Muscodor Fengyangensis Sp. Nov. from Southeast China: Morphology, Physiology and Production of Volatile Compounds

fungal biology 114 (2010) 797e808

journal homepage: www.elsevier.com/locate/funbio

Muscodor fengyangensis sp. nov. from southeast China: morphology, physiology and production of volatile compounds

Chu-Long ZHANGa, Guo-Ping WANGb, Li-Juan MAOc, Monika KOMON-ZELAZOWSKAd, Zhi-Lin YUANa, Fu-Cheng LINa,*, Irina S. DRUZHININAd, Christian P. KUBICEKd,* aState Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China bZhejiang Dayang Chemical Co. LTD, Jiande 311616, China c985-Institute of Agrobiology and Environmental Sciences, Zhejiang University, Hangzhou 310029, China dInstitute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Vienna University of Technology, 1060 Vienna, Austria article info abstract

Article history: The fungal genus Muscodor was erected on the basis of Muscodor albus, an endophytic fun- Received 24 April 2010 gus originally isolated from Cinnamomum zeylanicum. It produces a mixture of volatile or- Received in revised form ganic compounds (VOCs) with antimicrobial activity that can be used as mycofumigants. 12 July 2010 The genus currently comprises five species. Here we describe the isolation and character- Accepted 20 July 2010 ization of a new species of Muscodor on the basis of five endophytic fungal strains from Available online 29 July 2010 leaves of Actinidia chinensis, Pseudotaxus chienii and an unidentified broad leaf tree in the Corresponding Editor: Marc Stadler Fengyangshan Nature Reserve, Zhejiang Province, Southeast of China. They exhibit white colonies on potato dextrose agar (PDA) media, rope-like mycelial strands, but did not spor- Keywords: ulate. The optimum growth temperature is 25 C. The results of a phylogenetic analysis Antimicrobial activity based on four loci (ITS1e5.8SeITS2, 28S rRNA, rpb2 and tub1) are consistent with the hy- ITS rRNA pothesis that these five strains belong to a single taxon. All five strains also produce volatile Muscodor chemical components with antimicrobial activity in vitro, which were different from those Optimum growth temperature previously described for other Muscodor species. rpb2 ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. 28S rRNA tub1 Volatile metabolites

Introduction Mitchell et al. 2008; Sopalun et al. 2003; Strobel et al. 2007; Worapong et al. 2001, 2002). So far ﬁve species have, on the Muscodor is a genus of endophytic fungi that are known from basis of morphological, phenetic and genetic features, been certain tropical tree and vine species in Central/South Amer- described in the genus (i.e. Muscodor albus, Muscodor roseus, ica, and South Eastern Asia and Australia (Atmosukarto et al. Muscodor vitigenus, Muscodor crispans and Muscodor yucatanen- 2005; Daisy et al. 2002b; Ezra et al. 2004; Gonza´lez et al. 2009; sis)(Daisy et al. 2002a, 2002b; Gonza´lez et al. 2009; Mitchell

* Corresponding authors. E-mail addresses: [email protected], [email protected] 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.07.006 798 C.-L. Zhang et al.

et al. 2008; Worapong et al. 2001, 2002). Muscodor spp. also char- Japan), operating between 10 and 15 kV on samples frozen in acteristically produce a mixture of volatile organic com- liquid nitrogen and coated with a thin layer of gold sputter. pounds (VOCs) that consist primarily of various alcohols, acids, esters, ketones, and lipophilic substances that are lethal Determination of the optimum growth temperature to a wide variety of plant- and human-pathogenic fungi and bacteria, as well as to nematodes and certain insects (Daisy The optimum growth temperature was determined on PDA et al. 2002a; Strobel et al. 2001). Muscodor spp. are therefore of and MEA. The fungal strains were pre-grown on PDA medium high value and promise for biocontrol (Strobel 2006) and the for 15 d in darkness at 25 C. Then an agar disk (5 mm diame- discovery of new isolates and taxa of this genus is of major in- ter) was excised from the growing front of the fungus and terest to mycologists. placed into the middle of a new Petri dish and incubated in During a systematic study of the fungal endophytic popula- darkness for 15 d at 10, 15, 20, 25, 28, 30, 35 and 40 C, respec- tion of woody plants distributed in the Southeast of China, we tively. The diameter of the growing colony was measured. We isolated five strains that exhibited characteristics typical for shall note that because of the very slow growth of the tested Muscodor. Here we describe them as a new taxon of Muscodor, isolates, they still had not reached the border of the plates af- based on their morphology, the optimum growth tempera- ter 15 d, and the measurements of different growth rates are ture, production of volatile chemicals profiles and a multilocus thus comparable. phylogenetic analysis, thereby also expanding the concept of its genus. DNA extraction, gene fragment amplification, sequencing and phylogenetic analysis

