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Morphological and molecular evidence support a new endophytic fungus, Chaetomella endophytica from Japan
Mycoscience xxx (2018) 1e6
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Morphological and molecular evidence support a new endophytic fungus, Chaetomella endophytica from Japan Nakarin Suwannarach a, Jaturong Kumla a, Kenji Matsui b, Saisamorn Lumyong a, c, * a
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi, 753-8515, Japan c Center of Excellence on Bioresources for Agriculture, Industry and Medicine, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2017 Received in revised form 17 April 2018 Accepted 21 May 2018 Available online xxx
A new endophytic fungus, described herein as Chaetomella endophytica sp. nov., was isolated from stems of Rosa arvensis in Yamaguchi Prefecture, Japan. Morphological investigations revealed that its conidia are shorter and narrower than other known Chaetomella species. Phylogenetic analysis of three combined loci (large subunit, internal transcribed spacer, and small subunit regions of ribosomal DNA) confirmed that it is a new species within the family Chaetomellaceae. A full description, illustrations and a phylogenetic tree showing the position of C. endophytica are provided. © 2018 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Keywords: Chaetomellaceae Fungal endophyte Morphology Phylogeny
The genus Chaetomella was described by Fuckel (1870) with C. oblonga Fuckel as the type species. This genus belongs to the €rtel, order Helotiales, family Chaetomellaceae (Baral, 2015; Pa ~ldmaa1, 2017). Chaetomella species have been Baral, Tamm, & Po described as plant pathogens, and as saprophytes growing on litter, dead logs and soil distributed in both temperate and trop€rtel et al., 2017). Kirk, ical regions (Baral, 2015; Fuckel, 1870; Pa Cannon, Minter, and Stalpers (2008) suggested that there are five species of Chaetomella, but Index Fungorum (http://www. indexfungorum.org/Names/Names.asp, January 2018) lists 35 species. The statistics presented in Index Fungorum may include synonyms and misidentifications, and some species may not have been well documented. Traditional identification of microfungi is based on morphological characteristics, but morphological variations associated with environmental and cultural conditions, as well as the developmental stage of the fungi, may make morphological identification difficult among closely related genera and species (Baschien, Tsui, Gulis, Szewzyk, & Marvanova, 2013; Kohout et al., 2012). Therefore, the development of
* Corresponding author. Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand. E-mail addresses: [email protected], [email protected] (S. Lumyong).
molecular identification has become a powerful tool for fungal €rtel et al., 2017; Wang et al., 2006). identification (Baral, 2015; Pa During our investigation of endophytic fungi on wild rose (Rosa arvensis L.) in Japan, we found an interesting species of Chaetomella; morphological observation and phylogenetic analysis revealed it as a new species. Stem samples of R. arvensis were collected from a natural forest in Yoshida, Yamaguchi City, Yamaguchi Prefecture, Japan in May 2016. The samples were taken to the laboratory and processed within 24 h. Samples were washed in running tap water for 15 min and then cut into 10 mm long pieces. All pieces were surface sterilized in 75% ethanol for 30 s, followed by 2% sodium hypochlorite for 3 min and 95% ethanol for 30 s under a laminar flow hood (Suwannarach et al., 2012). The sterilized samples were placed in Petri dishes containing potato dextrose agar (PDA), 0.05% streptomycin sulfate and 0.03% Rose Bengal. Petri dishes were sealed with Parafilm M and incubated at 25 C in darkness for one week. Fungi growing out from the samples were aseptically transferred to fresh PDA. We obtained fourteen fungal isolates in resulted and the isolate CMU300 was used in this study. The holotype specimen (dried culture) and ex-type living culture in this study were deposited in the Culture Collection of Sustainable Development of Biological Resources Laboratory (SDBR), Faculty of Science, Chiang Mai University, Chiang Mai, and Thailand Bioresource Research Center (TBRC), Pathum Thani, Thailand, respectively.
https://doi.org/10.1016/j.myc.2018.05.001 1340-3540/© 2018 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Suwannarach, N., et al., Morphological and molecular evidence support a new endophytic fungus, Chaetomella endophytica from Japan, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.05.001
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Chaetomella endophytica Suwannarach, Kumla, Matsui & Lumyong, sp. nov. Fig. 1. MycoBank no: 823701. Diagnosis: Distinguished from other species of Chaetomella, except C. raphigera, by its conidia being shorter and narrower, and from C. raphigera by having straight, rather than curved setae. Etymology: referring to the fungus being isolated as an endophyte. Holotype: JAPAN, Yamaguchi Prefecture, Yamaguchi City, Yoshida, 34 080 50.76}N, 131280 33.70}E, isolated as an endophyte from stems of Rosa arvensis, May 2016, N. Suwannarach, SDBR-CMU300 (dried culture); TBRC 7238 (ex-type living). Gene sequences (from holotype): MG406984 (LSU), MG406985 (ITS) and MG406986 (SSU). Fungal colonies on PDA, oatmeal agar (OA) and malt extract agar (MEA) reaching 75e80, 73e75, and 72e73 mm diam, respectively at 25 C after 1 wk (Fig. 1AC). Colonies on PDA and OA flat, sparse, with white to cream mycelium and reverse white to light orange. Colonies on MEA, flat, with white mycelium and reverse cream.
