Endophytic fungi isolated from Khaya anthotheca in ...

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Oct 6, 2011 - nator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster. City, CA, USA) .... New Zealand .... Bank sequences were separated into a clearly distinct lineage. (F). ..... Yang YL, Phoulivong S, Liu ZY, Prihastuti H, Shivas RG,.
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Endophytic fungi isolated from Khaya anthotheca in Ghana5 Riikka LINNAKOSKIa,b,*, Helena PUHAKKA-TARVAINENb, Ari PAPPINENb a

Department of Biology, Section of Biodiversity and Environmental Sciences, University of Turku, FIN-20014 Turku, Finland Faculty of Science and Forestry, University of Eastern Finland, P.O. Box 111, FIN-80101 Joensuu, Finland

b

article info

abstract

Article history:

An important African timber tree species, white mahogany (Khaya anthotheca), is heavily

Received 5 August 2010

exploited and vulnerable. Endophytic fungi are thought to have an important role in plant

Revision received 23 June 2011

communities, e.g. by increasing the fitness of the host tree. The aim of the present study

Accepted 4 July 2011

was to isolate and characterize endophytic fungi collected from K. anthotheca in Ghana. In

Available online 6 October 2011

total, 64 fungal isolates were obtained from symptomless leaf samples. The fungi were

Corresponding editor:

grouped according to their morphology, and subjected to DNA sequence comparisons of

Kevin Hyde

the ribosomal ITS region. Based on phylogenetic analyses, the fungal isolates were grouped in Sordariomycetes and Dothideomycetes (Ascomycota). Species of Xylariales were the

Keywords:

most common fungi associated with K. anthotheca. The results of this study indicate that

Ascomycota

K. anthotheca serves as a host to numerous endophytic fungi. These fungi could have

Biodiversity

significance as a source of novel metabolites, and for the fitness of this tree species.

Conservation

ª 2011 Elsevier Ltd and The British Mycological Society. All rights reserved.

Endophytic fungi Funguseplant interactions Ghana forest decline Khaya anthotheca Molecular identification Phylogenetic analysis

Introduction The role of fungal species in forest ecosystems remains poorly understood (Saikkonen 2007; Arnold 2008). Basically every plant is invaded by endophytic fungi, but their classification and systematics are still largely unknown, and to date only the endophytes of herbaceous and woody plants have been explored in any detail (Redlin & Carris 1996; Saikkonen 2007). The common definition for endophytic fungi, or fungi living inside a plant, is based on the habitat of the organism (endophytes: “endo” ¼ within, “phyte” ¼ plant). From a broader perspective, endophytes are considered to be fungi living at

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least part of their life cycle inside plant tissues without causing any pathogenic symptoms to the host plant; the plant simply seems to provide a niche suitable for fungal growth (Wilson 1995; Carlile et al. 2001). As a consequence of this definition, endophytes are considered also to include pathogenic species having latent phases within their life cycles (Wilson 1995; Cheplick & Faeth 2009). Originally endophytes might have evolved from pathogens like Claviceps through an extended latency period (Clay 1988). Endophytes are considered to serve a significant role within the communities of plants (Ahlholm 2003; Arnold et al. 2003; Arnold & Engelbrecht 2003; Saikkonen 2007). The coexistence between an endophyte and

Fungi associated with white mahogany. * Corresponding author. Faculty of Science and Forestry, University of Eastern Finland, P.O. Box 111, FIN-80101 Joensuu, Finland. Tel.: þ358 44 5330212; fax: þ358 13 2514444. E-mail address: [email protected] (R. Linnakoski). 1754-5048/$ e see front matter ª 2011 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2011.08.006

Endophytic fungi isolated from K. anthotheca

the host plant is in most cases neither pathogenic nor mutualistic (Wilson 1995; Cheplick & Cho 2003), but sometimes host plants benefit from their endophytes, e.g. by using alkaloids produced by them as protective compounds against grazing herbivores (Wilson 1995; Cheplick & Faeth 2009). On the other hand, recent findings indicate that in some cases endophytes even exploit their host plant by expressing parasitic properties (Faeth & Sullivan 2003; Cheplick 2004; Sieber 2007; Faeth 2009). Particularly in tropical forests the endophytic fungi remain extremely poorly known components of tropical forest ecology. Every tropical plant studied to date harbours a remarkable diversity of endophytic fungi (Lodge et al. 1996, € chlich & Hyde 1999; Arnold et al. 2000; Gamboa & 2008; Fro Bayman 2001; Arnold et al. 2003; Arnold & Lutzoni 2007), of which the majority represent previously undescribed species (Arnold 2002). Unfortunately, tropical forests are experiencing rapid rates of depletion in several countries. One example of these countries is Ghana, where tropical forests have been decreasing rapidly and significantly, and are amongst the most depleted and fragmented forests in the world (Bossart et al. 2007). Reasons for forest loss are numerous, including illegal logging, pressures from mining interests and clearing for crop cultivation. The forests in Ghana are rich in biodiversity, and recognized as among the most biologically unique forests in the world, harbouring many species which are not found anywhere else. At the same time, these forests are also among the most poorly studied and ecologically understood in the world. The objective of our ongoing research (RIFLAG project funded by the Academy of Finland) is to conserve the indigenous tree species in Ghana. White mahogany (Khaya anthotheca) belongs to the family Meliaceae and together with African mahogany (Khaya ivorensis) is considered as one of the most valuable tropical timber tree species in Africa (Irvine 1961; Abbiw 1990). K. anthotheca is listed as vulnerable in the IUCN Red List of Threatened Species (International Union for Conservation of Nature and Natural Resources 2010), and this has turned the focus of the research concerning the species to conservation. One tool to reach that goal is to examine the site and environmental factors affecting the survival and competitiveness of the species. Symbionts like endophytic fungi might provide an advantage for mahogany in resistance to insect predation or plant diseases, and thus in competition against invasive tree species, which are predominantly used in tree-planting activities (Rodrigues 1996). Innovative ways to exploit indigenous tree species and raise their value might be found by screening for these same compounds. Endophytes are known as a significant cradle of various metabolites that are functional, including compounds with pharmaceutical potential (e.g. Ogawa et al. 1995; Desjardins et al. 1997; Li et al. 2001; Giese et al. 2008; Rukachaisirikul et al. 2009). For further research, it is also important to identify these potentially bioactive fungi. Endophytic fungi are typically identified through a comparison of morphological features. Typically, distinguishing between closely related or morphologically similar species is a complex task. The morphological characters of endophytic fungi can be growth medium-specific and the cultural conditions can impact the sexual and vegetative compatibility of the fungi (Zhang et al. 2006a; Hyde & Soytong