Materials and methods For DNA isolation, the fungal mycelium was scraped from the margin of the colonies with a sterile needle. Genomic DNA Study site was extracted using the Multisource Genomic DNA Miniprep Kit (Axygen Bioscience, Inc. China) following the manufac- The site of study was located in Fengyangshan Nature turer’s instructions. For amplification of the internal tran- Reserve, Zhejiang province, in the Southeast of China scribed spacer (ITS) and 28S ribosomal RNA (rRNA), the 0 0 0 0 (E119 06 e119 15 , N27 46 e27 58 ). primers ITS1 (50-TCCGTAGGTGAACCTGCGG-30) and ITS4 (50-TC CTCCGCTTATTGATATGC-30), LR0R (50-ACCCGCTGAACT- TAAGC-30) and LR5 (50-TCCTGAGGGAAACTTCG-30), respec- Sampling, isolation of endophytic fungi and maintenance of tively, were used (White et al. 1990). A fragment of the cultures RNA-polymerase subunit B encoding gene rpb2 was amplified with primer pairs RPB2-5f (50-GAYGAYMGWGATCAYTTYGG- Healthy and intact twigs and leaves of Actinidia chinensis, Pseu- 30) and RPB2-7cr (50-CCCATRGCTTGYTTRCCCAT-30)asde- dotaxus chienii and an unidentified broad leaf tree, respec- scribed by Liu et al. (1999). A fragment of the beta-tubulin coding tively, were sampled, placed in sterile plastic bags, stored in gene (tub1) was amplified with the primers bena-T1 (50 -AACA an ice box and transported to laboratory within 48 h of TGCGTGAGATTGTAAGT-30) and bena-T22 (50-TCTGGATG sampling. TTGTTGGGAATCC -30)(O’Donnell & Cigelnik 1997). ITS rRNA The plants were then rinsed softly with tap water, thereaf- polymerase chain reaction (PCR) products were separated in ter immersed in ethanol (75 %, v/w; 30 s), followed by immer- 1.0 % (w/v) agarose gels, purified by the aid of a gel band purifi- sion in sodium hypochlorite (1 %, w/v; 10 min) and finally cation kit (Axygen Bioscience, Inc. China), and then sequenced rinsed thrice with 30 ml of sterile distilled water. The plant tis- in an ABI 3730 sequencer (Applied Biosystems, USA), using the sues were then cut into pieces of 0.6 cm length and six of them same primers as for PCR. PCR products of 28S rRNA, rpb2 and placed on a plate containing malt extract agar (2 % w/v; MEA), tub1 were purified as above and ligated into pGEM-T Easy vector supplemented with chloromycetin (50 mg l 1) to prevent bac- (Promega, USA), and transformed into Escherichia coli JM109 terial growth, and incubated at 25 C in darkness. After the (Promega) according to the manufacturer’s instructions. Posi- emerging of fungal hyphae from the tissues, they were trans- tive clones subjected to sequencing with ABI 3730 sequencer. ferred to new agar plates and purified by subculturing. For cul- All sequences were subjected to Basic Local Alignment ture maintenance, the isolates were grown on potato dextrose Search Tool (BLAST) analysis at the National Center for Bio- agar (PDA) and either covered with sterile liquid paraffin technology Information (NCBI) server. The sequences with (at 25 C) or suspended in GYCG (glucose, 10 g l 1; yeast extract highest similarity to each locus were retrieved, combined 1gl 1; acid casein hydrolysate, 1 g l 1; and glycerol 15 %, v/v) with the sequences obtained during this work, aligned using and stored at 70 C. Clustal X1.8 (Thompson et al. 1997), and the alignment finally manually corrected using GENEDOC (Nicholas & Nicholas Scanning electron microscopy (SEM) analysis of the 1997). The alignments were then exported as a NEXUS format endophytic isolates and a maximum parsimony analysis was performed in PAUP* 4.0 b 10 (Swofford 2003), using the heuristic search option For SEM analysis, the fungal strains were grown on PDA me- with tree bisection-reconnection (TBR) branch swapping; sta- dium (10 d, darkness, 25 C). The peripheral front of the radial bility of clades was tested using 1000 bootstrap replications. cultures was then carefully removed with a scalpel. SEM was Gaps were coded as missing data. Sequences newly performed using cryo-SEM (HITACHI S-3000N microscope, obtained during this work were deposited in NCBI GenBank M. fengyangensis sp. nov. from southeast China 799

(HM034839eHM034865, EU636700, GQ220337, GQ241929, (5 % phenyl methyl siloxane: 30 m 0.25 mm, 0.25 mm film FJ480346). thickness) was used for the separation of the volatiles, using helium as the carrier gas. The fiber was conditioned at 240 C Calculation of genetic distances for 20 min in the GC under a stream of helium before extract- ing the volatiles. The SPME needle was inserted into the GC in- We used the p-distance, implemented in MEGA 4.0 (Tamura jection port and the sample was then desorbed from the fiber et al. 2007), to determine of genetic relatedness of isolates. for 30 s. After conditioning was completed, the fiber was The p-distance is the proportion (p) of nucleotide sites at retracted and the needle removed from the infection port. which two sequences being compared are different, and is The column was programmed as follows: Initial temperature obtained by dividing the number of nucleotide differences 30 C for 3 min, then increasing to 220 Cat5Cmin 1, post by the total number of nucleotides compared. Gaps were run at 270 C for 1 min. The mass spectrum was scanned at treated as missing characters, and Maximum Composite Like- a rate of 3.35 scans/s over a mass range of 20e450 a.m.u. Iden- lihood was chosen as a model for the analysis. tification of the volatile compounds was performed by com- Nucleotide properties (variable sites h, haplotypes, nucleo- parison with a chemical compound library using the NIST05 tide diversity p, evolutionary models) were calculated with database on the mass spectrometer. Controls, using plates DNASp 5 (Librado & Rozas 2009). without the fungus, were also analyzed and the corresponding compounds withdrawn from the analysis. Antimicrobial activity of volatile compounds

