Fungal Biomass Associated with the Phyllosphere of Bryophytes and Vascular Plants

mycological research 113 (2009) 1254–1260

journal homepage: www.elsevier.com/locate/mycres

Fungal biomass associated with the phyllosphere of bryophytes and vascular plants

M. L. DAVEYa,b,*, L. NYBAKKENa,1, H. KAUSERUDb, M. OHLSONa aDepartment of Ecology and Natural Resource Management, Norwegian University of Life Sciences, PO Box 5003, 1432 A˚ s, Norway bMicrobial Evolution Research Group, Department of Biology, University of Oslo, PO Box 1066, Blindern, N-0316 Oslo, Norway article info abstract

Article history: Little is known about the amount of fungal biomass in the phyllosphere of bryophytes Received 24 April 2009 compared to higher plants. In this study, fungal biomass associated with the phyllosphere Received in revised form of three bryophytes (Hylocomium splendens, Pleurozium schreberi, Polytrichum commune) and 31 July 2009 three vascular plants (Avenella flexuosa, Gymnocarpium dryopteris, Vaccinium myrtillus) was Accepted 4 August 2009 investigated using ergosterol content as a proxy for fungal biomass. Phyllosphere fungi ac- Available online 12 August 2009 counted for 0.2–4.0 % of the dry mass of moss gametophytes, representing the first estima- Corresponding Editor: tion of fungal biomass associated with bryophytes. Significantly more fungal biomass was John W. G. Cairney associated with the phyllosphere of bryophytes than co-occurring vascular plants. The ergosterol present in moss gametophytic tissues differed significantly between species, while Keywords: the ergosterol present in vascular plant leaf tissues did not. The photosynthetic tissues of Bryophytes mosses had less associated fungal biomass than their senescent tissues, and the magni- Endophytes tude of this difference varied in a species-specific manner. The fungal biomass associated Epiphytes with the vascular plants studied varied significantly between localities, while that of Ergosterol mosses did not. The observed differences in phyllosphere community biomass suggest Erogsterol extraction their size could be affected by host anatomical and physiological attributes, including mi- Fungal biomass cro-niche availability and chemical host defenses, in addition to abiotic factors like mois- Phyllosphere ture and nutrient availability. Vascular plants ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction phyllosphere fungi has been characterized in a variety of vascular plants (Arnold 2007; Arnold & Lutzoni 2007; Osono 2007, 2008; The term ‘phyllosphere’ has been used to describe the aerial, Osono & Mori 2003; Santamaria´ &Bayman2005). These fungi, above-ground portions of plants that provide habitats for both particularly as endophytes, have been shown to fill important epiphytic and endophytic microorganisms (Whipps et al. 2008). ecological roles (Rodriguez et al. 2009), including affecting host The phyllosphere of vascular plants is known to host a diverse susceptibility to pathogens (Arnold et al. 2003; Lehtonen et al. microbiological community including bacteria, archaea, yeast, 2006), acting as herbivore deterrents (Bing & Lewis 1991; Gonth- and filamentous fungi, both as epiphytes on the plant surfaces, ier et al. 2008), and affecting host stress tolerance (Arnold & andasendophytescolonizingtheinternaltissuesofleavesand Engelbrecht 2007; Mu¨ ller & Krauss 2005; Redman et al. 2002). De- stems (Arnold 2007; Inaćio et al. 2002; Rodriguez et al. 2009; spite the presence of phyllosphere fungi, particularly endo- Whipps et al. 2008). The diversity of endophytic and epiphytic phytes, in all land plant lineages, the study of these fungi has

