ORIGINAL ARTICLE

New isolation method for endophytes based on enzyme digestion

René Prior & Katharina Görges & Andrey Yurkov &

Dominik Begerow

Received: 21 November 2013 /Revised: 4 February 2014 /Accepted: 12 February 2014# German Mycological Society and Springer-Verlag Berlin Heidelberg 2014

Abstract Investigation of the diversity and ecology of endo-phytic fungi has gained popularity in recent decades. Thereby,culture-independent methods were very instrumental as theyrevealed greater species richness and challenged the efficiencyof isolation methods. However, culture-based methods remainthe only possibility to provide strains for future studies. Toimprove the efficiency of endophyte isolation, we comparedseven different artificial media that included a variety of plantextracts. Furthermore, we developed a method based on en-zymatic digestion of plant tissue to improve the isolationefficiency in terms of species richness. The effect of additionalplant extract in media that contained yeast and malt extractwas of minor significance, whereas isolations using enzymaticdigestion of plant leaf tissue combined with a 1:10 dilution ofnutrients revealed a much higher diversity of species. Usingthis protocol, we isolated three times as many species from thesame leaves than with previously described methods. This isreflected by the large number of singletons obtained inculture-independent approaches to study the diversity of en-dophytic fungi.

Keywords Endophytes . Fungi . Strain cultivation . Isolationmethod . Enzyme digestion

Introduction

Almost all plants harbour numerous fungi known as endo-phytes in various amounts in almost all plant tissues (Schulzand Boyle 2005; Rodriguez et al. 2009; Peršoh 2013; Rajalaet al. 2013). Besides pathogenic or mycorrhizal fungi, endo-phytes are able to alter the metabolic activity of their hosts.Hence, endophytes are often classified as beneficial symbiontsof their hosts (Schulz and Boyle 2005; Moore et al. 2011;Pérez et al. 2013; Xiao et al. 2013). In particular, resistance toherbivores, pathogens, drought and other abiotic stresses areoften increased in the presence of endophytes (Clay andSchardl 2002; Redman et al. 2002; Ban et al. 2012;Hamilton and Bauerle 2012). While root endophytes are oftenanalysed in the context of direct promotion of growth, leafendophytes are studied mainly in the context of species diver-sity or in relevance to the antagonists mentioned above.Therefore, the identity, diversity and community compositionof endophytes is of major interest for functional studies ofplant fitness.

In principle, two different approaches studying fungal en-dophytic communities are distinguished. While culture-independent methods allow high-throughput analyses andprovide the greatest species diversity (e.g. Unterseher et al.2013), culture-based methods are restricted to cultivable spe-cies, and obligate biotrophs are regularly missing amongisolates. Obviously, both strategies have advantages and dis-advantages, depending on underlying questions and requests(Impullitti and Malvick 2013). Although it is not possible tocultivate obligate biotrophs on artificial media, some impor-tant and useful features come along with cultivation-basedmethods (Giovannoni and Stingl 2007). Living cultures aresuitable for morphological and physiological tests, analyses ofbioactivity, or experimental approaches (Arnold et al. 2003;Schulz and Boyle 2005; Burmeister and Hau 2009; Rodriguezet al. 2009; Bernardi-Wenzel et al. 2010). Also, collection of

R. Prior (*) :K. Görges :D. BegerowGeobotany, Department of Evolution and Biodiversity of Plants,Faculty of Biology and Biotechnology, Ruhr-Universität Bochum,Bochum, Germanye-mail: rene.prior@rub.deURL: http://www.ruhr-uni-bochum.de/geobot/index.html

A. YurkovLeibniz Institute DSMZ-German Collection of Microorganisms andCell Cultures, Inhoffenstraße 7B, 38124 Brunswick, Germany

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living strains allows polyphasic or multi-gene identification offungi, which is advantageous in the case when the selectedDNA-marker does not have equal resolution for all taxonom-ical groups. However, for culture-based approaches, it is rel-evant to identify the most appropriate isolation method andculture medium to obtain maximum diversity.

