Download Schedonorus pratensis

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No effects of Epichloë endophyte infection on nitrogen cycling in meadow
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fescue (Schedonorus pratensis) grassland
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Juha Mikola 1, Marjo Helander 2,3, Kari Saikkonen 3
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Finland
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Section of Ecology, Department of Biology, University of Turku, 20014 Turku, Finland
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Management and Production of Renewable Resources, Natural Resources Institute Finland
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Department of Environmental Sciences, University of Helsinki, Niemenkatu 73, 15140 Lahti,
(Luke), Itäinen Pitkäkatu 3, 20520 Turku, Finland
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Abstract
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Background and aims Systemic Epichloë endophytes produce alkaloids that protect their grass
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hosts against pathogens and herbivores. These alkaloids, together with other endophyte induced
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changes in litter quality, may decelerate the decomposition of infected grass litter, but so far no
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study has tested whether the effects on decomposition rate translate into changes in N cycling in
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infected grasslands. Here we test if the Epichloë uncinata infection of meadow fescue, Schedonorus
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pratensis decelerates litter decomposition and N release, increases soil C and N accumulation and
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lowers the availability of mineral N in the soil under infected grass.
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Methods To analyze grass litter and soil attributes, samples were collected from endophyte
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infected (E+) and non-infected (E-) field plots, established seven years earlier. At the time of the
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study, the frequency of E+ plants was 80-90 % and 0-3 % in the E+ and E- plots, respectively.
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Litter decomposition rate and litter N release were examined using litter mesh bags, placed on field
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ground. Soil mineral N availability was estimated using ion exchange resin bags that were buried in
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the soil.
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Results Epichloë uncinata infection did not affect meadow fescue litter N%, litter mass loss or
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litter N release. Neither did soil C and N content and resin NH4 and NO3 contents differ between the
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E+ and E- grass plots. E+ litter did not decompose faster in E+ than E- plots, i.e. no home-field
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advantage was observed.
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Conclusions We did not find evidence that Epichloë uncinata infection would decelerate N cycling
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and reduce N mineralization in meadow fescue grasslands. This suggests that the infection may not
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decrease the benefit of the endophyte-grass symbiosis by reducing soil fertility.
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Keywords Epichloë uncinata, soil, plant litter, decomposition, systemic endophyte, mineralization,
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symbiosis.
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Introduction
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Plant litter decomposition and nutrient mineralization are fundamental processes in all ecosystems.
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A great share of the variation in these processes within and among ecosystems can be explained by
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plant trait variation (Wardle 2002). Due to the adaptation of plants and their foliage to different
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habitats, plant species differ in the quality of leaf litter they produce and such differences can have
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significant consequences on litter decomposition and nutrient cycling in terrestrial ecosystems
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(Hobbie 1992, Wardle et al. 1997). Significant intra-specific genetic variation has also been shown
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to exist in both woody (Madritch et al. 2006, Silfver et al. 2007) and herbaceous species (Crutsinger
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et al. 2009), which allows natural selection to drive litter decomposition through the effects on
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foliage traits in plant populations (Whitham et al. 2006). Much of the interspecific variation in plant
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traits and litter quality is due to plant adaptation to trophic interactions (Wardle 2002), but
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interactions can also modify litter quality in the ecological time scale. For instance, herbivore
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feeding can induce secondary metabolite production in plant leaves (Nykänen and Koricheva 2004)
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and because these metabolites can remain through leaf senescence and reduce litter decomposition
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rate (Findlay et al. 1996, Schweitzer et al. 2005), herbivore-induced changes in green leaf chemistry
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can have after-life effects on litter decomposition. Although all herbivore effects on litter
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decomposition are not explainable by induced production of secondary metabolites (Silfver et al.
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2015) and the effects can vary across plant genotypes (Schweitzer et al. 2005, Silfver et al. 2015),
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these effects show how organisms that interact with live plants can significantly contribute to plant
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litter decomposition.
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Like the herbivores, foliar endophytes can modify plant green leaf chemistry (Huitu et al.
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2014). The systemic Epichloë endophytes (including the fungi earlier classified in Neotyphodium;
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Leuchtmann et al. 2014), which as common among the cool season Poaceae grasses (subfamily
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Pooideae) (Schardl et al. 2011), are particularly well known for their abundant production of
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alkaloids (Lehtonen et al. 2005, Saikkonen et al. 2013a) that protect their grass hosts against
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pathogens and herbivores (Clay 1988, Clay and Schardl 2002). Like the secondary metabolites of
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plant leaves, these alkaloids remain in senescent grass leaves (Siegrist et al. 2010) and are therefore
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assumed to have effects on litter decomposition and nutrient cycling (Saikkonen et al. 2015). There
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are few studies that have examined the effects of Epichloë infection on grass litter decomposition,
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but so far all published studies have found a weak, negative effect on litter mass loss (Omacini et al.