* Corresponding author. Department of Ecology and Natural Resource Management, Norwegian University of Life Sciences, PO Box 5003, 1432 A˚ s, Norway. Tel.: þ47 64 96 53 47; fax: þ47 64 96 58 01. E-mail addresses: [email protected] (M. L. Davey), line.nybakken@joensuu.ﬁ (L. Nybakken), [email protected] (H. Kauserud), [email protected] (M. Ohlson). 1 Present address: Department of Biology, University of Joensuu, Box 111, FI-80101 Joensuu, Finland. 0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2009.08.001 Fungal biomass in the phyllosphere 1255

been largely limited to spermatophyte lineages (Pinophyta, photosynthetic and brown senescent sections and the inter- Magnoliophyta) and little research has been undertaken to char- vening parts discarded. Green gametophyte parts, senescent acterize the composition, structure, and ecological significance gametophyte parts, and vascular plant leaves were freeze- of endophytic fungal communities associated with other line- dried overnight, crushed to a powder using a Retsch ball ages (Davey & Currah 2006; Davis & Shaw 2008; Fisher 1996; mill, and stored at 80 C until analyses were conducted. Total Read et al. 2000). In particular, bryophytes are a dominant and free and esterified ergosterol was extracted with saponifica- functionally important vegetation component that contributes tion using a modified protocol after Gessner & Schmitt considerable biomass in boreal and tundra biomes (Bach et al. (1996). The powdered plant material (100–200 mg) was com- 2009; Benscoter & Vitt 2007), yet the prevalence, diversity, and bined with 8 mL 3 M KOH in ethanol and incubated at 80 C function of their epiphytic and endophytic fungal communities on a shaker in a water bath for 1 h. Extraction mixtures were is virtually unknown (Racovitza 1959; Felix 1988; Davey & Currah centrifuged for 15 min at 4000 rpm and the supernatant di- 2006; Kauserud et al. 2008). luted with 2 mL distilled water. Ergosterol was extracted Ergosterol has been widely used as a specific biomarker for from the supernatant by two successive applications of 5 mL fungi (Bermingham et al. 1995) and an estimator of living fun- hexane. The organic fractions were combined and evaporated gal biomass in complex abiotic and biotic systems, including under N2, then re-suspended in 500 mL of methanol and ana- soil, water, compost, seeds, and mycorrhizal roots (Djajakir- lysed for ergosterol content using HPLC. ana et al. 1996; Grant & West 1986; Joergensen & Wichern Both living and senescent bryophyte materials were sub- 2008; Klamer & Ba˚a˚th 2004; Martin et al. 1990; Montgomery jected to ergosterol extraction without saponification (Dahl- et al. 2000; Nylund & Wallander 1992; Richardson & Logendra man et al. 2002). Between 100 and 200 mg of powdered plant 1997; Seitz et al. 1977; Stahl & Parkin 1996). The biomass of material was combined with 1 mL of 99.5 % EtOH, vortexed phyllosphere or endophyte communities has been measured for 5 s, and placed on a shaker in the dark at room tempera- in relatively few studies, all of which have focused on vascular ture for 30 min. The suspension was then centrifuged plants (Jurc et al. 1997; Osswald et al. 1986; Richardson & at 18 000 rpm for 15 min, and the supernatant analysed for Logendra 1997; Verma et al. 2002; Young et al. 2005). These ergosterol content using HPLC. studies indicate that phyllosphere fungi typically account for only a small proportion (0.0013–2.5 %) of the biomass of vascu- HPLC analysis lar plant tissues, despite their diversity, and ecological significance. In light of recent studies suggesting comparable levels The extracts were analysed according to Dahlman et al. (2002) of biodiversity between the endophyte communities of bryo- on an 1100 Series HPLC (Agilent Technologies, Waldbronn, phytes and vascular plants (Davey & Currah, Unpublished Germany). Ergosterol was separated on a reversed phase data; Kauserud et al. 2008), ergosterol was used as a proxy to ODS ultrasphere column, 250 cm 4.6 mm, particle size estimate the biomass of fungal phyllosphere communities as- 5 mm (Beckman, Fullerton, USA), using MeOH as the mobile sociated with bryophyte and vascular plant species that are phase. The flow rate was 1.5 mL min1 and the total analysis abundant in a broad range of boreal forest ecosystems. time 12 min. The detection wavelength was 280 nm, and the identification of ergosterol was based on retention time, Materials and methods online UV-spectra and co-chromatography of commercial standard of ergosterol (Sigma, St. Louis, USA). Sampling Biomass estimates The bryophytes Hylocomium splendens, Pleurozium schreberi, and 1 Polytrichum commune, and the vascular plant species Avenella An average biomass conversion factor of 5.8 mg ergosterol g flexuosa, Gymnocarpium dryopteris, and Vaccinium myrtillus DW fungus was calculated from the literature using 189 re- were sampled in early Jul. of 2008 from each of five plots in cords reported for 82 species in 21 studies (Table S1) and two mature Picea abies spruce forests near A˚ s in south-east was used to estimate the fungal biomass associated with Norway. The vegetation in the two forests was typical bil- each phyllosphere community. berry-Norway spruce forest type, which is a major forest type in boreal Eurasia (Schmidt-Vogt 1977). In each plot, green, Results photosynthetic and brown, senescent parts of apparently healthy moss gametophytes, and the apical green leaves of Ergosterol was present in significantly larger quantities in the apparently healthy vascular plants were collected within photosynthetic tissues of bryophytes, than in those of vascu- a 6 m diameter circular plot. Moss gametophytes were lar plants (Fig 1), with almost an order of magnitude difference collected in their entirety, while whole leaves of V. myrtillus, between mean ergosterol contents of the two, i.e. 48.5 versus G. dryopteris leaflets with their associated petioloules, and 6.0 mg ergosterol g1 dry weight plant material (DW) (P < 0.001). leaf lamina tissue of A. flexuousa were collected. In the bryophytes, ergosterol levels were significantly higher in the senescent lower portions than in the green portions of Ergosterol extraction the gametophytes (P < 0.001), and the magnitude of this difference varied significantly between species. Also the average er- All plant material was cleaned of coarse debris. Individual gosterol content in the bryophytes was species-specific and stems of each bryophyte species were separated into green varied by a factor of two between Polytrichum commune, which 1256 M. L. Davey et al.

