Communities of fungal endophytes in leaves of Fraxinus ornus are highly diverse

Fungal Ecology 29 (2017) 10e19

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Communities of fungal endophytes in leaves of Fraxinus ornus are highly diverse Mohammed Ibrahim, Thomas N. Sieber*, Markus Schlegel €tstrasse 16, 8092 ETH Zurich, Department of Environmental Systems Science, Institute of Integrative Biology, Forest Pathology and Dendrology, Universita Zurich, Switzerland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 July 2016 Received in revised form 18 April 2017 Accepted 4 May 2017

Communities of endophytic fungi in leaves of Manna ash (Fraxinus ornus) were examined to both the north and south of the Alps, i.e. within and beyond the native range of this tree species. Almost all leaves examined had been colonized by endophytic fungi. One hundred and two morphotypes were found, and 62 of them were identified to genus or species level using ITS sequencing and micromorphology. Venturia orni was most frequent and occurred in almost one third (32%) of the 1536 examined leaf segments. It was five times more abundant than Colletotrichum acutatum, the second most frequent endophyte. Other frequently isolated endophytes include Paraconiothyrium sp. 1, Mycosphaerella aurantia, Septoria cretae, Botryosphaeria dothidea and Boeremia exigua. The ash dieback pathogen was not isolated. The endophyte communities differed between the north and south of the Alps and the individual tree types had a distinct influence within sites. © 2017 Elsevier Ltd and British Mycological Society. All rights reserved.

Corresponding Editor: Tom Bultman Keywords: Fraxinus excelsior Ash dieback Hymenoscyphus fraxineus Host jump Plant resistance Global warming

1. Introduction Endophytic fungi colonize plant tissues without causing disease symptoms and occur in a wide range of plant species and habitats, including tropical, temperate, and boreal forests (Carroll, 1988; Sieber, 1988; Petrini, 1991; Petrini et al., 1992; Sieber, 2007; Grünig et al., 2011; Sieber and Grünig, 2013; Bonfim et al., 2016). Their effects on host plants are diverse and range from plant growth promotion to protection against herbivores and pathogens (Latch et al., 1985; Schulz et al., 1995; Arnold et al., 2003; Doty, 2011; Tellenbach and Sieber, 2012; Witzell et al., 2014; Khan et al., 2015). Manna ash or South European flowering ash (Fraxinus ornus) is a deciduous, medium-sized tree (maximal height 25 m) native to southern Europe and southwestern Asia. In Switzerland, it is native only to regions south of the Alps (Info Flora, 2014). Manna ash is insect-pollinated, whereas Fraxinus excelsior and Fraxinus angustifolia, the other two ash species native to Europe, are wind-pollinated. F. ornus are often planted as ornamentals in parks and gardens in Central Europe, north of the Alps, from where they sometimes escape and establish themselves naturally in * Corresponding author. E-mail address: [email protected] (T.N. Sieber). URL: http://www.forestpathology.ethz.ch http://dx.doi.org/10.1016/j.funeco.2017.05.001 1754-5048/© 2017 Elsevier Ltd and British Mycological Society. All rights reserved.

thermophile habitats (Brandes, 2006). F. ornus occurs preferentially at dry, warm sites, but also in hardwood forests in southern European floodplains (Lippert et al., 1995; Info Flora, 2014). It is likely that F. ornus will become a permanent fixture in forests north of the Alps due to global warming. Descriptions of fungi associated with F. ornus are scarce (Farr and Rossman, 2016), and the communities of endophytic fungi on this host have yet to be studied. The aim of this study is to determine the communities of F. ornus leaf endophytes originating from its native terrain (south of the Alps) and from where it has been planted as an ornamental (north of the Alps). Studying endophyte communities of F. ornus is especially intriguing because this tree species is largely resistant to the ash dieback pathogen Hymenoscyphus fraxineus which made its way to Europe from southeast Asia and has resulted in the current high mortality of common ash (F. excelsior) throughout Europe (Gross et al., 2014). With this in mind, we hypothesize that H. fraxineus is either a harmless endophyte with regards to F. ornus leaves or is simply unable to infect them.

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Fig. 1. Map depicting the study sites. Monte Caslano and Lago di Ledro are situated south of the Alps and Berne, Thun and Zurich north of the Alps.

2. Materials and methods 2.1. Sites and sample collection Samples were collected at six different sites (Fig. 1). Two of the sites were located south of the Alps and four to the north (Table 1). The two southern sites are located within F. ornus' native range and represent natural regeneration, whereas the northern sites lie outside the species' native range. Despite this, F. ornus appears to regenerate naturally north of the Alps (Info Flora, 2014; LFI, 2016). However, only planted trees were examined there in this case. Leaves were collected between September 3rd and September 18th, 2013, from 16 trees at each of the two sites located south of the Alps (Lago di Ledro and Monte Caslano), from four trees each in

Thun and Bern and from four trees each at two sites in Zurich. All leaves appeared healthy except for a handful of leaves that had been attacked by powdery mildew (Phyllactinia fraxini) collected north of the Alps. Eight leaves were collected per tree. The leaves were immediately stored in a cool box and processed within 72 h.

2.2. Surface-sterilization and incubation of the leaf samples One leaflet, as well as the petiole, were severed from each leaf and surface-sterilized using the following sequence of immersions: 1 min 70% ethanol, 3 min NaClO (4% active chlorine), 1 min sterile water and 30 s 70% ethanol. The concentration and immersion duration were evaluated in preliminary experiments (Schulz et al., 1993).