Typically, surface-sterilized leaves are cut into small stripesor squares and laid flat onto Petri dishes containing artificialmedia (e.g. Arnold and Lutzoni 2007; Arnold et al. 2000,2003; Helander et al. 2007; Fröhlich et al. 2000; Guo et al.2008). Less often, scissors or blender shafts are used(Glushakova and Chernov 2004; Unterseher and Schnittler2009) and grinding with a mortar and pestle is also conceiv-able. The common feature of all these methods is that theendophytes must grow out of the fragmented leaf tissue toaccess the media. Different fungal species have differentgrowth rates, and optimal in vitro growth conditions will varybetween endophyte species. Therefore, the resulting endo-phyte community structure is often biased toward fast-growing species. To prevent fungi from overgrowing eachother, the dilution-to-extinction method was established byPaulus et al. (2003) and Unterseher and Schnittler (2009). Inaddition, this issue is often addressed by using different arti-ficial media and temperatures for isolation and cultivation. Awide range of media, including nutrient-pure media, is oftenselected to support slow-growing species (e.g., Guo et al.2008; Musetti et al. 2006; Arnold and Herre 2003; Arnoldand Lutzoni 2007; Osono 2008). To establish growth ofbiotrophic species, several authors have tested media basedon plant extracts or even put sterilized plant particles in themedium (Arnold and Herre 2003; Arnold et al. 2003).

Here, we introduce a new isolation method for endophytesand study its performance in comparison to establishedmethods. In addition, we test the usability of seven differentculture media. The aim is to contrast the outcome in terms ofspecies richness of endophytes, regardless of other influences.We adapted a method for protoplast isolation releasing endo-phytic fungal cells from the leaf tissue by enzymatic digestion.To optimize the culture conditions, we employed yeastextract/malt extract agar (YM), potato dextrose agar (PDA)and cornmeal agar (CMA). Further on, we used tap-water-agar (TWA) complemented either with glucose or two differ-ent plant extracts, as described by Arnold and Herre (2003).

Material and methods

Artificial media

Yeast malt agar (YM): 1.0 % (w/v) glucose, 0.3 % (w/v) yeastextract, 0.5 % (w/v) peptone, 0.3 % (w/v) malt extract and2.0 % (w/v) agar filled to volume with deionized water; after

autoclaving, 80 % lactic acid was added to a concentration of0.1 % to prevent bacterial growth.

Yeast malt agar diluted by 10 (YM10): 0.1 % (w/v) glu-cose, 0.03 % (w/v) yeast extract, 0.05 % (w/v) peptone,0.03 % (w/v) malt extract and 2.0 % (w/v) agar filled tovolume with deionized water; after autoclaving, 80 % lacticacid was added to a concentration of 0.1 % to prevent bacterialgrowth.

Potato dextrose agar (PDA): 3.9 % (w/v) potato dextroseagar (Difco) and 1 % (v/v) 1 M Tris–HCl pH 8.0 filled tovolume with deionized water; after autoclaving, 80 % lacticacid was added to a concentration of 0.1 % to prevent bacterialgrowth.

Cornmeal agar (CMA): 4.2 % (w/v) cornmeal (RapunzelNaturkost GmbH, Legau, Germany) was added to 800 ml ofdeionized water and heated to 60 °C for one hour, afterwardsthe medium was filtered, 2 % (w/v) agar was added and filledagain to 800 ml with deionized water; after autoclaving, 80 %lactic acid was added to a concentration of 0.1 % to preventbacterial growth.

Tap water agar + plant extract I (TWA + plant extract I):leaves of Oxalis acetosella were collected at the same site asthe analysed plants (see below). Leaves (16 g per 800 mlmedium) were rinsed twice with running tap water, added to200ml of deionized water and shredded using a sterile blendershaft. The plant suspension was then filtered with a large-meshed filter to exclude long or solid fragments. Prior studiesrevealed that the medium does not solidify, since the agardegrades at low pH. Hence, to neutralize the medium, KOHwas added to obtain a pH value of 7. To get a sterile, cell-freemedium without thermal treatment of the plant extract, thefiltrate was centrifuged (3220rcf / 30 min) and the supernatantwas pipetted into a sterile tube. To check whether the sterili-zation via centrifugation was efficient, the supernatant wasanalysed with a light microscope and growing tests onartificial media were performed. Both procedures con-firmed the suspension being completely sterile. Theplant extract and 80 % lactic acid were added to aconcentration of 0.1 % to sterile tap water agar (TWA,tap water + 2 % agar) at ca. 50 °C, and poured intoPetri dishes immediately.