Fig 1 – Ergosterol content of three bryophytes (Hylocomium splendens, Pleurozium schreberi, and Polytrichum commune) and three vascular plants (Avenella ﬂexuosa, Gymnocarpium dryopteris, and Vaccinium myrtillus), distributed on locality and type of tissue (brown, senescent versus green, photosynthetic tissues). Each value is the average of ﬁve samples ± standard error of mean.

had the lowest level, and Hylocomium splendens, which had the Living fungal biomass was found to account for 0.2–1.2 % of highest level (P < 0.001) (Fig 1 and Table 1). Conversely, ergos- the dry weight of photosynthetic bryophyte tissues, and 1.3– terol content did not vary significantly among the vascular 4.0 % of senescent bryophyte tissues. In the vascular plants plant species (Fig 1 and Table 2). studied, 0.05–0.2 % of the dry weight of photosynthetic tissues Total ergosterol content in the bryophyte gametophytes was comprised of living fungal biomass (Table 3). did not differ between the forest sites (Fig 1 and Table 1). In No significant difference (P > 0.1) was observed in the contrast, the vascular plants had generally higher levels of er- amount of ergosterol extracted from bryophyte tissues using gosterol at one of the sites (Fig 1), but the difference between ethanol extraction (mean ergosterol extracted ¼ 105.7 mgg1 the sites was only marginally non-significant (P ¼ 0.07, see DW) or ethanol extraction with saponification (mean ergos- Table 2). terol extracted ¼ 86.9 mgg1 DW).

