Phytotaxa 233 (1): 027–048 www.mapress.com/phytotaxa/ Copyright © 2015 Magnolia Press ISSN 1179-3155 (print edition) Article PHYTOTAXA ISSN 1179-3163 (online edition) http://dx.doi.org/10.11646/phytotaxa.233.1.2 Taxonomy and phylogeny of Cercospora spp. from Northern Thailand JEERAPA NGUANHOM1, RATCHADAWAN CHEEWANGKOON1, JOHANNES Z. GROENEWALD2, UWE BRAUN3, CHAIWAT TO-ANUN1* & PEDRO W. CROUS2,4 1 Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, 50200, Thailand *email: chaiwat.toanun@gmail.com 2 CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands 3 Martin-Luther-Universität, Institut für Biologie, Bereich Geobotanik und Botanischer Garten, Herbarium, Neuwerk 21, 06099 Halle (Saale), Germany 4 Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria 0002, South Africa Abstract The genus Cercospora represents a group of important plant pathogenic fungi with a wide geographic distribution, being commonly associated with leaf spots on a broad range of plant hosts. The goal of the present study was to conduct a morphological and molecular phylogenetic analysis of the Cercospora spp. occurring on various plants growing in Northern Thailand, an area with a tropical savannah climate, and a rich diversity of vascular plants. Sixty Cercospora isolates were collected from 29 host species (representing 16 plant families). Partial nucleotide sequence data for two gene loci (ITS and cmdA), were generated for all isolates. Results from this study indicate that members of the genus Cercospora vary regarding host specificity, with some taxa having wide host ranges, and others being host-specific. Based on cultural, morphological and phylogenetic data, four new species of Cercospora could be identified: C. glycinicola (from Glycine max), C. cyperacearum and C. cyperina (from Cyperus alternifolius), and C. musigena (from Musa sp.). The most common Cercospora sp. found in Northern Thailand was C. cf. malloti, which occurred on a wide host range. Several collections could not be resolved to species level due to the lack of reference cultures and DNA data for morphologically similar species. Further collections from other countries are needed to help resolve the taxonomy of some species complexes occurring on various plant hosts in Thailand. Key words: biodiversity, cercosporoid hyphomycetes, Mycosphaerellaceae, phylogeny Introduction Species of Cercospora (Mycosphaerellaceae, Capnodiales) commonly occur associated with leaf and fruit spots on a range of cultivated and wild plants worldwide (Crous & Braun 2003, Groenewald et al. 2013, Amaradasa et al. 2014, Bakhshi et al. 2015b). To date there have been several studies focused on these fungi in Thailand, and more than 500 cercosporoid species have been identified (Giatgong 1980, Sontirat et al. 1980, Petcharat & Kanjanamaneesathian 1989, Braun et al. 2006, Meeboon et al. 2007a, 2007b, 2007c, 2008, Nakashima et al. 2007, Phengsintham et al. 2013). However, almost all these studies have thus far relied exclusively on morphological data, and very few records are supported by cultures and DNA data. The first application of DNA phylogenetic analysis (ITS) to distinguish Cercospora species from Thailand was published by To-anun et al. (2010, 2011). In other studies multi-locus DNA data proved highly effective to distinguish among species of cercosporoid fungi (Groenewald et al. 2013, Crous et al. 2013, Bakhshi et al. 2015a, 2015b). The same approach also