Tap water agar + plant extract II (TWA + plant extract II):To evaluate the effect of thermal treatment, leaves (16 g per800 ml medium) ofO. acetosellawere treated equally as plantextract I (see above), but instead of sterilization via centrifu-gation, the suspension was added to tap water agar beforeautoclaving.

Tap water agar + plant extract II + glucose (TWA + plantextract II + glucose): The medium is nearly identical to “TWA+plant extract II” (see above), but 1 % glucose was added toenhance the nutritional conditions.

Yeast malt agar + plant extract II (YM + plant extract II):The medium was compounded similar to “TWA + plant

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extract II”, but the medium was enriched with all nutrients ofYM (see above) and filled to volume with deionized water.

Plant species, study site and experimental design

Oxalis acetosella L. (wood sorrel) was used as model becauseit is a perennial, evergreen, early flowering geophyteconsisting of tuberous roots and annual, ternate leaves.Leaves usually sprout in spring and live up to 1 year, depend-ing on the environmental conditions. Fresh leaves ofO. acetosella were sampled 13 June 2012 and 18 April 2013from a population in a deciduous forest near the Ruhr-Universität Bochum, Germany (51.445 N, 7.273E). Data setsfrom each year were analysed separately, due to differentexperimental setups. We analysed 52 leaves in the first exper-iment, consisting of four different isolationmethods and sevenartificial media. For each treatment, we used entire leavesincluding leaf veins. Endophytes obtained in this experimentwere assessed by means of morphotypes, as suggested byWatrud et al. (2006). To analyse in more detail the improve-ment of enzymatic digestion, the second experiment made useof species identification of endophytic fungi by ITS rDNAsequencing of strains isolated from five different leaves.

Isolation methods for endophytic fungi

Symptomless leaves, i.e., without any kind of disease, damageor senescence, were harvested and processed on the same day.All leaves were about 1-year old, thus emerged in spring orsummer of the previous year. Each leaf was surface sterilizedby sequential washes in 96 % ethanol (5 sec), 0.5 % NaOCl(1 min) and 70 % ethanol (1 min) (comp. Arnold and Lutzoni2007). The sterile leaves were then shaken by hand in 50 mltubes filled with 30ml of sterile tap water for ca. 5 min (Larranet al. 2002, modified). To check the efficiency of sterilization,200 μl of the supernatant was spread on YM-plates andincubated at room temperature (about 23 °C) for 4 weeks. Inthe first experiment, thirteen leaves per isolation method weretreated following the procedures described below to isolateendophytic fungi. All steps of isolation were conducted understerile conditions. Each combination of isolation method andartificial media led to five replicates of identical petri dishes,resulting in 140 petri dishes in total.

Isolation by leaf-cutting

Leaves were cut into narrow stripes (ca. 1 mm) using a sterilerazor blade. The stripes were then put onto the seven artificialmedia, and wet with two drops of sterile tap water to preventdesiccation of the leaf tissue.

Isolation by blender shaft

Leaves were added to 250 ml of sterile tap water in a beakerand shredded with an ethanol-sterilized blender shaft. For thecultivation of endophytes, 200 μl of the leaf suspension in-cluding the sediment was spread on the seven artificial media.

Isolation by mortar and pestle

A mortar was prepared with 1 ml of tap water and some sterilesand. Leaves were added to the mortar and crushed with apestle. The leaf suspension was then filled up with tap water toa volume of 8 ml and 200 μl of the resulting suspension wasspread on the seven artificial media.

Isolation by enzymatic digestion

Leaves were cut into stripes (ca. 1 mm) with a razor blade asdescribed above and put into 10 ml test tubes filled with 2 mlof the prepared enzymatic solution. The enzymatic solutionconsisted of 1.5 % cellulase “Onozuka R10” (ServaElectrophoresis GmbH, Heidelberg, Germany); 0.4 %macerozyme R10 (Serva Electrophoresis GmbH,Heidelberg, Germany); 0.4 M mannitol (Merk, Darmstadt,Germany); 20 mM KCl (J.T. Baker, Deventer, theNetherlands); 20 mM MES pH=5.7 (AppliChem,Darmstadt, Germany); heat to 55 °C for 10 min; 10 mMCaCl2 (J.T. Baker, Deventer, the Netherlands); 0.1 % BSA(Fraktion V pH=7.0: AppliChem, Darmstadt, Germany). Forthe thirteen leaves, we used four tubes in total and distributedthe leaf stripes among the tubes. Tubes were covered withParafilm and put into a desiccator to incubate the solutionovernight under low pressure. The next day, 200 μl of the cell-containing solution was spread on the seven artificial media.

