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Basic and Applied Ecology 14 (2013) 146–154

ungal endophytes of a forage grass reduce faecal degradation ratesichael G. Cripps∗, Grant R. Edwards

griculture and Life Sciences Faculty, Lincoln University, PO Box 84, Lincoln 7647, New Zealand

eceived 18 September 2012; accepted 21 December 2012vailable online 20 January 2013

bstract

Mutualisms between fungal endophytes and forage grasses can exert broad-reaching effects on grassland communities andcosystem processes. We hypothesised that endophytes of grasses would retard the process of faecal degradation since grazingnimals consume primarily live plant material and excrete a large portion of the herbage they consume as faeces. We examinedhe degradation rates of faeces from sheep that had consumed pure swards of perennial ryegrass containing a range of uniquetrains (AR1, AR37, or Wild type) of the fungal endophyte, Neotyphodium lolii, or no endophyte. Ultimately, the presencef endophytes in perennial ryegrass resulted in slower faecal decay rates compared to the nil endophyte treatment, althoughnly consistently for the C concentration decay rates that were approximately 2× to 4× slower in the endophyte-derived faecalatter. The decay rate of dry matter content was significantly slower (ca. 1.5×) in the novel endophyte-derived faeces (AR1

nd AR37) compared to the nil endophyte-derived faeces. The N decay rates differed significantly only in the AR1 treatmenthat was approximately 4× slower than the nil endophyte group. The reduced decay rates are attributed to the presence ofndophyte-derived alkaloids in the faeces, and a greater proportion of more easily degraded hemicellulose in faeces from sheephat consumed the endophyte-free grass. There were no significant differences in the faecal carbon and nitrogen decay ratesmong the three endophyte strain treatments. This suggests that all the strain-specific alkaloids might have similar effects, orhat N. lolii has a general effect that is not strain-specific, such as altered fibre composition, as reported here. This is the firsteport of a fungal endophyte affecting the rate of faecal degradation, and the first report of the alkaloids peramine, lolitrem

and epoxy–janthitrems in faecal matter. This study shows that a common agronomic grass–endophyte mutualism can haveffects on ecosystem processes that have not previously been considered.

usammenfassung

Mutualismus zwischen endophytischen Pilzen und Weidegräsern kann weitreichende Auswirkungen auf Graslandgemein-chaften und Ökosystemprozesse haben. Wir nahmen an, dass Grasendophyten den Abbau von Dung verzögern würden, weil

rasende Tiere vorwiegend lebendes Pflanzenmaterial aufnehmen und einen großen Teil des konsumierten Futters als Fäzes usscheiden. Wir untersuchten die Abbauraten des Dungs von Schafen, die auf geschlossenen Rasen von Deutschem Weidelgraseweidet hatten, wobei das Gras einzelne Stämme (AR1, AR37 bzw. Wildtyp) des endophytischen Pilzes Neotyphodium loliider keinen Endophyten enthielt. Letztendlich verursachte der Endophyt eine langsamere Abbaurate des Dungs verglichen miter Variante ohne Endophyten, wenn auch durchgehend nur bei den Kohlenstoffabbauraten, die zwei- bis viermal langsamerm vom endophythaltigen Futter stammenden Dung waren. Verglichen mit endophytfreiem Futter, war die Abbaurate der Troc-enmasse des Dungs signifikant langsamer (ca.1.5-fach) bei Futter, das die neuartigen Endophyten AR1 und AR37 enthielt.ie Stickstoffabbauraten differierten nur beim AR1-haltigen Futter signifikant, wobei sie ungefähr viermal langsamer als nach

∗Corresponding author at: AgResearch Lincoln, Private Bag 4749, Christchurch, New Zealand. Tel.: +64 3 325 9926.E-mail addresses: mike.cripps@agresearch.co.nz, michael cripps@hotmail.com (M.G. Cripps).

