Research Article |
Corresponding author: Li-Zhou Tang ( biologytang@163.com ) Corresponding author: Chao Liu ( liuchao_80@163.com ) Academic editor: Pedro Crous
© 2019 Dong-Qin Dai, Nalin N. Wijayawardene, Li-Zhou Tang, Chao Liu, Li-Hong Han, Hong-Long Chu, Hai-Bo Wang, Chun-Fang Liao, Er-Fu Yang, Rui-Fang Xu, Yun-Min Li, Kevin D. Hyde, D. Jayarama Bhat, Paul F. Cannon.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Dai D-Q, Wijayawardene NN, Tang L-Z, Liu C, Han L-H, Chu H-L, Wang H-B, Liao C-F, Yang E-F, Xu R-F, Li Y-M, Hyde KD, Bhat DJ, Cannon PF (2019) Rubroshiraia gen. nov., a second hypocrellin-producing genus in Shiraiaceae (Pleosporales). MycoKeys 58: 1-26. https://doi.org/10.3897/mycokeys.58.36723
|
Shiraiaceae is an important family in Pleosporales (Dothideomycetes), which includes medical fungi and plant pathogens. Two hypocrellin-producing taxa, Shiraia bambusicola and a novel genus Rubroshiraia gen. nov., typified by Rubroshiraia bambusae are treated in this article. Maximum likelihood analysis, generated via RAxML (GTR+G model), using a combined SSU, LSU, TEF1 and RPB2 sequence dataset, shows that Rubroshiraia is close to Shiraia and belongs to the family Shiraiaceae. Descriptions, illustrations and a taxonomic key are provided for the genera in Shiraiaceae. Rubroshiraia morphologically differs from Shiraia in having small and dark ascostromata and filiform ascospores. Production of the ascostromatal metabolites, hypocrellin A and B, were examined by HPLC and spectrophotometer. The content of hypocrellin A and B of specimen HKAS 102255 (R. bambusae) is twice that produced by HKAS 102253 (S. bambusicola). To clarify the relationship between R. bambusae and Hypocrella bambusae, type material of the latter was examined and provided the illustration.
HPLC, metabolite, new genus, phylogeny, taxonomy
Shiraia is typified by S. bambusicola Henn. (
Distribution | References | |
---|---|---|
Country | Province | |
China | Anhui |
|
Guangxi |
|
|
Guizhou |
|
|
Henan |
|
|
Hubei |
|
|
Hunan |
|
|
Jiangsu |
|
|
Jiangxi |
|
|
Sichuan |
|
|
Yunan |
|
|
Zhejiang |
|
|
Japan | Tokyo |
|
Osaka |
|
Shiraia has previously been placed in several families, depending on the opinions of authors.
Earlier classifications of Shiraia were based on morphological characters. The first attempt of DNA-based taxonomy (
Shiraia bambusicola has been reported as a pathogen on various bamboo species (Table
Bamboo host | References |
---|---|
Brachystachyum densiflorum (Rendle) Keng |
|
Brachystachyum albostriatum G.H. Lai |
|
Brachystachyum ensiflorum (Pendle) Keng |
|
Brachystachyum yixingense |
|
Phyllostachys nidularia Munro | GenBank |
Phyllostachys praecox f. prevernalis S.Y. Chen & C.Y. Yao | GenBank |
Pleioblastus amarus (Keng) Keng f. | GenBank |
Shiraia bambusicola produces hypocrellins. Four hypocrellins have been extracted from the fungal stromata (
A Chinese medical fungus named “Zhuhongjun” in Chinese, was identified as Hypocrella bambusae (Berk. & Broome) Sacc. by
The monotypic genus Grandigallia, collected on Polylepis sericea Wedd. (Rosaceae), was introduced by
In this study, ten specimens of S. bambusicola and a hypocrellin producing taxon (“Zhuhongjun” in Chinese) were collected from Yunnan Province in China. Morphological and phylogenetic studies were carried out to determine the taxonomic status of these taxa. Sequences from endophytic strains, named as Shiraia spp., were also downloaded from GenBank and included in the phylogenetic analyses. The metabolite content of hypocrellin extracted from the specimens was determined by HPLC (
Bamboo culms with large, reddish to pale yellow ascostromata were collected from Yunnan, China and brought to the laboratory in 2017. Samples were examined following the methods described in
The surface of fungal fruiting bodies was sterilised by 75% alcohol and rinsed three times in sterile water. The internal tissue with locules was cut into pieces and ground in a mortar into powder with liquid nitrogen. The powder was used to directly extract DNA with an OMEGA E.Z.N.A. Forensic DNA Kit, following the manufacturer’s instructions.
