Stachybotrys from soil in China, identified by morphology and molecular phylogeny Chun-Yu Jie, Kun Geng, Yu-Lan Jiang, Jun-Jie Xu, Kevin D. Hyde, Eric H. C. McKenzie, Tian-Yu Zhang, Ali H. Bahkali, De-Wei Li, et al. Mycological Progress ISSN 1617-416X Mycol Progress DOI 10.1007/s11557-012-0878-y 1 23 Your article is protected by copyright and all rights are held exclusively by German Mycological Society and Springer-Verlag Berlin Heidelberg. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy Mycol Progress DOI 10.1007/s11557-012-0878-y ORIGINAL ARTICLE Stachybotrys from soil in China, identified by morphology and molecular phylogeny Chun-Yu Jie & Kun Geng & Yu-Lan Jiang & Jun-Jie Xu & Kevin D. Hyde & Eric H. C. McKenzie & Tian-Yu Zhang & Ali H. Bahkali & De-Wei Li & Yong Wang Received: 27 October 2012 / Revised: 3 December 2012 / Accepted: 7 December 2012 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2012 Abstract Four Stachybotrys strains were isolated from soil in China. One was identified as a novel species by morphological characters of phialides and conidia. It produced cylindrical conidia with irregular striations and smooth, hyaline conidiophores. Phylogenetic analysis of three DNA markers, the internal transcribed spacer region of rDNA (ITS1–5.8S–ITS2), the translation elongation factor 1 alpha (tef1) and RNA polymerase II subunit (rpb2), supported the morphological results. The correlation between morphological and molecular-based clustering demonstrated that the studied isolate was a new species. Two other isolates were identified as S. cf. elegans. Keywords Phylogenetic analysis . Soil fungi . Taxonomy Introduction Stachybotrys species are saprobes, common in soil (Ellis 1971, 1976), decaying plant material (Whitton et al. 2001) and wild fruits (Tang et al. 2003), and have also been recorded on submerged wood in mangroves (Maria and Sridhar 2003). Stachybotrys species have also been demonstrated to be a health risk in buildings with long-term water damage (Etzel et al. 1998; Hintikka 2004; Al-Ahmad et al. 2010; Karunasena et al. 2004; Pestka et al. 2008). Pinruan et al. (2004) summarized previous studies and provided a key to Stachybotrys and to the closely morphologically related Memnoniella species. Nine Stachybotrys species have been reported from China, with eight isolated from soil (Kong et al. 2007; Wang et al. 2009; Jiang and Zhang 2009; Li and Jiang 2011; Wu and Zhang 2009, 2010). The ninth species originated from a cardboard box in Yunnan Province (Kong 1997). During a survey of hyphomycetes in China, various taxa were isolated from soil in different environments. Among them were four isolates that produced single-celled conidia aggregated in slimy heads, fitting typical morphological characteristics of Stachybotrys Corda (Jong and Davis Chun-Yu Jie and Kun Geng contributed equally to the manuscript and should be considered as joint first authors. C.-Y. Jie : K. Geng : Y.-L. Jiang : Y. Wang (*) Department of Plant Pathology, College of Agriculture, Guizhou University, Guiyang, Guizhou 550025, China e-mail: yongwangbis@yahoo.cn K. Geng Plant Protection and Quarantine Station, Guiyang, Guizhou 550081, China J.-J. Xu College of Life Sciences, Linyi University, Linyi, Shandong 276005, China K. D. Hyde Institute of Excellence in Fungal Research, School of Science, Mae Fah Luang University, Chiang Rai, Thailand E. H. C. McKenzie Landcare Research, Private Bag 92170, Auckland, New Zealand K. D. Hyde : A. H. Bahkali Botany and Microbiology Department, College of Sciences, King Saud University, P.O. Box: 2455, Riyadh 1145, Saudi Arabia T.-Y. Zhang Department of Plant Pathology, Shandong Agricultural University, Taian, Shandong 271018, China D.