VOLUME 4 DECEMBER 2019 Fungal Systematics and Evolution PAGES 43–57 doi.org/10.3114/fuse.2019.04.05 Setophoma spp. on Camellia sinensis F. Liu1, J. Wang1,2, H. Li3, W. Wang4, L. Cai1, 2* 1State key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China 2College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China 3College of Life Sciences, Hebei University, Baoding, Hebei Province, 071002, China 4Shandong Hetian Wang Biological Technology Co., Ltd., WeiFang, 261300, China *Corresponding author: [email protected] Key words: Abstract: During our investigation of Camellia sinensis diseases (2013–2018), a new leaf spot disease was found in seven five new taxa provinces of China (Anhui, Fujian, Guangxi, Guizhou, Jiangxi, Tibet and Yunnan), occurring on both arboreal and terraced fungal pathogen tea plants. The leaf spots were round to irregular, brown to dark brown, with grey or tangerine margins. Multi-locus (LSU, phylogeny ITS, gapdh, tef-1α, tub2) phylogenetic analyses combined with morphological observations revealed four new species taxonomy belonging to the genus Setophoma, i.e. S. antiqua, S. longinqua, S. yingyisheniae and S. yunnanensis. Of these four species, tea plants S. yingyisheniae was found to be present on diseased terraced tea plants in six of the seven sampled provinces (excluding Yunnan). The other three species only occurred on arboreal tea plants in Yunnan Province. In addition to the four species isolated from diseased leaves, S. endophytica sp. nov. was isolated from healthy leaves of terraced tea plants. Effectively published online: 15 May 2019. INTRODUCTION morphological comparison, host association and geographical Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. distribution. E-mail: [email protected] During our investigation of diseases of tea plants (Camellia sinensis) cultivated in China in 2013, a new leaf spot disease was found to cause severe yield losses in Guangxi Province. The MATERIALS AND METHODS associated leaf spots were circular to irregular, brown to dark brown in colour, with grey or tangerine margins. A subsequent Isolates collection effort of similarly affected tea plant leaves has been ongoing in many tea plantations located in six provinces of China, Isolates were obtained from either diseased or healthy tea i.e. Anhui, Fujian, Guizhou, Jiangxi, Tibet and Yunnan. Preliminary plant tissues collected from 18 locations in seven provinces of morphological observations and molecular analyses identified China, following the single spore isolation and tissue isolation the associated fungi as Setophoma spp. To our knowledge, this methods described in Liu et al. (2015). Type specimens of new is the first report of Setophoma on tea plants. Considering the species were deposited in the Mycological Herbarium of the potential commercial yield losses in tea plantations and the Institute of Microbiology, Chinese Academy of Sciences, Beijing, limited knowledge of this disease, accurate identification of the China (HMAS), with the ex-type living cultures being deposited causal organisms is of great importance. in the China General Microbiological Culture Collection Center The genus Setophoma (Phaeosphaeriaceae) was introduced (CGMCC). to accommodate Phoma terrestris and Pyrenochaeta sacchari (de Gruyter et al. 2010). Species of Setophoma are characterised DNA extraction, PCR amplification and phylogenetic as having setose pycnidia, phialidic conidiogenous cells and analyses hyaline, ellipsoidal to subcylindrical, aseptate conidia (de Gruyter et al. 2010, Quaedvlieg et al. 2013). According to Index Total genomic DNA was extracted from fresh mycelia using Fungorum and MycoBank, four additional Setophoma species the CTAB method. Five partial loci including the large subunit have been described since the genus was introduced in 2010. of the nrDNA (LSU), the 5.8S nuclear ribosomal RNA gene with The currently recognised species are: S. chromolaenae, S. the two flanking internally transcribed spacer regions (ITS), cyperi, S. poaceicola, S. sacchari, S. terrestris, and S. vernoniae. partial glyceraldehyde-3-phosphate dehydrogenase (gapdh), All except S. terrestris are reported to occur on unique host translation elongation factor 1-alpha (tef-1α) and β-tubulin plants (Table 1). (tub2) were amplified and sequenced using the following primer The aim of the present study was to investigate the taxonomic pairs: ITS1/ITS4 for ITS (White et al. 1990), LR0R/LR5 for LSU and phylogenetic relationships of Setophoma spp. associated (Vilgalys & Hester 1990, Rehner & Samuels 1994), gpd1/gpd2 for