The antimicrobial action of the volatile compounds (VOCs) Results produced by the Muscodor strains was tested as described by Strobel et al. (2001). For testing the antimicrobial action, Botry- Identification of the endophytic isolates as new species of tis cinerea (strain ZJUP10; ZJUP numbers refer to the collection Muscodor of the Fungal Laboratory of Zhejiang University), Aspergillus clavatus (strain ZJUP204), Colletotrichum fragariae (strain We isolated two (ZJLQ023, ZJLQ024), two (ZJLQ151 and ZJUP45), Didymella bryoniae (strain ZJUP11), Fusarium oxysporum ZJLQ374) and one strain (ZJLQ070), respectively, in a total of (strain ZJUP28), Magnaporthe oryzae (Guy-11), Penicillum digita- four, 36 and 13 endophytic fungal isolates from Actinidia chi- tum (strain ZJUP42), Pythium ultimum (strain ZJUP22), Rhizocto- nensis, Pseudotaxus chienii and an unidentified broad leaf tree. nia solani (strain ZJUP05), Sclerotium rolfsii (strain ZJUP14), These five isolates grew essentially in the form of white myce- Verticillum dahliae (strain ZJUP218), Saccharomyces cerevisiae, lium on all media tested. Only Strain ZJLQ374 produced a yel- Escherichia coli and Phyllobacterium sp. were used. To this end, low pigment. All the cultures emitted a musty odor. These a disk (5 mm diameter) of the growing front of Muscodor myce- phenotypic characteristics therefore suggested that the five lia was excised and inoculated on PDA on one section of a two- isolates could belong to the genus Muscodor. section Petri dish. After incubation at 25 C, a disk of the grow- To test whether these strains may indeed belong to Musco- ing front of the test microorganisms (5 mm diameter) was in- dor and represent one or more species, we used gene sequence oculated onto PDA on another section of the Petri dish. The analysis of the internal transcribed spacers ITS1 and 2 and the Petri dishes were then wrapped with two layers of Parafilm 5.8 rRNA, the D1 and D2 fragment of 28S rRNA, a fragment and incubated at 25 C. The increase in diameter of the grow- of the RNA-polymerase subunit B (rpb2) and a fragment of b- ing colonies was measured at least three times for a period of tubulin (tub1). The number of variable sites, nt diversity, and 3 d. After the experiment, the microorganisms were placed number of haplotypes of these loci are shown in Table 1. Fu onto new PDA plates to evaluate their viability. Controls in- & Li’s (1993) D* and F* statistics, and Tajimas test (Tajima cluded the test microorganisms, subjected to the same growth 1989) revealed that all these loci evolved neutral and were conditions, but in the absence of Muscodor. thus suitable for the phylogenetic analysis. Although these four genes grouped the five isolates into 3e5 haplotypes, the Solid phase microextraction/Gas chromatograph/Mass nucleotide variation was low (0.5 %, 2.7 %, 1.6 %, 4.5 % to 28S, spectra (SPME/GC/MS) analysis of volatile compounds ITS, rpb2, tub1, respectively; cf. Table 1). This variation is in the range of intraspecific variation in other fungal genera The chemical nature of the volatile compounds produced by such as Hypocrea, Gibberella or Neurospora (Druzhinina et al. the five Muscodor strains was investigated by SPME/GC/MS as 2005; Dettman et al. 2003; O’Donnell et al. 2009). We therefore described by Strobel et al. (2001), and compared to a reference considered the five isolates of Muscodor to be a single taxon. isolate 9-6 of Muscodor albus. The VOCs by the mycelia of Mus- To identify the relationship of this putative taxon to other codor growing on PDA in Petri dishes were extracted with Muscodor spp., we submitted the sequences of the four genes a SPME syringe (SUPELCO, USA), 50/30 mm divinylbenzene/car- from all five isolates to a BLAST similarity search. Unfortu- boxen/polydimethylsiloxane (DVB/CAR/PDMS) on StableFlex nately, Muscodor spp. were represented in the database only fiber. A needle was passed through a small drilled hole in the by their ITS1e5.8S rRNAeITS2 sequences, and no sequence in- side of the Petri plate and the fiber was then exposed to the formation was available for the other loci. Best hits for the for- air space above the mycelium for 45 min. Volatile compounds mer (negative probability values (E) ¼ 0.0) were obtained with were analyzed by GC equipped with a mass detector (Agilent 21 isolates of Muscodor, a single unknown endophytic isolates, 6890N/5975B) and the data processed in an MSD ChemStation one unknown fungal species, one unknown species from the software G1701DA (Agilent, USA). A HP-5MS capillary column Xylariaceae and one uncultured Sordariomycete clone 800 C.-L. Zhang et al.

Table 1 e Nucleotide properties of the four loci to the ﬁve new strains in this study. Loci 28S ITS1 þ 5.8S þ ITS2 rpb2 tub1

No. of sites 742 565 878 1660 h (Variable sites) 4 15 14 74 No. of haplotypes 3 5 5 5

Haplotype Hap_1: 2 [ZJLQ023 ZJLQ070] Hap_1: 1 [ZJLQ023] Hap_1: 1 [ZJLQ023] Hap_1: 1 [ZJLQ151] Hap_2: 2 [ZJLQ024 ZJLQ374] Hap_2: 1 [ZJLQ024] Hap_2: 1 [ZJLQ024] Hap_2: 1 [ZJLQ374] Hap_3: 1 [ZJLQ151] Hap_3: 1 [ZJLQ070] Hap_3: 1 [ZJLQ070] Hap_3: 1 [ZJLQ070] Hap_4: 1 [ZJLQ151] Hap_4: 1 [ZJLQ151] Hap_4: 1 [ZJLQ024] Hap_5: 1 [ZJLQ374] Hap_5: 1 [ZJLQ374] Hap_5: 1 [ZJLQ023]

Haplotype diversity 0.800 0.164 1.000 0.126 1.000 0.126 1.000 0.126 p (nt diversity) 0.00270 0.00075 0.01352 0.00255 0.00740 0.00123 0.01971 0.00436 Tajima’s D n.s.a n.s. n.s. n.s. Fu and Li’s D* n.s. n.s. n.s. n.s. Fu and Li’s F* n.s. n.s. n.s. n.s.

a Not signiﬁcant.