All 140 plates were incubated for 1 day at room tempera-ture (about 23 °C) and then transferred to a refrigerator (7 °C).Characterization based on culture morphology was conductedafter 4 weeks of incubation.

We conducted a follow-up experiment using enzymaticdigestion. Five leaves were collected in 2013 at the same siteas in 2012 and surface sterilized as described above. Each leafwas cut into two equal pieces. The first half was treated withthe leaf-cutting method, and the isolation by enzymatic diges-tion was applied to the second half as described above. Weincubated the partly digested leaf stripes on agar plates as athird treatment. For this second experiment, we chose YM10as artificial medium, i.e., 10 % of nutrient concentrations ofYM medium. Plates were incubated for 2 months at 7 °C.

Identification of cultures in the second experiment

DNA was isolated from 2–10 day old pure liquid cultures(YM) using a phenol-chloroform method, as described in

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Mülhardt (2009). Strains were primarily grouped on the basisof their culture morphology and for each group, DNA finger-printingwas performed using themicrosatellite-specific primer(GTG)5 as a single PCR primer (Gadanho and Sampaio 2002).Identical electrophoretic profiles were assumed to be clonal orconspecific. A representative of each DNA profile permorphotype was chosen for amplification of ITS and 5’LSUrDNA-region using primers ITS1f andNL4 (Gardes and Bruns1993; O’Donnell 1993). Initial denaturation was performed at96 °C for 2 min, followed by 35 cycles of 20 s at 96 °C, 50 s at52 °C and 1.5 min at 72 °C, respectively, followed by a finalextension step of 7 min at 72 °C. PCR products were purifiedwith Sephadex (Sigma-Aldrich, Steinheim, Germany) and se-quencing was conducted by GATC Biotech AG (Konstanz,Germany) using ITS4 primer (White et al. 1990).Chromatograms were checked with Sequencher 5.0 (GeneCodes Corp., USA). For identification of the obtained nucleo-tide sequences, we used the BLAST-algorithm implemented inNCBI (www.ncbi.nih.gov) and CBS (www.cbs.knaw.nl)databases, respectively. Strains with identity higher than98 % were considered as conspecific. Representative strainswere deposited in the DSMZ (comp. Table 1).

Data analysis

In the first experiment, we counted morphotypes per plate toevaluate performance in dependence of isolation method andartificial medium. Based on the five replicates, average valuesand standard deviation were calculated for the number ofmorphotypes per plate. One-way analysis of variance(ANOVA) and tests for significance were performed withSPSS 21 (IBM, Ehningen, Germany).

In the second experiment, we used presence/absence of adistinct species per leaf. Non metric multidimensional scaling(NMDS) was calculated with “R” (http://www.r-project.org/),“vegan” (http://vegan.r-forge.r-project.org/) package, utilizingdissimilarity index “binomial” as recommended for differentsample sizes in the “R” documentation. Calculation ofFriedman rank sum test (“stats” package), Kruskal-Wallis ranksum test (“stats” package) and species estimator “Chao1”(“vegan” package, Chao 1987) was conducted with “R”.Effects were considered to be statistically significant at thelevel p<0.05. Boxplots (“graphics” package) for speciesabundance within the three tested treatments and for Chao1estimates were performed with “R” as well.

Results

Efficiency of surface sterilization

Surface sterilized leaves showed no growth after the incuba-tion period, which indicated the technique was efficient.

Additionally, the plant tissue still remained intact aftersterilization.

Features of artificial media

The various combinations of isolation methods and artificialmedia in the first experiment resulted in 0–200 colonies perpetri dish. In total, we isolated 452 strains corresponding to atotal of 53 morphotypes. The comparison of the seven differ-ent media showed a significant increase in endophytic diver-sity from media containing low nutrients to nutrient-rich me-dia. The average number of morphotypes per plate was 1.8 forYM + plant extract II, 1.6 for PDA and YM, 1.3 for CMA, 1.2for TWA + plant extract II, 1.05 for TWA + plant extract II +glucose and 0.85 for TWA + plant extract I. The TWA-medium including only plant extract or plant extract + glucoseperformed poorly. No significant difference between plantextract I and plant extract II could be found, although theobserved species richness was slightly higher on plant extractI. PDA and YM media showed similar results, whereas CMAperformed worse, but still better than any of the TWA-media.