Table 1 Site characteristics and sample sizes. Site

Monte Caslanoa

Lago di Ledrob

Thuna

Berna

Zurich-Triemlia

Zurich-Nordheima

Region Stand type Altitude [m] Coordinates

South of the Alps Natural regeneration 446 45
570 40.500 N 8
520 53.300 E 2.5 21.1 11.5 1559 98.1

South of the Alps Natural regeneration 713 45
520 1700 N 10
440 2400 E 0.4 19.6 9.8 1123 112.8

North of the Alps Plantation 558 46
450 0300 N 7
380 1300 E 0.4 18.3 8.8 1059 124.2

North of the Alps Plantation 572 46
570 2900 N 7
300 0500 E 0.5 18.2 8.7 1059 124.2

North of the Alps Plantation 461 47
210 54.600 N 8
290 59.800 E 0.1 18.6 9.2 1054 128.4

North of the Alps Plantation 475 47
240 2000 N 8
310 4900 E 0.1 18.6 9.2 1054 128.4

16 128 256 256

16 128 256 256

4 32 64 64

4 32 64 64

4 32 64 64

4 32 64 64

Mean temperature in January [
C] Mean temperature in July [
C] Mean annual temperature [
C] Annual precipitation [mm] Mean number of days with rain per year [d] Number of trees Number of leaves Number of leaf discs Number of petiole pieces

a Weather data of Swiss sites: 30-year means (1981e2010), MeteoSchweiz (http://www.meteoschweiz.admin.ch/home/klima/vergangenheit/klima-normwerte/ klimadiagramme-und-normwerte-pro-station.html; accessed March 10, 2017). b Weather data of Italian site: temperature: 14-year means (1999e2012); precipitation: 18-year mean (1995e2012), Davis Vantage Pro2 Station (http://www. altogardameteo.com/; accessed November 27, 2013).

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M. Ibrahim et al. / Fungal Ecology 29 (2017) 10e19

Two leaf discs 7 mm in diameter were cut out from each leaflet with a cooled-down flame-treated punch and two 3-mm-long segments were excised from the central part of each petiole. The four tissue pieces originating from the same leaf were transferred to a Petri dish containing terramycine-malt-extract agar (TMA, 20 g l-1 malt extract, 15 g l-1 agar, 50 mg l1 terramycine (oxytetracycline)). The resulting 384 Petri dishes were incubated at 20
C and inspected every second to third day. Emerging mycelia were transferred to slants containing malt-extract agar (MA, 20 g l-1 malt extract, 15 g l-1 agar) so as to obtain pure cultures. 2.3. Morphotyping and identification Pure cultures were assigned to morphotypes based on growth rate, colony colour (reverse of Petri dish and aerial mycelium), presence/absence and texture of the aerial mycelium. The colony morphology of some cultures changed over time. Consequently, colony morphology had to be monitored for several weeks to reach a satisfactory classification. In case of doubt, a morphotype was further subdivided into additional morphotypes. If spores or conidia were present, the morphotype was identified down to the genus or species level through the use of identification keys and relevant literature (Müller and von Arx, 1962; Ellis, 1971, 1976; von Arx and Müller, 1975; Hermanides-Nijhof, 1977; Sutton, 1980; von Arx, 1981; Petrini and Mueller, 1986; Ellis and Ellis, 1997; Domsch et al., 2007; Seifert et al., 2011). Sterile mycelia were incubated under near-UV light to stimulate sporulation (Leach, 1967). The nucleotide sequence of the internal transcribed spacer (ITS) regions of each morphotype was determined to verify the morphology-based identification and to identify non-sporulating morphotypes. Several strains of the most frequent morphotypes were sequenced to verify the classification power of morphotyping. Sequences were deposited in GenBank (Table 2). 2.4. Amplification and sequencing of the internal transcribed spacer (ITS) regions For each colony, a small piece of mycelium was transferred to a 2-ml tube and lyophilized for 48 h. Subsequently, DNA was extracted using the NucleoSpin® 96 Plant II Kit (Macherey-Nagel, Düren, Germany) in line with the manufacturer's instructions. The ITS region was amplified in a final volume of 15 ml using 0.3 U of GoTaq polymerase (Promega, Dübendorf, Switzerland). The mix contained 2 ml DNA, 0.5 mM of the primers ITS1F and ITS4 (White et al., 1990; Gardes and Bruns, 1993), 0.2 mM of each dNTP (Fermentas, St. Leon-Rot, Germany) and 1 x reaction buffer. The running conditions were as follows: initial denaturation at 94
C for 2 min, followed by 35 cycles of denaturation at 94
C for 30 s, annealing at 56
C for 30 s, and extension at 72
C for 105 s and a final elongation of 5 min at 72
C. Five ml of each PCR product (diluted 1:10) were mixed with 0.5 ml exonuclease I (20 U/ml) (Fermentas, St. Leon-Rot, Germany) and 1 ml shrimp alkaline phosphatase (SAM) (1 U/ml) (Fermentas, St. Leon-Rot, Germany) and incubated for 15 min at 37
C to purify the DNA. The enzymes were deactivated at 80
C for 15 min thereafter. The cycle sequencing reactions were performed in a volume of 10 ml with 1 ml of purified PCR product, 0.5 ml BigDye (Applied Biosystems, Rotkreuz, Switzerland), 1 ml of the ITS4 primer (10 mM) and 1 x reaction buffer. The reaction conditions were as follows: 1 min at 96
C, 55 cycles of 10 s at 95
C, 5 s at 50
C and 4 min at 60
C. For purification, 22.5 ml SAM buffer and 5 ml BDXTerminator (BigDye XTerminator Purification Kit, Applied Biosystems, Rotkreuz, Switzerland) were added to the products, and the mixtures agitated for 30 min on a shaker. Sequencing took place after the addition of 40 ml ddH2O using an ABI 3130xl Genetic Analyzer (Applied Biosystems, Rotkreuz, Switzerland) at the