439-1791/$ – see front matter © 2013 Gesellschaft für Ökologie. Published by Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.baae.2012.12.004

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M.G. Cripps, G.R. Edwards / Basic and Applied Ecology 14 (2013) 146–154 147

ütterung mit endophytfreiem Gras waren. Die verringerten Abbauraten werden auf das Vorhandensein von Alkaloiden, die vomndophyten stammen, zurückgeführt, sowie auf einen höheren Gehalt an leicht abbaubarer Hemicellulose im Dung von Schafen,ie endophytfreies Gras gefressen hatten. Es gab keine signifikanten Unterschiede zwischen den drei Endophytenstämmeninsichtlich der Abbauraten von Kohlen- und Stickstoff im Schafdung. Dies legt den Schluss nahe, dass alle stammspezifischenlkaloide ähnliche Effekte haben könnten oder dass N. lolii eine generelle Wirkung hat, die nicht stammspezifisch ist, wie z.B.ie veränderte Faserzusammensetzung, die wir hier festgestellt haben. Dies ist der erste Bericht darüber, wie ein endophytischerilz Dungabbauraten beeinflusst, und der erste Bericht über die Alkaloide Peramin, Lolitrem B und Epoxy-Janthitreme inungmaterial. Diese Untersuchung zeigt, dass ein häufiger Mutualismus zwischen einem Ackergras und einem Endophytenuswirkungen auf Ökosystenprozesse haben kann, die zuvor noch nicht in Betracht gezogen wurden.2013 Gesellschaft für Ökologie. Published by Elsevier GmbH. All rights reserved.

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eywords: Decomposition; Ecosystem process; Lolium perenne; M

ntroduction

An estimated 20% of the earth’s land surface is man-ged grasslands that support grazing livestock (Snaydon981; Seré, Steinfeld, & Gronewold 1996). These grasslandsre primarily composed of annual and perennial grasses,f which many, through the process of coevolution, haveormed close associations with endophytic fungi of the fam-ly Clavicipitaceae (Leuchtmann 1992). The most thoroughlytudied endophytes are the Neotyphodium species, whichre frequently found in most pastoral grasses. The endo-hyte resides symptomless within its host grass where itolonises all aboveground plant tissues, remains inside itsost for the entire life of the plant, and is transmitted solelyia seed (Siegel, Latch, & Johnson 1987; Clay 1990). Theeotyphodium endophytes are obligate symbionts, and their

uccess is tightly linked to the survival and reproductionf their host grass, which is thought to result in a strongutualistic relationship (Gundel et al. 2008). The endophyte

eceives protection and nutrients from the grass, and the grassenefits from the endophyte-derived alkaloids that provideefence against herbivores (Siegel et al. 1987; Clay 1990;ut see Faeth 2002).

Little was known of the functional role of endophytes untilhe late 1970s and early 1980s, when a causal link betweenndophyte-derived alkaloids and livestock toxicosis was dis-overed for tall fescue (Schedonorus phoenix (Scop.) Holub)Bacon, Porter, Robbins, & Luttrell 1977) and perennial rye-rass (Lolium perenne L.) (Fletcher & Harvey 1981). Sincehis discovery, rapid progress was made to isolate and selecttrains of endophytes that avoided the problem of livestockoxicity, but conveyed resistance to invertebrate pests. Now,ost commercial grass cultivars contain selected endophyte

trains that produce desirable alkaloids. The incorporation ofelected strains of ‘novel’ endophytes into grass cultivars hasffectively resulted in landscape scale distribution of a smallumber of genotypes that express some desirable traits (e.g.articular alkaloid production).