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2007). Furthermore, a large number of endophytic fungi fail to sporulate in culture (Lacap et al. 2003). Molecular methods have greatly increased our knowledge regarding fungal diversity and taxonomy. Several recent studies have shown that molecular methods can be successfully used in the study of endophytic fungi (Johnston & Jones 1997; Mazzaglia et al. 2001; Vega et al. 2010). The most widely used gene regions for detection of endophytic fungi are the ribosomal DNA sequences, especially the internal transcribed spacer region (ITS1, 5.8S, ITS2). In the present study, the main focus was on the isolation and classification of cultivable foliar endophytic fungi from white mahogany (K. anthotheca) in Ghana by using the ITS region as an indicator of the phylogenetic relationships.

Materials and methods Sampling sites White mahogany (K. anthotheca) was sampled for endophytic fungi in 2007. Sampling sites included locations in Bobiri forest reserve (6o410 N; 1o210 W) in Ghana, approximately 20 km south-east of Kumasi. The forest reserve lies inside the tropical moist semi-deciduous area of the forest and has a total land area of 54.6 km2. The reserve is actively managed for timber, but also has significant sections of mostly intact forest canopy (Bossart et al. 2007). The forests are also used by local villagers to collect palm wine and firewood.

Isolations of endophytic fungi The endophytic fungi were isolated from leaves of asymptomatic K. anthotheca. Samples from fresh leaves were taken to the laboratory where they were processed within 24 hr of collection, following standard isolation methods (Posada et al. 2007). Leaves were rinsed with tap water before being cut into smaller pieces under sterile conditions. Segments of surface sterilized leaves were plated on three different media: the A, B and C agar containing antibiotics (A: 400 mg L-sorbose, 50 mg Difco Bacto yeast extract from Becton, Dickinson & Company, Sparks, USA and 1.5 g Difco Bacto agar in 100 ml of Milli-Q water, 10 mg tetracycline in 100 ml of ethanol added after autoclaving; B: 1.5 g Difco Bacto malt extract and 1.5 g Difco Bacto agar in 100 ml of Milli-Q water, 0.2 mg benomyl and 0.2 mg dichloran in 100 ml of ethanol added after autoclaving; C: 3 g sucrose, 200 mg NaNO3, 100 mg K2HPO4, 1 mg MgSO4 [7H2O], 50 mg KCl, 1 mg FeSO4 [7H2O] and 1.5 g Difco Bacto agar in 100 ml of Milli-Q water, 10 mg chloramphenicol and 0.2 mg dichloran in 100 ml of ethanol added after autoclaving; pH 5.6 adjusted for all). The plates were incubated at 22  C for several weeks, and any fungal growth was subcultured onto malt extract agar plates (2 % MEA: 20 g Difco Bacto malt extract, 20 g Difco Bacto agar and 1 l of Milli-Q water). The resulting fungal isolates were further purified and grouped as morphospecies based on culture morphology. Fungal species were characterized using molecular sequence data. The fungi were deposited at the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands (Table 1).

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Table 1 e Endophytic fungal isolates obtained from leaves of Khaya anthotheca and used for DNA sequencing. Group

Species identity

A

Xylaria grammica

B

Xylaria cf. ianthinovelutina

C

Xylaria sp.

D

Pestalotiopsis theae

E

Pestalotiopsis sp.

F

Biscogniauxia sp. 1

G H I J

Biscogniauxia sp. 2 Hypoxylon sp. 1 Hypoxylon sp. 2 Phomopsis sp.

K

Fusarium sp.

L

Colletotrichum sp.

M N O

Colletotrichum cf. gloeosporioides Corynespora cassiicola Lasiodiplodia cf. theobromae

Isolate no.

CBS 130699a CBS 130701a CBS 130698a CBS 130704a CBS 130706a CBS 130707a GHA23b CBS 130708a GHA09b CBS 130710a CBS 130709a CBS 130711a CBS 130695a CBS 130697a CBS 130700a GHA06b GHA30b CBS 130705a GHA28b CBS 130693a CBS 130694a GHA13b CBS 130703a CBS 130702a CBS 130696a GHA20b

GenBank accession no.