proved successful to study other, related, cercosporoid genera from Thailand (Hunter et al. 2006, Cheewangkoon et al. 2008). To date, however, most cercosporoid records from Thailand cannot be substantiated based on a lack of cultures and DNA data. The main objective of the present study was therefore to confirm the identification of different Cercospora spp. associated with various plant diseases from Northern Thailand, and to resolve their taxonomy and DNA phylogeny. Accepted by Eric McKenzie: 24 Aug. 2015; published: 30 Oct. 2015 Licensed under a Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0 27 Materials and methods Isolates Specimens with disease symptoms were collected in the field and taken to the laboratory for fungal isolation. Leaves were examined directly using a dissecting microscope to observe Cercospora conidiophore fascicles, or when insufficiently developed, incubated in moist chambers for 1–2 d to induce sporulation. Single conidium colonies were established on Petri dishes containing 2% malt extract agar (MEA) as described by Crous et al. (2009). Reference strains are maintained at the working collection of P.W. Crous (CPC), with representative isolates deposited in the CBS-KNAW Fungal Biodiversity Centre (CBS), Utrecht, The Netherlands (Table 1). DNA extraction, amplification and sequencing Genomic DNA was extracted from fungal mycelium growing on MEA, placed in a 2-ml Eppendorf tube with 600 μl hexadecyltrimethyl ammonium bromide (CTAB) extraction buffer (500 μl of TES Buffer (100 mM Tris pH 8; 10 mM EDTA pH 8; 2% SDS), 140 μl of 5 M NaCl and 65 μl of 10% CTAB solution) and mixed well (protocol modified from Möller et al. 1992). To break the cells, the tube was placed in a boiling water bath for 3 min, after which it was chilled directly on ice for 10 min. Four hundred microliters of chloroform:isoamyl alcohol (24:1) were added and mixed properly by inversion, and centrifuged at 14,000 rpm for 5 min at room temperature to separate the phases. The upper phase was carefully collected and transferred to a new 2 ml tube. An equal volume of cold 5 M ammonium acetate was added and the gDNA precipitated with 600 μl of cold isopropanol and inverted. After 15 min incubation on ice, the solution was centrifuged at 14,000 rpm for 5 min and the supernatant discarded. The pellet was washed with 70% ethanol, air-dried and resuspended in 100 μl of TE buffer. All isolates were sequenced for two genomic loci, namely the internal transcribed spacer region with intervening 5.8S nrRNA gene (ITS) and partial calmodulin gene (cmdA). The primer ITS5 (White et al. 1990) or V9G (de Hoog & Gerrits van den Ende 1998) and ITS4 (White et al. 1990) were used to amplify the ITS and the primer set CAL-228F and CAL-737R (Carbone & Kohn 1999) or CAL2Rd (Groenewald et al. 2013) for cmdA. The reaction mixture had a total volume of 12.5 μl containing 1 μl diluted DNA, 1× PCR buffer, 2 mM MgCl2, 25 μM of each dNTPs, 1 μM of each primer, and 0.5 U Taq DNA polymerase (GoTaq, Promega). The amplification reactions were done on a 2720 Thermal Cycler (Applied Biosystems). PCR amplification conditions for ITS were set as follows: an initial denaturation temperature of 94°C for 5 min, followed by 35 cycles of denaturation temperature of 94°C for 45 s, primer annealing at 48°C for 45 s, primer extension at 72°C for 2 min and a final extension step at 72°C for 7 min. PCR amplification conditions for cmdA were