Although the proximate agronomic benefits of these

elected endophyte strains are compelling, the possiblextended effects on grassland communities and ecosystemrocesses are unclear. At the community level, endophytic

(sd

sm; Neotyphodium; Nutrient cycling

ungi have been implicated in reduced plant diversity dueo directly or indirectly enhancing competitive ability of theost species (Clay & Holah 1999), and to reduced herbi-ore abundance and altered patterns of herbivory as a resultf plant resistance (Omacini, Chaneton, Ghersa, & Muller001; Rudgers & Clay 2008). At the ecosystem level, endo-hytes have been shown to reduce the rate of plant residueecomposition, thus retarding the process of nutrient cyclingOmacini, Chaneton, Ghersa, & Otero 2004; Lemons, Clay,

Rudgers 2005), although the mechanism of this effect isncertain (Siegrist, McCulley, Bush, & Phillips 2010).

In addition to plant residues, another important source ofutrient returns to the soil is through the decomposition ofnimal excrement. Grazing livestock primarily consume livereen leaves, and excrete a large portion of the herbage theyonsume in faeces (Parsons, Orr, Penning, Lockyer, & Ryden991; Haynes & Williams 1993). Faecal returns are an impor-ant source of soil organic matter, nitrogen, phosphorous,otassium, several secondary nutrients, and micronutrientsShand & Coutts 2006; Aarons, O’Connor, Hosseini, &ourley 2009; Bosshard et al. 2011). Degradation of faeces

nd incorporation into the soil is necessary before final min-ralisation of this organic matter into plant available formsan occur. Furthermore, persistent faecal patches can result inirect productivity losses through unutilised areas of pasturesue to animal avoidance (Marsh & Campling 1970), and indi-ect losses through the enhanced survival of dung-inhabitingarasites and subsequent increases in livestock infectionsWilliams & Warren 2004). Thus, the efficient degradationf faeces in grasslands is a critical component of nutrientycling and healthy ecosystem functioning. The possibilityhat endophytes might have an effect on this avenue of nutri-nt returns has not previously been investigated, but couldlso have important ecosystem consequences.

Here, for the first time, we tested for differences in theegradation of faeces from sheep that had grazed perennialyegrass containing different strains of the endophyte Neoty-hodium lolii (Latch, Christensen & Samuels) Glenn, BaconHanlin, or no endophyte. Specifically, we hypothesised that

1) the presence of endophyte in perennial ryegrass wouldubsequently inhibit faecal degradation, and (2) that faecalegradation rates would differ among the endophyte strains.

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48 M.G. Cripps, G.R. Edwards / Basic

ethods

ollection of faecal samples

Fresh faecal samples were collected on 14 April 2011rom four different paddocks that were grazed by 15 to 20wes at Lincoln, New Zealand. Each of the four paddocks0.18 ha) was a pure sward of perennial ryegrass (Loliumerenne) cv. Grasslands Samson (Agricom NZ Ltd.). Threef the perennial ryegrass paddocks contained a unique strainAR1, AR37, or Wild type) of the fungal endophyte, N. lolii,nd the fourth paddock had no endophyte (Nil). The percentndophyte infection in each paddock was determined by themmunoblot technique (Hiatt, Hill, Bouton, & Stuedemann999), and found to be 85% for AR1, 78% for AR37, 80%or wild type, and <5% for endophyte-free.

In perennial ryegrass, four major types of alkaloids arenown to be produced by different strains of N. lolii. StrainR1 produces peramine, AR37 produces epoxy–janthitrems,

nd wild type (WT) produces peramine, ergovaline, andolitrems (Powell & Petroski 1992). On the same day as theaecal collections, ca. 20 whole tiller perennial ryegrass sam-les were taken from each paddock and tested for their knownlkaloids, and was found that AR1 had peramine at 17.5 ppm,R37 had epoxy–janthitrems at 14.8 ppm, and Wild type haderamine at 20.1 ppm, ergovaline at 0.3 ppm, and lolitremat 1.0 ppm. This therefore confirmed the presence of the

espective endophyte strains in each paddock.Five individual faecal samples were collected from each

addock, placed in plastic bags, and refrigerated (4 ◦C) untilhe fresh weights were recorded on 20 April. From each indi-idual sample, four approximately equally sized subsamplesone teaspoon) were taken, and the fresh weight recorded tohe nearest 0.01 g. The fresh weight of the remainder (5thegment) of each sample was also recorded, and then freeze-ried to estimate the initial dry matter (DM) proportion ofach individual faecal sample. Thus, the initial DM contentf each of the four subsamples was an estimate based on theM proportion recorded from the remaining 5th segment,

nd assumed all segments of the same faecal sample had theame proportional DM.