Mycelium superficial and immersed, hyphae branched, septate, hyaline, 2e3 mm wide. Conidiomata pycnidial or sporodochial were observed in all agar media after incubation at 25 C for 1 wk. Pycnidia 120e340 170e380 mm, subglobose to elongate or reniform, dark brown to black, with a short stalk of hyaline textura angularis cells, pycnidial wall with three regions: outer region hyaline, 7e10 mm thick, 1e2 cell layers; middle region dark brown to black, 7e10 mm thick, 2e4 cell layers; inner region pale yellow, 4e5 mm thick, 3e4 cell layers, cells more or less square at base of conidiomata, rectangular along sides and top (Fig. 1DeF). Setae 50e82.5 3.75e6.25 mm, tapering, pale to dark brown, becoming paler towards apex, thick-walled, but walls thinner towards apex, septate, straight with rounded end. Sporodochia 170e370 150e350 mm, pale yellow, setose on outer wall, setae 87.5e142.5 2.5e5 mm, similar in shape to setae on pycnidia; spodorochial wall similar in shape to middle region of the pycnidia; conidiophores, conidiogenous cells and conidia same in pycnidia and in sporodochia (Fig. 1G and H). Conidiophores up to 150 0.5e1 mm, hyaline, smooth, septate, cylindrical, branched (Fig. 1I). Conidiogenous cells terminal and lateral, monophialidic,
Fig. 1. Chaetomella endophytica SDBR-CMU300 (holotype). AeC: Colonies on different media after 1 wk at 25 C. A: Potato dextrose agar; B: Oatmeal agar; C: Malt extract agar. DeF: Pycnidia. G: Sporodochium with stalk. H. Setae on sporodochium. I: Conidiophores and conidiogenous cells. J: Conidia. Bars: AC 10 mm; D 100 mm; EG 50 mm; H, J 10 mm; I 5 mm.
Please cite this article in press as: Suwannarach, N., et al., Morphological and molecular evidence support a new endophytic fungus, Chaetomella endophytica from Japan, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.05.001
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subcylindrical, straight to curved, smooth, hyaline, 5e25 0.5e1.5 mm. Conidia 3e6 1e2 mm (n ¼ 50), hyaline, smooth, aseptate, cylindrical to cymbiform, guttulate (Fig. 1J). The conidia of C. endophytica and other Chaetomella species are compared (Table 1). The shorter conidia of C. endophytica clearly distinguishes it from most other Chaetomella species, with the exception of C. andropogonis Cooke & Ellis, C. brachyspora Sacc. & Speg., C. flavoviridis Torrend and C. raphigera Swift. However, the conidia of C. andropogonis (3.5e4 mm wide), C. brachyspora (3 mm wide) and C. flavoviridis (3.5e4 mm wide) are broader than those of C. endophytica (Cooke & Ellis, 1878; Saccardo, 1878; Swift, 1930; Torrend, 1913). The conidia of C. raphigera (4e8.1 1.4e3 mm) are within the range of those of C. endophytica (3e6 1e2 mm) (Rossman et al., 2004; Swift, 1930), but the setae of C. raphigera are curved or hooked at the apex, whereas they are straight in C. endophytica (Rossman et al., 2004; Swift, 1930). Genomic DNA was extracted from one wk-old fungal mycelia (1e5 mg) grown on MEA using a DNA Extraction Mini Kit (FAVORGEN, Taiwan) following the manufacturer's protocol. The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR) using ITS4 and ITS5 primers (White, Burns, Lee, & Taylor, 1990), under the following thermal conditions: 95 C for 2 min, 30 cycles of 95 C for 30 s, 50 C for 30 s, 72 C for 1 min, and 72 C for 10 min on a peqSTAR 2X Thermocycler (PEQLAB, UK). The large subunit (LSU) region was amplified with LROR and LR5 primers (Vilgalys & Hester, 1990) under the following thermal conditions: 94 C for 2 min, 30 cycles of 95 C for 30 s, 52 C for 30 s, 72 C for 1 min, and 72 C for 10 min. The small subunit (SSU) region was amplified using NS1 and NS4 primers (White et al., 1990) under the same conditions as the LSU amplification. PCR products were checked on 1% agarose
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gels stained with ethidium bromide under UV light and purified using NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany). The purified PCR products were directly sequenced by 1ST BASE Company (Kembangan, Malaysia) using the PCR primers mentioned above. The LSU, ITS and SSU sequences of C. endophytica were deposited in GenBank as numbers MG406984, MG406985 and MG406986, respectively. For the phylogenetic analysis, details of sequences from the present study and those from previous studies, obtained from the GenBank database, are provided (Table 2). The multiple sequence alignment was carried out using MUSCLE (Edgar, 2004), and the alignment of the combined LSU, ITS and SSU sequences was deposited in TreeBASE under the number 21793. A maximum likelihood (ML) phylogenetic tree was constructed using RAxML v7.0.3 (Stamatakis, 2006). The optimal ML tree search was conducted with 1000 separate runs, using the default algorithm of the program from a random starting tree for each run. The final tree was selected among suboptimal trees from each run by comparing likelihood scores under the GTRGAMMA nucleotide substitution model. The ML trees were viewed with TreeView32 (Page, 2001). Clades with bootstrap values (BS) 70% were considered significantly supported (Hillis & Bull, 1993). Bayesian phylogenetic analyses were carried out using the Metropolis-coupled Markov chain Monte Carlo (MCMCMC) method in MrBayes version 3.2 (Ronquist et al., 2012), under a GRT þ I þ G model. Markov chains were run for 1000000 generations, with six chains and random starting trees. The chains were sampled every 100 generations. Among these, the first 2000 trees were discarded as the burn-in phase of each analysis and the resulting trees were used to calculate Bayesian posterior probabilities. Bayesian posterior probabilities (PP) 0.95 were considered significantly supported (Alfaro, Zoller, & Lutzoni, 2003).