ITS5 and ITS4, NS1 and NS4 (
The final volume of the polymerase chain reaction (PCR) was prepared following
The BLAST searches in GenBank, using LSU and ITS sequence data were carried out to obtain the close strains. Additional sequences were downloaded from GenBank based on recent publications (
Single gene sequence alignments were carried out with MAFFT v. 7.215 (
Maximum-likelihood (ML) analyses, including 1000 bootstrap replicates, were run using RAxMLGUI v.1.0. (
Maximum-parsimony (MP) analyses were carried out in PAUP v. 4.0b10 (
Bayesian analyses were performed using MrBayes v. 3.0b4 (
Trees were visualised with TreeView (
Standards of hypocrellin A and hypocrellin B were purchased from Shanghai Tauto Biotech CO., Ltd. (http://www.tautobiotech.com) and used as received. Their purity is ≥ 98% (HPLC) and their structures are redrawn based on references (
Instrument | Condition |
---|---|
Reverse phase-column | CAPCELL PAK C18 (4.6 mm × 25 cm, 5 µm) |
Oven temp. (°C) | 35 |
Flow rate (ml/min) | 1 |
Mobile phase (%) | 38% solvent A: H2O + 0.5% formic acid; 62% solvent B: acetonitrile |
UV Absorbance (nm) | 265 |
Gradient elution | isocratic elution |
Run time (min) | 30–40 |
List of newly generated sequences with their culture collection numbers and GenBank accession numbers.
To clarify the family placement of newly established taxa, maximum likelihood phylogenetic analysis was generated from RAxML (GTR+G model), based on combined SSU, LSU, TEF1 and RPB2 sequences data (Fig.
Maximum likelihood phylogenetic tree generated from RAxML (GTR+G model), based on combined LSU, SSU, TEF1 and RPB2 sequences data. ML values (MLBP) (> 50%), resulting from 1000 bootstrap replicates and Bayesian posterior probabilities (BYPP) greater than 0.90, are given at the nodes. The original isolate numbers’ codes are noted after the species names. The tree is rooted to Dothidea insculpta (CBS 189.58). Ex-type or ex-epitype strains are in bold. Newly generated strains are in red and the new genus is in yellow background.
To clarify the relationship between endophytic strains named as shiraia-like (Shiraia spp.) and Shiraiaceae, a phylogenetic tree was constructed (RAxML (GTR+G model), based on combined LSU and ITS sequences data and compared. The combined alignment comprises 1442 characters including gaps for 57 ingroup taxa and one outgroup taxon Pleospora herbarum (CBS 191.86). Of the 1442 characters of the combined matrix, 1116 were constant and 220 were parsimony informative. The endophytic strains separated into two lineages (Group A and group B) forming at the base clade of Shiraiaceae (Fig.
Maximum likelihood phylogenetic tree generated from RAxML (GTR+G model), based on combined LSU and ITS sequences data. ML and MP values (MLBP/MPBP) (> 50%), resulting from 1000 bootstrap replicates, are given at the nodes. The original isolate numbers’ codes are noted after the species names. The tree is rooted to Pleospora herbarum (CBS 191.86). Ex-type or ex-epitype strains are in bold. Newly generated strains are in red.
Stromatal extracts from specimens of S. bambusicola (HKAS102266) and R. bambusae (HKAS102270) contained high quantities of hypocrellin A (304.03 ng/ul and 790.86 ng/ul, respectively). Stromatal extracts from specimens of S. bambusicola contained 42.55 ng/ul hypocrellin B, whereas R. bambusae produces a higher quantity (204.60 ng/ul). The HPLC profiles of S. bambusicola and R. bambusae are depicted in Figure
The family Shiraiaceae was introduced by
Shiraia Henn., Bot. Jb. 28(3): 274 (1900).