Genetic Diversity Center (GDC, ETH Zurich, Switzerland). The resulting sequences were edited and aligned (within morphotypes) using Geneious 6.0.6 (Biomatters, Auckland, New Zealand) and compared with the NCBI nucleotide sequence collection (GenBank) using BLAST searches (BLAST, 2016). 2.5. Statistical analyses The software R was used for all statistical analyses (R_Development_Core_Team, 2013). The vegan package (Oksanen et al., 2016) was used to construct species accumulation curves and to calculate the expected number of species using the Chao, first-order-jacknife and bootstrap estimators (Smith and van Belle, 1984; Chao, 1987). The differences among the trees with regards to their endophyte communities were explored using the metaMDS routine with the Bray-Curtis distance measure in vegan. The effects of various factor combinations (geographic origin, tree individual (random factor), leaf tissue) on the frequency of the most abundant endophyte species were tested using generalized linear mixed models (GLMM) in lme4 (Bates et al., 2016). The ggplot2 package was used for plotting isolate counts (Wickham, 2009). 3. Results It was possible to isolate endophytic fungi from almost all leaves (97e99%) at all sites except in Zurich, where the frequency of isolation was ‘only’ 80%. However, this comparatively low average colonization rate seen in Zurich was due to two of the eight trees examined being characterized by a low number of colonized leaves. 102 morphotypes were successfully discriminated based on culture morphology, and 62 of them could be further identified down to the genus or species level through use of ITS sequencing and/or micromorphology (Table 2). The remaining 40 morphotypes neither sporulated nor showed their ITS sequences to a minimum identity accuracy of 95% to those deposited in GenBank. As a result, they were considered ‘unidentified fungi’. However, these fungi occurred in less than 0.5% of the tissue samples. H. fraxineus, the ash dieback pathogen, was not isolated. A total of 102 species were found, assuming that each of the 54 unidentified isolates represents a separate species. However, the resulting species accumulation (rarefaction) curve was not distinctly asymptotic (Fig. 2), indicating that sampling was not exhaustive. In contrast, the three estimators delivered relatively satisfactory approximations of the 102 assumed species (Chao estimator: 115.1 ± 20.0; first-order-jacknife estimator: 104.4 ± 7.1; bootstrap estimator: 87.8 ± 3.7). Species richness was highest at Monte Caslano, where 47 different taxa were detected, followed by Lago di Ledro with 38 taxa. Only 32 taxa were found north of the Alps. Venturia orni (Ibrahim et al., 2016) was the most frequent endophyte and occurred five times more frequently than the second most abundant Colletotrichum acutatum (Table 3). The frequency of many species varied between sites. V. orni was more abundant north of the Alps but the difference was only statistically significant for the subsite Zurich-Nordheim (p ¼ 0.008) (Fig. 3 and Table 4). C. acutatum, Botryosphaeria dothidea, Discula quercina, Diaporthe oncostoma and Amphiporthe castanea almost only occurred at Monte Caslano. Paraconiothyrium sp. 1 and Mycosphaerella aurantia occurred most frequently at Lago di Ledro, whereas Septoria cretae and Boeremia exigua were mainly found at the sites north of the Alps (Table 3). Differences in endophyte community composition among sites are clearly evident when multivariate statistics are applied (Fig. 4), indicating that the site effect on species composition of endophyte communities was much stronger than the effect of the individual tree type (p < 0.001). Differences among the sites

Table 2 Micromorphology-based and ITS-sequence-based identification of endophytic fungi isolated from Fraxinus ornus leaves. The fungi are ordered according to their frequency of isolation. Morphotype (OTU)

Proposed taxon nameb

ITS-sequence-based identification

Taxon name

Closest matching taxon in GenBank

Order

Minimum identity

Maximum identity

Mean identity

Count

GenBank accession of closest match

Vo

Fusicladium sp.

Venturia fraxini

Venturiales

92.5

98.6

95.55

19

EU852360

Venturia orni

Ca1 Di1 Di1 Pa1

Colletotrichum sp. Phomopsis sp. Phomopsis sp. n.s.a

Glomerellales Diaporthales Diaporthales Pleosporales

99.4 98.4 98.7 94

100 99.8 99.6 94.7

99.7 99.1 99.15 94.35

30 7 13 12

KP064135 KC343073 JX515701 JX496099

My1

n.s.

Colletotrichum acutatum Diaporthe eres Diaporthe viticola Paraconiothyrium variabile Mycosphaerella aurantia

Capnodiales

98.8

99.6

99.2

40

EU853468

Colletotrichum acutatum Diaporthe eres Diaporthe viticola Paraconiothyrium sp. 1 Mycosphaerella aurantia

My2 Bd

Septoria sp. n.s.

Septoria cretae Botryosphaeria dothidea

100 99.5

100 100

100 99.75

1 86

KF251233 KF766151

Be Ns

Boeremia exigua Nemania serpens

99.2 92.1

99.4 98.1

99.3 95.1

3 56

Dq Aa Di2 Bn Ac My3 Kd

Phoma sp. Geniculosporium serpens Discula sp. Alternaria sp. Phomopsis sp. Nodulisporium sp. Discula sp. n.s. n.s.

Capnodiales Botryosphaeriales, Pleosporales Pleosporales Xylariales

Discula quercina Alternaria alternata Diaporthe oncostoma Biscogniauxia nummularia Gnomoniopsis smithogilvyi Mycosphaerella sp. Kretzschmaria deusta

Diaporthales Pleosporales Diaporthales Xylariales Diaporthales Capnodiales Xylariales

95.8 100 99.6 95.4 99.6 97.3 99.5

100 100 99.6 99.8 100 98.5 100

97.9 100 99.6 97.6 99.8 97.9 99.75

Na

n.s.