set as follows: an initial denaturation temperature of 94°C for 3 min, followed by 35 cycles of denaturation temperature of 94°C for 30 s, primer annealing at 58°C for 40 s, primer extension at 72°C for 50 s and a final extension step at 72°C for 5 min. The PCR products were separated by electrophoresis at 100 V for 30 min on a 1% (w/v) agarose gel stained with GelRed in 1 × TAE buffer (0.4 M Tris, 0.05 M NaAc, and 0.01 M EDTA, pH 7.85) and visualized under UV light. The resulting fragments were sequenced in both directions with the various PCR primers using a BigDye® Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems, Foster City, CA) and analysed on an ABI Prism 3100 DNA Sequencer (Perkin-Elmer, Norwalk, CN). Phylogenetic analyses A consensus sequence was computed from the forward and reverse sequences using SeqMan from the Lasergene package (DNASTAR, Madison, Wisconsin). The consensus sequence was added to the alignment using MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html; Katoh & Standley 2013) and manually improved in MEGA v. 5 (Tamura et al. 2011). MrModeltest v. 2.3 (Nylander 2004) was used to determine the best nucleotide substitution model setting for each locus. The phylogenetic analyses of sequence data were performed in MrBayes v. 3.2.1 (Ronquist et al. 2012). The optimal substitution model for each locus, as recommended by MrModeltest, was implemented. The heating parameter was set at 0.3 and the Markov Chain Monte Carlo (MCMC) analysis of four chains was started in parallel from a random tree topology and lasted until the average standard deviation of split frequencies reached 0.01. Trees were saved each 1,000 generations and the resulting phylogenetic tree was printed with Geneious v. 5.5.4 (Drummond et al. 2011). New sequences generated in this study were submitted to GenBank (accession numbers listed in Table 1) and the alignment and phylogenetic tree to TreeBASE (ID 17818; www.treebase.org). 28 • Phytotaxa 233 (1) © 2015 Magnolia Press NGUANHOM ET AL. TAXONOMY AND PHYLOGENY OF CERCOSPORA TABLE 1. Names, accession numbers and collection details of isolates studied. Species and Culture accession number(s)1 Host name and family or isolation source Country Collector(s) GenBank accession numbers (ITS, cmdA)2 Cercospora agavicola CBS 117292; CPC 11774 (ex-type) Agave tequilana var. azul (Agavaceae) Mexico: Penjamo V. Ayala-Escobar & Ma. de Jesús Yáñez-Morales AY647237; AY966899 Althaea rosea (Malvaceae) Romania: Fundulea O. Constantinescu JX143530; JX142792 CBS 132683; CPC 16663 Moluccella laevis (Lamiaceae) Zimbabwe S. Dimbi JX143531; JX142793 CBS 252.67; CPC 5084 Plantago lanceolata (Plantaginaceae) Romania: Domnesti O. Constantinescu DQ233318; DQ233394 CPC 5260 Glebionis coronaria (≡ Chrysanthemum coronarium, Asteraceae) New Zealand: Auckland C.F. Hill JX143533; JX142795 CPC 23816 Apium graveolens (Apiaceae) Thailand: Mae Wang K. Wongsopa KT193650; KT193710 CPC 24837 — Thailand S. Seekanha KT193651; KT193711 Apium sp. (Apiaceae) Venezuela: Caripe N. Pons AY840536; AY840434 CBS 250.67; CPC 5088 (ex-type) Armoracia rusticana (= A. lapathifolia, Brassicaceae) Romania: Fundulea O. Constantinescu JX143545; JX142807 CBS 555.71; IMI 161117; CPC 5082 Coronilla varia (Fabaceae) Romania: Hagieni O. Constantinescu JX143550; JX142812 Beta vulgaris (Chenopodiaceae) Germany S. Mittler AY840526; AY840424 Cercospora althaeina CBS 248.67; CPC 5117 (ex-type) Cercospora apii