xperimental design and recovery of subsamples

The experiment was established on 21 April 2011 athe Lincoln University Research Dairy farm, Lincoln NZ.he four subsamples from each faecal replicate werelaced into mesh litter bags made of polyvinyl chloridePVC)-coated fibreglass. The litter bags were approximately0 cm × 10 cm, and had a mesh size of 1.2 mm × 1.6 mm.ach litter bag was labelled and nailed to the ground sur-

ace between the drill rows of a perennial ryegrass (cv. Altoontaining AR1 endophyte) pasture on Paparua sandy loamoil. The experiment was set up as a randomised block designith five blocks, each block containing four subsamples of

Tpe

plied Ecology 14 (2013) 146–154

ach replicate for each treatment (i.e. 16 litter bags per blocknd a total of 80 litter bags in the experiment). One sub-ample of each replicate from each treatment was collectedequentially on, 5 May, 19 May, 16 June, and 14 July 2011,orresponding to 14, 28, 56, and 84 days after placement ofhe faecal samples in the experiment. The recovered subsam-les were freeze-dried, their dry weights were recorded, andhe proportion DM remaining was calculated. The propor-ional change in DM (�DM) of each subsample recoveredver time was calculated as �DM = (DM0 − DMt)/DM0,here DM0 is the estimated initial DM, and DMt is the

ecorded DM at the respective collection date. Each fae-al sample was then manually ground using a mortar andestle in preparation for carbon, nitrogen, fibre content,nd alkaloid analyses. The total carbon and nitrogen con-entrations (%) of the faecal samples were analysed usingn Elementar Vario-Max CN elemental analyser (Elemen-ar Analysensysteme GmbH, Hanau, Germany). The meanplus minimum and maximum) daily temperatures, accu-ulated rainfall, and number of days with rainfall >1 mm

ver the duration of each recovery period were recorded (seeppendix A).

etermination of fibre content

The percent acid detergent fibre (ADF) and neutral deter-ent fibre (NDF) of each initial faecal sample (i.e. athe starting point of time zero) was determined accord-ng to the methods of Van Soest, Robertson, & Lewis1991), but without the addition of �-amylase since fae-al samples contain minimal starch. The ADF fraction isomposed of only the most recalcitrant fibres, primarily cel-ulose and lignin; and the NDF fraction is composed of the

ore easily decomposed hemicelluloses, plus cellulose andignin.

lkaloid analyses

For the analyses of peramine and ergovaline, 50 mg ofried, ground material was extracted with 1 ml of 50% aque-us methanol containing internal standards of ergotaminend homoperamine and analysed by high pressure liquidhromatography (HPLC) as described by Spiering, Davies,apper, Schmid, & Lane (2002). Lolitrem B was analysedsing an adaptation of the method of Gallagher, Hawkes,nd Stewart (1985) as described by Hunt, Rasmussen,ewton, Parsons, and Newman (2005) and estimatedy comparison of peak areas with those obtained withuthentic external standard lolitrem B. Janthitrems werextracted using 50 mg dried ground material extracted with0% acetone and analysed by HPLC as described by

apper and Lane (2004) and estimated by comparison ofeak areas with those obtained with authentic standardpoxy–janthitrem I.