HM752504 HM752505 HM752502 HM752503 HM752498 HM752499 HM752500 HM752501 HM752508 HM752509 HM752506 HM752507 HM752510 HM752511 HM752523 HM752513 HM752512 HM752519 HM752520 HM752514 HM752515 HM752516 HM752517 HM752518 HM752522 HM752521

BLAST match sequence

Family

Order

Xylariaceae Xylariaceae Xylariaceae Xylariacea Xylariaceae Xylariaceae Xylariaceae Xylariaceae Amphisphaeriacea Amphisphaeriacea Amphisphaeriacea Amphisphaeriacea Xylariaceae Xylariaceae Xylariaceae Xylariaceae Xylariaceae Diaporthaceae Diaporthaceae Nectriaceae Nectriaceae Glomerellaceae Glomerellaceae Glomerellaceae Corynesporascaceae Botryosphaeriaceae

Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Xylariales Diaporthales Diaporthales Hypocreales Hypocreales Incertae sedis Incertae sedis Incertae sedis Pleosporales Botryoshaeriales

Accession Coverage/Max no. ident % AB524025 AB524025 GU322441 GU322441 EU010004 EU010004 EU010004 EU010004 DQ812932 DQ812917 HQ248207 HQ248207 EF026132 EF026132 EU686174 DQ201131 HM192912 GU595054 GU595054 HQ332533 HQ332533 FJ434135 FJ434135 HQ022504 GU066728 HQ022463

100/99 100/99 100/98 100/98 100/99 100/99 100/99 100/99 100/99 100/100 100/100 100/100 97/83 97/83 100/88 96/98 61/90 100/98 100/98 100/100 100/100 100/99 100/100 100/99 100/100 100/100

a CBS: Centraalbureau voor Schimmelcultures, Utrecth, The Netherlands. b GHA: the private culture collection of the School of Forest Sciences, University of Eastern Finland, Finland.

DNA sequencing In preparation for DNA extraction, fungal isolates were grown on 2 % MEA plates. A total of 26 representative isolates of different morphological groups were selected for molecular identification (Table 1). Fungal genomic DNA was extracted with the PrepMan Ultra Sample Preparation reagent (Applied Biosystems, Foster City, USA) following the Kit User’s Manual. The extracted DNA was stored at 4  C until further use. For the ribosomal DNA operon, the internal transcribed spacer regions (ITS1 and ITS2), including the 5.8 gene, were amplified using primers ITS1-F (Gardes & Bruns 1993) and ITS4 (White et al. 1990). Amplification was performed in a 25 ml reaction mixture containing 0.25 ml of Super-Therm DNA Polymerase mixture (250 U) (HoffmanneLa Roche Ltd., Nutley, USA), 2.5 ml of reaction buffer (10) and 2.5 ml of dNTP’s (2 mM each) and 0.25 ml of each primer (10 mM). The PCR conditions were: an initial denaturation step at 98  C for 30 s, followed by 35 cycles of 10 s denaturation at 98  C, 30 s primers annealing at 57  C, and 30 s extension at 72  C, and 8 min at 72  C for a final chain elongation. The products from PCR were analyzed under the impact of the UV-light after they were separated through a 1 % ethidium bromide (10 mg ml1) stained agarose gel. The amplification products went through a process of purification using the High Pure PCR Product Purification Kit (Roche Molecular Biochemicals, Indianapolis, USA) following the manufacturer’s directions. The purified products were eluted in 50 ml of sterilized Milli-Q water and

again checked on a 1 % agarose gel. The purified PCR-products were sequenced in both directions with the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on the ABI Prism 377 Autosequencer (Applied Biosystems, Foster City, CA, USA), primers from the initial amplification were utilized again.

Identification of isolates For each isolate, sequences obtained using the forward and reverse primers were assembled and edited in Geneious Pro version 4.0.4 for MacIntosh (Biomatters, Auckland, New Zealand). Preliminary identifications of all sequences were obtained by sequence similarity searches (Zhang et al. 2000) from BLAST searches in GenBank (http://blast.ncbi.nlm.nih. gov/blast.cgi). Identification of fungi based on a BLAST search was applied with caution due to the existence of misidentified sequences in GenBank (Vilgalys 2003; Arnold & Lutzoni 2007; Cai et al. 2009). The dataset included the best BLAST match sequence identified through a Mega BLAST search (Zhang et al. 2000) (Table 1). A conservative approach was applied as described by Sakadilis et al. (2011). If a similarity match of 98 % was reported for the isolate sequence, and there were multiple matching sequences, the isolate identity was confirmed by gathering multiple matching sequences obtained ideally from different studies. Possible references that were associated with the matching sequences were also checked (Table 2). When possible, only sequences obtained

Endophytic fungi isolated from K. anthotheca

301

Table 2 e Reference sequences used in the phylogenetic study and their accession numbers Species Biscogniauxia anceps Biscogniauxia capnodes Botryosphaeria populi Botryosphaeria protearum Colletotrichum cf. gloeosporioides Colletotrichum cliviae Colletotrichum gloeosporioides Colletotrichum gloeosporioides Corynespora cassiicola Corynespora cassiicola Diaporthe melonis Fungal endophyte Fusarium annulatum Fusarium proliferatum Fusarium proliferatum Fusarium proliferatum Glomerella magna Glomerella sp. Hypoxylon macrocarpum Hypoxylon sp. Lasiodiplodia hormozganensis Lasiodiplodia parva Lasiodiplodia pseudotheobromae Lasiodiplodia pseudotheobromae Lasiodiplodia plurivora Lasiodiplogia theobromae Pestalotiopsis microspora Pestalotiopsis rhododendri Pestalotiopsis sydowiana Pestalotiopsis sydowiana Pestalotiopsis theae Pestalotiopsis theae Pestalotiopsis theae Phomopsis sp. Phomopsis longicolla Phomopsis longicolla Protomyces inouye Taphrina deformans Taphrina wiesneri Xylariaceae sp. Xylaria ianthinovelutina Xylaria grammica Xylaria grammica

Culture collection no.a

CBS 110300 MUCC 497

ICMP 17941

CBS 507.78 CBS 258.54

CBS 494.78 CBS 111530 CBS 120832 CBS 111530 HKUCC 8316 BRIP 25628 HKUCC 8326

HKUCC 7982

CBS 356.35 CBS 275.28

Host

Origin

GenBank accession no.