Cercospora apii complex Phytotaxa 233 (1) © 2015 Magnolia Press • 29 Cercospora apiicola CBS 116457; CPC 10267 (ex-type) Cercospora armoraciae Cercospora beticola CBS 116454; CPC 11558 ...Continued on next page 30 • Phytotaxa 233 (1) © 2015 Magnolia Press TABLE 1. (Continued) Species and Culture accession number(s)1 Host name and family or isolation source Country Collector(s) GenBank accession numbers (ITS, cmdA)2 CBS 116456; CPC 11557 (ex-type) Beta vulgaris (Chenopodiaceae) Italy: Ravenna V. Rossi AY840527; AY840425 CBS 117.47 Beta vulgaris (Chenopodiaceae) Czech Republic G.E. Bunschoten DQ233322; DQ233400 CPC 23815 Beta vulgaris (Chenopodiaceae) Thailand: Chiang Mai K. Wongsopa KT193652; KT193712 CPC 24825 Apium graveolens (Apiaceae) Thailand: Mae Rim S. Seekanha KT193653; KT193713 Geranium thunbergii (≡ G. nepalense var. thunbergii, Geraniaceae) South Korea: Namyangju H.D. Shin JX143559; JX142821 CBS 118712 Lesions on calyx attached to fruit Fiji P. Tyler GU214653; JX142830 MUCC 574; MUCNS 810; MAFF 238227 Capsicum annuum (Solanaceae) Japan: Chiba S. Uematsu JX143569; JX142833 CPC 22000 Capsicum frutescens (Solanaceae) Thailand: San Sai J. Nguanhom KT193654; KT193714 CPC 22007 Capsicum frutescens (Solanaceae) Thailand: Mae Klang Loung J. Nguanhom KT193655; KT193715 CPC 22008 Capsicum frutescens (Solanaceae) Thailand: Chiang Dao J. Nguanhom KT193656; KT193716 CPC 22009 Capsicum frutescens (Solanaceae) Thailand: Li, Lamphun J. Nguanhom KT193657; KT193717 CPC 22011 Capsicum frutescens (Solanaceae) Thailand: Li, Lamphun J. Nguanhom KT193658; KT193718 CPC 22012 Capsicum frutescens (Solanaceae) Thailand: Li, Lamphun J. Nguanhom KT193659; KT193719 CPC 22013 Capsicum frutescens (Solanaceae) Thailand: Li, Lamphun J. Nguanhom KT193660; KT193720 CPC 22015 Capsicum annuum var. acuminatum (Solanaceae) Thailand: Kalayaniwattana J. Nguanhom KT193661; KT193721 Celosia argentea var. cristata (≡ C. cristata, Amaranthaceae) South Korea: Chuncheon H.D. Shin JX143570; JX142834 Cercospora cf. brunkii CBS 132657; CPC 11598 Cercospora capsici Cercospora celosiae NGUANHOM ET AL. CBS 132600; CPC 10660 Cercospora chenopodii ...Continued on next page TAXONOMY AND PHYLOGENY OF CERCOSPORA TABLE 1. (Continued) Species and Culture accession number(s)1 Host name and family or isolation source Country Collector(s) GenBank accession numbers (ITS, cmdA)2 Chenopodium cf. album (Chenopodiaceae) France: Ardeche P.W. Crous JX143571; JX142835 CBS 119395; CPC 12682 Musa sp. (Musaceae) Bangladesh: Western I. Buddenhagen EU514222; JX142843 CBS 132669; CPC 12683 Musa sp. (Musaceae) Bangladesh: Western I. Buddenhagen EU514223; JX142844 CPC 20714 Cyathula prostrata (Amaranthaceae) Thailand: Hang Dong J. Nguanhom KT193662; KT193722 CPC 20740 Momordica charantia (Cucurbitaceae) Thailand: Ban Ti, Lamphun J. Nguanhom KT193663; KT193723 CPC 23937 Coccinia grandis (Cucurbitaceae) Thailand: Doi Saket J. Nguanhom KT193664; KT193724 CPC 24842 Coccinia grandis (Cucurbitaceae) Thailand: Chiang Mai S. Seekanha KT193665; KT193725 MUCC 576; MUCNS 300; MAFF 237913 Citrullus lanatus (Cucurbitaceae) Japan: Okinawa T. Kobayashion et al. JX143579; JX142845 MUCC 577; MUCNS 254; MAFF 238205 Momordica charantia (Cucurbitaceae) Japan: Kagoshima E. Imaizumi & C. Nomi JX143580; JX142846 Corchorus olitorius (Tiliaceae) Japan: Shimane T. Mikami JX143584; JX142850 CPC 22014 Solanum mammosum (Solanaceae) Thailand: Li, Lamphun J. Nguanhom KT193666; KT193726 CPC 