Table 1 Origin and conidia size of Chaetomella species. Chaetomella spices
Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella
acutiseta andropogonis artemisiae beticola brychyspora brassicae cinnamomea circinata circinoseta cycadina flavoviridis endophytica gasteriae helicotricha horrida indica longiseta lygei madeirensis oblonga ochracea raphigera raripila sacchari stevensonii tinosporae tortilis tritici viridescens viridi-olivacea zambiensis
Origin
Pakistan e South Africa Netherlands Italy e India e Brazil India e Japan Austria, Italy e Netherlands India France Southwest Europe e Austria,Canada, USA Italy Brazil, Congo, India, Pakistan, UK, USA Western Europe South Africa USA India France USA e e Zambia
Conidia size (mm)
Reference
Length
Wide
7.5e10 5e6 8e10 10e12 5e6 ND 5.5e8 9e14 8.5e10.5 9.4e11 5e7 3e5.5 9.5e19 8e10 5.5e7 8e11.5 6 9e12 8e10 7e12 10e13 4e8.1 13e14 10e18 19e26 6.65e8.5 12.5 ND 18 10e12 7e9
1.5e2 3.5e4 4 6e7 3 5e10 1.5e2.5 7e9 1.5e2 2.35 3e4 1e2 2 7e8 3.5e4 1.7e2.5 4 2.5e3 6e8 1.5e2.5 ND 1.4e3 2 9 2.5e3 1.90 6.5 4.4e5 8 9e10 1.5e2
Sutton and Sarbhoy (1976) Cooke and Ellis (1878), Swift (1930) Cooke (1882), Swift (1930) Oudemans (1902) Saccardo (1878) €ck (1894) Starba Mathur and Thirumalachar (1964), Petrak (1965) Torrend (1913) Stolk (1963) Bahekar (1966) Torrend (1913) In this study Saccardo (1913) Torrend (1913) Oudemans (1902) Sydow, Mitter, and Tandon (1937), Bahekar (1966) Delacroix (1891) Gonz alez (1926) Torrend (1913) Fuckel (1870), Rossman et al. (2004) Torrend (1913) Swift (1930), Rossman et al. (2004) Saccardo (1884) Delacroix (1897) Ellis and Martin (1882) Bahekar (1966) Delacroix (1891) Tehon and Daniels (1925) Torrend (1912) Torrend (1912) Crous et al. (2014)
ND ¼ not described.
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Taxa
Chaetomella acutiseta Chaetomella endophytica Chaetomella oblonga Chaetomella raphigera Chaetomella raphigera Chaetomella raphigera Chaetomella zambiensis Cudonia sichunensis Cudoniella clavus Cudoniella indica Hymenoscyphus fructigenus Hymenoscyphus scutula Mycofalcella calcarata Ombrophila violacea Phaeohelotium epiphyllum Pilidium acerinum Pilidium acerinum Pilidium concavum Pilidium eucalyptorum Pilidium lythri Pilidium pseudoconcavum Spathularia flavida Sphaerographium nyssicola Sphaerographium nyssicola Synchaetomella acerina Tricladium obesum Tricladium splendens Tricladium splendens Xeropilidium dennisii Xeropilidium dennisii Zoellneria rosarum Zoellneria rosarum
Voucher/Strain
AFTOL-ID 207 SDBR-CMU300 BPI 843552 BPI 843551 BPI 843541 JCM 9995 CBS 137978 HMAS 75140 AFTOL-ID 166 CBS 430.94 M159 MBH29259 CCM F-10289 WZ0024 TNS:F-40042 CBS 736.68 PBI 843554 BPI 1107274 CPC 26594 CCTU: PN6 CPC 21642 KUS-F52331 CBS 128284 AR4654 DAOM 242271 CCM F-14598 CCM F-16599 CCM F-19087 KL251 KL159 PDD 102789 HB7919
Origin
e Japan USA India USA e South Africa China USA Thailand Finland e e China Japan Netherlands Netherlands Japan France Iran South Africa Korea USA USA France Czech Republic Czech Republic Canada France Germany Germany Germany
GenBank accession number
Reference
ITS
LSU
SSU
e MG406985 AY487079 AY487085 AY487076 LC228653 KJ869130 NR137533 DQ491502 DQ202513 EU940233 AY789432 KC834065 AY789366 AB926061 NR119500 AY487088 AY487097 KT950854 KX639607 KF777184 JN033405 NR119916 HQ338472 NR111811 KC834068 AY204635 AY204632 LT158441 LT158422 KF661532 KF588378
AY544679 MG406984 AY487080 AY487086 AY487077 LC228710 KJ869187 AF433137 DQ470944 e EU940157 AY789431 KC834033 AY789365 AB926130 AY487092 AY487089 AY487098 KT950868 KX639613 KF777236 JN086708 e e NG042747 KC834035 GQ47733 GQ477334 KX090824 KX090807 KF661533 e
AY544728 MG406986 AY487081 AY487087 AY487078 e e e DQ470992 e EU940081 AY789430 e AY789364 e AY487093 AY487090 AY487099 e e e JN086781 e e JX989832 e e e KX090876 KX090859 KF661534 e