Reference

Unknown Unknown Populus nigra Santalum acuminatum Hevea guianensis Clivia miniata Diospyros kaki Fragaria ananassa Dimocarpus longan Hevea brasiliensis Cucumis melo Gackstroemia alpina Oryza sativa Lycopersicon esculentum Havea brasiliensis Zea mays Citrullus lanatus Syzygium paniculatum Salix alba Unknown Mangifera indica Cassava-field soil Hevea guianensis Coffea sp. Prunus salicina Leucospermum sp. Aegiceras corniculatum Antidesma ghaesembilla Protea mellifera Elaeis sp. Camellia sinensis Camellia sinensis Protea mellifera Rhizophoraceae sp. Glycine max Abutilon theophrasti Unknown Prunus persica Prunus avium Coffea arabica Swietenia macrophylla Unknown Wood

France Thailand Portugal Australia Peru China Japan USA Malaysia Malaysia USA New Zealand New Caledonia India Peru Italy USA USA Czech Republic Thailand Iran Colombia Peru Zaire South Africa USA Hong Kong Australia South Africa Colombia China China South Africa China China Croatia Unknown Netherlands Unknown Colombia Martinique Thailand Thailand

EF026132 DQ631933 AY640253 EF591912 HQ022504b GQ485607 GU174547 AF411774 GU066728b FJ52662 FJ889447 EU686174b AY213654 HQ332533b FJ884099 EU151490 DQ003103 FJ434135b HM192912 DQ201131b GU945357 EF622084 HQ022463b EF622080 EF445362 FJ150695 AF409958 AF409986 AF409970 HQ248207b DQ812932b DQ812917b AF405297 GU595054b EU650789 AY857868 DQ497617 AF492095 AF492127 EU010004b GU322441 AB524025b GU300097

Hsieh et al. 2010 Tang et al. 2007 Phillips et al. 2005 Taylor et al. 2009 Unpublished Yang et al. 2009 Weir & Johnston 2010 Vinnere et al. 2002 Unpublished Dixon et al. 2009 Santos et al. 2010 Davis & Shaw 2008 Rakeman et al. 2005 Unpublished Gazis & Chaverri 2010 Visentin et al. 2009 Du et al. 2005 Unpublished Pazoutova et al. 2010 Unpublished Abdollahzadeh et al. 2010 Abdollahzadeh et al. 2010 Unpublished Alves et al. 2008 Abdollahzadeh et al. 2010 Marincowitz et al. 2008 Jeewon et al. 2003 Jeewon et al. 2003 Jeewon et al. 2003 Unpublished Unpublished Unpublished Jeewon et al. 2003 Unpublished Cui et al. 2009 Vrandecic et al. 2004 Unpublished Rodriques & Fonseca 2003 Rodriques & Fonseca 2003 Vega et al. 2010 Hsieh et al. 2010 Unpublished Hsieh et al. 2010

a CBS, Centraalbureau voor Schimmelcultures; HKUCC, The University of Hong Kong Culture Collection; ICMP, International Collection of Microorganisms from Plants; MUCC, The Culture Collection of Murdoch university. b Closest related sequences in GenBank. These sequences include results from unpublished studies, and species may have not been correctly named.

from isolates deposited in public culture collections and/or published in peer reviewed journals were selected. The identities were further confirmed by phylogenetic analysis. The alignment of sequences was achieved through MAFFT v6 online (Katoh et al. 2002), using the E-INS-i method with a gap opening penalty of 1.53 along with an offset value of 0.00. All sequences obtained in this study were deposited in GenBank (Table 1). Molecular Evolutionary Genetic Analyses (MEGA) Version 3.1 (Kumar et al. 2008) was used for compiling datasets and conducting preliminary analyses. The datasets were analyzed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian (BI) methods. MP

analyses were conducted using TNT v1.1 (Goloboff et al. 2008). Heuristic searches with 10 000 replicates of random addition sequences (RAS) and tree bisection and reconnection (TBR) branch swapping were carried out. Gaps were treated as a fifth character. A Jackknife test (JK) with 10 000 replicates was used for counting the support values. ML analyses were conducted using RAxML 7.2.5 (Stamatakis et al. 2008) assuming the GTRCAT substitution model, run on the CIPRES Portal 2.0 at the San Diego Supercomputing Center (Miller et al. 2009). Support for the nodes was assessed from 1 000 bootstrap replicates (Felsenstein 1985). BI analyses based on a Markov Chain Monte Carlo (MCMC) were conducted using the

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program MrBayes version 3.1.2 (Ronquist & Huelsenbeck 2003). Four independent MCMC chains were run for five million generations using the best fitting model chosen through the AIC in MrModeltest v2.3 (Nylander 2004). Trees were sampled every 100 generations resulting in 50 000 trees from both runs. From these, the first 150 trees were discarded as burn-in. The remaining trees were used to construct a majority rule consensus tree.