23918 (ex-type) Cyperus alternifolius (Cyperaceae) Thailand: Kun Chang Kien S. Seekanha KT193667; KT193727 CPC 24811 — Thailand S. Seekanha KT193668; KT193728 Cyperus alternifolius (Cyperaceae) Thailand: Kun Chang Kien S. Seekanha KT193669; KT193729 Delairea odorata (= Senecio mikanioides, Asteraceae) South Africa: Long Tom Pass S. Neser JX143587; JX142853 Euphorbia sieboldiana (Euphorbiaceae) South Korea: Samcheok H.D. Shin JX143593; JX142859 CBS 132620; CPC 14237 Cercospora cf. citrulina Cercospora corchori MUCC 585; MUCNS 72; MAFF 238191 (extype) Cercospora cyperacearum Phytotaxa 233 (1) © 2015 Magnolia Press • 31 Cercospora cyperina CPC 23919 (ex-type) Cercospora delaireae CBS 132595; CPC 10455; GV2 PPRI number: C558 (ex-type) Cercospora euphorbiae-sieboldianae CBS 113306 (ex-type) ...Continued on next page 32 • Phytotaxa 233 (1) © 2015 Magnolia Press TABLE 1. (Continued) Species and Culture accession number(s)1 Host name and family or isolation source Country Collector(s) GenBank accession numbers (ITS, cmdA)2 Fagopyrum esculentum (Polygonaceae) South Korea: Yangpyeong H.D. Shin JX143594; JX142860 CBS 113127; RC3766; TX-18 Eichhornia crassipes (Pontederiaceae) USA: Texas D. Tessmann & R. Charudattan DQ835075; DQ835148 CBS 132637; CPC 10079 Trachelium sp. (Campanulaceae) Israel E. Tzul-Abad JX143600; JX142866 CPC 23911 (ex-type) Glycine max (Fabaceae) Thailand: Mae Hia J. Nguanhom KT193670; KT193730 CPC 23912 Glycine max (Fabaceae) Thailand: Mae Hia J. Nguanhom KT193671; KT193731 Helianthus tuberosus (Asteraceae) Japan: Wakayama C. Nakashima & I. Araki JX143615; JX142882 CBS 132639; CPC 10102 Persicaria thunbergii (Polygonaceae) South Korea: Pocheon H.D. Shin JX143616; JX142883 CBS 132652; CPC 10833 Ipomoea nil (= I. hederacea, Convolvulaceae) South Korea: Chuncheon H.D. Shin JX143617; JX142884 Glycine soja (Fabaceae) Japan T. Matsumoto DQ835070; DQ835134 CBS 132604; CPC 10728 Ixeris chinensis subsp. strigosa (≡ Ixeris strigosa, Asteraceae) South Korea: Chuncheon H.D. Shin JX143621; JX142888 CPC 10082 Ixeris chinensis subsp. strigosa (≡ Ixeris strigosa, Asteraceae) South Korea: Chuncheon H.D. Shin JX143622; JX142889 CPC 20719 Lactuca sativa var. longifolia (Asteraceae) Thailand: Chiang Mai J. Nguanhom KT193672; KT193732 CPC 23817 Lactuca sativa (Asteraceae) Thailand: Chiang Mai K. Wongsopa KT193673; KT193733 CPC 23818 Lactuca sativa (Asteraceae) Thailand: Chiang Mai K. Wongsopa KT193674; KT193734 Cercospora fagopyri CBS 132623; CPC 14541 (ex-type) Cercospora cf. flagellaris Cercospora glycinicola Cercospora cf. helianthicola MUCC 716 Cercospora cf. ipomoeae Cercospora kikuchii CBS 128.27; CPC 5068 (ex-type) Cercospora lactucae-sativae NGUANHOM ET AL. ...Continued on next page TAXONOMY AND PHYLOGENY OF CERCOSPORA TABLE 1. (Continued) Species and Culture accession number(s)1 Host name and family or isolation source Country Collector(s) GenBank accession numbers (ITS, cmdA)2 Phytotaxa 233 (1) © 2015 Magnolia Press • 33 CPC 23819 Lactuca sativa (Asteraceae) Thailand: Chiang Mai K. Wongsopa KT193675; KT193735 CPC 24838 — Thailand: Chiang Mai S. Seekanha KT193676; KT193736 MUCC 570; MUCNS 463; MAFF 238209 Lactuca sativa (Asteraceae) Japan: Chiba C. Nakashima JX143623; JX142890 CPC 20729 Melampodium divaricatum (Asteraceae) Thailand: Mae On J. Nguanhom KT193677; KT193737 CPC 20737 Asystasia salicifolia (Acanthaceae) Thailand: Mae Hia J. Nguanhom KT193678; KT193738 CPC 22010 Physalis peruviana (Solanaceae) Thailand: Li, Lamphun J. Nguanhom KT193679; KT193739 CPC 22023 Nicotiana tabacum (Solanaceae) Thailand: Phu Phing Palace J. Nguanhom KT193680; KT193740 CPC 22024 Phlox drummondii (Polemoniaceae) Thailand: Phu Phing Palace J. Nguanhom KT193681; KT193741 CPC 23821 Brassica alboglabra (Brassicaceae) Thailand: Chiang Mai K. Wongsopa KT193682; KT193742 CPC 23826 Codiaeum variegatum (Euphorbiaceae) Thailand: Chiang Rai K. Wongsopa KT193683; KT193743 CPC 23828 Jatropha integerrima (Euphorbiaceae) Thailand: Chiang Mai K. Wongsopa KT193684; KT193744 CPC 23834 Abelmoschus esculentus (Malvaceae) Thailand: Chiang Mai K. Wongsopa KT193685; KT193745 CPC 23835 Abelmoschus esculentus (Malvaceae) Thailand: Chiang Mai K. Wongsopa KT193686; KT193746 CPC 23920 Plantago major (Plantaginaceae) Thailand: Kun Chang Kien J. Nguanhom KT193687; KT193747 CPC 24820 Eupatorium odoratum (Asteraceae) Thailand: Mae Hia S. Seekanha KT193688; KT193748 CPC 24822 — Thailand: Suthep-Pui S. Seekanha KT193689; KT193749 CPC 24827 Musa sapientum (Musaceae) Thailand: Sa Moeng S. Seekanha KT193690; KT193750 CPC 24828 Musa sapientum (Musaceae) Thailand: Sa Moeng S. Seekanha KT193691; KT193751 CPC 24845 — Thailand S. Seekanha KT193692; KT193752 MUCC 575; MUCNS 582; MAFF 237872 Cucumis melo (Cucurbitaceae) Japan: Okinawa K. Uehara JX143625; JX142892 MUCC 787 Mallotus japonicus (Euphorbiaceae) Japan: Okinawa C. Nakashima & T. Akashi JX143626; JX142893 Cercospora cf. malloti ...Continued on next page 34 • Phytotaxa 233 (1) © 2015 Magnolia Press TABLE 1. (Continued) Species and Culture accession number(s)1 Host name and family or isolation source Country Collector(s) GenBank accession numbers (ITS, cmdA)2 Cercospora mercurialis CBS 550.71 (ex-type) Mercurialis perennis (Euphorbiaceae) Romania: Cheia O. Constantinescu JX143628; JX142895 CPC 20741 Mikania cordata (Asteraceae) Thailand: Mae Hia J. Nguanhom KT193693; KT193753 CPC 22030 Mikania cordata (Asteraceae) Thailand: Lamphun J. Nguanhom KT193694; KT193754 CPC 22031 Mikania cordata (Asteraceae) Thailand: Lamphun J. Nguanhom KT193695; KT193755 CPC 23908 Mikania cordata (Asteraceae) Thailand: Sa Moeng J. Nguanhom KT193696; KT193756 CPC 23909 Mikania cordata (Asteraceae) Thailand: Sa Moeng J. Nguanhom KT193697; KT193757 CPC 24809 (ex-type) Musa sp. (Musaceae) Thailand: Fang, Chiang Mai S. Seekanha KT193698; KT193758 CPC 24831 Musa sp. (Musaceae) Thailand: Fang, Chiang Mai S. Seekanha KT193699; KT193759 CBS 131.32; CPC 5076 Nicotiana tabacum (Solanaceae) Indonesia: Medan H. Diddens & A. Jaarsveld DQ835073; DQ835146 CBS 132632; CPC 15918 Glycine max (Fabaceae) Mexico: Tamaulipas Ma. de Jesús Yáñez-Morales JX143631; JX142898 CBS 570.69; CPC 5075 Nicotiana tabacum (Solanaceae) Nigeria S.O. Alasoadura DQ835074; DQ835147 CPC 20715 Nicotiana tabacum (Solanaceae) Thailand: Mae Tang J. Nguanhom KT193700; KT193760 CPC 20730 Houttuynia cordata (Saururaceae) Thailand: Mae Hia J. Nguanhom KT193701; KT193761 CPC 22006 Nicotiana tabacum (Solanaceae) Thailand: Wiang Pa Pao J. Nguanhom KT193702; KT193762 CPC 22019 Petunia hybrida (Solanaceae) Thailand: Chiang Mai Univ. J. Nguanhom KT193703; KT193763 Aristolochia clematitis (Aristolochiaceae) Romania: Cazanele Dunarii O. Constantinescu JX143632; JX142899 Cercospora cf. mikaniicola Cercospora musigena Cercospora cf. nicotianae NGUANHOM ET AL. Cercospora olivascens CBS 253.67; IMI 124975; CPC 5085 (ex-type) ...Continued on next page TAXONOMY AND