The YM10 medium used in the second experiment yieldedgreater species richness, and it was possible to incubate thepetri dishes for up to 2 months (at 7 °C).

Performance of isolation methods

While the first experiment was mainly conducted to comparevarious media, the analyses of the various isolation methodsrevealed mortar and pestle being preferable compared to leaf-cutting, blender shaft and enzymatic digestion methods interms of number of morphotypes (data not shown). A strongcontrast is given in the second experiment using sequence-based identification of the isolated strains. All emerging col-onies were isolated, and a total of 238 strains were transferredinto pure cultures. The various isolation methods supportedgrowth of 60 (leaf-cutting), 121 (enzyme) and 57 (plant resi-dues) strains, respectively. They grouped in 103 DNA finger-printing patterns, and thus ITS rDNA was sequenced for atotal of 103 strains. The enzymemethod yielded roughly threetimes as many species as the leaf-cutting method (Fig. 1).Communities found in the leaf stripes after enzymatic treat-ment (residues) were very similar to those isolated with leaf-cutting alone (Fig. 3). The number of species isolated with thethree different methods demonstrated that enzyme digestionresulted in the highest species richness (Table 1). Some spe-cies frequently isolated with the leaf-cutting method were alsoisolated with the other methods. Five species were isolatedonce, namely Phoma viburnicola, Colletotrichium sp.,Alternaria infectoria, Epicoccum nigrum, and a species fromHelotiales, which were restricted to the leaf-cutting method.Species that appeared in all treatments often belonged to thegenus Colletotrichum. The enzyme method also provided the

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most diverse communities out of the five leaves (Fig. 3). TheNMDS analysis illustrated that the five communities, corre-sponding to each of the treatments, were very similar within

and between the leaf-cutting and the enzyme residuemethod. Incontrast, the endophytic communities that emerged from theenzymatic digestion were variable between the different leaves.

Table 1 Frequency of isolatedspecies by three different treat-ments. Species’ names are alwaysgiven as best hit to GenBankmatches (NCBI); percental iden-tity is provided in brackets.Frequency is counted as numberof leaves from which therespective species was isolated(maximum=5). (unc. = uncultured;aff. = [species affinis] resemblance)