Neofabraea alba

Helotiales

96.5

98.7

Aq Rc

n.s. n.s.

Ampelomyces quisqualis Rosellinia corticium

Pleosporales Xylariales

99.3 98.6

99.6 99.4

GenBank accession number

KT823569 KT823575 KT823566 KT823572 KY367488 KY367489 KY367490 KY367491

Number of colonized leaf segments

Percentage (%) colonized leaf segments

n ¼ 1536

n ¼ 1536

480

31.3

96 92 92 71

6.3 6.0 6.0 4.6

62

4.0

Septoria cretae Botryosphaeria dothidea

KY367492 KY367493 KY367494 KY367495

57 46

3.7 3.0

JX241664 EU686070

Boeremia exigua Nemania serpens

KY367496 KY367497

28 21

1.8 1.4

55 21 2 16 13 8 7

EU254836 KP127978 LN714541 AJ390415 KC145851 JF495202 KT281901

Discula quercina Alternaria alternata Diaporthe oncostoma Biscogniauxia nummularia Amphiporthe castanea Mycosphaerella sp. 3 Kretzschmaria deusta

17 13 12 12 12 10 10

1.1 0.8 0.8 0.8 0.8 0.7 0.7

97.6

58

HQ166296

Neofabraea albac

8

0.5

99.45 99

72 3

HM124894 KC311485

Ampelomyces quisqualis Rosellinia corticium

KY367498 KY367499 KY367500 KY367501 KY367502 KY367503 KY367504 KY367505 KY367506 KY367507 KY367508 KY367509

8 8

0.5 0.5

M. Ibrahim et al. / Fungal Ecology 29 (2017) 10e19

Morphology-based identification

Endophytic fungi occurring in <0.5% of leaf segments ordered according to frequency (GenBank accession numbers in brackets): Diaporthe phaseolorum (KY367510); Mycosphaerella sp. 4 (KY367511). Fusicoccum quercus (KY367512); Elsinoe sp. (KY367513); Leptosphaeria biglobosa (KY367514); Phoma herbarum (KY367515); Phoma macrostoma; Rosellinia sp. (KY367516); Pyrenochaeta cava (KY367517). Aureobasidium pullulans (KY367518); Didymella pisi (KY367519); Daldinia childiae (KY367520); Lachnum sp.; Mycosphaerella punctiformis (KY367521); Sphaerulina menispermi (KY367522); Cladosporium cladosporioides. Diatrypaceae 1; Fusarium oxysporum (KY367523); Hyalodendriella sp.; Didymella vitalbina (KY367524); Alternaria infectoria (KY367525); Calosphaeriaceae 1; Colletotrichum sp.; Preussia sp.; Annulohypoxylon sp.. Phyllosticta sp. (KY367526); Phoma sp. (KY367527); Mycosphaerellaceae 2; Creosphaeria sp.; Hypoxylon rubiginosum (KY367528); Paraconiothyrium sp. 2; Nemania sp. 1; Nemania sp. 2. Biscogniauxia mediterranea (KY367529); Microstroma juglandis (KY367530); Mycosphaerella sp. 5 (KY367531); Ophiognomonia setacea; Periconia macrospinosa (KY367532); Sphaceloma sp. (KY367533). Venturia sp. 1 (KT823544); Ramularia calcea (KY367534); Xylaria sp. (KY367535). a n.s. ¼ non-sporulating. b Minimum identity required for species level 95%, for genus level 90%. c As Phlyctema vagabunda in GenBank.

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M. Ibrahim et al. / Fungal Ecology 29 (2017) 10e19

Fig. 2. Species accumulation curve (rarefaction) depicting the expected species richness in dependence on the number of trees examined. The standard deviations are given as box plots and the grey area indicates the 95% confidence interval.

Table 3 Number and frequency (%) of colonized leaf segments (leaflet and petiole segements combined) originating from trees North and South of the Alps. Taxon name

Venturia orni Colletotrichum acutatum Diaporthe spp.a Paraconiothyrium sp. 1 Mycosphaerella aurantia Septoria cretae Botryosphaeria dothidea Boeremia exigua Nemania serpens Discula quercina Alternaria alternata Diaporthe oncostoma Biscogniauxia nummularia Amphiporthe castanea Mycosphaerella sp. 3 Kretzschmaria deusta Neofabraea alba Ampelomyces quisqualis Rosellinia corticium a

South of the Alps (Monte Caslano) (n ¼ 512)

South of the Alps (Lago di Ledro) (n ¼ 512)

North of the Alps (all sites combined) (n ¼ 512)

Overall frequency (n ¼ 1536)

Number of colonized segments

Frequency (%) of colonized segments

Number of colonized segments

Frequency (%) of colonized segments

Number of colonized segments

Frequency (%) of colonized segments

Number of colonized segments

Frequency (%) of colonized segments

133 95 46 1 11 6 46 1 3 17 6 12 0 11 1 1 3 0 8

26.0 18.6 9.0 0.2 2.1 1.2 9.0 0.2 0.6 3.3 1.2 2.3 0.0 2.1 0.2 0.2 0.6 0.0 1.6

158 0 40 70 51 11 0 0 9 0 6 0 2 1 9 2 1 0 0

30.9 0.0 7.8 13.7 10.0 2.1 0.0 0.0 1.8 0.0 1.2 0.0 0.4 0.2 1.8 0.4 0.2 0.0 0.0

189 1 6 0 0 40 0 27 9 0 1 0 10 0 0 7 4 8 0

36.9 0.2 1.2 0.0 0.0 7.8 0.0 5.3 1.8 0.0 0.2 0.0 2.0 0.0 0.0 1.4 0.8 1.6 0.0

480 96 92 71 62 57 46 28 21 17 13 12 12 12 10 10 8 8 8

31.3 6.3 6.0 4.6 4.0 3.7 3.0 1.8 1.4 1.1 0.8 0.8 0.8 0.8 0.7 0.7 0.5 0.5 0.5

Two different species: D. eres and D. viticola.