Unpublished In this study Rossman et al. (2004) Rossman et al. (2004) Rossman et al. (2004) Unpublished Crous et al. (2014) Wang, Binder, and Hibbett (2002) Spatafora et al. (2006) Unpublished Baral, de Sloover, Huhtinen, Laukka, and Stenroos (2009) Wang, Binder, and Hibbet (2005) Baschien et al. (2013) Wang et al. (2005) Unpublished Rossman et al. (2004) Rossman et al. (2004) Rossman et al. (2004) Crous et al. (2015) Karimi, Arzanlou, Babai-Ahari, and Pertot (2016) Crous et al. (2013) Han, Hosoya, Sung, and Shin (2014) Minnis, Rossman, Farr, and Olsen (2010) Minnis et al. (2010) €rtel et al. (2017) Pa Baschien et al. (2013) Baschien et al. (2013) , and Gulis (2009) Campbell, Marvanova €rtel et al. (2017) Pa €rtel et al. (2017) Pa Johnston et al. (2014) Johnston et al. (2014)
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Please cite this article in press as: Suwannarach, N., et al., Morphological and molecular evidence support a new endophytic fungus, Chaetomella endophytica from Japan, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.05.001
Table 2 Details of sequences used in molecular phylogenetic analyses.
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Fig. 2. Phylogram derived from maximum likelihood analysis of combined LSU, ITS and SSU regions from rDNA. Cudonia sichuanensis and Spathularia flavida were used as the outgroup. The numbers above branches represent maximum likelihood bootstrap percentages (left) and Bayesian posterior probabilities (right). Only bootstrap values 50% are shown, and the bar represents the substitutions per nucleotide position. The novel species described in this study is shown in bold.
The combined LSU, ITS and SSU dataset consisted of 32 isolates with the aligned dataset comprising 2533 characters including gaps (LSU: 1e907, ITS: 908e1489 and SSU: 1490e2533). A phylogram of a combination of the LSU, ITS and SSU sequences is shown in Fig. 2. Two main clades of the order Helotiales, families Chaetomellaceae and Helotiaceae, were assigned according to previous phylogenetic €rtel et al., 2017; Rossman et al., 2004). Four studies (Baral, 2015; Pa sub-clades of the family Chaetomellaceae were assigned in this study. Sub-clade I contained the genera Chaetomella, Synchetomalla and Zoellneria. Sub-clades II, III and IV contained the single genera Pilidium, Sphaerographium and Xeropilidium, respectively. Chaetomella endophytica stood within sub-clade I and as sister taxon to C. zambiensis Crous with a bootstrap support of 90% and a Bayesian posterior probability of 0.99. Morphologically, C. zambiensis differs from C. endophytica in having longer conidia (7e9 mm) (Crous et al., 2014). Therefore, the combinations of morphological and molecular characteristics strongly support the recognition of a new endophytic fungus, C. endophytica from Japan. Previously, Chaetomella species have been reported as both plant pathogenic and sapro€rtel et al., 2017; Rossman et al., phytic (Baral, 2015; Fuckel, 1870; Pa 2004). Endophytic fungi may develop as saprophytes once a plant senesces or a leaf dies, and they may also become latent pathogens
that develop to cause plant diseases under certain conditions (Brown, Hyde, & Guest, 1998; Promputtha, Hyde, McKenzie, Peberdy, & Lumyong, 2010). However, there is no indication that C. endophytica is a fungal pathogen. Acknowledgments This work was supported by grants from Chiang Mai University, Thailand Research Fund Research-Team Association Grant (RTA5880006), Thailand and partly by New Core to Core Program (Establishment of an international research core for new bioresearch fields with microbes from tropical areas) by JSPS, Japan. We are grateful to Mr. Russell K. Hollis and Dr. Eric HC McKenzie for English proofreading. References Alfaro, M. E., Zoller, S., & Lutzoni, F. (2003). Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. Molecular Biology and Evolution, 20, 255e266. https://doi.org/10.1093/molbev/msg028. Bahekar, V. S. (1966). Notes on some new fungi from India. Mycopathologia et Mycologia Applicata, 30, 152e154.