Molecular identification

A B C D E F G H I J K L M N O

Species identity

No. of isolates

Xylaria grammica Xylaria cf. ianthinovelutina Xylaria sp. Pestalotiopsis theae Pestalotiopsis sp. Biscogniauxia sp. 1 Biscogniauxia sp. 2 Hypoxylon sp. 1 Hypoxylon sp. 2 Phomopsis sp. Fusarium sp. Colletotrichum sp. Colletotrichum cf. gloeosporioides Corynespora cassiicola Lasiodiplodia cf. theobromae

2 3 23 2 8 1 2 2 1 10 4 2 1 1 2

Dothideomycetes

Group

Dothideomycetidae

100

Botryosphaeriales O

Pleosporomycetidae

All the isolated fungi from K. anthotheca were included in Ascomycota and subphylum Pezizomycotina within two

GU237879 Phoma viburnicola 100GU237872 P. viburnicola EF591912 B. protearum AY640253 Botryosphaeria populi EF622080 Lasiodiplodia pseudotheobromae EF445362 L. plurivora 100 EF622084 L. parva G 98 FJ150695 L. theobromae HQ022463 L. pseudotheobromae GU945357 L. hormozganensis DQ497617 Protomyces inouyei AF492095 Taphrina deformans 100 AF492127 T. wiesneri

Hypocreomycetidae

I GHA30 HM192912 H. macrocarpum CBS 130705 J GHA28 Diaporthales GU595054 Phomopsis sp. 100 EU650789 P. longicolla AY857868 P. longicolla FJ889447 Diaporthe melonis CBS 130693 HQ332533 Fusarium proliferatum CBS 130693 Hypocreales 76 K 100 AY213654 F. annulatum FJ884099 F. proliferatum EU151490 F. proliferatum GHA13 95 100 FJ434135 Glomerella sp. CBS 130703 L DQ003103 G. magna GQ485607 Colletotrichum cliviae 100 FJ972607 C. kahawae Glomerellaceae GQ485600 C. hymenocallidis 96 DQ003093 C. fragariae M 77 EU371022 C. gloeosporioides CBS 130702 HQ022504 C. cf gloeosporioides 90 FJ972611 C. fructicola GU066728 Corynespora cassiicola 100 N CBS 130696 Pleosporales 100 FJ852662 Corynespora cassiicola

Phylogeny based on the ITS region

Table 3 e Number of endophytic fungal cultures obtained from K. anthotheca during the course of this study

Sordariomycetes

EF026132 Biscogniuxia anceps CBS 130700 G DQ631933 B. capnodes 99 EU686174 Fungal endophyte 96 GHA06 H DQ201131 Hypoxylon sp. 99

Sordariomycetidae

In total, 64 endophytic fungal isolates were obtained from healthy K. anthotheca leaves collected from Bobiri forest reserve in Ghana (Table 3). These represented 26 different morphotypes according to their morphological characters. The ITS rDNA region was sequenced for all morphotypes and identified by comparing with the published GenBank sequences (Table 1). The amplification products of the ITS region generated a single fragment approximately 600 bp in size. The sequencing of the PCR-products yielded 486e707 bp of informative sequence. A maximum likelihood tree (Fig 1) including all sequences obtained in this research, is given here to show the diversity and the phylogenetic placement of the endophytic fungi associated with K. anthotheca. The ITS data matrix contained 74 taxa and 825 characters including gaps. Species of Taphrinomycotina were selected for an outgroup based on phylogenetic relationships reported in Spatafora et al. (2006). The Bayesian analyses produced trees with topologies similar to ML and MP analyses. The best fitting substitution model selected for the Bayesian analysis was GTR þ I þ G.

Xylariales

Xylariomycetidae

Results and discussion

CBS 130701 A 99 CBS 130699 AB524025 Xylaria grammica 99 BI 100 GU300097 X. grammica GU322441 X. ianthinovelutina B 98 CBS 130704 CBS 130698 EU010004 Xylariaceae sp. 96 CBS 130707 BI 100 100 CBS 130708 C GHA23 CBS 130706 CBS 130710 DQ812917 Pestalotiopsis theae D 96 GHA09 DQ812932 P. theae 100 AF405297 P. theae AF409970 P. sydowiana AF409986 P. rhododendri 100 99 E CBS 130709 93 CBS 130711 HQ248207 P. sydowiana 90 AF409958 P. microspora 100 CBS 130695 F CBS 130697

0.1

Fig 1 e Phylogram obtained from ML analyses of the ITS region. Isolate numbers of sequences obtained in this study are printed in bold type. ML bootstrap support values (1 000 replicates) (normal type) and MP Jackknife values (5 000 replicates) (bold type) above 75 % are indicated at the nodes. Posterior probabilities (above 90 %) obtained from BI are indicated by bold lines at the relevant branching points. * [ bootstrap values lower than 75 %. AeO [ phylogenetic lineages. Scale bar [ total nucleotide difference between taxa. classes, the Sordariomycetes and the Dothideomycetes (Fig 1). Sordariomycetes was represented by all currently recognized subclasses, the Xylariomycetidae, the Sordariomycetidae and the Hypocreomycetidae (Zhang et al. 2006b). Also