Discussion Table 1 – ANOVA results comparing ergosterol content in photosynthetic and senescent shoot fragments of the Signiﬁcantly more ergosterol was detected in the photosyn- mosses Hylocomium splendens, Pleurozium schreberi and thetic tissues of bryophytes than vascular plants from the Polytrichum commune collected at two forest localities in same understory community, indicating that greater fungal Norway. biomass is associated with the phyllosphere of moss Factor Sum of Degrees of Mean F-ratio P-value squares freedom square

Species 2.491 2 1.245 54.957 0.000 Table 2 – ANOVA results comparing ergosterol content in Tissue Type 3.646 1 3.646 160.910 0.000 green leaves of Avenella ﬂexuosa, Gymnocarpium Locality 0.060 1 0.060 2.639 0.111 dryopteris, and Vaccinium myrtillus collected at two forest Species 0.585 2 0.293 12.909 0.000 localities in Norway. Tissue Type Factor Sum of Degrees of Mean F-ratio P-value Species Locality 0.031 2 0.016 0.693 0.505 squares freedom square Tissue Type 0.059 1 0.059 2.588 0.114 Locality Species 0.024 2 0.012 0.073 0.929 Species Tissue 0.001 2 0.001 0.024 0.977 Locality 0.595 1 0.595 3.572 0.071 Type Locality Species Locality 0.015 2 0.008 0.046 0.955 Error 1.088 48 0.023 Error 3.997 24 0.167 Fungal biomass in the phyllosphere 1257

Table 3 – Estimates of phyllosphere biomass in plant tissues. Reference Tissue source Quantiﬁcation method % fungal biomass in plant tissue

Osswald et al. (1986) Green spruce and ﬁr needles Ergosterol analysis 0.0013–0.0026 Richardson & Logendra (1997) Endophyte infected seeds Ergosterol analysis 0.0216 of Festuca rubra ssp. Fallax Young et al. (2005) Endophyte infected seeds Real time PCR and fungal:plant 0.3–1.9 of Lolium perenne DNA comparison Verma et al. (2002) Green stems of Scirpus Ergosterol analysis 0.4 Jurc et al. (1997) Pinus nigra buds and needles Ergosterol analysis 0.025–2.5 This study Photosynthetic bryophyte tissue Ergosterol analysis 0.20–1.22 (mean: 0.75) This study Senescent bryophyte tissue Ergosterol analysis 1.28–3.98 (mean: 2.57) This study Photosynthetic vascular plant tissue Ergosterol analysis 0.05–0.22 (mean: 0.44)