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2004, Lemons et al. 2005, Siegrist et al. 2010). There is also evidence of lower microbial activity
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and accumulation of organic matter under endophyte infected grass (Franzluebbers et al. 1999).
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These effects are assumed to be due to the alkaloids having adverse effects on soil decomposers
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(Franzluebbers et al. 1999), but this assumption has also been questioned in a study, where high
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alkaloid concentrations of green grass leaves had only a small negative effect on leaf decomposition
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in comparison to green leaves with no alkaloids (Siegrist et al. 2010). Further investigations of the
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effects of Epichloë infection on grass litter decomposition are therefore needed (Omacini et al.
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2012), and so far no study has tested whether the Epichloë effects on litter mass loss translate to
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changes in nutrient cycling in plant communities with Epichloë infected grasses. These effects
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might be particularly interesting as the symbiotic interaction between the grass and its endophyte
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fungus appears to be mutualistic in fertile soils only (Saikkonen et al. 2006). The Epichloë fungi are
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commonly considered as strong plant mutualists as their fitness depends on host fitness and the
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fungi can significantly increase the vigor, environmental tolerance and pathogen and herbivore
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resistance of the host (Clay 1988, Clay and Schardl 2002, Saikkonen et al. 2004, 2010). However, if
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the endophyte infection could significantly decrease nutrient return from grass litter, the fertility of
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the soil along with the benefits of the infection might decrease to the level, where selection would
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start acting against the infection. It should be noted though that any effects of litter quality on litter
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nutrient release can quickly vanish if the decomposer communities are able to adapt to the
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chemistry of the litter. In such case, the ability of local decomposer communities to degrade litters
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of different quality would not differ, a phenomenon called a home-field advantage (Austin et al.
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2014). Home-field advantage has been shown to evolve under different plant species (Vivanco and
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Austin 2008) and plant genotypes (Madritch and Lindroth 2011), and the faster decomposition of
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Epichloë infected litter in infected than non-infected tall fescue plots (Lemons et al. 2005) suggests
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that this phenomenon can also be induced by the chemistry of endophyte-infected litter.
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Here we report results from a study, where we examined the effects of the systemic Epichloë
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uncinata endophyte on grassland nitrogen (N) cycling in infected (E+) and non-infected (E-)
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meadow fescue, Schedonorus pratensis (ex. Lolium pratense and Festuca pratensis) field plots.
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Meadow fescue is a widely used perennial forage grass in Nordic countries, but also grows in
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meadows, roadsides and wastelands (Hämet-Ahti et al., 1988). Both agricultural and wild
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populations are often colonized by E. uncinata, a heritable systemic fungus, which grows
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asymptomatically throughout the plant and is transmitted to new plant generations in plant seed
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(Saikkonen et al. 1998, Cheplick and Faeth 2009, Leuchtmann et al. 2014). Epichloë uncinata is
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known to produce loline alkaloids and their derivatives (Lehtonen et al. 2005). These appear to be
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non-toxic to large mammal herbivores (Clay and Schardl 2002), but can be noxious to invertebrates
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and small vertebrates (Conover 2003, Saikkonen et al. 2006, Huitu et al. 2008). Infected plants also
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have higher silicon concentration than uninfected plants (Huitu et al. 2014), but whether the
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differences in leaf chemistry affect litter decomposition and N cycling in meadow fescue grasslands
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has not earlier been investigated. Based on the earlier findings of reduced litter mass loss in
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Epichloë infected Italian ryegrass, Lolium multiflorum (Omacini et al. 2004) and tall fescue,
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Schedonorus arundinaceus (syn. Festuca arundinacea and Lolium arundinaceum) (Lemons et al.
