional Supporting Information may be found online at: http://onlinelibrary.wiley.com/doi/10.1002/ecs2. 1710/full


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