with tea plants based on multi-locus phylogenetic analyses, gapdh (Berbee et al. 1999), EF-1/EF-2 for tef-1α (O’Donnell et al. Fungal Systematics and Evolution is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License © 2019 Westerdijk Fungal Biodiversity Institute 43 Liu et al. Table 1. Host and distribution of Setophoma species. Species Host Distribution References S. antiqua Camellia sinensis China This study S. chromolaenae Chromolaena odorata Brazil Quaedvlieg et al. (2013) S. cyperi Cyperus sphaerocephalus South Africa, Eastern Cape Crous et al. (2016) S. endophytica Camellia sinensis China This study S. longinqua Camellia sinensis Editor-in-Chief China This study Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] S. poaceicola Grass Thailand Thambugala et al. (2017) S. sacchari Saccharum officinarum Brazil de Gruyter et al. (2010) S. terrestris Allium cepa North America, Senegal de Gruyter et al. (2010) Allium sativum United States de Gruyter et al. (2010) Brassica sp. Canada, Alberta Yang et al. (2017) Cucurbita maxima USA, Oregon Rivedal et al. (2018) Cucurbita moschata Japan Ikeda et al. (2012) Solanum lycopersicum Canada, Ontario Johnston-Monje et al. (2017) S. vernoniae Vernonia polyanthes Brazil Crous et al. (2014) S. yingyisheniae Camellia sinensis China This study S. yunnanensis Camellia sinensis China This study Table 2. GenBank accession numbers from NCBI database. Name Strain/specimen No.a LSU ITS Acericola italica MFLUCC 13-0609* MF167429 MF167428 Allophaeosphaeria muriformia MFLUCC 13-0349* KP765681 KP765680 Allophaeosphaeria subcylindrospora MFLUCC 13-0380* KT314183 KT314184 Amarenomyces ammophilae CBS 114595 GU301859 KF766146 Ampelomyces quisqualis CBS 129.79* EU754128 HQ108038 Bhatiellae rosae MFLUCC 17-0664* MG828989 MG828873 Chaetosphaeronema achilleae MFLUCC 16-0476 KX765266 KX765265 Chaetosphaeronema hispidulum CBS 216.75 KF251652 KF251148 Coniothyrium carteri CBS 105.91 KF251712 KF251209 Dactylidina dactylidis MFLUCC 14-0966* MG829002 MG828886 Dematiopleospora rosicola MFLU 16-0232* MG829006 MG828888 Dematiopleospora salsolae MFLUCC 17-0828* MG829007 MG828889 Didymocyrtis consimilis Gardiennet 12041 KT383796 KT383813 Didymocyrtis ramalinae Ertz 16399 KT383802 KT383838 Embarria clematidis MFLUCC 14-0976* MG828987 MG828871 Galiicola pseudophaeosphaeria MFLUCC 14-0524* KT326693 KT326692 Hawksworthiana alliariae MFLUCC 13-0070 KX494877 KX494876 Hawksworthiana clematidicola MFLUCC 14-0910* MG829011 MG828901 Italica achilleae MFLUCC 14-0959* MG829013 MG828903 Juncaceicola achilleae MFLUCC 13-0606* KX449526 KX449525 Juncaceicola luzulae MFLUCC 16-0780* KX449530 KX449529 Juncaceicola typharum CBS 296.54 KF251695 KF251192 Leptospora galii KUMCC 15-0521 KX599548 KX599547 Leptospora rubella CPC 11006 DQ195792 DQ195780 Muriphaeosphaeria galatellae MFLUCC 14-0614* KT438329 KT438333 Neosetophoma samarorum CBS 138.96* KF251664 KF251160 Neostagonospora caricis CBS 135092* KF251667 KF251163 Neostagonospora elegiae CBS 135101* KF251668 KF251164 44 © 2019 Westerdijk Fungal Biodiversity Institute Setophoma spp. on Camellia sinensis Table 2. (Continued). Name Strain/specimen No.a LSU ITS Neosulcatispora agaves CPC 26407* KT950867 KT950853 Nodulosphaeria hirta MFLUCC 13-0867 KU708845 KU708849 Nodulosphaeria scabiosae MFLUCC 14-1111* KU708846 KU708850 Ophiobolopsis italica MFLUCC 17-1791* MG520959 MG520939 Ophiobolus artemisiae MFLUCC 14-1156* KT315509 KT315508 Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] Ophiobolus disseminans MFLUCC 17-1787* MG520961 MG520941 Ophiosimulans tanaceti MFLUCC 14-0525 KU738891 KU738890 Paraophiobolus arundinis MFLUCC 17-1789* MG520965 MG520945 Paraophiobolus plantaginis MFLUCC 17-0245* KY815010 KY797641 Paraphoma chrysanthemicola CBS 172.70 KF251669 KF251165 Paraphoma radicina CBS 102875 KF251677 KF251173 Paraphoma rhaphiolepidis CBS 142524* KY979813 KY979758 Paraphoma vinacea UMPV001 = BRIP 63684 KU176888 KU176884 Parastagonospora caricis CBS 135671 KF251680 KF251176 Parastagonospora nodorum CBS 110109 KF251681 KF251177 Parastagonospora poagena CBS 136776* KJ869174 KJ869116 Parastagonosporella fallopiae CBS 135981* MH460545 MH460543 CCTU 1151.1 MH460546 MH460544 Phaeosphaeria oryzae CBS 110110* KF251689 KF251186 Phaeosphaeria papayae CBS 135416 KF251690 KF251187 Phaeosphaeriopsis glaucopunctata CBS 653.86 KF251702 KF251199 Poaceicola arundinis MFLU 15-0702* KU058726 KU058716 Poaceicola italica MFLUCC 13-0267* KX910094 KX926421 Populocrescentia forlicesenensis MFLU 15-0651* KT306952 KT306948 Pseudoophiobolus achilleae MFLU 17-0925* MG520966 MG520946 Pseudoophiobolus