Endophytic fungi isolated from K. anthotheca

Dothideomycetes was represented by both currently recognized subclasses, the Pleosporomycetidae and the Dothidiomycetidae (Schoch et al. 2006). Based on the number of the isolates, the majority of the fungal isolates belonged to the Sordariomycetes (Table 3). The sixty-one isolates belonging to Sordariomycetes included species in the orders Xylariales (nine species), Diaporthales (one species), and Hypocreales (one species) (Fig 1). Species of Glomerellaceae are currently placed within Hypocreomycetidae incertae sedis (Zhang et al. 2006b). Three isolates were related to the Pleosporales (one species) and the Botryosphaeriales (one species) within the Dothideomycetes. Xylariales was the dominant group of fungi associated with mahogany in this study (Fig 1, Table 3). Forty-four isolates were distributed in the genera Xylaria, Pestalotiopsis, Biscogniauxia and Hypoxylon. The most numerous species in this study, represented by twenty-three strains, was similar to Xylariaceae sp. (C) originated from coffee (Coffea arabica) (Vega et al. 2010). Three isolates were similar to Xylaria ianthinovelutina (B), and two strains formed a well-supported clade with Xylaria grammica (A). Two strains had highly similar sequences to Pestalotiopsis theae (D). Another Pestalotiopsis species was closely related to Pestalotiopsis sydowiana and Pestalotiopsis rhododendri (E). The genus Biscogniauxia included two species. Two isolates showing low similarity (83 %) to available GenBank sequences were separated into a clearly distinct lineage (F). Also the other Biscogniauxia species showed only low similarity (88 %) to available reference sequences, and was separated into another lineage together with Biscogniauxia anceps and Biscogniauxia capnodes (G). Isolate GHA06 (98 % similarity) formed a lineage with Hypoxylon sp. DQ201131 (H). In addition, another species represented by a single isolate grouped together with other Hypoxylon species (I), but its sequence similarity to available reference sequences in GenBank was low (90 %). Four isolates were distributed in the order Hypocreales (Fig 1). These isolates formed a lineage with species of Fusarium/Gibberella (K). The ITS region of Fusarium species is widely studied. The ITS sequences revealed 100 % sequence similarity to numerous Fusarium species, including Fusarium proliferatum and Fusarium annulatum. Based on the ITS sequences alone, the species level identification of Fusarium is unreliable (O’Donnell & Cigelnik 1997). A more accurate identification should be confirmed by sequence analyses of multiple gene regions. Two species belonging to the family Glomerellaceae (the Hypocreomycetidae i.s.) were recognized (Fig 1, LeM). In both cases, the BLAST search resulted in 100 % sequence similarity to numerous Colletotrichum sequences, including Colletotrichum gloeosporioides. In most cases, names under C. gloeosporioides based on ITS sequence data in GenBank have been wrongly applied (Cai et al. 2009). In addition, the type cultures of different Colletotrichum species have not been subjected to studies of other gene regions. Recent multigene studies have shown that the ITS sequence data can be used for resolving the different species complexes within Colletotrichum (Du et al. 2005; Cai et al. 2009). For preliminary identification and constructing the final dataset, we followed the Colletotrichum backbone tree provided by Cai et al. (2009). The first species of Colletotrichum found in this study formed a distinct clade with

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Glomerella magna and Glomerella cliviae. This is inconsistent with a previous study, which reported that C. magna can easily be distinguished from the morphologically similar C. gloeosporioides based on the ITS sequences (Du et al. 2005). The second Colletotrichum species represented by a single isolate grouped within the C. gloeosporioides species complex (M). The complex includes several species, such as Colletotrichum fragariae and Colletotrichum musae, which cannot be separated from C. gloeosporioides based on the ITS sequence data only (Du et al. 2005; Cai et al. 2009). Ten isolates belonging to the order Diaporthales were recognized (Fig 1, Table 3). The monophyly of the order was strongly supported, as a recent multigene study has reported (Zhang et al. 2006b). The isolates found in the present study were closely related to Phomopsis longicolla (J). Previous studies have shown that the ITS region in Diaporthe/Phomopsis is evolving faster compared to other fungal genera, and can be considered as a relatively sufficient method for distinguishing species (Santos et al. 2010). The Dothideomycetes found in the present study included two species (Fig 1, Table 3). The first species was similar to Corynespora cassiicola (N). The ITS sequence data are known to be relatively sufficient for a species level identification of C. cassiicola, and even studying genetic variation within the species (Dixon et al. 2009). The other species grouped within the Lasiodiplodia theobromae complex (O), as first recognized by Alves et al. (2008). The complex currently accommodates eight species, which cannot be distinguished based on their ITS sequences only (Abdollahzadeh et al. 2010). The current research suggests that the ITS sequence analyses provided a sufficient resolution to identify endophytic fungi to the genus level at least. The identification of endophytic fungi faces certain challenges. Several endophytic fungi remain uncharacterized, and their phylogenetic relationships are poorly understood. The ITS region is most widely studied and in many cases a sufficient method, but caution should be taken with numerous erroneously named sequences in GenBank (Vilgalys 2003; Arnold & Lutzoni 2007; Cai et al. 2009). Ideally, studies should include comparisons made with type specimens. In several cases type specimens cannot be used, because they are missing or in poor condition (Cai et al. 2009). Only some genera such as Fusarium are more closely studied (Kvas et al. 2009). Several other commonly reported genera, such as Botryosphaeria, Phomopsis and Pestalotiopsis need further investigations before the status of several species is clarified (Jeewon et al. 2002, 2003; Alves et al. 2008; Santos et al. 2010). Consequently, more accurate identification should be confirmed by sequence analyses of multiple gene regions in combination with other characters, such as morphological and biochemical data (e.g. Jeewon et al. 2002; Schoch et al. 2006; Arnold 2007; Cai et al. 2009; Prihastuti et al. 2009; Abdollahzadeh et al. 2010; Santos et al. 2010). This study offers a framework for further investigations on endophytic fungi associated with K. anthotheca.