gametophytes than vascular plant leaves. For the purpose of plant lineages share common anatomical host defenses. How- this study, the uppermost green photosynthetic portions of ever, bryophytes and vascular plants synthesize different fun- moss gametophytes and the leaves of vascular plants are con- gistatic and fungitoxic compounds that are chemical defenses sidered to be equivalent and comparable, given that they are against fungal infection (Ferreira et al. 2006; Frahm 2004; Ham- functionally identical, only the most recently produced tis- merschmidt 1999; Mekuria et al. 2005; Van Hoof et al. 1981; sues of all species were sampled, and all occupy similar niches Wang et al. 2005), and the relative efficacy of these compounds as understory plants. The observed difference in biomass may in preventing and controlling colonization by phyllosphere be attributed to anatomical or ecophysiological differences be- fungi may also account for the observed differences in tween vascular plants and bryophytes that enable mosses to biomass. sustain larger amounts of fungi. For instance, the gameto- Statistically significant variation was observed in fungal phytes of the mosses studied are anatomically complex in biomass associated with entire gametophytes of Hylocomium comparison to the flat, glabrous leaves of the vascular plants splendens, Pleurozium schreberi, and Polytrichum commune, with studied. The many microniches created by inrolled leaves, H. splendens hosting the greatest fungal biomass, and Polytri- dense tomenta of rhizoids and pseudoparaphyllia, complex chum commune the least. This suggests that there are species- leaf anatomy, and leaf axils (Do¨ bbeler 1985, 2002; Nyholm specific differences in host suitability among bryophytes. 1975), combined with a more nutrient rich leaf surface envi- Despite the fact that Polytrichum commune has a larger number ronment due to the release of nutrients during successive of microniches than H. splendens and Pleurozium schreberi wetting and drying cycles (Carleton & Read 1991) may support (Do¨bbeler 1985, 2002), and would be expected to host a bigger a larger population of epiphytic fungi on bryophytes than vas- community of epiphytic fungi, the moss was found to have cular plants. Bryophyte gametophytes represent a heteroge- the smallest fungal biomass associated with its tissues. The neous substrate in which senescent and moribund tissues low biomass of the phyllosphere communities of Polytrichum are directly connected to the metabolically active photosyn- commune could be attributed to the moss being of lower nutri- thetic parts of the moss, and are in close proximity to the ent content than the other species studied (Smith et al. 2001). rich fungal community of the soil, forming a continuum Alternatively, the chemical defenses of this species may be with it (Lindahl et al. 2007). The mosaic nature of bryophyte ga- more effective at suppressing epiphytic and endophytic fungal metophytes allows for opportunistic host entry for endophytic growth than the defenses of H. splendens and Pleurozium schre- fungi, and creates an additional phyllosphere micro-niche for beri. In particular, Polytrichum commune has been shown to saprophytic fungi and mycorrhizal fungi acting as saprobes on host epiphytic bacteria that produce antifungal compounds mosses (Carleton & Read 1991). Furthermore, the gameto- (Vandamme et al. 2007), which could reduce epiphytic fungal phytes’ close proximity to and intergradation with the soil, growth and members lower the overall biomass of the phyllo- a large inoculum reservoir, would allow for ready dispersal sphere community. Significant differences in fungal biomass to and colonization of the phyllosphere by fungi. The unique associated with the leaves of Avenella flexuosa, Gymnocarpium composition and location of moss gametophytes may result dryopteris, and Vaccinium myrtillus were not observed, suggest- in their ability to sustain larger phyllosphere fungal communi- ing uniformity in both anatomy and physiological suitability ties than vascular plants, whose phyllosphere is more for colonization by endophytic and epiphytic fungi. Further- spatially isolated and homogenous. Alternatively, the physio- more, the fungal biomasses associated with these plants logical environment of the bryophyte cell may be more hospi- were consistent with those reported for other species in the lit- table to epiphytes and endophytes, accounting for the greater erature (see Table 3) suggesting the magnitude of fungal phyl- biomass of phyllosphere fungi observed in mosses. Greater losphere communities of vascular plants is relatively constant. availability of nutritional resources, relative moisture avail- A nearly statistically significant difference (P ¼ 0.07) was ability or fewer, less effective host defenses could enable bryo- detected in the ergosterol content of vascular plants from phytes to host larger populations of phyllosphere fungi. the two different forest sites. This may be reflective of site dif- Genetic data (Akita & Valkonen 2002; Andersson et al. 2005; ferences affecting the physiological suitability of the plants for De Leon et al. 2007) and comparisons of disease etiology in vas- colonization by endophytic and epiphytic fungi either directly, cular plants versus bryophytes (Davey et al. 2009) indicate both through overall nutrient availability in the system, or 1258 M. L. Davey et al.