(Table 2). Maximum parsimony analysis of the ITS1e5.8S Muscodor sp.1 isolate S18-3-1. The results, shown in Fig 2,docu- rRNAeITS2 sequences resulted in a well resolved tree which ment that the five new isolates from this study displayed a high- displayed two major, highly supported clades A and B (Fig 1): est growth rate at 25 C, the colony diameter is 23e28 mm and clade A forming two subclades, one (subclade I) contained 26e35 mm for 15 d in MEA and PDA, respectively. The growth all known species of Muscodor and 11 fungal endophytes; rates decreased significantly when the temperature rose above and a second one (subclade II) containing all the isolates 25 C, and they did not grow above 30 C. M. albus and Muscodor from this study, one uncultured Sordariomycete clone, one sp.1, in contrast, displayed a slightly higher optimum tempera- unknown fungal sp. and several unknown Xylariaceae spp. ture than the five new isolates (25e28 C). Also their colony di- These data suggest that Muscodor is monophyletic, but also in- ameter was larger, and they were able to grow at 30 C. dicate the presence of infrageneric clusters. To confirm this position of the new isolates within the Antagonistic properties of the novel Muscodor species known Muscodor spp., we sequenced rpb2, tub1 and 28S rRNA from two isolates of Muscodor albus and one Muscodor sp.1 All five isolates exhibited volatile antimicrobial activity, al- (¼the only living cultures of Muscodor that were available to though to a varying degree, ZJLQ023, ZJLQ024 and ZJLQ070 being us for this study) and all of the five isolates of the putative most effective (Table 4). Most of the tested fungi were com- new taxon. Phylogenetic analysis of these three additional pletely inhibited and killed by the VOCs produced by these three loci confirmed the monophyly of Muscodor, but also consis- isolates. Only Fusarium oxysporum was partially resistant to the tently separated of M. albus and Muscodor sp.1 from the five volatile compounds from ZJLQ023, ZILQ024 and ZJLQ070, and isolates from this study (Supplementary Figs 1e3). We also Penicillum digitatum to ZJLQ023 and ZJLQ070, respectively. noted that this analysis further split the five Muscodor spp. Strains ZJLQ151 and ZJLQ374 showed the weakest antimicrobial into 2e3 branches, but they were supported by low bootstrap activity, leading to partial resistance in all of the fungi tested. values only and the occurrence of strains in these clades was inconsistent between different loci. We therefore conclude Chemistry of the volatile components of the novel Muscodor that these five isolates belong to the same species. species Data consistent with the phylogenetic analysis were also obtained when the p-distance proportion of nucleotide sites SPME/GC/MS analysis revealed a strikingly different pattern of among the sequences of the four loci was compared between VOCs of the five Muscodor isolates when compared with Mus- the five isolates and other Muscodor spp. (Table 3): the p-value codor albus 9-6 (Table 5). Naphthalene derivatives with a Mr for the present isolates was always 10- to 20-fold lower than of 204 were the most abundant components in the volatile that between these isolates and Muscodor spp., and e in the mixture. Apart of the naphthalene derivatives, propanoic case of ITS where sequences for all named Muscodor spp. acid, its 2-methyl-, and methyl ester and 2-methyl-propanoic were available e was in the same range. acid were present in the five new isolates as well as in M albus. The new strains accumulated a-phellandrene, ß-phellan- Temperature-dependence of growth of the novel Muscodor drene and 2-cyclohexen derivatives that were not produced species in M. albus, whereas they lacked formation of 4-nonanone and 2-nonanone produced by the latter. Temperature-dependence of growth has become a sensitive physiological means to differentiate between species (Chaverri Taxonomic description of Muscodor fengyangensis sp. nov. & Samuels 2004). We therefore tested the growth of the five new Muscodor isolates from this study at eight different temper- Based on the data reported above, we conclude that the five atures, and compared it to Muscodor albus isolate 9-6 and isolates from this study represent a new taxon of Muscodor, M. fengyangensis sp. nov. from southeast China 801

Table 2 e ITS1e5.8S rDNAeITS2 sequences of Muscodor related species used in this study. No. GenBank Species Strain Origin Host Reference accession