GenBank matches DSM Nr. cutting enzyme residues

Colletotrichum truncatum, AJ301937 (99 %) 27735 4 2 5

Colletotrichum acutatum, FJ455524 (100 %) 27732 4 2 4

Diaporthe eres, GU062205 (100 %) 27738 3 2 4

Colletotrichum kahawae, JX010227 (99 %) 27734 3 1 4

Paraconiothyrium sporulosum, JF340257 (100 %) 27745 2 4 2

Colletotrichum fuscum, EU886770 (100 %) 27740 1 2 1

Phoma macrostoma var. macrostoma GU237885 (100 %) 27731 1 2 1

Phomopsis viticola, AF230765 (100 %) 27733 4 2

Lambertella tubulosa, EU543256 (99 %) 27780 1 4

Plectosphaerella cucumerina, AB425979 (100 %) 27779 1 4

Colletotrichum clavatum, JQ754371 (100 %) 27741 1 1

Xylaria castorea, JF908802 (100 %) 28261 4 1

Neofabraea malicorticis, KC916706 (98 %) 2 1

Leptosphaeria sp., AM262434 (100 %) 28268 1 1

Pseudaegerita viridis, EF029235 (99 %) 27781 1 1

Epicoccum nigrum, EU566259 (99 %) 27747 1 1

Phoma viburnicola, GU237879 (100 %) 27749 1

Colletotrichum sp., JX624299 (99 %) 28260 1

Alternaria infectoria, FJ440601 (99 %) 27743 1

Helotiales sp., DQ914733 (97 %) 27795 1

Discosia sp., FJ861386(100 %) 1

unc. Alternaria sp., KC920885 (99 %) 1

Fusarium sp., EF601653 (100 %) 28267 1

Biscogniauxia nummularia, EF155488 (99 %) 4

Pyrenochaeta nobilis, NR103598 (99 %) 4

Hyaloscyphaceae sp., GU393951 (96 %) 28262 3

Dasyscyphus fuscescens, AY853231 (96 %) 27797 2

Dasyscyphella longistipitata, AB481239 (92 %) 2

Hypoxylon fragiforme, EF155524 (100 %) 2

unc. Helotiales sp., DQ974826 (99 %) 2

Cadophora sp.,KF156297 (97) 28265 2

Ceratobasidiaceae sp., JF449713 (99 %) 28263 1

Diaporte toxica, KC343220 (94 %) 1

Dictyochaeta sp., HE998757 (100 %) 1

Ascomycota cf. Helotiales sp., HE998754 (100 %) 1

Hypoxylon rubiginosum, KC477232 (100 %) 1

Ascomycota sp. HQ623451 (96 %) 27796 1

Mollisia cinerea, JF514855 (99 %) 1

Nemania diffusa, JN198514 (99 %) 28266 1

Obolarina dryophila, GQ428315 (99 %) 28264 1

Phoma eupyrena, JX537965 (100 %) 1

Rosellinia corticinum, KC311485 (99 %) 1

cf. Stagnospora sp. KF251268 (99 %) 1

Tetracladium marchalianum, EU883423 (99 %) 1

unc. Leotiomycetes sp., JF449683 (100 %) 1

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Species estimator Chao1 was applied to each of the fivereplicates of the three different treatments. It suggested that thehigher species recovery rate was based on the enzymaticdigestion treatment compared to leaf-cutting and residual leafstripes (Fig. 2).

Discussion

The aim of our experiments was to improve culture-basedspecies richness studies of endophytes and to analyse effectsof various isolation methods and different culture media. Thefirst experiment compared seven different media for speciesrichness recovery, expressed as a number of isolatedmorphotypes per plate. This experiment was performed to

assess the impact of different artificial media containing plantextracts as proposed byArnold and Herre (2003). Plant extractfrom the host plant is thought to promote a greater diversity ofcultivable fungi, as it promotes effects in endophyte metabo-lism (Arnold and Herre 2003; Padhi and Tayung 2013).However, our results indicated that nutrient-rich media likePDA, YM and CMA performed better than TWA media,including plant extracts. Yurkov et al. (2011) reported similarresults for forest soils, where nutrient-rich media yielded thehighest number of species.

The plant extract showed a beneficial effect in combinationwith YM; namely, a slightly higher number of morphotypesthan on pure YM medium. Moreover, we identified a smalleffect of the thermal treatment on the plant extract, which wasnot significant at the end. Thereby, our experiments demon-strated that removal of plant debris via centrifugation is highlyefficient, and sterile plant extract can be produced withoutthermal treatment of the artificial medium.

Taking these results into account, we used YMmedium forthe second experiment, as it performed well in our studies andis very similar to the commonly used MEA medium becauseof its composition (Arnold and Herre 2003; Arnold andLutzoni 2007; Arnold et al. 2000, 2003; Wang and Guo2007). To support slow-growing fungi rather than fast-growing moulds, we modified the medium by diluting it andused YM10 for the second experiment. This increased thenumber of isolated strains, and additionally made it easier todistinguish between different morphotypes due to the in-creased incubation time on the primary plates. The dilution-to-extinction cultivation technique (Unterseher and Schnittler2009) in this case seemed inappropriate to us, because of thegreater effort required for preparation of plates and samples incontrast to simple plating of dilutions on Petri dishes. Asshown above, the second experiment yielded higher speciesrichness, mainly due to the strain identification based onsequence data (comp. Begerow et al. 2010). Using methodo-logical improvements after the first experiment and applyingthe newly proposed method using enzymatic digestion, wewere able to isolate species numbers that were three timeshigher than with other the conventional methods (Fig. 1).Thus, based on our observations, we expect a higher speciesrichness of endophytic fungi in wood sorrel than calculated onthe basis of other isolation methods. The Chao1 species rich-ness estimator (Fig. 2) confirms that species richness estima-tions are heavily influenced by the isolation method, androughly mirrors the threefold number of species (comp.Fig. 1). Also, both analyses showed that enzymatic digestionleads to isolation of a higher number of species than theconventional method for each single leaf (Figs. 1 and 2).