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Please cite this article in press as: Suwannarach, N., et al., Morphological and molecular evidence support a new endophytic fungus, Chaetomella endophytica from Japan, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.05.001
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Short communication
Morphological and molecular evidence support a new endophytic fungus, Chaetomella endophytica from Japan Nakarin Suwannarach a, Jaturong Kumla a, Kenji Matsui b, Saisamorn Lumyong a, c, * a
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi, 753-8515, Japan c Center of Excellence on Bioresources for Agriculture, Industry and Medicine, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2017 Received in revised form 17 April 2018 Accepted 21 May 2018 Available online xxx
A new endophytic fungus, described herein as Chaetomella endophytica sp. nov., was isolated from stems of Rosa arvensis in Yamaguchi Prefecture, Japan. Morphological investigations revealed that its conidia are shorter and narrower than other known Chaetomella species. Phylogenetic analysis of three combined loci (large subunit, internal transcribed spacer, and small subunit regions of ribosomal DNA) confirmed that it is a new species within the family Chaetomellaceae. A full description, illustrations and a phylogenetic tree showing the position of C. endophytica are provided. © 2018 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Keywords: Chaetomellaceae Fungal endophyte Morphology Phylogeny
The genus Chaetomella was described by Fuckel (1870) with C. oblonga Fuckel as the type species. This genus belongs to the €rtel, order Helotiales, family Chaetomellaceae (Baral, 2015; Pa ~ldmaa1, 2017). Chaetomella species have been Baral, Tamm, & Po described as plant pathogens, and as saprophytes growing on litter, dead logs and soil distributed in both temperate and trop€rtel et al., 2017). Kirk, ical regions (Baral, 2015; Fuckel, 1870; Pa Cannon, Minter, and Stalpers (2008) suggested that there are five species of Chaetomella, but Index Fungorum (http://www. indexfungorum.org/Names/Names.asp, January 2018) lists 35 species. The statistics presented in Index Fungorum may include synonyms and misidentifications, and some species may not have been well documented. Traditional identification of microfungi is based on morphological characteristics, but morphological variations associated with environmental and cultural conditions, as well as the developmental stage of the fungi, may make morphological identification difficult among closely related genera and species (Baschien, Tsui, Gulis, Szewzyk, & Marvanova, 2013; Kohout et al., 2012). Therefore, the development of
* Corresponding author. Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand. E-mail addresses: [email protected], [email protected] (S. Lumyong).
molecular identification has become a powerful tool for fungal €rtel et al., 2017; Wang et al., 2006). identification (Baral, 2015; Pa During our investigation of endophytic fungi on wild rose (Rosa arvensis L.) in Japan, we found an interesting species of Chaetomella; morphological observation and phylogenetic analysis revealed it as a new species. Stem samples of R. arvensis were collected from a natural forest in Yoshida, Yamaguchi City, Yamaguchi Prefecture, Japan in May 2016. The samples were taken to the laboratory and processed within 24 h. Samples were washed in running tap water for 15 min and then cut into 10 mm long pieces. All pieces were surface sterilized in 75% ethanol for 30 s, followed by 2% sodium hypochlorite for 3 min and 95% ethanol for 30 s under a laminar flow hood (Suwannarach et al., 2012). The sterilized samples were placed in Petri dishes containing potato dextrose agar (PDA), 0.05% streptomycin sulfate and 0.03% Rose Bengal. Petri dishes were sealed with Parafilm M and incubated at 25 C in darkness for one week. Fungi growing out from the samples were aseptically transferred to fresh PDA. We obtained fourteen fungal isolates in resulted and the isolate CMU300 was used in this study. The holotype specimen (dried culture) and ex-type living culture in this study were deposited in the Culture Collection of Sustainable Development of Biological Resources Laboratory (SDBR), Faculty of Science, Chiang Mai University, Chiang Mai, and Thailand Bioresource Research Center (TBRC), Pathum Thani, Thailand, respectively.
https://doi.org/10.1016/j.myc.2018.05.001 1340-3540/© 2018 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Suwannarach, N., et al., Morphological and molecular evidence support a new endophytic fungus, Chaetomella endophytica from Japan, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.05.001
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Chaetomella endophytica Suwannarach, Kumla, Matsui & Lumyong, sp. nov. Fig. 1. MycoBank no: 823701. Diagnosis: Distinguished from other species of Chaetomella, except C. raphigera, by its conidia being shorter and narrower, and from C. raphigera by having straight, rather than curved setae. Etymology: referring to the fungus being isolated as an endophyte. Holotype: JAPAN, Yamaguchi Prefecture, Yamaguchi City, Yoshida, 34 080 50.76}N, 131280 33.70}E, isolated as an endophyte from stems of Rosa arvensis, May 2016, N. Suwannarach, SDBR-CMU300 (dried culture); TBRC 7238 (ex-type living). Gene sequences (from holotype): MG406984 (LSU), MG406985 (ITS) and MG406986 (SSU). Fungal colonies on PDA, oatmeal agar (OA) and malt extract agar (MEA) reaching 75e80, 73e75, and 72e73 mm diam, respectively at 25 C after 1 wk (Fig. 1AC). Colonies on PDA and OA flat, sparse, with white to cream mycelium and reverse white to light orange. Colonies on MEA, flat, with white mycelium and reverse cream.