1 EU686811 Fungal endophyte 1157 Unknown Rhipidocladum Unpublished racemiflorum 2 EU686808 Fungal endophyte 1138 Unknown Streptochaeta spicata Unpublished 3 AY100022 Muscodor vitigenus P15 Lake Sandoval of Peru Paullinia paullinioides Daisy et al. (2002a, 2002b) 4 EU687018 Fungal endophyte 2067 Unknown Panicum pilosum Unpublished 5 EF564149 Muscodor sp. M7 South of Thailand Garcinia sp. Phongpaichit et al. (2007) 6 EF564150 Muscodor sp. N28 South of Thailand Garcinia sp. Phongpaichit et al. (2007) 7 EF564148 Muscodor sp. D31 South of Thailand Garcinia sp. Phongpaichit et al. (2007) 8 FJ664551 Muscodor sp. WG-2009a Malaysia Unknown Unpublished 9 EU687035 Fungal endophyte 2161 Unknown Panicum pilosum Unpublished 10 FJ917287 M. yucatanensis MEXU Yucatan Peninsula of Bursera simaruba Gonza´lez et al. (2009) 25511 Mexico 11 EU686807 Fungal endophyte 1128 Unknown Chusquea simpliciflora Unpublished 12 EU686810 Fungal endophyte 1155 Unknown Rhipidocladum Unpublished racemiflorum 13 EU636700 Muscodor sp.1 S18-3-1 Yunnan Province of Wild rice Unpublished Southwest China (Oryza granulata) 14 EU686946 Fungal endophyte 1730 Unknown Oplismenus hirtellus Unpublished 15 EU686949 Fungal endophyte 1744 Unknown Olyra latifolia Unpublished 16 HM034852 M. fengyangensis ZJLQ151 Zhejiang Province of Pseudotaxus chienii This study Southeast China 17 HM034853 M. fengyangensis ZJLQ070 Zhejiang Province of Unknown broad This study Southeast China leaf tree 18 HM034854 M. fengyangensis ZJLQ374 Zhejiang Province of Pseudotaxus chienii This study Southeast China 19 HM034855 M. fengyangensis ZJLQ024 Zhejiang Province of Actinidia chinensis This study Southeast China 20 HM034856 M. fengyangensis ZJLQ023 Zhejiang Province of Actinidia chinensis This study Southeast China 21 AY699660 Fungal sp. R15 Queensland, Australia Rhododendron lochiae Bougoure & Cairney (2005) 22 DQ273343 Uncultured D11 California, USA Lithocarpus densiflorus Bergemann & sordariomycete clone Garbelotto (2006) 23 AM921731 Xylariaceae sp. IZ-1249 Unknown Ammophila arenaria Unpublished 24 EU686114 Fungal endophyte sp. ECD-2008 New Zealand Lepidozia sp. Davis & Shaw (2008) 25 EU977208 Fungal endophyte sp. P913A Border of Peru and Bolivia Hibiscus adscensionis Smith et al. (2008) 26 EF183509 M. albus E-6 Guayaquil, Ecuador Guazuma ulmifolia Strobel et al. (2007) 27 EU195297 M. crispans B-23 Bolivian Amazon Basin Ananas ananassoides Mitchell et al. (2008) 28 GQ220337 M. albus S13-1-2 Yunnan Province of Wild rice Unpublished Southwest China (Oryza granulata) 29 AY244622 M. albus MFC2 Thailand Myristica fragrans Sopalun et al. (2003) 30 AY927993 M. albus I41-3s West Sumatra, Indonesia Unidentified small vine Atmosukarto et al. (2005) 31 AF324336 M. albus CZ-620 Northern Territory of Cinnamomum zeylanicum Worapong et al. (2001) Australia 32 HM034857 M. albus 9-6 / / Reference strain send by Gary Strobel 33 AY034665 M. roseus A3-5 Northern territory of Australia Grevillea pteridifolia Worapong et al. (2002) 34 EU977281 Fungal endophyte sp. P1907B Border of Peru and Bolivia Vernonanthura Smith et al. (2008) membranacea 35 AY527045 M. albus TP 21 Northern Territory of Australia Terminalia prostrata Ezra et al. (2004) 36 AY527044 M. albus KN 26 Northern Territory of Australia Kennedia nigriscans Ezra et al. (2004) 37 AY527046 M. albus KN 27 Northern Territory of Australia Kennedia nigriscans Ezra et al. (2004) 38 AY527048 M. albus GP 206 Northern Territory of Australia Grevillea pteridifolia Ezra et al. (2004) 39 AY527047 M. albus GP 115 Northern Territory of Australia Grevillea pteridifolia Ezra et al. (2004) 40 AY555731 M. albus GP 100 Northern Territory of Australia Grevillea pteridifolia Ezra et al. (2004)

M. fengyangensis Zhang CL, anam. sp. nov., which is herewith Hyphae hyalinae, septa, ramosa, tenuitunicata 1.0e1.9 mm. formally described: Mycelium ﬁla funalia 15e20 mm latae formans. Conidia vel Muscodor fengyangensis C.L. Zhang, sp. nov. Fig 3. structurae sporulationis non observa. Fungus ﬁlamentosus. Incrementum optimum ad 25 C, in Colonies (Fig 3) slowly growing on PDA and MEA, the colony agaro MEA 23e28 mm diam, in agaro PDA 26e35 mm post diameter 23e28 mm on MEA, 26e35 mm on PDA 15 d at the op- 15 d. Coloniae albae vel roseae, odoratae proprie mucide. timum growth temperature 25 C. The fungal colonies are 802 C.-L. Zhang et al.

Fig 1 e Maximum parsimony tree of Muscodor spp. based on ITS1e5.8S rDNAeITS2 sequence. Bootstrap values >50 % are indicated above branch nodes. Tree length [ 611; Consistency index (CI) [ 0.74; Homoplasy index (HI) [ 0.26; Retention index (RI) [ 0.87; Hypocrea lixii was used as outgroup.

whitish, except for ZJLQ374 with pink colour. All fungal strains unidentiﬁed broad leaf tree. Known from Zhejiang Province, produce a striking sour odor. Pigment not present in tested me- Southeast of China. dia, except in ZJLQ374 (brown pigment). The aerial mycelium Holotype: China, Fengyangshan Nature Reserve, comes from occurring on MEA is denser than on PDA. Hyphae hyaline, sep- leaves tissues of wild plants in May, 2008 by CL Zhang; Lyophi- tate, branched, thin-walled, 1.0e1.9 mm. Mycelium frequently lized cultures ZJLQ023, ZJLQ024 and ZJLQ070 are deposited in intertwining, forming rope-like strands (Fig 3), 15e20 mm coiled China General Microbiological Culture Collection Center structure occasionally observed. No conidia and sporulation (CGMCC, accession numbers: 2862, 2863, 2864) and State Key Lab- structure were observed under laboratory conditions. oratory for Rice Biology, Institute of Biotechnology, Zhejiang Uni- Habitat and distribution: Endophytic in healthy stems and versity. And lyophilized culture of ZJLQ070 was also deposited at leaves of Actinidia chinensis, Pseudotaxus chienii and an Centraalbureau voor Schimmelcultures (CBS 126601). The M. fengyangensis sp. nov. from southeast China 803