PHYLOGENY OF CERCOSPORA TABLE 1. (Continued) Species and Culture accession number(s)1 Host name and family or isolation source Country Collector(s) GenBank accession numbers (ITS, cmdA)2 Cercospora cf. physalidis CBS 765.79 Solanum tuberosum (Solanaceae) Peru L.J. Turkensteen JX143633; JX142900 Pilea pumila (= P. mongolica, Urticaceae) South Korea: Dongducheon H.D. Shin JX143634; JX142901 Persicaria longiseta (≡ P. blumei, Polygonaceae) South Korea: Cheongju H.D. Shin JX143637; JX142904 Cynanchum wilfordii (Asclepiadaceae) South Korea: Bonghwa H.D. Shin JX143638; JX142905 CBS 118793 Reseda odorata (Resedaceae) New Zealand: Auckland C.F. Hill JX143639; JX142906 CBS 257.67; CPC 5057 Helianthemum sp. (Cistaceae) Romania: Bucuresti O. Constantinescu DQ233319; DQ233395 Rumex sanguineus (Polygonaceae) New Zealand: Manurewa C.F. Hill JX143648; JX142915 Senecio walkeri (Asteraceae) Laos P. Phengsintham JX143649; JX142916 Glycine soja (Fabaceae) South Korea: Hongcheon H.D. Shin JX143659; JX142927 CPC 23905 Crassocephalum crepidioides (Asteraceae) Thailand: Hang Dong J. Nguanhom KT193704; KT193764 CPC 23906 Crassocephalum crepidioides (Asteraceae) Thailand: Hang Dong J. Nguanhom KT193705; KT193765 Zea mays (Poaceae) South Africa P. Caldwell DQ185071; DQ185107 Cercospora pileicola CBS 132607; CPC 10749 (ex-type) Cercospora polygonacea CBS 132614; CPC 11318 Cercospora punctiformis CBS 132626; CPC 14606 Cercospora cf. resedae Cercospora rumicis CPC 5439 Phytotaxa 233 (1) © 2015 Magnolia Press • 35 Cercospora senecionis-walkeri CBS 132636; CPC 19196 Cercospora sojina CBS 132615; CPC 11353 (ex-type) Cercospora sp. Cercospora sp. F CBS 132618; CPC 12062 ...Continued on next page 36 • Phytotaxa 233 (1) © 2015 Magnolia Press TABLE 1. (Continued) Species and Culture accession number(s)1 Host name and family or isolation source Country Collector(s) GenBank accession numbers (ITS, cmdA)2 Myoporum laetum (Myoporaceae) New Zealand: Grey Lynn C.F. Hill JX143732; JX142999 Crepidiastrum denticulatum (≡ Youngia denticulata, Asteraceae) South Korea: Yangpyeong H.D. Shin JX143733; JX143000 Vigna unguiculata (= V. sinensis, Fabaceae) South Korea: Jeongeup H.D. Shin JX143734; JX143001 CBS 251.67; CPC 5079 (ex-type) Viola tricolor (Violaceae) Romania: Cazanele Dunarii O. Constantinescu JX143737; JX143004 CPC 5368 Viola odorata (Violaceae) New Zealand C.F. Hill JX143738; JX143005 MUCC 129 Viola sp. (Violaceae) Japan: Kochi J. Nishikawa JX143739; JX143006 CBS 132624; CPC 14549 Zinnia elegans (Asteraceae) South Korea: Yangpyeong H.D. Shin JX143756; JX143026 CBS 132676; CPC 15075 — Brazil: Valverde A.C. Alfenas JX143757; JX143027 CPC 22027 Zinnia elegans (Asteraceae) Thailand: Lamphun J. Nguanhom KT193706; KT193766 CPC 22040 Zinnia elegans (Asteraceae) Thailand: Doi Pui J. Nguanhom KT193707; KT193767 CPC 22041 Zinnia elegans (Asteraceae) Thailand: Doi Pui J. Nguanhom KT193708; KT193768 CPC 23910 Zinnia elegans (Asteraceae) Thailand: Sa Moeng J. Nguanhom KT193709; KT193769 MUCC 131 Zinnia elegans (Asteraceae) Japan: Shizuoka J. Nishikawa JX143758; JX143028 MUCC 572; MUCNS 215; MAFF 237718 Zinnia elegans (Asteraceae) Japan: Chiba S. Uematsu JX143759; JX143029 Cercospora sp. R CBS 114644 Cercospora sp. S CBS 132599; CPC 10656 Cercospora vignigena CBS 132611; CPC 10812 (ex-type) Cercospora violae Cercospora cf. zinniae NGUANHOM ET AL. 1 CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS; IMI: International Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, U.K.; MAFF: Ministry of Agticulture, Forestry and Fisheries, Tsukuba, Ibaraki, Japan; MUCC: Culture Collection, Laboratory of Plant Pathology, Mie University, Tsu, Mie Prefecture, Japan; MUCNS: Active cultures & specimens of Chiharu Nakashima, housed at Mie University; PPRI: Plant Protection Research Institute, Pretoria, South Africa. 