Mycelium superficial and immersed, hyphae branched, septate, hyaline, 2e3 mm wide. Conidiomata pycnidial or sporodochial were observed in all agar media after incubation at 25 C for 1 wk. Pycnidia 120e340 170e380 mm, subglobose to elongate or reniform, dark brown to black, with a short stalk of hyaline textura angularis cells, pycnidial wall with three regions: outer region hyaline, 7e10 mm thick, 1e2 cell layers; middle region dark brown to black, 7e10 mm thick, 2e4 cell layers; inner region pale yellow, 4e5 mm thick, 3e4 cell layers, cells more or less square at base of conidiomata, rectangular along sides and top (Fig. 1DeF). Setae 50e82.5 3.75e6.25 mm, tapering, pale to dark brown, becoming paler towards apex, thick-walled, but walls thinner towards apex, septate, straight with rounded end. Sporodochia 170e370 150e350 mm, pale yellow, setose on outer wall, setae 87.5e142.5 2.5e5 mm, similar in shape to setae on pycnidia; spodorochial wall similar in shape to middle region of the pycnidia; conidiophores, conidiogenous cells and conidia same in pycnidia and in sporodochia (Fig. 1G and H). Conidiophores up to 150 0.5e1 mm, hyaline, smooth, septate, cylindrical, branched (Fig. 1I). Conidiogenous cells terminal and lateral, monophialidic,
Fig. 1. Chaetomella endophytica SDBR-CMU300 (holotype). AeC: Colonies on different media after 1 wk at 25 C. A: Potato dextrose agar; B: Oatmeal agar; C: Malt extract agar. DeF: Pycnidia. G: Sporodochium with stalk. H. Setae on sporodochium. I: Conidiophores and conidiogenous cells. J: Conidia. Bars: AC 10 mm; D 100 mm; EG 50 mm; H, J 10 mm; I 5 mm.
Please cite this article in press as: Suwannarach, N., et al., Morphological and molecular evidence support a new endophytic fungus, Chaetomella endophytica from Japan, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.05.001
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subcylindrical, straight to curved, smooth, hyaline, 5e25 0.5e1.5 mm. Conidia 3e6 1e2 mm (n ¼ 50), hyaline, smooth, aseptate, cylindrical to cymbiform, guttulate (Fig. 1J). The conidia of C. endophytica and other Chaetomella species are compared (Table 1). The shorter conidia of C. endophytica clearly distinguishes it from most other Chaetomella species, with the exception of C. andropogonis Cooke & Ellis, C. brachyspora Sacc. & Speg., C. flavoviridis Torrend and C. raphigera Swift. However, the conidia of C. andropogonis (3.5e4 mm wide), C. brachyspora (3 mm wide) and C. flavoviridis (3.5e4 mm wide) are broader than those of C. endophytica (Cooke & Ellis, 1878; Saccardo, 1878; Swift, 1930; Torrend, 1913). The conidia of C. raphigera (4e8.1 1.4e3 mm) are within the range of those of C. endophytica (3e6 1e2 mm) (Rossman et al., 2004; Swift, 1930), but the setae of C. raphigera are curved or hooked at the apex, whereas they are straight in C. endophytica (Rossman et al., 2004; Swift, 1930). Genomic DNA was extracted from one wk-old fungal mycelia (1e5 mg) grown on MEA using a DNA Extraction Mini Kit (FAVORGEN, Taiwan) following the manufacturer's protocol. The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR) using ITS4 and ITS5 primers (White, Burns, Lee, & Taylor, 1990), under the following thermal conditions: 95 C for 2 min, 30 cycles of 95 C for 30 s, 50 C for 30 s, 72 C for 1 min, and 72 C for 10 min on a peqSTAR 2X Thermocycler (PEQLAB, UK). The large subunit (LSU) region was amplified with LROR and LR5 primers (Vilgalys & Hester, 1990) under the following thermal conditions: 94 C for 2 min, 30 cycles of 95 C for 30 s, 52 C for 30 s, 72 C for 1 min, and 72 C for 10 min. The small subunit (SSU) region was amplified using NS1 and NS4 primers (White et al., 1990) under the same conditions as the LSU amplification. PCR products were checked on 1% agarose
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gels stained with ethidium bromide under UV light and purified using NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany). The purified PCR products were directly sequenced by 1ST BASE Company (Kembangan, Malaysia) using the PCR primers mentioned above. The LSU, ITS and SSU sequences of C. endophytica were deposited in GenBank as numbers MG406984, MG406985 and MG406986, respectively. For the phylogenetic analysis, details of sequences from the present study and those from previous studies, obtained from the GenBank database, are provided (Table 2). The multiple sequence alignment was carried out using MUSCLE (Edgar, 2004), and the alignment of the combined LSU, ITS and SSU sequences was deposited in TreeBASE under the number 21793. A maximum likelihood (ML) phylogenetic tree was constructed using RAxML v7.0.3 (Stamatakis, 2006). The optimal ML tree search was conducted with 1000 separate runs, using the default algorithm of the program from a random starting tree for each run. The final tree was selected among suboptimal trees from each run by comparing likelihood scores under the GTRGAMMA nucleotide substitution model. The ML trees were viewed with TreeView32 (Page, 2001). Clades with bootstrap values (BS) 70% were considered significantly supported (Hillis & Bull, 1993). Bayesian phylogenetic analyses were carried out using the Metropolis-coupled Markov chain Monte Carlo (MCMCMC) method in MrBayes version 3.2 (Ronquist et al., 2012), under a GRT þ I þ G model. Markov chains were run for 1000000 generations, with six chains and random starting trees. The chains were sampled every 100 generations. Among these, the first 2000 trees were discarded as the burn-in phase of each analysis and the resulting trees were used to calculate Bayesian posterior probabilities. Bayesian posterior probabilities (PP) 0.95 were considered significantly supported (Alfaro, Zoller, & Lutzoni, 2003).