M.G. Cripps, G.R. Edwards / Basic and Applied Ecology 14 (2013) 146–154 149

Table 1. Mean initial percent dry matter (DM), carbon (C), nitrogen(N), and carbon–nitrogen ratio (C:N) of faeces from ewes that grazedperennial ryegrass (cv. Grasslands Samson) with three strains of theendophyte Neotyphodium lolii (AR1, AR37, WT, Nil), or no endo-phyte (Nil). The least significant difference (LSD) values are givenfor statistical comparison among the three endophyte treatments(AR1, AR37, and WT), or no endophyte (Nil) for each variable.Significant differences within a variable are indicated by differentletters.

Variable AR1 AR37 WT Nil LSD (5%)

DM 26.4a 27.3a 23.3a 25.3a 10.1C 46.1a 41.7c 45.4a 42.9b 0.902N 2.95a 2.36b 2.78a 2.31b 0.365C

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Fig. 1. Decay curves for the proportion dry matter (DM), carbon(C), and nitrogen (N)remaining in faeces collected from ewes thatgrazed perennial ryegrass (Lolium perenne cv. Grasslands Samson)containing strains (AR1, AR37, and WT) of the fungal endophyte,N

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tatistical analyses

Each degradation variable (DM, C, and N content) for eachreatment was modelled as a decay curve, and the mean decayates from the fitted curves were compared among the endo-hyte treatments. The change in DM, C and N over time weretted to simple linear regression models (y = 1 − kt, where k is

he decay constant expressed in day−1) since they providedbetter goodness of fit than a standard negative exponen-

ial decay model. Linear decay models are often appropriatehen the material being examined loses little mass over the

nvestigation time period (Wieder & Lang 1982). Regressionsere performed by fitting treatment-group-specific slopes

nd comparing each slope to the reference slope of the nilndophyte group. The ADF and NDF were also analysedsing ANOVA followed by LSD comparisons among thereatment means if the overall ANOVA indicated significantifferences.

esults

There was no significant difference in the initial DMroportion among the four endophyte (AR1, AR37, WT,il) treatments (P = 0.844). The initial total C concen-

ration (%) was significantly different among the fourreatments, and ranked from greatest to least concentrations AR1 = WT > Nil > AR37 (Table 1). The initial N con-entration among the treatments also significantly differed,nd ranked from greatest to least as AR1 = WT > AR37 = NilTable 1).

The decay rates (k) for the proportional change in DM con-ent were significantly slower in the novel endophyte (AR1nd AR37) compared to WT and nil endophyte derived faecesTable 2; Fig. 1A). The decay rates for C concentration wereignificantly slower in all endophyte treatments compared

o the nil endophyte group, and differed by 1.8×, 3.9×, and.7× for AR1, AR37, and WT, respectively (Table 2; Fig. 1B).he decay rates for N concentration differed significantly

orf

eotyphodium lolii, or no endophyte (Nil).

nly in the AR1 treatment that was 3.7× slower comparedo the nil endophyte group (Table 2; Fig. 1C). There were noignificant differences among the decay rates of DM, C and Nrom the faeces derived from the three endophyte-containingerennial ryegrasses (AR1, AR37, and WT) except for the Cecay rate between AR37 and WT (P = 0.046).

There were no significant differences in the proportions

f ADF among the faeces derived from the four perennialyegrass treatments (P = 0.094; Fig. 2A). However, faecesrom nil endophyte perennial ryegrass had a significantly

150 M.G. Cripps, G.R. Edwards / Basic and Applied Ecology 14 (2013) 146–154

Table 2. Mean decay rates (day−1) for dry matter (DM), carbon (C), and nitrogen (N) for each endophyte treatment (AR1, AR37, WT, andNil). Rates significantly different from the nil endophyte reference group are indicated by asterisks.

Variable AR1 AR37 WT Nil

DM 6.06 × 10−3* 6.35 × 10−3* 7.32 × 10−3 8.85 × 10−3

C 1.25 × 10−3** 5.87 × 10−4*** 1.34 × 10−3* 2.27 × 10−3

N 9.51 × 10−4* 1.78 × 10−3 2.16 × 10−3 3.48 × 10−3

*P < 0.05.**P < 0.01.