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2005, Siegrist et al. 2010), we hypothesized that (1) E. uncinata infection will decelerate the
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decomposition rate of meadow fescue shoot litter, and as a result, (2) more organic C and N will
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accumulate in the soil in E+ than E- plots (Franzluebbers et al. 1999), (3) less N will be released
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from E+ than E- litter and (4) less mineralized N (NH4 and NO3) will be available in the soil under
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E+ than E- plants. However, since microbial communities can express home field advantage
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(Austin et al. 2014), we also predicted that the mass loss and N release of E+ litter would be higher
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in the E+ than E- plots. Finally, since the Epichloë infected grass often grow more vigorously than
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non-infected grass (Clay 1998), we followed soil temperature in our E+ and E- plots to assess the
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possibility that (6) any observed effects of E. uncinata infection on litter decomposition and N
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cycling could simply be explained by differences in the degree of shade and soil temperature
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between the E+ and E- plots.
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Materials and methods
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Experimental site and endophyte treatment
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The experiment was conducted in Jokioinen, South-West Finland (60° 49´ N, 23° 30´ E) in an old
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agricultural field, which was tilled and fertilized with cow manure (30 000 kg ha-1). Twenty metal
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fenced enclosures (25 m × 39 m) were established in May 2006: ten of the enclosures (later called
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plots) were sown with endophyte-free (E-; 0% endophyte frequency) and another ten with
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endophyte infected (E+; 79% infection frequency) seeds of the meadow fescue (Schedonorus
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pratensis) cultivar ‘Kasper’ (20 kg seeds ha-1). The E+ and E- treatments were randomly assigned
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to 10 plot pairs (used as replicate blocks in statistical analyses). The seeds were obtained from seed
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production farms via the Finnish Food Safety Authority (EVIRA, Seed Certification Unit, Loimaa,
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Finland). In June 2007, the plots were fertilized with a commercial fertilizer (16:9:22 N:P:K with
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micronutrients; Kemira, product number 0647334). In 2012, six years after establishment, the
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frequency of E+ plants was 80-90 % and 0-3 % in the E+ and E- plots, respectively, and the aerial
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cover of meadow fescue had decreased from 100 % to 75% in E− and 98% in E+ plots due to weed
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invasion (Saikkonen et al. 2013b).
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Sampling and analyses
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For the samples and measurements of the present study, a triangular area (each side 3.5 m) with
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high meadow fescue coverage was selected in each plot in mid-May 2013. Overwintered, erect
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meadow fescue shoot litter, composed of senescent leaves and stems, was collected from each of
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the 20 plots (not from the area of the triangle) at the same time and stored at room temperature until
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placed into litter mesh bags (7 cm × 11 cm, mesh size 1 mm, 5 g fresh mass in each bag). Six bags
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were established from each of the 20 litter samples: two of the bags were later placed on the plot,
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where the litter was collected (i.e. E- litter placed on an E- plot, E+ litter placed on an E+ plot), two
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were placed on the adjacent plot in the same replicate block (i.e. E- litter placed on an E+ plot, E+
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litter placed on an E- plot), and the remaining two bags were placed outside the experimental area
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amongst a dense species-rich herbaceous vegetation. The latter area was used as a comparison site,
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where the decomposers had not been subjected to, and potentially adapted to either E- or E+ litter
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and is called as a “neutral” plot. The litter bags were placed on soil surface (in E+ and E- plots in
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the middle of the triangular subplot) on 11th of June 2013, covered using a white mesh and
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collected on 21st of August 2013. The collected bags were kept frozen (-18 °C) until the litter in
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each bag was dried (48 h, 70 °C), weighed and ground and its N concentration analyzed using a
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LECO CNS-2000 analyzer (LECO Corporation, USA). To be able to calculate litter mass loss and
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the amount of N released from each litter bag during the decomposition period, subsamples of the
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original 20 field samples were weighed before and after drying (to determine their dry mass
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content), ground and analyzed for N concentration. The amount of N released from each litter bag
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was then calculated using the initial and final dry masses and N concentrations.
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To analyze the effect of the grass endophyte infection on the soil N and C content, three
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random soil cores (depth 10 cm, diameter 3 cm) were collected from each subplot in mid-May
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2013. After removing visible roots, the samples were pooled within each subplot, dried (48 h, 70
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°C) and the soil C and N concentrations were determined using the LECO CNS-2000 analyzer. The
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availability of mineral N (NH4+ and NO3-) in the soil was estimated using mesh bags (5 cm × 5 cm,
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mesh size 200 µm) filled with 1 g of ion exchange resin (Amberlite® MB-150 Mixed Bed
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Exchanger, Rohm and Haas, France). Nine bags were buried in the depth of 5 cm in each triangle
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on 11th of June 2013.The bags were collected 5, 7 and 14 weeks later, three random bags at each
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date. In the laboratory, the bags were rinsed using distilled water, the three replicate bags of each
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date were pooled and their NH4+ and NO3- contents extracted in 50 ml of 2M KCl. The KCl solution
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was filtered through a glass microfiber filter (Whatman, GE Healthcare Europe GmbH) and the
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NH4 and NO3 concentrations were analyzed using a Lachat QuikChem 8000 analyzer (Zallweger
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Analytics, Inc., Lachat Instruments Division, USA).