Table 3 e p-Distance of nucleotide sites among the four loci sequences compared between Muscodor related sequences. This distance is the proportion of nucleotide sites at which two sequences being compared are different. It is obtained by dividing the number of nucleotide differences by the total number of nucleotides compared. ITS sequence [1e7]a [8e15] [25e40] [24] [16e20] [21e23]

[1e7] 0.000e0.004 [8e15] 0.017e0.027 0.000e0.015 [25e40] 0.036e0.044 0.032e0.042 0.000e0.013 [24] 0.042e0.044 0.045e0.049 0.042e0.049 [16e20] 0.079e0.091 0.071e0.088 0.081e0.097 0.069e0.078 0.000e0.007 [21e23] 0.092e0.096 0.084e0.093 0.085e0.100 0.073e0.083 0.035e0.047 0.010e0.020 ZJLQ070 ZJLQ151 ZJLQ023 ZJLQ374 ZJLQ024 S18-3-1 M. albus 9-6 M. albus S13-1-2

28S sequence ZJLQ070 ZJLQ151 0.003 ZJLQ023 0.000 0.003 ZJLQ374 0.003 0.005 0.003 ZJLQ024 0.003 0.005 0.003 0.000 S18-3-1 0.017 0.017 0.018 0.014 0.014 M. albus 9-6 0.015 0.015 0.015 0.012 0.012 0.010 M. albus S13-1-2 0.018 0.018 0.018 0.015 0.015 0.001 0.011

rpb2 sequence ZJLQ070 ZJLQ151 0.003 ZJLQ023 0.007 0.007 ZJLQ374 0.007 0.007 0.007 ZJLQ024 0.008 0.010 0.008 0.010 S18-3-1 0.196 0.196 0.197 0.201 0.189 M. albus 9-6 0.235 0.235 0.240 0.240 0.232 0.177 M. albus S13-1-2 0.233 0.233 0.239 0.238 0.230 0.176 0.001

tub1 sequence ZJLQ070 ZJLQ151 0.016 ZJLQ023 0.034 0.020 ZJLQ374 0.017 0.012 0.024 ZJLQ024 0.019 0.006 0.023 0.016 S18-3-1 0.169 0.170 0.173 0.177 0.172 M. albus 9-6 0.167 0.165 0.169 0.172 0.168 0.115 M. albus S13-1-2 0.166 0.164 0.167 0.170 0.166 0.117 0.018

a Numbers in brackets refer to ITS sequence number indicated in Table 2. Nos. 16e20 representing the ﬁve new strains of this study.

Fig 2 e The curve of growth temperature: - Muscodor albus 9-6 in PDA, , Muscodor albus 9-6 in MEA; : Muscodor sp.1 S18-3-1 in PDA, 6 Muscodor sp.1 S18-3-1 in MEA; C Muscodor fengyangensis in PDA, B Muscodor fengyangensis in MEA. For Muscodor fengyangensis, here is the average temperature of ﬁve isolates. 804 C.-L. Zhang et al.

Table 4 e Effects of the volatile compounds of the novel Muscodor species on a group of the test microorganisms. Test microorganisms Growth after 3 d exposure to Muscodor (% vs control) Viability after 3 d exposure to Muscodor culture

ZJLQ023 ZJLQ024 ZJLQ070 ZJLQ151 ZJLQ374 ZJLQ023 ZJLQ024 ZJLQ070 ZJLQ151 ZJLQ374

Botrytis cinerea 0 0 0 50.0 3.1 % 61.1 2.8 % Dead Dead Dead Alive Alive Aspergillus clavatus 0 0 0 16.4 0.9 % 27.5 1.6 % Dead Dead Dead Alive Alive Colletotrichum fragariae 0 0 0 21.3 2.7 % 46.9 2.4 % Dead Dead Dead Alive Alive Didymella bryoniae 0 0 0 74.6 1.6 % 49.8 0.9 % Dead Dead Dead Alive Alive Fusarium oxysporum 64.0 2.0 % 73.3 1.2 % 73.0 1.0 % 83.2 2.9 % 93.1 1.7 % Alive Alive Alive Alive Alive Magnaporthe oryzae 0 0 0 47.3 1.9 % 56.1 2.5 % Dead Dead Dead Alive Alive Pythium ultimum 0 0 0 74.7 2.0 % 46.7 3.4 % Dead Dead Dead Alive Alive Rhizoctonia solani 0 0 0 70.2 2.1 % 25.0 3.6 % Dead Dead Dead Alive Alive Sclerotium rolfsii 0 0 0 19.4 2.9 % 27.8 1.7 % Dead Dead Dead Alive Alive Verticillum dahliae 0 0 0 74.6 2.1 % 63.7 1.6 % Dead Dead Dead Alive Alive Penicillum digitatum 66.7 3.4 % 0 28.9 3.8 % 81.2 2.4 % 83.1 1.7 % Alive Dead Alive Alive Alive Saccharomyces cerevisiae 0 0 0 NT NT Dead Dead Dead NT NT Escherichia coli 0 0 0 NT NT Dead Dead Dead NT NT Phyllobacterium sp. 0 0 0 NT NT Dead Dead Dead NT NT

Note: Tests were repeated three times and means SD were calculated. “NT” means not tested.