Fungal diversity within white mahogany In total, 64 endophytic fungal cultures were isolated from the leaves of asymptomatic K. anthotheca trees in Ghana. A high number of endophytic species were encountered during this

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relatively small survey, despite the fact that the methodology used in our study is culture specific and slow growing and nonculturable species are likely to be missed (Hyde & Soytong 2007). All of the endophytic fungi within this research were distributed among Ascomycota, in the classes Sordariomycetes and Dothideomycetes. The majority of the species were grouped into the Sordariomycetes, agreeing with previous studies with tropical plants (Arnold & Lutzoni 2007). The Xylariaceae consist of a single order, the Xylariales (Zhang et al. 2006b). The Xylariales were the dominant endophytes in K. anthotheca, including species in the genera Xylaria, Pestalotiopsis, Biscogniauxia and Hypoxylon. Xylaria is the first described and probably the largest genus within the family Xylariaceae (Kirk et al. 2001). Xylaria is found on decaying wood of living or dead angiosperms (Rogers 1979; Petrini & Petrini 1985; Weber & Anke 2006). The occurrence of these species on K. anthotheca was not a surprise, since they are commonly found as endophytes of various tropical host plants (Gamboa & Bayman 2001; Crozier et al. 2006). In this study, three species of Xylaria were isolated. The most frequently collected isolates were closely related to endophytes isolated from coffee (C. arabica) in Colombia (Vega et al. 2010). These isolates formed a clearly distinct lineage, and might represent a novel species. The second species was similar to X. ianthinovelutina. Currently, only two ITS sequences are available for the species in GenBank. The third species was closely related to X. grammica. X. grammica has been reported to occur widely in the tropics in both Africa and America (Lodge et al. 2008). Two species of Pestalotiopsis were isolated in this study. Species of Pestalotiopsis are widely distributed, occurring on a variety of substrata (Jeewon et al. 2003). Most Pestalotiopsis species are pathogens or endophytes of living plants (Lee et al. 1995; Tuset et al. 1999; Rivera & Wright 2000; Karaca & Erper 2001; Taylor et al. 2001) and many occur as saprobes (Wu et al. 1982). Endophytic Pestalotiopsis species are particularly common organisms in the global rainforest systems, and are known as a source of secondary metabolites (Ogawa et al. 1995; Li et al. 2001, 2008; Li & Strobel 2001; Liu et al. 2009; Xu et al. 2010). Pestalotiopsis microspora, a common species found in association with a number of tree species (Metz et al. 2000), is a good example of the importance and potential of endophytes. Metz et al. (2000) suggested that the species may play a significant role in rainforest ecosystems. In most cases, the fungus seems to be either an endophytic symbiont or a weak pathogen of its host tree (Metz et al. 2000). The fungus has also been studied as a potential agent of the endangered Florida torreya’s (Torreya taxifolia) decline in North America (Lee et al. 1995; Schwartz et al. 1995, 1996). This dramatic decline of the Florida torreya is an example of how environmental changes, such as climate change or intensive forestry practices, can increase plant stress and trigger the pathological activity of common fungal associates. Besides the importance of P. microspora in nature, its potential as a producer of taxol (a chemotherapeutic drug) as well as a biological and biochemical model organism has been studied (Metz et al. 2000). Two Pestalotiopsis isolates found in the present study were similar to P. theae, which is a causal agent of grey blight disease of tea (Camellia sinensis) (Koh et al. 2001; Li et al. 2008). Leaf blight on the persimmon tree (Diospyros kaki) in changed

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environmental conditions has also been reported (Tuset et al. 1999). The other isolates were similar to P. sydowiana. P. sydowiana has been reported to infect ornamentals such as ericaceous plants (McQuilken & Hopkins 2004). Biscogniauxia and Hypoxylon are commonly found in dead plant material (Ju et al. 1998). Species of Hypoxylon have widespread occurrence and they are particularly common fungal species in subtropical and tropical regions (Mazzaglia et al. 2001). These species occur as saprobes, pathogens causing bark necrosis or wood decay on angiosperms (Manion & Griffin 1986; Rayner & Boddy 1986; Whalley 1996), or as endophytes (Petrini & Petrini 1985). Typical to Biscogniauxia species is that they are well adapted to dry or seasonally dry habitats, and are capable of persisting for long periods on dead wood (Ju et al. 1998; Nugent et al. 2005). Two species related to species of Biscogniauxia, and two species related to Hypoxylon were discovered in this study. None of the Biscogniauxia or Hypoxylon species matched with any known species represented in GenBank. These isolates might represent undescribed species, or currently poorly studied species not yet present in GenBank. Gibberella and Fusarium (its anamorph) species are frequently isolated fungal endophytes of several plant species (Bills & Polishook 1992; Suryanarayanan & Kumaresan 2000; Photita et al. 2001; Gong & Guo 2009). Four isolates similar to F. proliferatum were found in the present study. F. proliferatum is a common pathogen of numerous crops (Elmer 1990; Logrieco et al. 1995; Desjardins et al. 1997) and an agent of wilt, blight and diebacks of palm trees (Abdalla et al. 2000; Polizzi & Vitale 2003; Armengol et al. 2005). F. proliferatum produces numerous toxins (Logrieco et al. 1995), and has strong antimicrobial properties against human pathogens (Gong & Guo 2009). Glomerella and Colletotrichum (its anamorph) are known as plant pathogens, causing economically important diseases (anthracnoses) on a wide range of hosts (Bailey & Jeger 1992). The species are widely distributed, but found mainly in subtropical and tropical regions. Our phylogenetic analysis, based on the ITS sequences, did not differentiate distinct lineages amongst the isolates. The C. gloeosporioides species complex is challenging to resolve due to the high number of greatly diverse species complexes and species, the statuses of which are questionable (Du et al. 2005). The ITS sequence data were not useful for separating other C. fragariae from C. gloeosporioides (Sreenivasaprasad et al. 1992, 1994; Johnston & Jones 1997; Du et al. 2005). Therefore, additional gene regions should be explored to distinguish between these taxa. The Sordariomycetes also included isolates closely related to P. longicolla. P. longicolla together with other species in the Diaporthe and Phomopsis (its anamorph) complex are causal agents of seed decay, and cause blight and canker diseases of soybean (Glycine max) (Kulik & Sinclair 1999). Of the species in the complex, P. longicolla is known as the most aggressive seed pathogen occurring in soybean production areas worldwide (Hobbs et al. 1985; Brown et al. 1987). The species-rich genus Botryosphaeriaceae (Dothideomycetes), and its several anamorphic genera, include species that have a cosmopolitan distribution and are found in association with a wide variety of host plants (von Arx 1987; Crous et al. 2006; Phillips et al. 2008). These fungi are responsible for symptoms such as fruit rots, shoot blights, diebacks and