Table 1 Origin and conidia size of Chaetomella species. Chaetomella spices
Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella Chaetomella
acutiseta andropogonis artemisiae beticola brychyspora brassicae cinnamomea circinata circinoseta cycadina flavoviridis endophytica gasteriae helicotricha horrida indica longiseta lygei madeirensis oblonga ochracea raphigera raripila sacchari stevensonii tinosporae tortilis tritici viridescens viridi-olivacea zambiensis
Origin
Pakistan e South Africa Netherlands Italy e India e Brazil India e Japan Austria, Italy e Netherlands India France Southwest Europe e Austria,Canada, USA Italy Brazil, Congo, India, Pakistan, UK, USA Western Europe South Africa USA India France USA e e Zambia
Conidia size (mm)
Reference
Length
Wide
7.5e10 5e6 8e10 10e12 5e6 ND 5.5e8 9e14 8.5e10.5 9.4e11 5e7 3e5.5 9.5e19 8e10 5.5e7 8e11.5 6 9e12 8e10 7e12 10e13 4e8.1 13e14 10e18 19e26 6.65e8.5 12.5 ND 18 10e12 7e9
1.5e2 3.5e4 4 6e7 3 5e10 1.5e2.5 7e9 1.5e2 2.35 3e4 1e2 2 7e8 3.5e4 1.7e2.5 4 2.5e3 6e8 1.5e2.5 ND 1.4e3 2 9 2.5e3 1.90 6.5 4.4e5 8 9e10 1.5e2
Sutton and Sarbhoy (1976) Cooke and Ellis (1878), Swift (1930) Cooke (1882), Swift (1930) Oudemans (1902) Saccardo (1878) €ck (1894) Starba Mathur and Thirumalachar (1964), Petrak (1965) Torrend (1913) Stolk (1963) Bahekar (1966) Torrend (1913) In this study Saccardo (1913) Torrend (1913) Oudemans (1902) Sydow, Mitter, and Tandon (1937), Bahekar (1966) Delacroix (1891) Gonz alez (1926) Torrend (1913) Fuckel (1870), Rossman et al. (2004) Torrend (1913) Swift (1930), Rossman et al. (2004) Saccardo (1884) Delacroix (1897) Ellis and Martin (1882) Bahekar (1966) Delacroix (1891) Tehon and Daniels (1925) Torrend (1912) Torrend (1912) Crous et al. (2014)
ND ¼ not described.
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Taxa
Chaetomella acutiseta Chaetomella endophytica Chaetomella oblonga Chaetomella raphigera Chaetomella raphigera Chaetomella raphigera Chaetomella zambiensis Cudonia sichunensis Cudoniella clavus Cudoniella indica Hymenoscyphus fructigenus Hymenoscyphus scutula Mycofalcella calcarata Ombrophila violacea Phaeohelotium epiphyllum Pilidium acerinum Pilidium acerinum Pilidium concavum Pilidium eucalyptorum Pilidium lythri Pilidium pseudoconcavum Spathularia flavida Sphaerographium nyssicola Sphaerographium nyssicola Synchaetomella acerina Tricladium obesum Tricladium splendens Tricladium splendens Xeropilidium dennisii Xeropilidium dennisii Zoellneria rosarum Zoellneria rosarum
Voucher/Strain
AFTOL-ID 207 SDBR-CMU300 BPI 843552 BPI 843551 BPI 843541 JCM 9995 CBS 137978 HMAS 75140 AFTOL-ID 166 CBS 430.94 M159 MBH29259 CCM F-10289 WZ0024 TNS:F-40042 CBS 736.68 PBI 843554 BPI 1107274 CPC 26594 CCTU: PN6 CPC 21642 KUS-F52331 CBS 128284 AR4654 DAOM 242271 CCM F-14598 CCM F-16599 CCM F-19087 KL251 KL159 PDD 102789 HB7919
Origin
e Japan USA India USA e South Africa China USA Thailand Finland e e China Japan Netherlands Netherlands Japan France Iran South Africa Korea USA USA France Czech Republic Czech Republic Canada France Germany Germany Germany
GenBank accession number
Reference
ITS
LSU
SSU
e MG406985 AY487079 AY487085 AY487076 LC228653 KJ869130 NR137533 DQ491502 DQ202513 EU940233 AY789432 KC834065 AY789366 AB926061 NR119500 AY487088 AY487097 KT950854 KX639607 KF777184 JN033405 NR119916 HQ338472 NR111811 KC834068 AY204635 AY204632 LT158441 LT158422 KF661532 KF588378
AY544679 MG406984 AY487080 AY487086 AY487077 LC228710 KJ869187 AF433137 DQ470944 e EU940157 AY789431 KC834033 AY789365 AB926130 AY487092 AY487089 AY487098 KT950868 KX639613 KF777236 JN086708 e e NG042747 KC834035 GQ47733 GQ477334 KX090824 KX090807 KF661533 e
AY544728 MG406986 AY487081 AY487087 AY487078 e e e DQ470992 e EU940081 AY789430 e AY789364 e AY487093 AY487090 AY487099 e e e JN086781 e e JX989832 e e e KX090876 KX090859 KF661534 e