north of the Alps were less pronounced than those between the two sites south of the Alps. Within the sites, the individual tree type (genotype) had a distinct influence on the species composition of the endophyte community (Fig. 3). For example, C. acutatum and B. dothidea were abundant on the leaves of five out of 16 trees at Monte Caslano, apparently substituting V. orni, which was either rare in these trees or quickly overgrown by the fast-growing C. acutatum. Similarly, no V. orni was found in two of the trees at Lago di Ledro and the frequency of colonization was very low in two of the trees north of the Alps (Fig. 3). No intra-tree effect could be observed when comparing two subgroups of leaves (1e4 and 5e8) (p ¼ 0.329), whereas the leaf tissue (petiole versus leaflet) saw a significant effect (p < 0.001). In particular, V. orni was significantly more frequent in the petioles than the leaflets (p ¼ 0.006), as

demonstrated by applying a generalized linear mixed model in which sites and leaf tissue were entered as fixed components and the tree individuals as random variables (Table 4). Diaporthe eres, Diaporthe viticola, C. acutatum, Paraconiothyrium sp. 1, M. aurantia, S. cretae, B. dothidea and B. exigua occurred more frequently in leaflets than in petioles, whereas only D. eres and D. viticola (besides V. orni) occurred more frequently in the petioles. However, all these differences in colonization frequency were statistically insignificant. 4. Discussion Rates of colonization by endophytic fungi ranging as high as 80e100% are common for leaves of deciduous trees. In contrast, the number of species found during the current study was

M. Ibrahim et al. / Fungal Ecology 29 (2017) 10e19

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Table 4 Effects of site and leaf tissue on the frequency of Venturia orni as identified by fitting a generalized linear mixed model by maximum likelihood. * ¼ statistically significant at p  0.05.

(Intercept)

Estimate

Std.Error

z-value

p-value

0.3957

0.1861

2.126

0.03347*

Effect of site (compared to Monte Caslano) Lago di Ledro 0.101 0.2574 Thun 0.1132 0.4003 Bern 0.6461 0.3798 Zurich-Nordheim 0.9521 0.3611 Zurich-Triemli 0.5435 0.4403

0.392 0.283 1.701 2.636 1.235

0.69476 0.7774 0.08888 0.00838* 0.21699

Effect of leaf tissue (compared to petioles) Leaflets 0.2943 0.107

2.751

0.00595*

Fig. 4. NMDS map defined by the first two axes using the metaMDS routine with the Bray-Curtis distance in vegan of the software R showing differences among endophyte communities north (all sites combined) and south (Monte Caslano and Lago di Ledro) of the Alps.

Fig. 3. Frequencies of the most frequently isolated endophytic fungi in each of the examined trees north (third panel at the bottom of this figure) and south (Monte Caslano and Lago di Ledro) of the Alps. The four trees each originating from Bern, Thun and the two sites in Zurich are separated by vertical lines.

comparatively high (Sieber, 2007). The number of morphotypes can be considered a reasonable estimate of the number of species present in this study since the number of isolates erroneously assigned to the same morphotype was roughly of the same proportion as those erroneously assigned to different morphotypes. For example, isolates of the fairly abundant morphotype Di1 consistently possessed the same colony morphology but proved to belong to at least two different species, i.e. D. eres and D. viticola, according to the ITS DNA sequences (Table 2). In contrast, other isolates differed in colony morphology and were, thus, assigned to differing morphotypes, but ITS sequencing demonstrated that they

represented the same species. For instance, the colony morphology of C. acutatum varied considerably among isolates despite the ITS sequences being identical. The species accumulation (rarefaction) curve was not distinctly asymptotic at 100 species (Fig. 2), whereas the estimates delivered by the three estimators indicated that most of the species had been found. However, it must be kept in mind that the method used in this study allowed for the detection of cultivable endophytes only, while obligate biotrophs ‘escaped’ detection. In addition, it is conceivable that some species characterized by slow growth on TMA were overgrown by fast-growing species and therefore escaped as well. The next generation sequencing of DNA extracted directly from surface-sterilized plant tissues could help in compiling a more complete picture of endophyte diversity. V. orni dominated the community of endophytic fungi in F. ornus leaves and occurred five times more frequently than the second most frequent C. acutatum. An abundance of only one or a handful of species accompanied by a multitude of rare species is the most common endophyte community structure, independent of the ecosystem considered (Fisher and Petrini, 1990; Izzo et al., 2005; Cai et al., 2006; Grünig et al., 2006; Queloz et al., 2011). This structure also holds true for endophyte communities in leaves of other deciduous trees. For example, a Venturia species dominated in leaves of Betula pubescens in Finland and Switzerland (Helander et al., 1993; Barengo et al., 2000) and Apiognomonia errabunda in beech (Fagus sylvatica) leaves (Sieber and Hugentobler, 1987). Cryptosporella suffusa (syn. Ophiovalsa suffusa) has been reported to