-W. Li (*) The Connecticut Agricultural Experiment Station, Valley Laboratory, 153 Cook Hill Road, Windsor, CT 06095, USA e-mail: Dewei.Li@ct.gov Author's personal copy Mycol Progress 1976; Mercado-Sierra et al. 1997). However, one isolate differed from known species by the morphological characters of conidia and conidiophores. In the present paper, we provide detailed illustrations and a description of this fungus. DNA was extracted and sequence data for ITS (ITS1– 5.8S–ITS2), the translation elongation factor 1 alpha (tef1) and RNA polymerase II subunit b (rpb2) were obtained and analyzed to evaluate the morphological results. Based on morphology and DNA sequence comparison, the strain is proposed as Stachybotrys subcylindrospora sp. nov. Materials and methods Fungal strains and morphology The four cultures of Stachybotrys isolated in the study are conserved in Herbarium of Guizhou University, Plant Pathology (HGUP) and Herbarium of Shandong Agricultural University, Plant Pathology (HSAUP). The fungi are described from cultures grown at 25 °C on corn meal agar (CMA). Conidia and conidiophores were placed in a drop of 85 % lactic acid, and examined and photographed using a Nikon 80i microscope (Nikon, Japan) at 400× and 1,000× magnification. DNA extraction, amplification and DNA sequencing Genomic DNA was extracted from colonies grown on potato-dextrose agar (PDA), using the Fungal gDNA Kit GD2416 (Biomiga, CA, USA), following the manufacturer’s instructions. The universal primers ITS1/ITS4 (White et al. 1990) were used for the ITS region (ITS1– 5.8S–ITS2) amplification, and primers EF1-983F and EF12218R (Rehner 2001) were used for partial translation elongation factor 1 alpha (tef1) amplification, while part of the second largest subunit of RNA polymerase II (rpb2) gene was amplified using primers fRPB2-5f/fRPB2-7cr (Liu et al. 1999). Amplification reactions were performed in a BioRAD PTC-200 thermocycler, in a 25 μl reaction mixture using the following final concentrations or total amounts: 5 ng DNA, 1× PCR buffer (20 mM Tris/HCl pH 8.4, 50 mM KCl), 1 μM of each primer, 2.5 mM MgCl2, 0.25 mM of each dNTP, 0.5 U of Taq polymerase. The PCR amplified DNA fragments were fractionated in 1 % agarose gels in 0.5× TBE buffer, and DNA was visualized by ethidium bromide staining and UV illumination. Sequencing was performed with an ABI PRISM 3730 DNA autosequencer using either dRhodamine terminator or Big Dye Terminator chemistry (Life Technologies™, USA). Sequence data of the isolates used in the study were deposited in GenBank (Table 1). Alignments are available in TreeBASE (www.treebase.org/treebase-web/home.html) under the study ID 13555. Phylogenetic analyses Preliminary nucleotide sequence alignments were constructed using Clustal X 1.81 (Thompson et al. 1997). A partition homogeneity test (Farris et al. 1994) was applied to evaluate the feasibility of combining the data sets. Phylogenetic analysis of ITS sequence and combined rpb2 and tef1 sequence were computed using MP analysis in PAUP* (Swofford 2002). In the MP analyses, trees were inferred using heuristic search option with tree bisection reconnection (TBR) branch swapping and 1,000 random sequence additions; maxtrees were 5,000; branches of zero length were collapsed, and all parsimonious trees were saved. Measures calculated for parsimony included tree length (TL), consistency index (CI), retention index (RI) and rescaled consistence index (RC). Bootstrap analyses (Hillis and Bull 1993) were conducted with 1,000 replications. Results Phylogenetic analyses The aligned sequence matrix contained 16 ITS data and 28 rpb2/tef1 data downloaded from GenBank. In the ITS tree, 117 parsimony-informative characters included in the parsimony analyses yielded four parsimonious trees (TL0395, CI00.67, RI00.70, RC00.32), one of which is presented (Fig. 1). In the combined tree, 310 parsimony-informative characters in 1,229 characters included in the parsimony analyses yielded three most parsimonious trees (TL 0 1,089, CI00.53, RI00.60, RC00.32), one of which is presented (Fig. 2). All 19 Stachybotrys/Memnoniella isolates clustered together as a strong clade with 100 % bootstrap support (Fig. 1). In this tree, S. subsimplex Cooke (0 Memnoniella subsimplex (Cooke) Deighton) showed a distant relationship with other isolates. The other isolates were further divided into two clades with high bootstrap support (79 %, 88 %). Among our four isolates, HGUP 0103 and 0201 grouped into a branch