Unpublished In this study Rossman et al. (2004) Rossman et al. (2004) Rossman et al. (2004) Unpublished Crous et al. (2014) Wang, Binder, and Hibbett (2002) Spatafora et al. (2006) Unpublished Baral, de Sloover, Huhtinen, Laukka, and Stenroos (2009) Wang, Binder, and Hibbet (2005) Baschien et al. (2013) Wang et al. (2005) Unpublished Rossman et al. (2004) Rossman et al. (2004) Rossman et al. (2004) Crous et al. (2015) Karimi, Arzanlou, Babai-Ahari, and Pertot (2016) Crous et al. (2013) Han, Hosoya, Sung, and Shin (2014) Minnis, Rossman, Farr, and Olsen (2010) Minnis et al. (2010) €rtel et al. (2017) Pa Baschien et al. (2013) Baschien et al. (2013) , and Gulis (2009) Campbell, Marvanova €rtel et al. (2017) Pa €rtel et al. (2017) Pa Johnston et al. (2014) Johnston et al. (2014)
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Please cite this article in press as: Suwannarach, N., et al., Morphological and molecular evidence support a new endophytic fungus, Chaetomella endophytica from Japan, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.05.001
Table 2 Details of sequences used in molecular phylogenetic analyses.
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Fig. 2. Phylogram derived from maximum likelihood analysis of combined LSU, ITS and SSU regions from rDNA. Cudonia sichuanensis and Spathularia flavida were used as the outgroup. The numbers above branches represent maximum likelihood bootstrap percentages (left) and Bayesian posterior probabilities (right). Only bootstrap values 50% are shown, and the bar represents the substitutions per nucleotide position. The novel species described in this study is shown in bold.
The combined LSU, ITS and SSU dataset consisted of 32 isolates with the aligned dataset comprising 2533 characters including gaps (LSU: 1e907, ITS: 908e1489 and SSU: 1490e2533). A phylogram of a combination of the LSU, ITS and SSU sequences is shown in Fig. 2. Two main clades of the order Helotiales, families Chaetomellaceae and Helotiaceae, were assigned according to previous phylogenetic €rtel et al., 2017; Rossman et al., 2004). Four studies (Baral, 2015; Pa sub-clades of the family Chaetomellaceae were assigned in this study. Sub-clade I contained the genera Chaetomella, Synchetomalla and Zoellneria. Sub-clades II, III and IV contained the single genera Pilidium, Sphaerographium and Xeropilidium, respectively. Chaetomella endophytica stood within sub-clade I and as sister taxon to C. zambiensis Crous with a bootstrap support of 90% and a Bayesian posterior probability of 0.99. Morphologically, C. zambiensis differs from C. endophytica in having longer conidia (7e9 mm) (Crous et al., 2014). Therefore, the combinations of morphological and molecular characteristics strongly support the recognition of a new endophytic fungus, C. endophytica from Japan. Previously, Chaetomella species have been reported as both plant pathogenic and sapro€rtel et al., 2017; Rossman et al., phytic (Baral, 2015; Fuckel, 1870; Pa 2004). Endophytic fungi may develop as saprophytes once a plant senesces or a leaf dies, and they may also become latent pathogens
that develop to cause plant diseases under certain conditions (Brown, Hyde, & Guest, 1998; Promputtha, Hyde, McKenzie, Peberdy, & Lumyong, 2010). However, there is no indication that C. endophytica is a fungal pathogen. Acknowledgments This work was supported by grants from Chiang Mai University, Thailand Research Fund Research-Team Association Grant (RTA5880006), Thailand and partly by New Core to Core Program (Establishment of an international research core for new bioresearch fields with microbes from tropical areas) by JSPS, Japan. We are grateful to Mr. Russell K. Hollis and Dr. Eric HC McKenzie for English proofreading. References Alfaro, M. E., Zoller, S., & Lutzoni, F. (2003). Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. Molecular Biology and Evolution, 20, 255e266. https://doi.org/10.1093/molbev/msg028. Bahekar, V. S. (1966). Notes on some new fungi from India. Mycopathologia et Mycologia Applicata, 30, 152e154.
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Please cite this article in press as: Suwannarach, N., et al., Morphological and molecular evidence support a new endophytic fungus, Chaetomella endophytica from Japan, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.05.001