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Soil temperature was measured in mid-July, early and late August and mid-September 2013 in
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the top and 5-10 cm layer. For each measurement in each layer, the value is based on five readings
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at random spots within the triangle.
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Statistical analyses
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The effects of grass endophyte infection and plot endophyte status on plant and soil variables were
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analyzed using ANOVA models. The replicate block was included in the models to explain field
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variation and repeated measures ANOVA was used for those response variables, which had
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repeated measures in time (resin NH4 and NO3 contents, soil moisture and temperature) or space,
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i.e. repeated measurements in E+, E- and neutral plots (litter mass loss and litter N release). The
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homogeneity of variances was tested using the Levene’s test and the normality using model
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residuals and the Shapiro-Wilk test. None of the response variables needed transformations to fulfil
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these requirements. When the sphericity of data was violated in the repeated measures ANOVA, the
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degrees of freedom were corrected using the Greenhouse-Geisser ε.
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Results
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The endophyte infection did not affect the grass litter attributes, i.e. shoot litter N%, litter mass loss
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and litter N release (Table 1). Litter N release was higher in the neutral than E+ and E- plots, while
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litter mass loss was not affected by the identity of the placement plot (Table 2). Grass endophyte
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status × placement plot identity interactions were not significant for either litter N release (F=0.09,
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P=0.913) or litter mass loss (F=0.10, 0.821). Litter N% correlated positively with litter N release (in
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E+ plots r=0.54, P=0.015, n=20; E- plots r=0.53, P=0.016; neutral plots r=0.72, P<0.001) and litter
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mass loss (in E+ plots r=0.58, P=0.007; E- plots r=0.79, P<0.001; neutral plots r=0.53, P=0.017).
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Soil temperature and N and C contents did not differ between the E+ and E- plots (Table 3).
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The resin NH4 content varied across the summer (F=7.20, P=0.005; July 72±6, August 105±9,
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September 78±8 mg g-1 resin), while the NO3 content did not (F=0.10, P=0.904), and neither was
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affected by plot endophyte status (Table 3). The mean NO3 content of the three resin harvests of a
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plot correlated positively with plot litter N% (r=0.58, P=0.008, n=20), and the resin NH4 and NO3
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contents correlated positively with each other in August (r=0.50, P=0.023) and September (r=0.74,
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P<0.001). The August temperatures of the 5-10 cm soil layer correlated negatively with the August
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resin NH4 (r=-0.67, P=0.001, n=20) and NO3 (r=-0.59, P=0.006) contents and the September
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temperatures with the September NH4 (r=-0.51, P=0.021) and NO3 (r=-0.44, P=0.052) contents.
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Discussion
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We hypothesized that the meadow fescue grass infected by Epichloë uncinata would produce litter
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that decomposes more slowly and releases less N than the litter produced by non-infected grass and
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that this would lead to more organic C and N accumulating and less mineral N being available in
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the soil under infected than non-infected plants. These hypotheses were, however, falsified by our
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data: although the mean N release was lower from E+ (0.26 mg N g-1 dry litter) than E- (1.06 mg)
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litter as we predicted, this difference was not statistically significant and the other variables showed
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no signs of endophyte effects.
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The absence of the endophyte effect on litter mass loss in our study is in contrast to earlier
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observations with other fungus-grass combinations. Omacini et al. (2004) found that the litter of E+
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Italian ryegrass had 18% lower mean decay rate than the E- litter. Similarly, Lemons et al. (2005)
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showed that E+ tall fescue (S. arundinaceus) litter had 4.4% and 7.9% more mass remaining than E-
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litter after 197 and 256 d of decomposition. In the latter study, the endophyte effect got stronger
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with time and no difference was observed after 135 d of decomposition and ~33% mass loss. Our
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meadow fescue litter was subjected to decomposition for 70 d with an average mass loss of 33%. If
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the endophyte effect on litter mass loss only appeared at the later stage of decomposition as in
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Lemons et al. (2005), this would suggest that we missed the effect in our shorter test. However, late
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effects do not seem to be a rule as the significant reduction in litter decay rate in Omacini et al.