S. bambusicola Henn., Bot. Jb. 28(3): 274 (1900).
Parasitic on living branches of bamboo. Sexual morph: Ascostromata 1–6 cm long × 1–4 cm wide, solitary, superficial, subglobose, long ellipsoid to irregular, tuberculate, fleshy, white to pinkish, with locules lining the periphery, with dark ostiolate points appearing on surface. Ascostromatic tissue thick, pinkish, composed of wide, woven hyphae of textura intricata. Locules in vertical section 370–700 µm high × 370–700 µm diam. (x̄ = 541 × 513 µm, n = 20), globose to subglobose, immersed in the peripheral layer of ascostromata, with 100–200 µm wide ostioles. Peridium 20–45 µm thick, composed of several layers of hyaline to light brown, small cells of textura angularis to textura intricata. Hamathecium composed of interthecial, hyaline septate, branched pseudoparaphyses, 1–2.5 µm wide. Asci 200–370 × 20–35 µm (x̄ = 291.6 × 26.6 µm, n = 20), 4–6-spored, thick-walled, bitunicate, fissitunicate, cylindrical, short-pedicellate, with an ocular chamber. Ascospores 50–77 × 15–24 µm (x̄ = 62.3 × 18.1 µm, n = 20), 1-seriate, overlapped, fusiform, muriform, hyaline, with 7 transverse septa, constricted at the septum, smooth-walled. Asexual morph: Conidiomata 200–500 µm high, 300–400 µm wide, loculate, forming within ascostromata, globose to subglobose or irregular. Wall of locules 20–40 µm thick, composed of several layers of hyaline to light brown, small cells of textura intricata. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 3–6 × 2–3 µm (x̄ = 4.7 × 2.1 µm, n = 10), blastic, cylindrical, hyaline, smooth-walled. Conidia 60–80 × 19–25 µm (x̄ = 75.4 × 23.1 µm, n = 20), fusiform, muriform, hyaline, with irregularly transverse and longitudinal septa, straight to curved, smooth-walled.
Shiraia bambusicola A–J sexual morph A fruiting bodies (HKAS102253, HKAS102254, HKAS102257, HKAS102261, HKAS102262) B–J photographs from material HKAS102253 B Surface of ascostromata showing the dark openings of ostiole C vertical section of ascostromata D vertical section of locule E pseudoparaphyses F, G asci (G Showing the fissitunicate asci) H–J ascospores K–M asexual morph K vertical section of asexual locules L–M conidia. Scale bars: 2 cm (A), 5 mm (B), 1 mm (C), 100 μm (D, K), 50 μm (F, G), 20 μm (H–J, L, M).
Colonies growing slowly, attaining 30 mm diam. after 2 weeks at 27 °C under dark, circular, with even margin, floccose at the centre, drift white at margin, light greenish at centre, dark from below.
CHINA, Yunnan province, Lijiang, on living branches of Brachystachyum densiflorum (Rendle) Keng, 3 May 2017, Dong-Qin Dai, DDQ00409 (HKAS102253), Ibid. (duplicate specimen deposited in HMAS 290446), Ibid. DDQ00410 (HKAS102254), Ibid. DDQ00413 (HKAS102257), Ibid. 10 June 2017, Dong-Qin Dai, DDQ00418 (HKAS102261), Ibid. DDQ00419 (HKAS102262), Ibid. DDQ00420 (HKAS102263), Ibid. DDQ00421 (HKAS102264), Ibid. DDQ00422 (HKAS102265), Ibid. DDQ00423 (HKAS102266), Ibid. DDQ00424 (HKAS102267).
Shiraia bambusicola was erected by
See
Grandigallia dictyospora M.E. Barr et al., Mycotaxon 29: 196 (1987)
The monotypic genus Grandigallia was introduced by Barr (1987) and is typified by G. dictyospora. The fungus infects branches of Polylepis sericea Wedd. (Rosaceae) and produces conspicuous (3–14 cm in diam.) and black ascostromata. Grandigallia closely resembles Shiraia in having muriform ascospores, however, it differs by its black and larger ascostromata.