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be the most frequently isolated endophyte species in Alnus glutinosa bark in Europe and in Alnus rubra in North America (Fisher and Petrini, 1990; Sieber et al., 1991). Similarly, Cryptosporella betulae is the most frequently isolated endophyte in branches of Betula pendula and B. pubescens in Europe (Kowalski and Kehr, 1992; Barengo et al., 2000). Phomopsis (Diaporthe) species often dominate endophyte communities, e.g. those in leaves of bigleaf maple (Acer macrophyllum) or Rubus spp. (Sieber and Dorworth, 1994; Shamoun and Sieber, 2000). Likewise, the endophyte community in leaves of F. excelsior was dominated by Alternaria alternata and Alternaria infectoria (Scholtysik et al., 2013). Interestingly, Venturia fraxini, a species closely related to V. orni (Ibrahim et al., 2016), was only the sixth most frequent endophyte in F. excelsior and occurred five times less frequently than the two dominant Alternaria species in the study of Scholtysik et al. (2013). In the current study, A. alternata occurred in only 13 and A. infectoria in only one leaf segment (n ¼ 1536). V. fraxini was the second most frequent colonizer of overwintered F. excelsior leaf petioles in the Ukraine (Davydenko et al., 2013). V. fraxini was likely already present as an endophyte in the healthy leaves. V. fraxini and V. orni were also the most frequent endophytes in F. excelsior seedling leaves and F. ornus seedlings, respectively, upon experimental inoculation in a climate chamber using leaf litter as inoculum source (Schlegel et al., 2016). A. castanea, B. exigua, B. dothidea, C. acutatum, D. oncostoma, D. quercina, M. aurantia, Paraconiothyrium sp. 1 and S. cretae were among the most abundant endophytes but occurred in only one region or only very rarely in another region, probably due to specific biogeographic factors (weather conditions, vegetation). Biogeographic factors are known to have a strong influence on the composition of endophyte communities (Sieber, 1989; Sieber et al., 1999; Pautasso et al., 2015). Plant community composition is presumably one of the main drivers for the composition of endophyte communities. For example, Quercus petraea and Quercus pubescens €ch, 2005) where the oak endoare abundant at Monte Caslano (Za phyte/pathogen D. quercina (Wilson and Carroll, 1994; Moricca and Ragazzi, 2008) was most frequently isolated from F. ornus during this study. The same is also true for D. oncostoma, a fungus associated with black locust (Robinia pseudoacacia) (Vajna, 2002). The abundance of C. acutatum in F. ornus leaves at Monte Caslano and its absence from the other sites was conspicuous. C. acutatum is a species complex containing both important anthracnose-causing pathogens and beneficial endophytes of a wide range of host plants, especially herbaceous plants, worldwide (Sette et al., 2006; Mejia et al., 2008; Rojas et al., 2010; Damm et al., 2012). It has been isolated as an endophyte from Castanea sativa bark (Bissegger and Sieber, 1994) one of the most abundant tree species on Monte Caslano (Z€ ach, 2005). However, the abundance of C. acutatum in F. ornus leaves at Monte Caslano cannot be explained by its occurrence in C. sativa alone. Most probably, the main source of this endophyte is another, as yet unidentified (herbaceous), plant species present and/or abundant only at Monte Caslano but not at the other study sites. B. dothidea is a cosmopolitan generalist e acting as both an endophyte and pathogen on the bark of many deciduous tree species (von Arx and Müller, 1954; Farr and Rossman, 2016). Slippers et al. (2004) epitypified B. dothidea so as to reduce taxonomic confusion surrounding this taxon. They collected specimens from Fraxinus sp., Prunus sp. and Ostrya sp. at the border between Switzerland and Italy in the same region where Cesati and De Notaris (1863) gathered their collections, i.e. in the vicinity of Monte Caslano. A sample from Prunus sp. was then designated as the epitype specimen. F. excelsior, F. ornus, Prunus mahaleb, Prunus spinosa and Ostrya carpinifolia are highly abundant on Monte Cas€ggli, 1928). lano and may have served as inoculum sources (Ja Paraconiothyrium sp. 1 was the second most frequent endophyte