with a 92 % bootstrap value, while HGUP 0310 and 0208 showed a close relationship with two strains of S. elegans (Pidopl.) Gams. Both subclades had high bootstrap support (70 %, 87 %). In the rpb2 and tef1 combined tree (Fig. 2), only three isolates (HGUP 0103, 0201 and 0310) were included, because the tef1 and rpb2 gene regions of HGUP 0208 were not successfully amplified. Isolates HGUP 0103 and 0201 showed a close relationship (100 % bootstrap support) with S. microspora (Mathur & Sankhla) Jong & Davis (Fig. 2), Author's personal copy Mycol Progress Table 1 Strains used in phylogenetic analyses and their corresponding GenBank accession numbers Species Fusarium sporotrichioides Sherbakoff Melanopsamma pomiformis (Persoon)Saccardo Stachybotrys subcylindrospora C.Y. Jie, Y.L. Jiang, D.W. Li, Mckenzie & Yong Wang bis S. chartarum (Ehrenberg) Hughes S. chlorohalonata Andersen & Thrane S. dichroa Grove S. echinata (Rivolta) Smith S. elegans (Pidoplichko) Gams S. cf. elegans S. cf. elegans S. eucylindrospora D.W.Li S. kampalensis Hansford S. longispora Matsush S. microspora (Mathur & Sankhla)Jong & Davis S. nephrospora Hansford S. oenanthes Ellis S. parvispora Hughes S. sansevieriae Agarwal & Sharma S. subsimplex Cooke S. theobromae Hansford Xepicula leucotricha (Peck) Nag Raj Accession number Substratum/origin GenBank accession numbers ITS tef1 rpb2 NRRL 3299 UAMH 7750/ATCC 18873 Plant/France Plant/Yorkshire, U.K. AF081478 DQ676612 DQ676587 DQ676622 DQ676597 HGUP 0201 Soil/Hainan, China JX998163 JX998166 UAMH 10150 UAMH 10153 CBS 109285ex-type UAMH 7748/ATCC 18913 CBS 949.72 UAMH 3195 CBS 399.65/ATCC 22173 UAMH 1526 DAOM 225565 HGUP 0208 Paper/Toronto, Canada Plant/Germany Build/Denmark Plant/England /Izmir, Turkey Build/Solomon Islands Plant/Germany Soil/Ontario, Canada AY095975 DQ676624 DQ676625 AY180261 AF081472 DQ676620 DQ676609 DQ676615 AF081480 DQ676608 DQ676614 JN942885 JX978445 HGUP 0310 CBS 203.61/ATCC 18851 ex-type UAMH 7122 DAOM 186941 UAMH 7746 ATCC 32451 UAMH 7747/ATCC 18852 Soil/Guangxi, China Soil/Ontario, Canada Plant/Alberta, Canada Plant/Ontario, Canada Plant/New Guinea Plant/Japan Plant/Zaria, Nigeria AF081482 AF081475 DQ676619 DQ676594 ATCC 18839 ATCC 22844 ex-type UAMH 7749/ATCC 18877 HGUP 0103 Plant/Osaka, Japan Plant/Channel Islands Soil/Congo Soil/Shanxi, China AF081476 AF081473 AF081483 JX998165 DQ676621 DQ676596 JX987250 JX987249 ATCC 18838 ATCC 18905 CBS 131.64 /Osaka, Japan AF205441 Plant/Sabah, Malaysia AF081479 Soil/Andlhra Pradesh, India AJ302000 but in the ITS analysis (Fig. 1) they showed a closeness to S. chartarum (Ehrenb.) Hughes and S. chlorohalonata Andersen & Thrane. Isolate HGUP 0310 clustered together with S. elegans (ATCC 22173) supported by a high bootstrap value (98 %), which was consistent with the ITS analysis. Taxonomy Stachybotrys subcylindrospora C.Y. Jie, Y.L. Jiang, D.W. Li, McKenzie & Yong Wang bis, sp. nov. can be seen in Fig. 3. MYCOBANK MB 801902 Coloniae in CMA effusae, 3.5–5 cm diam a 14 d, 25 °C. Conidiophora erecta, simplicia, septata, macronemata, solitaria vel fasciculata, determinata, recta vel exigue curvata, deinde ramosa, laeves, 1–2 septata, prope basin hyalina et latvia, usque ad (52–)68(–88) μm longa et (2.4–)3.4(–4.3) Soil/Yunan, China JX998164 AF081474 JX998168 DQ676600 DQ676601 DQ676595 DQ676585 DQ676590 DQ676584 DQ676589 JX998167 JX998169 DQ676605 DQ676581 DQ676617 DQ676592 JN942887 DQ676618 DQ676593 μm crassa. Phialides 3–8 fasciculatae, exigue curvatae vel erecto-clavatae, laevese, 8(8.4–)9.6–12.6(–14.3) × (4.0–)4.3–5.5(–6.1) μm. Conidia cylindrica vel subcylindrica, apice rotundata, basi truncata, delicate et irregulariter striata, (9.7–)11.6–13.8(–14.7) × (2.9–)3.8–4.6 (–5.0) μm. Colonies on CMA at 25 °C for 14 d reaching 3.5–5 cm diam., effuse, downy to felty, colorless at first, becoming dark with a granulate surface as conidial production commences. Mycelium mostly superficial, partly immersed. Conidiophores determinate, macronematous, mononematous, solitary or in groups, erect or slightly curved, simple or irregularly branched, 1–2-septate, smooth, hyaline, (52–)68(–88) μm