accession number of GenBank database for 28S, ITS, rpb2, tub1 se- previously known taxa of Muscodor requires a re-evaluation quences were HM034858eHM034862, HM034852eHM034856, by an integrated multilocus approach. HM034847eHM034851, HM034839eHM034843, respectively. In this study we also attempted to introduce additional tools for species differentiation, such as e.g. BIOLOG pheno- type arrays, but due to lack of spores (for inoculum prepa- Discussion ration) and the very slow growth rate of mycelia this method did not provide reproducible results. Similarly, Distinguishing between species e and consequently the de- the application of matrix-assisted laser desorption/ioniza- tection of new species e in the genus Muscodor by morphology tion (MALDI) for strain differentiation (Dong et al. 2009)is is difficult, because this genus lacks conidia and conidio- not possible without formation of spores. However, the phores, and colony and mycelial characters are the only avail- use of temperature-dependent growth rates on plates, as able morphological traits for this purpose. Therefore, advocated by Chaverri & Samuels (2004) for Trichoderma/ physiological or molecular characters (such as production of Hypocrea was successful in differentiating between M. fen- VOCs) are essential complimentary tools for this process. In gyangensis and M. albus and thus introduced a new crite- this paper we have applied a multiphasic approach to identify rium into Muscodor taxonomy. We therefore recommend a new species e Muscodor fengyangensis sp. nov. e of this ge- that this trait is further used in species discrimination in nus. It has recently been suggested, based on gene sequence Muscodor in the future. analysis, that Muscodor is actually a member of the Xylarioi- M. fengyangensis sp. nov., as M. albus (Strobel et al. 2007) deae, i.e. Nemania and Xylaria (Tang et al. 2009). In this study, and M. crispans (Mitchell et al. 2010) produce a mixture of we used multilocus gene sequence analysis and evaluated VOCs that collectively act to kill other microbes and even the phylogenetic data in accordance with the genealogical lower eukaryotes. Strobel and coworkers (Mitchell et al. concordance phylogenetic species recognition (GCPSR) con- 2010) speculated that M. albus lives in its host in a symbiotic cept (Taylor et al. 2000). Thereby, we used the criteria of condition, providing protection from pathogens while sur- Dettman et al. (2003) (i.e. that a clade is an independent evolu- viving and growing on plant nutrients. While this hypothesis tionary lineage [and therefore a phylogenetic species] if it is is attractive, it has e to the best of our knowledge e not yet supported in at least one gene tree and not contradicted in been experimentally tested. Production of VOCs, which any of the others). By this means, we confirmed that Muscodor likely are released into the surrounding atmosphere of the is a member of the Xylarioideae but forms a distinct phyloge- plant, could be superior for protection because they would netic clade within them. GCPSR also clearly shows that the antagonize potential pathogens already before they enter five new isolates from Fengyangshan Nature Reserve of China the plant. In this regards, it is intriguing that different Mus- represent a new species of Muscodor because they consistently codor spp. reported so far produce a unique pattern of vola- formed a strongly supported a sister clade to Muscodor albus tile compounds, whereas their action spectrum is largely and other Muscodor spp. in all gene trees, whereas no consis- similar. We consider it less likely that this is the result of tent differentiation within them was possible. We also note presence of different genes, but rather speculate that the en- that M. albus, M. roseus and M. crispans were distinguished zymes involved in VOC formation have a relaxed substrate only by very few nucleotide differences, which were in the specificity and the actual substrate used depends on the in- same range as those encountered within the five new isolates tracellular available precurser pool concentration. This could of M. fengyangensis. Given the importance of sequence based easily be tested by cultivating Muscodor spp. under different taxonomy in this genus, the species concept of these conditions and comparing the profile of produced VOCs. M. fengyangensis sp. nov. from southeast China 805

Table 5 e Comparison of the volatile compounds produced by Muscodor species through SPME/GC/MS analysis.

RT Possible compound Mr Total area (%) (min:s) ZJLQ023 ZJLQ024 ZJLQ070 ZJLQ151 ZJLQ374 M. albus 9-6