in leaves originating from Lago di Ledro. Currently, we do not have any explanation for its high frequency at this site and its absence at other sites with the exception of one isolate from Monte Caslano. Paraconiothyrium sp. 1 is closely-related to P. variabile and P. brasiliense but not conspecific. Paraconiothyrium variabile and Paraconiothyrium brasiliense are well-known for their role in the production of the anti-cancer drug taxol (Garyali et al., 2014; Somjaipeng et al., 2015) and for their antibiotic effects against a s et al., 2012; Nicoletti et al., 2013). multitude of pathogens (Combe Paraconiothyrium sp. 1 might be a similarly effective producer of interesting secondary metabolites because all strains of this fungus have almost completely inhibited ascospore germination of the ash dieback pathogen (H. fraxineus) (Schlegel et al., 2016). The high frequencies of B. exigua (syn. Phoma exigua) and S. cretae at the sites north of the Alps are difficult to explain. Perhaps hosts of B. exigua that occur only north of the Alps served as inoculum sources for the infection of F. ornus. The cosmopolitan B. exigua and its numerous varieties are well-known causal agents of disease for a wide range of plant species (Aveskamp et al., 2010; Li et al., 2012). B. exigua is known to produce several phytotoxic compounds (Bottalico et al., 1994; Rai et al., 2009) which are harmful to crop plants, but useful in controlling invasive plants (Berner et al., 2015). S. cretae is a species newly observed by Quaedvlieg et al. (2013) on oleander leaves (Nerium oleander) from Crete, Greece. Consequently, high frequencies of this species would be expected to occur south of the Alps where winters are less harsh and oleander native or naturalized. In the cities north of the Alps, oleanders are popular as ornamentals and are kept outside in pots during the summer. Perhaps, endophytic S. cretae in F. ornus originated from fructification of these ornamentals. Alternatively, N. oleander is not the main host of S. cretae and the endophytic S. cretae of F. ornus originates from a different, as yet unidentified plant host. M. aurantia has been described as a fungus on diseased Eucalyptus globulus leaves in Australia (Maxwell et al., 2003) and was recently detected in Eucalyptus plantations in Spain (Aguin et al., 2013). Detection on F. ornus is probably the first record of this fungus on a host other than Eucalyptus spp. and the second record in Europe. Kretzschmaria deusta, a member of the Xylariales, was found as an endophyte for the first time. The fungus causes an aggressive soft rot on several deciduous tree species. Other xylariaceous fungi are well-known endophytes, e.g. species of Hypoxylon, Nemania, Rosellinia or Xylaria (Petrini and Petrini, 1985). Nemania serpens is rarely dominant but occurs sporadically as an endophyte in a wide range of plant species (including conifers), i.e. its host specificity is low. However, it only sporulates on one or a few of these hosts. Although an apparent dead end, the principle of ‘sporadic endophytism’ could constitute a survival strategy of sorts and serve as reinsurance against host extinction. Moreover, ‘host jumps’ can be associated with species radiation driven by the adaptation to newly-emerged habitats and niches and/or environmental changes (Chaverri and Samuels, 2013). Site conditions certainly have a great influence on the communities of endophytic fungi. Not only are weather conditions decisive for fungal prosperity, reproduction and propagation, but also the edaphic conditions and the interactions with abiotic factors (Colhoun, 1973). The sampling sites differ in all these factors. Even Lago di Ledro and Monte Caslano differ not only in the climatic conditions, but also with regards to edaphic factors. At Monte Caslano, the soils are shallower, steeper and more exposed to the south, and the rainwater is drained quickly. This combination makes Monte Caslano a much drier site than Lago di Ledro, although the sum of the annual precipitation is higher at Monte Caslano (Table 1).

M. Ibrahim et al. / Fungal Ecology 29 (2017) 10e19

F. ornus is phenotypically a very plastic tree species and is able to vary the attributes of its leaves (thickness, specific leaf mass, photosynthetic activity, osmotic potential, etc.) according to its environmental conditions, e.g. different light conditions (Kalapos and Csontos, 2003). This plasticity enables it to adapt to a wide range of site conditions. Accordingly, differences in abiotic conditions, e.g. full sunlight versus shade and, consequently, leaf characteristics, may influence the community of leaf endophytes (Unterseher et al., 2007). It was expected that the sites north of the Alps would differ distinctly from those south of the Alps because F. ornus is not native to the north of the Alps. All trees north of the Alps were planted and, consequently, there were no F. ornus specific fungi at these sites. Numerous studies have shown that trees in their native range are colonized by more host-specific fungi than trees outside their range (Chen, 2012). As a result, it was expected that opportunistic generalists found on other plants would colonize the F. ornus planted outside its natural range. However, this was not the case since V. orni, a F. ornus-specific endophyte, was most frequently isolated irrespective of the side of the Alps on which the tree was growing. It must have arrived either in the form of spores carried by southerly winds over the Alps or together with F. ornus seedlings and/or saplings. Introduction of fungi by means of hitchhiking on their host plants to other regions or continents was shown for C. acutatum, Neofabraea alba and V. fraxini which were unintentionally introduced together with F. excelsior in New Zealand (Chen, 2012). Thus, introduction of V. orni on F. ornus seedlings and/or saplings in northern Switzerland cannot be excluded. F. ornus is an abundant and widespread tree species on calcareous bedrock in southern Europe where it regenerates naturally and has rarely been planted. In all likelihood, the F. ornus trees at Lago di Ledro and Monte Caslano belong to the same population, although the two sites lie 145 km apart. In contrast, F. ornus of many different provenances have been planted as ornamentals in parks, cemeteries and other public areas outside its native range. For example, most of the F. ornus planted in Zurich are cultivars (Baumkataster, 2016). These genotypes differ from those in the native range. These differences can affect the composition of endophyte communities (Todd, 1988; Cordier et al., 2012; Balint et al., 2013; Martín et al., 2013), as also suggested by the intratree variability of the endophyte communities observed in the current study (Fig. 3). Another crucial factor could be the removal of leaf litter in parks, cemeteries and public recreational areas by employees of the parks department, leading to a reduction of inoculum or the complete disruption of the endophytes' life cycle. Most endophytes overwinter and mature in the litter and, ideally, sporulation coincides with leaf flush in spring. Although care was taken to select trees only from places where leaf litter removal was improbable, anthropogenic disturbances could not be excluded north of the Alps. The effects of habitat fragmentation and litter removal on endophyte communities were nicely demonstrated by Helander et al. (2007). The authors examined the effect of habitat size and distance from the inoculum source on endophyte communities in birch leaves (B. pendula and B. pubescens) in an archipelago in southwestern Finland. The frequency of colonization by endophytic fungi was significantly affected by the size and remoteness of the islands. On small islands, there is less leaf litter and the probability of leaf litter being lost to the sea due to crosswinds is higher, and on remote islands, instances of immigration of inoculum from the mainland are fewer. Consequently, the lowest frequency of colonization by endophytic fungi was observed on small, remote islands. The endophyte communities varied between different leaf tissues, e.g. V. orni occurred preferentially in the petioles. Tissue