long, (2.4–)3.4(–4.3) μm wide at middle (Fig. 3a). Phialides borne in groups of 3–8 at the apices of conidiophores, discrete, slightly curved or erect, clavate, smooth, with conspicuous collarettes, (8.4–)9.6–12.6(– Author's personal copy Mycol Progress Fig. 1 One of the four equally most parsimonious trees of the analyzed ITS region (117 of the 677 characters were parsimony informative). Bootstrap support values less than 50 % are not shown. The tree is rooted with Xepicula leucotricha 14.3) (mean 0 11.1±1.5, n030) μm long, (4.0–)4.3–5.5 (–6.1) (mean 0 4.9±0.6) μm thick in the broadest part (Fig. 3b–c). Conidia acrogenous, aggregated in slimy masses, unicellular, cylindrical or subcylindrical, truncate at the base and rounded at the apex, surface of both young and mature spores show delicate and irregular striations under oil lens, (9.7–)11.6–13.8(–14.7) (mean 0 12.7 ± 1.1, n 040) × (2.9–)3.8–4.6(–5.0) (mean 0 3.9±0.5) μm, ratio of length/ width 2.6–3.9 (mean 0 3.1), usually containing one to three oil drops, especially when young (Fig. 3d–e). Holotype: CHINA, Hainan Province, Jianfengling, from tropical primordial rain forest soil, collected by Yue-Li Zhang on 5 Nov 2005, HGUPd0201. Isotype HSAUP052494; Ex-type: HGUP 0201. Etymology: to indicate the cylindrical or subcylindrical conidia. Discussion Stachybotrys subcylindrospora (HGUP0201) is similar to S. longispora Matsush. and S. eucylindrospora D.W. Li in producing cylindrical conidia (Matsushima 1971, 1975; Li 2007). However, the conidial size of S. subcylindrospora is somewhat different from that of S. longispora (8.8–12×2– 2.4 μm, L/W ratio > 4) and S. eucylindrospora (12.8–16× 3.4–5.5 μm, L/W ratio 0 3.4). More importantly, conidia of S. subcylindrospora have irregular striations, while those of S. eucylindrospora have longitudinal striations, and those of Author's personal copy Mycol Progress Fig. 2 Phylogram based on the combined data set of partial rpb2 and tef1 gene sequences, and analyzed using most parsimonious. The tree is rooted with Fusarium sporotrichioides S. longispora are smooth. Additionally, the phialides of S. subcylindrospora (4.3–5.5 μm wide) are wider than those of S. eucylindrospora (2.9–4.5 μm). The conidiophores of S. subcylindrospora are smooth and hyaline, but those of S. eucylindrospora are slightly olivaceous and possess ornamentation at the top. Fig. 3 Stachybotrys subcylindrospora (HGUP0201– holotype) on corn meal agar (CMA). a. Conidiophore, phialides and conidium. b–d. Phialides and conidia. e. Conidia. Scale Bars: a 0 30 μm; b–e 0 15 μm The genus Stachybotrys and its type species, S. chartarum, have been subject to controversy since they were proposed. For example, S. chartarum sensu stricto is a species with a great variation in morphology (Li and Yang 2005). Haugland et al. (2001) analyzed phylogenetic results and supported the previously proposed relegation of Author's personal copy Mycol Progress Memnoniella to synonymy with Stachybotrys (Smith 1962). Castlebury et al. (2004) revealed a new lineage for S. chartarum based on multigene phylogeny. Thus, Li and Yang (2005) believed the taxonomic problem of Stachybotrys had to be solved by examination of type specimens, morphology and phylogenetic analyses. In this study, we used ITS and combined tef1 and rpb2 sequence analyses to complement the morphological identification (Figs. 1 and 2). According to morphological comparison, HAUP0103 (conidia navicular, dark brown, 7.0–8.8 × 3.2–4.0 μm) should be S. sansevieriae G.P. Agarwal & N.D. Sharma (Pinruan et al. 2004), which showed a close relationship with HGUP0201 in ITS and combined tef1 and rpb2 sequence analyses. The phylogenetic results partly support the morphological comparison, but there is enough proof to discriminate HGUP0201 as a related species. Meanwhile, both HGUP0310 and 0208, which show a high similarity with S. elegans in morphology, should be determined as S. cf. elegans. Combining morphology and restricted phylogenetic analysis, we propose that S. subcylindrospora is a novel species. 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