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(2004) was found in an 83-d study with a mean mass loss of ~23%. Another explanation for the
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discrepancy between ours and earlier results might be the fact that we used overwintered litter and
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the alkaloids had partly degraded during the winter (see Kallenbach et al. 2003). Using
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overwintered litter to examine litter effects on N cycling is, however, justified as most of the litter
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enters the soil at this stage. Moreover, the absence of the endophyte effect on litter decomposition
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agrees well with our finding that soil C and N contents and N mineralization, which tell of longer-
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term endophyte effects, did not respond to the endophyte status of the field plots. The last piece of
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evidence of the absence of endophyte effects on N cycling in our field site comes from an earlier
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investigation carried out in the same site; in this study, the green leaf N% did not differ between the
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infected and non-infected meadow fescue (Huitu et al. 2014). If N cycling differed significantly
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between the E+ and E- plots in our site, the grass green leaf N% would most likely respond to the
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difference. Lastly, it is good to note that the theory of the effect of litter decomposition rate on soil
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organic matter accumulation is currently under revision (Cotrufo et al. 2013). In contrast to the
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earlier belief, slow decomposition of low-quality litter is now suggested to decrease the
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accumulation of soil organic matter due to lower accumulation of persistent microbial products
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(Cotrufo et al. 2013). This is not highly relevant in our study as the Epichloë infection had no effect
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on soil C and N accumulation, but it challenges the interpretation that alkaloids would be the cause
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for organic matter accumulation under endophyte infected grass (Franzluebbers et al. 1999).
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Litter quality is always a combination of attributes that enhance decomposition such as litter
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N% (Silfver et al. 2007) and those that decelerate decomposition such as lignin concentration
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(Melillo et al. 1982), but also depends on their interaction (Berg 2014). Besides producing
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alkaloids, the Epichloë fungi are known to have effects on many attributes of grass green leaf
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chemistry (Vázquez-de-Aldana 2013, Huitu et al. 2014, Saikkonen et al. 2015) and the variation in
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the relative magnitude of these effects may be one cause for the variation that appears in Epichloë
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effects on litter decomposition. In our study, the Epichloë infection did not affect litter N%, but the
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plot-to-plot variation in litter N% correlated positively with litter mass loss, litter N release and NO3
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availability in the plot soil. This shows that litter N%, even when originating from the limited
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spatial variation of a homogeneous agricultural soil, can be a main control of litter decomposition
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and N cycling in grasslands. Since litter N% was not affected by grass endophyte infection in our
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study, litter N% could not mask any potential alkaloid effects, but such mask is possible in other
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studies as the endophyte infection has been shown to increase grass green leaf N% (Siegrist et al.
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2010, Vázquez-de-Aldana 2013). In fact, Siegrist et al. (2010) speculated that the weak effect of
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high alkaloid concentrations on decomposition in their study with green leaf material was due to the
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high N% of the material, which potentially alleviated the adverse alkaloid effects. Considering that
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the alkaloid concentrations of green leaves vary widely among grass-endophyte symbioses
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(TePaske et al. 1993) and are low in the senescent leaves and litter of infected grasses (Eichenseer
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et al. 1991, Siegrist et al. 2010), leaf N% provides a noteworthy, additional mechanism to test in the
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studies of Epichloë endophyte effects on litter decomposition and grassland N cycling. It is likely
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though that variation in litter N% will also have its limitations in predicting the endophyte effects
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on decomposition rates; for example, while the increasing atmospheric CO2 concentrations have
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predictable effects on litter N% (reduced) and lignin concentrations (elevated), these effects do not
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appear to lead to slower litter decomposition rates as expected (Norby et al. 2001).