3.286 *Propanoic acid, 2-methyl-, methyl ester 102 10.18 0.70 8.02 4.71 5.12 4.39 4.510 1-Butanol, 3-methyl- 88 0.58 / / 0.62 3.01 0.47 4.634 1-Butanol, 2-methyl- 88 / / / / 5.11 5.316 *Propanoic acid, 2-methyl-, ethyl ester 116 / / / / / 1.97 5.763 Acetic acid, 2-methylpropyl ester 116 / / / / 14.61 7.86 7.092 *Propanoic acid, 2-methyl- 88 60.42 28.26 77.08 50.76 45.67 29.12 9.241 1-Butanol, 2-methyl-, acetate 130 / / / / 1.75 3.78 9.326 *1-Butanol, 3-methyl-, acetate 130 / / / / 3.39 18.53 13.515 {.alpha.-Phellandrene 136 0.32 2.58 0.36 0.38 0.42 / 13.843 Propanoic acid, 2-methyl-, 2-methylbutyl ester 158 / / / / / 0.75 13.928 Cyclohexene,1-methyl-4-(1-methylethylidene)- 136 / 1.05 / / / / 13.958 *Propanoic acid, 2-methyl-, 3-methylbutyl ester 158 / / / / / 0.70 14.321 {.beta.-Phellandrene 136 5.88 44.25 4.37 5.45 3.56 / 15.759 *4-Nonanone 142 / / / / / 2.45 16.256 Cyclohexene, 1-methyl-4-(1-methylethylidene)- 136 / 0.52 / / / / 16.366 *2-Nonanone 142 / / / / / 1.02 16.998 *Phenylethyl alcohol 122 0.11 / / 0.11 0.52 1.25 17.266 {2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, trans- 154 0.22 0.52 0.13 0.10 0.08 / 17.824 {2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, cis- 154 0.10 0.36 0.10 0.05 / / 19.500 {2-Cyclohexen-1-ol, 3-methyl-6-(1-methylethyl)-, trans- 154 0.29 3.91 0.32 0.27 0.58 / 19.854 {2-Cyclohexen-1-ol, 3-methyl-6-(1-methylethyl)-, cis- 154 0.06 0.24 0.04 / / / 21.187 {2-Cyclohexen-1-one, 3-methyl-6-(1-methylethyl)- 152 / 0.50 / / / / 21.242 *Acetic acid, 2-phenylethyl ester 164 / / / / 0.60 2.79 21.520 3,5-Dimethoxytoluene 152 0.13 / 0.54 0.13 0.18 / 23.883 Caryophyllene-[I1] 204 0.92 / 0.42 1.69 0.69 0.31 23.938 1,4-Methanoazulene,decahydro-4,8,8-trimethyl-9- 204 0.39 / 0.27 1.20 0.58 0.11 methylene,[1S-(1.alpha.,3a.alpha.,4.alpha.,8a.alpha)]- 24.386 Bicyclo[5.2.0]nonane,2-methylene-4,8,8-trimethyl-4-vinyl- 204 0.50 / / 1.05 0.41 / 24.545 1,1,4a-trimethyl-5,6-dimethylenedecahydro naphthalene 204 / / / / 0.67 / 24.958 Cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, 204 2.04 2.23 0.67 2.99 1.97 0.71 [1S-(1.alpha.,2.beta.,4.beta.)]- 25.690 Caryophyllene 204 0.38 0.12 0.11 0.62 0.86 0.18 26.058 Bicyclo[3.1.1]hept-2-ene, 2,6-dimethyl-6-(4-methyl- 204 0.95 0.15 0.07 6.88 1.12 0.65 3-pentenyl)- 26.147 *Azulene, 1,2,3,4,5,6,7,8-octahydro-1,4-dimethyl-7- 204 3.35 2.27 0.84 6.18 2.17 0.63 (1-methylethenyl)-, [1S-(1.alpha.,4.alpha.,7.alpha.)]- 27.103 *Naphthalene, 1,2,4a,5,6,8a-hexahydro-4,7-dimethyl-1- 204 / / / / / 0.28 (1-methylethyl)-, [1R-(1.alpha.,4a.alpha.,8a.alpha.)] 27.212 Naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-4a,8-dimethyl-2- 204 / 0.52 0.26 / / / (1-methylethyl)-, [2R-(2.alpha.,4a.alpha.,8a.beta.)] 27.331 Spiro[5,5]undec-2-ene,3,7,7-trimethyl-11-methylene-,(-)- 204 3.64 / / / / / 27.431 Azulene, 1,2,3,3a,4,5,6,7-octahydro-1,4-dimethyl-7- 204 0.25 0.24 / 0.42 / 0.49 (1-methylethenyl)-, [1R-(1.alpha.,4.alpha.,7.alpha.)]- 27.540 *Naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7- 204 0.19 / / / / 0.47 (1-methylethenyl)-, [1S-(1.alpha.,7.alpha.,8a.alpha.)]- 27.670 Naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-4a,8-dimethyl-2- 204 0.40 0.32 / 0.70 0.39 0.35 (1-methylethenyl)-, [2R-(2.alpha.,4a.alpha.,8a.beta.)]- 27.839 *Azulene, 1,2,3,5,6,7,8,8a-octahydro-1,4-dimethyl-7- 204 / 0.60 0.19 0.78 1.00 1.40 (1-methylethenyl)-, [1S-(1.alpha.,7.alpha.,8a.beta.)]- (common name: valencene) 27.859 Cyclohexene,1-methyl-4-[5-methyl-1-methylene- 204 1.00 / / / / / 4-hexenyl)-,[S]- 27.899 1H-Cycloprop[e]azulene, 1a,2,3,5,6,7,7a,7b-octahydro- 204 / / / 0.28 / / 1,1,4,7-tetramethyl-7-(1-methylethenyl)-, [1aR- (1a.alpha.,7.alpha.,7a.beta.,7b.alpha)]- 28.356 Isoaromadendrene epoxide 220 0.14 / / / 0.33 / 28.670 cis-alpha-Bisabolene 204 0.28 / / / 0.27 / 29.326 Diepicedrene-1-oxide 220 0.14 / / 0.11 0.18 / 29.426 Isoaromadendrene epoxide 220 0.21 29.705 1,4-Methanoazulen-3-ol,decahydro-1,5,5,8a-tetramethyl-, 222 0.29 [1S-1.alpha.,3.alpha.,3a.alpha.,4.alpha.,8a.alpha.)]

(continued on next page) 806 C.-L. Zhang et al.

Table 5 e (continued)

RT Possible compound Mr Total area (%) (min:s) ZJLQ023 ZJLQ024 ZJLQ070 ZJLQ151 ZJLQ374 M. albus 9-6

31.297 *Unknown 204 6.93 10.46 6.02 13.98 9.05 13.80 31.849 Tricyclo[6.3.0.0(2,4)]undec-8-ene, 3,3,7,11-tetramethyl 204 0.15 0.16 0.06 0.22 0.15 0.11 33.426 Caryophyllene oxide 220 0.20 0.23 0.08

Note: Several minor peaks, making up less than 0.6 % of the total detectable VOCs were deleted from the table. Compounds found in the control PDA plate are not included. Symbol * denoted that this component was observed in Strobel et al. (2001) and symbol { denoted that these components were observed in the novel Muscodor species, but not produced in M. albus.

Fig 3 e Colony morphology and scanning electron micrograph of the mycelium morphology of the ﬁve Muscodor fengyangensis isolates. M. fengyangensis sp. nov. from southeast China 807

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