***P < 0.001.

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Fig. 2. Mean (±SE) percent acid detergent fibre (ADF) and neutraldetergent fibre (NDF) in the faeces of ewes that grazed perennialryegrass (Lolium perenne cv. Grasslands Samson) containing strains(AR1, AR37, and WT) of the fungal endophyte, Neotyphodium lolii,or no endophyte (Nil). There was no overall significant differencein the ADF values among the treatments and therefore no multi-ple comparison test was conducted. Significant differences in theNDF among the treatments are compared using the least significantdifference (LSD) test, and indicated by different letters.

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reater proportion of NDF compared to all faeces derivedrom endophyte-containing perennial ryegrass (P = 0.024;ig. 2B).In the initial faecal samples (i.e. time 0), peramine and

rgovaline concentrations were present, but below the limitf detection for three replicates in AR1 and WT, respectivelyTable 3). Epoxy–janthitrems and lolitrem B were present inhe initial faecal samples (Table 3) at concentrations similarr greater to that found in the perennial ryegrass in April 2011see “Methods” section).

iscussion

This is, to our knowledge, the first empirical evidencef a fungal endophyte affecting the degradation of fae-al material. This corresponds well with other studieshat have found reduced decay rates of plant residuesrom endophyte-infected annual ryegrass (Lolium multiflo-um Lam.) and tall fescue, although it is possible thathe mechanisms behind the reduction in faecal decay ver-us plant residue decay rates are different. For example,iegrist et al. (2010) recently questioned the role of alka-

oids in the reduced decay rates of tall fescue residues, sinceinimal loline concentrations occur in dead or senesced

eaves. However, it is likely that alkaloids, and differences

n the fibre content, explain the reduced decay rates of fae-es from sheep that grazed endophyte-containing perennialyegrass.

able 3. Mean (±SE) alkaloid concentrations (ppm) in faeces fromwes that grazed monocultures of perennial ryegrass (cv. Grasslandsamson) containing strains (AR1, AR37, and WT) of the endophyteeotyphodium lolii, or no endophyte (Nil). NP = not present.

lkaloid AR1 AR37 WT Nil

eramine 2.3 ± 0.2a NP <2.0 NPpoxy–janthitrems NP 18.0 ± 3.24 NP NPolitrem B NP NP 1.9 ± 0.10 NPrgovaline NP NP 0.15 ± 0.05b NP

aPeramine was below the limit of detection (2.0 ppm) for faecal materialn three samples.

bErgovaline was below the limit of detection (0.1 ppm) for faecal materialn three samples.

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Previously, ergot (Westendorf et al., 1993), and lolinePatchett, 2007) alkaloids have been found in faeces fromivestock fed fescue containing the endophyte, Neotyphodiumoenophialum, but the possible broader ecosystem effects ofhese alkaloids were not considered. Several studies haveound wide-reaching effects of endophytes on communi-ies and ecosystems, and have implicated alkaloids in theseffects, although they were not explicitly quantified (e.g.macini et al. 2004; Lemons et al. 2005; Antunes, Miller,arvalho, Klironomos, & Newman 2008). Here, in conjunc-

ion with reduced decay rates, we provide the first report of,olitrem B, peramine and epoxy–janthitrems in faeces. Theoncentrations of epoxy–janthitrems and lolitrem B foundn the faeces was similar to that in the pasture grass, buteramine and ergovaline were detected at a much lower con-entration to that in the grass, which suggests that theselkaloids are more easily metabolised (De Lorme, Lodge-vey, & Craig 2007).