The epithet “Rubro” means red colour referring to reddish ascotromata similar to the genus Shiraia.
Parasitic on living branches of bamboo. Sexual morph: Ascostromata solitary, superficial, globose to subglobose, fleshy, reddish, with locules lining the periphery, with dark ostiolate tips appearing on surface. Ascostromatic tissue thick, pinkish, composed of wider woven hyphae of textura intricata. Locules globose to subglobose, immersed in the peripheral layer of ascostromata, with narrow ostiolate openings. Peridium composed of several layers of hyaline to dark brown, small cells of textura angularis to textura intricata. Hamathecium of interthecial, hyaline, septate, branched pseudoparaphyses above asci. Asci 8-spored, thick-walled, bitunicate, fissitunicate, cylindrical, short-pedicellate, with an ocular chamber. Ascospores spirally arranged in asci, filiform, hyaline, with transverse septa, smooth-walled. Asexual morph: Undetermined.
R. bambusae D.Q. Dai & K.D. Hyde.
The hypocrellin-producing fungus R. bambusae is a well-known taxon used in Chinese traditional medicine which is called “Zhuhongjun” or “Zhuxiaorouzhuojun” in Chinese. However, without molecular data, it was wrongly named as H. bambusae (
Hypocrella bambusae was combined by
New collections of “Zhuhongjun” were collected and sequenced. The phylogenetic analyses showed it belongs to Shiraiaceae and is separate from Shiraia with high bootstrap support (100/1.00 MLBS/BSPP) (Fig.
Rubroshiraia bambusae is often confused with S. bambusicola by Chinese traditional folk residents, probably because of the similarity of their ascostromata, parasitism on bamboo host and similar efficacy of medical treatment. However, it differs from S. bambusicola by its smaller sized ascostromata (0.7–1.2 cm long × 0.7–1 cm wide vs. 1–6 cm long × 1–4 cm wide) and distinct ascospores (filiform ascospores vs. fusiform and muriform ones). Both of the above species can produce the metabolites hypocrellin A and B, whereas R. bambusae contains almost double the content compared to S. bambusicola (Fig.
Rubroshiraia bambusae (HKAS102255, holotype) A fruiting bodies B, C surface of ascostromata showing the openings of ostiole D vertical section of ascostromata E, F vertical section of locule G peridium of locule H asci and pseudoparaphyses I asci and asci ocular chamber J ascospores K, L immature asci. Scale bars: 1 cm (A), 25 mm (B), 2 mm (C, D), 500 μm (E, F), 200 μm (G), 50 μm (H–L).
Refers the bamboo host.
HKAS102255.
Parasitic on living branches of bamboo. Sexual morph: Ascostromata 0.7–1.5 cm long × 0.7–1.3 cm wide, solitary, superficial, globose to subglobose, fleshy, reddish, with locules lining the periphery, with dark ostiolate points appearing on the surface. Ascostromatic tissue thick, pinkish, composed of wider woven hyphae of textura intricata. Locules in vertical section 800–1800 µm high × 1000–2000 µm diam. (x̄ = 1289.4 ×1368.8 µm, n = 20), globose to subglobose, immersed in the periphery layer of ascostromata, with 250–500 µm wide × 450–550 µm high ostioles. Peridium 20–35 µm thick, composed of several layers of hyaline to dark brown, small cells of textura angularis to textura intricata. Hamathecium of interthecial, hyaline septate, branched pseudoparaphyses, 1–3 µm wide. Asci 660–800 × 45–55 µm (x̄ = 751.6 × 49.5 µm, n = 20), 8-spored, thick-walled, bitunicate, fissitunicate, cylindrical, short-pedicellate, with an ocular chamber. Ascospores 600–750 × 5.5–11 µm (x̄ = 728.8 × 9.1 µm, n = 20), spirally arranged in asci, filiform, hyaline, with 15–18 transverse septa, smooth-walled. Asexual morph: Undetermined.