While some frequent species were isolated in all treat-ments, the newly proposed enzymatic digestion revealed 14additional singletons, i.e., species isolated from a single sam-ple only (see Table 1). This strongly affected the similarity

enzyme leaf cutting

leaf stripes after enzyme

510

1520 *

Fig. 1 Box plot of species richness values observed after applyingenzymatic treatment, leaf-cutting, and leaf stripes after enzymatic treat-ment. Friedman rank sum test confirmed statistical significance: chi-squared=7.6, df=2, p value=0.02237

50

100

150

200 *

enzyme leaf cutting

leaf stripes after enzyme

Fig. 2 Box plot of estimated species richness values (Chao1) based onfive replicates for each of the following methods: enzymatic treatment,leaf-cutting, and leaf stripes after enzymatic treatment. Kruskal-Wallisrank sum test confirmed statistical significance: chi-squared=8,8, df=2, pvalue=0.01217

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between single samples. Thereby, the enzymatic digestionrevealed more diverse fungal communities than leaf-cuttingor plant residues (Fig. 3). Actually, the singletons influencethe Chao1 estimations as well, and therefore, the absolutenumbers in Fig. 2 should be taken with care.

The partly digested leaf stripes allowed us to check micro-scopically whether some distinct endophytic fungi were dam-aged or not successfully released from the tissue by theenzymatic treatment. However, there were no indications forthis assumption, and the enzymatic digestion significantlyperformed best in terms of fungal species recovery (Figs. 1,2, and 3). Several abiotic and biotic factors may explain theseresults. For instance, the relevance of slow-growing fungi aspart of the endophytic community has been emphasized pre-viously (Unterseher and Schnittler 2009). The better dispersalof fungal cells over Petri dishes following the enzymaticdigestion method prevents slow-growing species from beingovergrown by fast-growing ones for a long period of time. Inaddition, the lower density of endophytic fungi on Petri dishesafter the enzymatic digestion compared to multiple strainsgrowing out of a single piece of leaf tissue (when leaf-cutting method is used) reduces the probability for competi-tion and possible antagonistic effects during cultivation.Furthermore, the effect of isolating more different strainscould also be based on very gentle processing of the surfacesterilized leaves without major physical disturbance of tissuesand cells and, consequently, substantially reduced damage offungal hyphae. Microscopic analyses displayed intact plantand fungal cells after the treatment. In our opinion, the highnumber of singletons and non-conventional endophytes ob-served with this treatment (Table 1) additionally supports thisassumption.

Overall, the comparison of isolation techniques showedthat five out of 45 species were not isolatedwith the enzymatictreatment, but were with the leaf-cutting method. We assumethese findings to be based on the great number of endophyticfungi colonizing Oxalis acetosella, which we did not gathercompletely with our approach (comp. Figs. 1 and 2), and not

on the isolationmethod used. This is also supported by the factthat all of these five species are singletons and three of themwere found in the residual pieces of the enzymatic treatment.Therefore, we do not assume our sampling to be exhaustive touncover all endophytes of the analysed plant species, butrather touching only the tip of the iceberg, and we expectmany more species using the appropriate isolation method.

Conclusion and outlook

Functional, organismic and experimental ecology all rely onthe availability of living cultures. Here we introduce a newisolation method based on enzymatic digestion of plant tissue,which resulted in the highest number of species in our study.Combined with improved artificial cultivation media, thismethod will allow the isolation and cultivation of many morefungal endophytes than has been possible to date. In additionto frequently isolated species, which were found in the controltreatments as well, we could identify a high number of single-tons being present in samples subjected to the enzymaticdigestion. Thus, our new method might enable isolation offormerly uncultivable fungi and will support further studies onfungal diversity and function.

Interestingly, enzymatic digestion of other plant species’tissue (data not shown) yields different results concerning thelevel of plant tissue degradation. Specifically, leaves ofPhaseolus vulgariswere almost completely dissolved, where-asMedicago lupulina orMalva sylvestris showed nearly intactleaf tissue after the treatment. Although the obtained speciesrichness values remained rather unaffected, the protocol mightneed adaptation for other plant species.

Acknowledgments We thank Danja Schünemann for providing prin-ciples and recipes of the enzymatic digestion method. We thank DominikSchmidt, Moritz Mittelbach, Alistair McTaggart and Derek Peršoh forcritically reading themanuscript and for their advice.We thank IlseWeßeland Tanja Rollnik for technical support and the Deutsche BundesstiftungUmwelt for financial support.

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