Lolitrem B and peramine were found in the faeces atean concentrations of 1.9 and 2.3 ppm, respectively. For

olitrem B, this is considered a reasonably high concentra-ion in forage grasses, capable of inducing mild toxicosis inivestock (Hovermale & Craig 2001), and protecting againstnvertebrate pest feeding (Siegel et al. 1990; Barker 2008).eramine, at a concentration less than 2 ppm has been found

o deter feeding of the argentine stem weevil, Listronotusonariensis (Rowan & Gaynor 1986). Failure to detect aose response of lolitrem B (Prestidge & Gallagher 1988)nd peramine (Goldson, Proffitt, Fletcher, & Baird 2000) tonsect pests suggests that the protective mechanism of theselkaloids is deterrence (antixenosis) rather than resistanceantibiosis) (Ball, Gwinn, Pless, & Popay 2011). Perennialyegrass containing the AR37 endophyte strain lacks thehree major alkaloid groups (ergot alkaloids, Loitrems, anderamine), but produces epoxy–janthitrems. There are sev-ral reports of AR37 endophyte protecting perennial ryegrassrom a wide range of pest species, but the concentrationsf the epoxy–janthitrems have not been reported (Pennell,opay, Ball, Hume, & Baird 2005; Popay & Gerard 2007;opay & Thom 2009). Since the alkaloids found in the fae-es of this study have only been studied in relation to defencegainst herbivores, it is uncertain to what extent they mighte involved in resistance or deterrence to the coprophagousnvertebrate community.

The feeding activity of coprophagous invertebrates, in par-icular earthworms, causes the breakdown and burial of faecesnd can to a large extent affect the rate of faecal decayWilliams & Warren, 2004; Lee & Wall 2006). Lee and

all (2006) found that excluding invertebrates from faecesor only the first two days after deposition caused a signifi-ant reduction in the faecal decay rates, which suggests thatapid colonisation is important. Therefore, even if chemi-

al residues in faeces are short-lived it may be enough toeter initial colonisation of decomposers, and reduce decayates. Loline alkaloids derived from tall fescue and artifi-ially applied to faeces at much higher concentrations than

gsfl

plied Ecology 14 (2013) 146–154 151

aturally found in faeces (Westendorf et al., 1993; Patchett,007), but lower concentrations than found in live plantsPatchett, Gooneratne, Fletcher, & Chapman 2011), wasound to impair the survival of horn fly (Haematobia irri-ans) larvae (Dougherty, Knapp, Bush, Maul, & Van Willigen998). No other endophyte-derived alkaloids have been stud-ed in relation to the presence or survival of dung-dwellingnvertebrates. In our study it is not clear if the alkaloids in theaeces inhibited coprophagous invertebrates, but it is knownhat other chemicals used to treat livestock against parasitenfections are excreted in faeces and cause reduced decayates attributable to the impaired survival of coprophagousnsects (Floate, Wardhaugh, Boxall, & Sherratt 2005). Sincehe alkaloids found in the faeces were at levels known tonhibit herbivorous insects, it is plausible that they mightlso inhibit coprophagous invertebrates. This would be worthurther investigation, particularly in New Zealand where 11pecies of dung beetles have been approved for introductionnd release with the aim of enhancing faecal degradation (S.orgie, Landcare Research NZ, personal communication). It

s unlikely that the mesh bags used in this study excludedany invertebrate decomposers since juvenile earthworms,

ollembola, and fly larvae were observed in the mesh bagsnd/or the faecal samples.

In comparison to invertebrates, much less is known abouthe effects of Neotyphodium endophytes on fungi. In vitroaboratory cultures of Neotyphodium endophytes have beenhown to inhibit the growth of several plant pathogenic fungiSiegel & Latch 1991; Li, Gao, & Nan 2007). Siegel andatch (1991) determined that the antibiosis effect was notue to loline, peramine or ergot alkaloids, and suggested thatther antimicrobial compounds may be produced by thesendophytes. Thus, although speculative, it is possible that N.olii produces other compounds in addition to alkaloids thatould inhibit fungi involved in the decomposition of faeces.