17

specificity of endophytic fungi has been shown for several plant species (Sieber et al., 1988; Fisher and Petrini, 1990; Petrini, 1991). This study shows that F. ornus hosts a highly diverse endophytic community. Its composition was dependent on factors such as geographic region, the individual tree and host tissue. Acknowledgments We would like to thank Christof Bigler for his help with statistical analyses and the Genetic Diversity Center (GDC) of ETH Zurich for providing laboratory facilities. This work was supported by SNF grant 31003A-146591/1 issued by the Swiss National Science Foundation, Bern, Switzerland. References Aguin, O., Sainz, M.J., Ares, A., Otero, L., Mansilla, J.P., 2013. Incidence, severity and causal fungal species of Mycosphaerella and Teratosphaeria diseases in Eucalyptus stands in Galicia (NW Spain). For. Ecol. Manag. 302, 379e389. Arnold, A.E., Mejia, L.C., Kyllo, D., Rojas, E.I., Maynard, Z., Robbins, N., Herre, E.A., 2003. Fungal endophytes limit pathogen damage in a tropical tree. P. Natl. Acad. Sci. U. S. A. 100, 15649e15654. Aveskamp, M.M., de Gruyter, J., Woudenberg, J.H.C., Verkley, G.J.M., Crous, P.W., 2010. Highlights of the Didymellaceae: a polyphasic approach to characterise Phoma and related pleosporalean genera. Stud. Mycol. 1e60. Balint, M., Tiffin, P., Hallstrom, B., O'Hara, R.B., Olson, M.S., Fankhauser, J.D., Piepenbring, M., Schmitt, I., 2013. Host genotype shapes the foliar fungal microbiome of balsam poplar (Populus balsamifera). PLoS One 8, e53987. Barengo, N., Sieber, T.N., Holdenrieder, O., 2000. Diversity of endophytic mycobiota in leaves and twigs of pubescent birch (Betula pubescens). Sydowia 52, 305e320. Bates, D., M€ achler, M., Bolker, B., Walker, S., 2016. lme4: Linear Mixed-effects Models Using 'Eigen' and S4. http://cran.r-project.org/package¼lme4. retrieved March 23, 2016. Baumkataster, 2016. Baumkataster - Stadt Zürich. https://www.stadt-zuerich.ch/ ted/de/index/gsz/planung_u_bau/inventare_und_grundlagen/baumkataster. html retrieved March 23, 2016. Berner, D., Cavin, C., Woudenberg, J.H.C., Tunali, B., Buyuk, O., Kansu, B., 2015. Assessment of Boeremia exigua var. rhapontica, as a biological control agent of Russian knapweed (Rhaponticum repens). Biol. Control 81, 65e75. Bissegger, M., Sieber, T.N., 1994. Assemblages of endophytic fungi in coppice shoots of Castanea sativa. Mycologia 86, 648e655. BLAST, 2016. Nucleotide BLAST: Search Nucleotide Databases Using a Nucleotide Query. http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM¼blastn&PAGE_ TYPE¼BlastSearch&LINK_LOC¼b. retrieved March 22, 2016. Bonfim, J.A., Vasconcellos, R.L.F., Baldesin, L.F., Sieber, T.N., Cardoso, E.J.B.N., 2016. Dark septate endophytic fungi of native plants along an altitudinal gradient in the Brazilian Atlantic forest. Fungal Ecol. 20, 202e210. Bottalico, A., Capasso, R., Evidente, A., Vurro, M., 1994. Process for the production and purification of cytochalasin B from Phoma exigua var. heteromorpha. Appl. Biochem. Biotech. 48, 33e36. Brandes, D., 2006. Zur Einbürgerung von Fraxinus ornus L. In: Braunschweig. Braunschweiger Naturkundl. Schriften, vol. 7, pp. 535e544. Cai, L., Ji, K.-F., Hyde, K.D., 2006. Variation between freshwater and terrestrial fungal communities on decaying bamboo culms. A. Leeuw 89, 293e301. Carroll, G.C., 1988. Fungal endophytes in stems and leaves - from latent pathogen to mutualistic symbiont. Ecology 69, 2e9. Cesati, V., De Notaris, G., 1863. Schema di classificazione degli sferiacei italici aschigeri piu' o meno appartenenti al genere Sphaeria nell'antico significato attribuitogli da Persoon. Comm. Soc. Critt. Ital. 1, 177e240. Chao, A., 1987. Estimating the population size for capture-recapture data with unequal catchability. Biometrics 43, 783e791. Chaverri, P., Samuels, G.J., 2013. Evolution of habitat preference and nutrition mode in a cosmopolitan fungal genus with evidence of interkingdom host jumps and major shifts in ecology. Evolution 67, 2823e2837. Chen, J., 2012. Fungal Community Survey of Fraxinus Excelsior in New Zealand. http://stud.epsilon.slu.se/4172/. Colhoun, J., 1973. Effects of environmental factors on plant disease. Annu. Rev. Phytopathol. 11, 343e364. s, A., Ndoye, I., Bance, C., Bruzaud, J., Djediat, C., Dupont, J., Nay, B., Prado, S., Combe 2012. Chemical communication between the endophytic fungus Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum. PLoS One 7, e47313. Cordier, T., Robin, C., Capdevielle, X., Desprez-Loustau, M.-L., Vacher, C., 2012. Spatial variability of phyllosphere fungal assemblages: genetic distance predominates over geographic distance in a European beech stand (Fagus sylvatica). Fungal Ecol. 5, 509e520. Damm, U., Cannon, P.F., Woudenberg, J.H.C., Crous, P.W., 2012. The Colletotrichum acutatum species complex. Stud. Mycol. 37e113. Davydenko, K., Vasaitis, R., Stenlid, J., Menkis, A., 2013. Fungi in foliage and shoots of

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