Endophytic fungi isolated from K. anthotheca

cankers of numerous woody hosts (von Arx 1987; Roux et al. 2001; Alves et al. 2004; Wang & Hsieh 2006). Species of Botryosphaeriaeceae have been reported as endophytes (Swart & Wingfield 1991; Smith et al. 1996). Despite their pathogenic nature, Botryosphaeria species are also a source of various types of bioactive compounds (Giese et al. 2008; Rukachaisirikul et al. 2009). One species belonging to L. theobromae complex (anamorph of Botryosphaeria rhodina) was found in the present study. Species belonging to the recently revealed L. theobromae complex are common pathogens (Punithalingam 1980; De Beer et al. 2001; Abdollahzadeh et al. 2010). The other species belonging to the class Dothideomycetes isolated in the present study was similar to C. cassiicola. The genus Corynespora includes species that are saprobes, pathogens and endophytes of woody and herbaceous plants, other fungi, nematodes and human skin (Dixon et al. 2009). C. cassiicola is an important plant pathogen, found in association with numerous host plants including economically important crops (Silva et al. 1995). Corynespora leaf fall disease (CLFD) caused by C. cassiicola is among the most devastating diseases affecting the rubber tree (Hevea brasiliensis).

Conclusions The aim of our study was to characterize endophytic fungi associated with white mahogany (K. anthotheca) in Ghana. K. anthotheca is considered to be an extremely important tree species in terms of the timber in Africa (Irvine 1961; Abbiw 1990), but is also found on the IUCN Red List of Threatened Species (International Union for Conservation of Nature and Natural Resources 2010). The present study forms a part of a larger project (RIFLAG project funded by Academy of Finland), which aims to conserve the indigenous tree species in Ghana, including K. anthotheca. Plants are hosts to a variety of microorganisms, of which many are host-specific, and in most cases these plant-microbe relationships are poorly understood. Arnold et al. (2003) and Ganley et al. (2008) have reported that some endophytic fungi produce protecting compounds mediating resistance against pathogens and increasing the fitness of the host tree. In addition, several other sources indicate that endophytes are rich in valuable compounds of pharmaceutical and industrial interest (Ogawa et al. 1995; Metz et al. 2000; Shimizu et al. 2000; Li et al. 2001, 2008; Li & Strobel 2001; Phongpaichit et al. 2006; Gong & Guo 2009; Liu et al. 2009; Sutjaritvorakul et al. 2010). This is, therefore, a strong argument for host tree conservation, since host plant extinction may also mean that the biosynthetic capability of the associated fungi will disappear as well. On the other hand, environmental disturbances such as climate change, intensive forestry practices, or large-scale cultivation of non-native trees surrounding the habitat of endangered species might also affect the hostefungus interaction, and physiological and environmental factors could trigger the pathological activity of normally latent endophytes. The forests in Ghana are among the most critically threatened forests in the world (Bossart et al. 2007). At the same time, they are very rich in biodiversity, but poorly

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understood ecosystems. The remaining forests are mainly situated in reserve regions, and are highly fragmented and degraded. As our results clearly show, forests in Ghana harbour species that have not previously been discovered. The number of endophytic species encountered during this relatively small survey indicates that K. anthotheca serves as a host to numerous endophytic fungi, many of which represent previously unidentified species and have great potential for novel metabolites. It is clearly very important to understand these unique forest ecosystems, their biodiversity and the plantemicrobe interactions. We will continue to work with hostefungus interactions in more detail and screen the possible bioactive compounds produced by the endophytes discovered in the present study. Taxonomic studies of more isolates and sequences of more gene regions, together with morphological approaches will be necessary to resolve the identity of several endophytic fungi isolated in this study, including characterization of potential novel species.

Acknowledgements This study was financially supported by the Academy of Finland and the Kone foundation. We are grateful to the Forestry Research Institute of Ghana (FORIG) for assistance in field work at the Bobiri Forest Reserve. We thank Dr. Jouni Ahlholm for valuable comments on the manuscript and Mr. Alain Joseph for the proofreading. We also thank the Finnish IT Centre for Science (CSC) for providing computational resources.

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