Contrary to our second hypothesis, there were no signifi-ant differences in the decay rates among the three differentndophyte treatments. This might indicate that the presencef any alkaloid had similar inhibitory effects on the decom-oser community, or that N. lolii in symbiosis with its hostaused a more general effect, regardless of endophyte strain.n our study we found no difference in the ADF fibre compo-ent, but a significantly greater NDF fibre component in theaeces from the nil endophyte treatment compared to all threendophyte-containing treatments. The ADF fibre componentomprises the most recalcitrant fibres, primarily cellulosend lignin. The NDF component comprises the more easilyegraded hemicelluloses plus cellulose and lignin. Since thery matter content of the nil endophyte-derived faeces con-ained a greater proportion of more easily degradable fibrehis could also explain the faster decay rates of this faeces.he fibre content of faeces derived from E+ vs. E− forage

rasses has not previously been compared. However, a fewtudies have analysed the fibre content of E+ vs. E− liveorage grasses and found fibre content to be either equiva-ent (Newman et al. 2003; Rasmussen, Parsons, Fraser, Xue,

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52 M.G. Cripps, G.R. Edwards / Basic

Newman 2008), or to have an elevated NDF componentn the absence of endophyte (Zabalgogeazcoa, Ciudad, deldana, & Criado 2006). Therefore the greater NDF fibre

omponent found in the nil endophyte-derived faeces in ourtudy could be a direct result of endophyte-induced changesn the fibre content of the plant. Or, the higher NDF in theil endophyte faeces could be a result of different consump-ion rates of sheep feeding on E+ vs. E− perennial ryegrass.or instance, if sheep had higher intake rates of nil endo-hyte grass (possibly because it is less toxic) the herbageould pass through their digestive tracks faster resulting

n less metabolised hemicellulose (i.e. more hemicellulosexcreted).

Finally, it is important to note that this experiment wasonducted in late autumn to mid-winter, a time that is knowno be more favourable for faecal degradation (Weeda 1967;owarth, Gillingham, Tillman, & Syers 1985; Allard et al.004) due to increased moisture levels and increased earth-orm activity (Yeates 1976). It is unclear whether or not

imilar differences in faecal decay rates would be expressednder dryer, less favourable degradation conditions, inummer. The vegetation background, which influences thebundance and diversity of invertebrates, can also alter theates of faecal decay (Williams & Warren, 2004). Our studyas carried out in a pure sward of perennial ryegrass contain-

ng AR1 endophyte and it is unclear if similar results woulde obtained in different or more diverse vegetation environ-ents. Thus, the generality of our result is uncertain, but

onetheless it indicates that under favourable environmentalonditions the mutualistic endophyte of perennial ryegrassan reduce the rate of faecal degradation. We also provide evi-ence for the mechanism of this effect, which is attributableo endophyte-derived alkaloids excreted in the faeces and areater proportion of more easily degradable fibres in fae-es from nil endophyte perennial ryegrass, but the relativeontribution of these factors is uncertain. This is the first evi-ence of a fungal endophyte causing a reduction in faecalegradation rates, and adds to a growing body of literaturehowing that mutualistic microbes can have extended effectsn ecosystem properties.

cknowledgements

This work was supported by the New Zealand Foundationf Research Science and Technology (CONC-20122-ENR-GR Wider ranging, novel, trait-based assessments for

ustainable resource use). We are grateful to Lester Fletcheror allowing us to collect faecal material from an AgResearchrial at Lincoln, NZ. We thank Diane Keaney for carrying outhe carbon and nitrogen analyses, Wade Mace (AgResearch,almerston North, NZ) for conducting the alkaloids analyses,

nd Shuang Jiang for assistance with the ADF and NDF pro-edures. We would also like to thank Racheal Bryant, Tonyarsons, and Susan Rasmussen for many helpful discussions,nd Chikako van Koten for statistical advice.

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plied Ecology 14 (2013) 146–154

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.aae.2012.12.004.

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