CHINA, Yunnan, Dali, on living branches of Fargesia spathacea Franch, 13 May 2017, Dong-Qin Dai, DDQ00411 (HKAS102255, holotype), Ibid. (HMAS 290447, isotype), Ibid. DDQ00412 (HKAS102256), Ibid. DDQ00416 (HKAS102260), Ibid. 20 June 2017, Dong-Qin Dai, DDQ00425 (HKAS102268), Ibid. DDQ00426 (HKAS102269), Ibid. DDQ00427 (HKAS102270), Ibid. DDQ00428 (HKAS102271), Ibid. DDQ00429 (HKAS102272), Ibid. DDQ00430 (HKAS102273), Ibid. DDQ00431 (HKAS102274).
1 | Parasitising bamboo branches, ascostromata are white to reddish | 2 |
– | Parasitising Rosaceae branches, ascostromata are black | Grandigallia |
2 | Ascospores muriform | Shiraia |
– | Ascospores filiform | Rubroshiraia |
Since the familial placement of H. bambusae is controversial in different studies (
Hypocrea bambusae Berk. & Broome, 1873
Parasitic on living inflorescence of bamboo. Sexual morph: Stromata around 0.14 cm diam., 0.06 cm high, solitary, superficial, subglobose, fleshy to coriaceous, black, with around 20 perithecia lining the periphery, with ostioles slightly raised above stroma surface. Stromatic tissue thick, brown to dark brown. Perithecia in vertical section around 100 µm diam., 200 µm high, pyriform, immersed in the periphery layer of stromata. Asci more than 220 µm long, 5–6 µm diam., 8-spored, unitunicate, cylindrical, with a glassy refractive cap around 3 µm from apex to base. Ascospores around 180 µm long, 1–1.5 µm diam., in a single fascicle but not significantly helically coiled, filiform, hyaline, with 9–10 transverse septa, with rounded ends, smooth-walled. Asexual morph: Undetermined.
Hypocrella bambusae (K(M)52469, isotype, images are accredited to the Royal Botanic Gardens, Kew) A, C fruiting bodies on inflorescence of bamboo B vertical section of stromata showing the perithecia locating D herbarium envelope E filiform ascospores F asci with caps (Staining by cotton blue). Scale bars: 5 mm (A), 200 μm (B), 2 mm (C), 20 μm (E, F).
SRI LANKA, on inflorescence of bamboo, January 1855, G.H.K. Thwaites s.n. (ex herb. M.J. Berkeley), K(M)52469, isotype.
This taxon has typical morphology of the Clavicipitaceae, which is pyriform perithecia with a gradually tapering upper part and cylindrical asci with a glassy refractive cap. New collections are required and need to be sequenced to clarify its placement.
Members of the family Shiraiaceae are distributed from Asia to South America but so far reported only from three countries, viz. China, Japan and Venezuela (
Stromatal methanol extracts of Rubroshiraia and Shiraia contain Hypocrellins (Fig.
Shiraia bambusicola has been used as a Chinese traditional folk-medicine, in curing rheumatoid arthritis, infantile convulsion and pertussis etc. for more than 400 years, because of its stromatal metabolites (
Hypocrellin has wide application prospects, but it was earlier only found existing in ascostromata of S. bambusicola and “Zhuhongjun” (R. bambusae in this paper) (
According to
This work was supported by the Key Laboratory of Yunnan Province Universities of the Diversity and Ecological Adaptive Evolution for Animals and plants on Yun-Gui Plateau, the National Natural Science Foundation of China (No. NSFC 31760013, 31950410558, 31260087, 31460561, 31860005, 31460179 and 31860057) and the Scientific Research Foundation of Yunnan Provincial Department of Education (2017ZZX186). Dong-Qin Dai would like to thank Yunnan Province Universities of the Science and Technology Innovation Team for the exploitation and utilisation of endophytes and the Thousand Talents Plan, Youth Project of Yunnan Provinces for support. Chao Liu thanks the Yunnan Local Colleges Applied Basic Research Projects (2017FH001-034). Dong-Qin Dai would like to thank Xiu Gao (Qujing Normal University) for the help with drawing chemical structures of hypocrellins and is grateful to Dr. Joanne E. Taylor for the help of the loaned herbarium.