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We did not find evidence of a home-field advantage (Austin et al. 2014), i.e. significant ‘litter
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endophyte status × plot endophyte status’ interaction effects on litter decomposition or litter N
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release, neither were there any signs of plot endophyte status affecting litter decomposition. These
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results provide further evidence of the absence of Epichloë uncinata effects on the activity or
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structure of the decomposer community in our meadow fescue grassland. Instead, we found that
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vegetation composition affected litter N release (the release was greater in the “neutral” than
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experimental plots) and that soil mineral N availability was linked to soil temperature. These results
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suggest two potential long-term mechanisms for endophyte effects on litter decomposition. First,
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since the grass endophyte infection can reduce the species richness of a plant community (Clay and
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Holah 1999) and prohibit weed invasion to a grass field (Saikkonen et al. 2013b), the species
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composition of infected and non-infected grass fields (wild or established) may eventually differ
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insomuch that this will have effects on the decomposition processes. In our study, we purposely
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eliminated this mechanism by choosing subplots (the triangles) that were dominated by meadow
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fescue. Secondly, the more vigorous growth of endophyte infected grasses (Clay 1988) and the
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concomitant changes in plant species richness (Clay and Holah 1999, Saikkonen et al. 2013b) may
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impact soil temperature, which will then reflect in N mineralization. Such effects would require a
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complex mixture of plant-plant competition, herbivory and community dynamics, but would still
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origin from the endophyte symbiosis.
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To conclude, we could not find evidence that Epichloë uncinata infection would decelerate N
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cycling and reduce N mineralization in meadow fescue grassland. This suggests that the infection
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may not decrease the benefit of the endophyte-grass symbiosis by reducing soil fertility as we
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speculated. Instead of the endophyte infection, the spatial variation of litter N% was a good
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predictor for the variation in litter mass loss, litter N release and soil mineral N concentrations in
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our field. When such clear link between litter N%, litter decomposition and N mineralization is
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combined with the earlier findings of low alkaloid concentrations in endophyte infected grass litter
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(Siegrist et al. 2010), varying effects of the infection on litter decomposition (Omacini et al. 2012)
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and the positive effects of the endophyte on grass amino acid and N concentrations (Lyons et al.
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1990, Siegrist et al. 2010), it appears that the endophyte effect on litter N% might be a worthwhile
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mechanism to examine to understand the endophyte effects on litter mass loss and N cycling in
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grassland ecosystems.
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Acknowledgements
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We thank Hillamari Lehikoinen, Serdar Dirihan and Tuija Koivisto for assisting with litter and resin
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bags, Merja Myllys for measuring soil temperatures and Viivi Toivio, Santeri Savolainen and
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Marianne Lehtonen for processing and analyzing the resin and litter samples. The study was
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supported by the Academy of Finland (grants no. 137909 and 281354) to Kari Saikkonen.
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Table 1. The means (± SE, n=10) of the attributes of E+ and E- meadow fescue litter and the F- and
P-statistics of ANOVA of the effect of the Epichloë uncinata infection.
Litter N%
Litter mass loss (% of dry mass) b
Litter N release (mg N g-1 dry litter) b
E+ grass
0.96 ± 0.04
33.2 ± 1.3
0.26 ± 0.28
E- grass
1.02 ± 0.10
31.8 ± 2.3
1.06 ± 0.48
F
0.45
0.64
2.40
P
0.520
0.446
0.156
b
means are based on the average of values obtained in E+, E- and neutral plots; statistics from
repeated measures ANOVA
Table 2. The mean attributes (± SE, n=20) of meadow fescue litter placed on E+, E- and neutral
plots and the F- and P-statistics of ANOVA of the effect of the placement plot identity.
a
Litter mass loss (% of dry mass)
Litter N release (mg N g-1 litter) a
a
E+ plot
32.3 ± 0.01
0.41 ± 0.29
E- plot
Neutral plot
33.0 ± 0.02 32.2 ± 0.01
0.66 ± 0.33 0.91 ± 0.30
F
0.47
4.34
P
0.556
0.029
means include both E+ and E- grass; statistics from repeated measures ANOVA
Table 3. The means (± SE, n=10) of soil attributes and resin NH4 and NO3 contents in E+ and Eplots and the F- and P-statistics of ANOVA of the effect of the plot endophyte status.
Soil N content (% of dry mass)
Soil C content (% of dry mass)
Resin NH4 content (mg g-1) a
Resin NO3 content (mg g-1) a
Soil temperature (°C, surface) b
Soil temperature (°C, 5-10 cm) b
a
E+ plot
0.38 ± 0.02
5.34 ± 0.23
83 ± 8
232 ± 39
17.2 ± 0.3
15.6 ± 0.2
E- plot
0.36 ± 0.01
5.16 ± 0.20
87 ± 8
233 ± 56
17.2 ± 0.3
15.6 ± 0.2
F
0.51
0.28
0.10
<0.01
0.07
0.18
P
0.493
0.609
0.758
0.995
0.798
0.685
means are based on the average of July, August and September values; statistics from repeated
measures ANOVA
b
means are based on the average of July, August (early and late) and September values; statistics
from repeated measures ANOVA