indirectly by affecting factors such as the production of anti- not a product of different levels of recalcitrance to ergosterol fungal defence compounds (Witzell & Shevtsova 2004). On extraction. This is consistent with the findings of Padgett & the contrary, there was no significant difference in ergosterol Posey (1993) and Martin et al. (1990) who found extraction sol- content in the bryophytes at the two forest sites studied. As vent, not saponification, has the greatest impact on ergosterol the two forests were quite similar in local climate and vegeta- recovery from cultured fungi and mycorrhizal root tips, and tion composition and structure, it is possible that the magni- contrary to the findings of Salmanowicz & Nylund (1988), tude of the phyllosphere communities of bryophytes remain who found that saponification increased ergosterol recovery relatively constant provided climate and vegetation features from mycorrhiza. It is unclear whether the lack of difference are alike. Analysis of ergosterol content of Pleurozium schreberi between extraction methods is a result of product loss during from a similar Picea–Vaccinium dominated site sampled during the extraction with saponification procedure, or is a result of winter found less living fungal biomass associated with the the membrane ergosterol of fungal endophytes of bryophytes moss (Stangeland 2009) compared to levels reported in this occurring in primarily neutral-extractable forms. study from the same species collected during the summer, Given the prevalence and importance of bryophytes in ma- suggesting that seasonal fluctuations in the biomass of bryo- jor northern ecosystems (Bach et al. 2009; Benscoter & Vitt phyte phyllosphere fungal communities may occur. 2007; Vitt 1990), and in light of the large amount of fungal bio- The green photosynthetic tissues of bryophytes contained mass associated with bryophyte phyllospheres in comparison significantly less ergosterol than brown senescent tissues to those of vascular plants, further research is needed to better from the same gametophytes. The senescent portions of characterize these communities. Preliminary studies suggest mosses are known to function as storage tissues (Hakala & high biodiversity within the fungal phyllosphere of bryo- Sewoń 1992; Skre et al. 1983) and are composed of a heteroge- phytes (Davey & Currah, Unpublished data; Kauserud et al. nous mixture of moribund, senescent, and storage cells. The 2008), and comparisons between the fungal communities as- fungal community may be larger in the senescent parts of the sociated with moss gametophytes and vascular plants are shoots due to greater nutrient availability in the form of abun- needed to determine if there are differences in taxonomic di- dant storage molecules, or the opportunistic colonization of versity between these hosts, and if bryophytes are capable of moribund cells as a micro-niche that is free of host defenses. Al- acting as inoculum reservoirs for horizontal transmission of ternatively, the difference could be attributed to environmental endophytes, or as alternate hosts for what are traditionally factors, which can greatly affect growth of the epiphytic com- thought of as vascular plant-associated fungi. Furthermore, munity in particular (Whipps et al. 2008). The senescent por- the ecological function of these fungi remains largely un- tions of a moss are frequently in the internal parts of a mat or known, and research is needed to determine if they play roles turve, where there are fewer fluctuations in temperature and in bryophyte host defenses and stress responses, as observed moisture (Bates 1998), potentially allowing for greater fungal in vascular plants. growth. Further research is needed to determine if the difference in biomass observed between photosynthetic and senescent tissues is also reflective of differences in endophyte and Acknowledgments epiphyte community biodiversity, with a gradient of community composition existing in the length of the bryophyte shoot. The authors thank the Research Council of Norway for The magnitude of the difference in fungal biomass associ- financial support. ated with the photosynthetic and senescent tissues of gametophytes varies significantly between moss species, with the greatest difference observed in Polytrichum commune.Thismay Supplementary material be a result of species-specific differences in phyllosphere phys- iology between the photosynthetic and senescent portions of Supplementary material may be found in the online version of the moss gametophyte. For example, in Polytrichum commune, this article at doi:10.1016/j.mycres.2009.08.001. phyllosphere fungi on photosynthetic tissues may be more sup- pressed than in other species, as a result of greater inhibition by references plant- and epiphytic bacteria-derived antifungal compounds (Mekuria et al. 2005; Vandamme et al. 2007). Alternatively, Poly- trichum commune may exhibit a larger phyllosphere community in its senescent portions because it possesses leptoids, special- Akita M, Valkonen JPT, 2002. A novel gene family in moss (Phys- comitrella patens) shows sequence homology and a phyloge- ized photosynthate transport cells, that are not found in the netic relationship with the TIR-NBS class of plant disease other host mosses studied. Polytrichum commune is able to rap- resistance genes. 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