2 ITS: internal transcribed spacers and intervening 5.8S nrDNA; cmdA: partial calmodulin gene. FIGURE 1. The Bayesian 50% majority rule consensus tree derived from the combined ITS/cmdA alignment. Bayesian posterior probabilities support values for the respective nodes are displayed in the tree. The scale bar indicates 0.01 expected changes per site and species are delimited by blocks of different colours. Strain accession numbers from Thailand and names of species containing Thai strains are printed in bold face. The tree was rooted to Septoria provencialis CPC 12226 (ITS GenBank DQ303096, cmdA GenBank JX143030). TAXONOMY AND PHYLOGENY OF CERCOSPORA Phytotaxa 233 (1) © 2015 Magnolia Press • 37 FIGURE 1. (Continued) The Bayesian 50% majority rule consensus tree derived from the combined ITS/cmdA alignment. Bayesian posterior probabilities support values for the respective nodes are displayed in the tree. The scale bar indicates 0.01 expected changes per site and species are delimited by blocks of different colours. Strain accession numbers from Thailand and names of species containing Thai strains are printed in bold face. The tree was rooted to Septoria provencialis CPC 12226 (ITS GenBank DQ303096, cmdA GenBank JX143030). 38 • Phytotaxa 233 (1) © 2015 Magnolia Press NGUANHOM ET AL. Taxonomy Morphological descriptions of Cercospora spp. were based on structures from herbarium material. Fungal structures were mounted in lactic acid and examined using a Nikon Eclipse 80i compound microscope (×1000), with 30 measurements taken for each structure, the 95% confidence intervals were determined, and extreme values given in parentheses. Colony colours on MEA, potato dextrose agar (PDA) and oatmeal agar (OA) (recipes according to Crous et al. 2009) were determined after 2 wk at 25°C in the dark in duplicate. The mycological colour charts of Rayner (1970) were used to define colours of the fungal colonies. Nomenclatural novelties and descriptions were deposited in MycoBank (www. MycoBank.org; Crous et al. 2004). The naming system employed by Groenewald et al. (2013) was used to simplify comparison between the studies. Results Phylogenetic analysis DNA data from the ITS and cmdA regions were combined in a MrBayes analysis. The sequence alignment consisted of 121 ingroup sequences and Septoria provencialis (CPC 12226) was used as outgroup. A combined dataset of a total of 731 characters was used in the phylogenetic analysis (470 and 261 characters for ITS and cmdA, respectively). Based on MrModeltest, a MrBayes analysis was conducted on the combined dataset using a symmetrical model (SYM) substitution model with equal rates. The dataset had fixed (equal) base frequencies implemented for ITS and had dirichlet base frequencies with gamma rates implemented for cmdA by using HKY+G model. A total of 731 characters were used for the Bayesian analysis; these contained 156 and 171 unique site patterns for ITS and cmdA, respectively. A total of 5,168 trees were saved, of which the last 3,876 were used to calculate the tree presented in Fig. 1. Taxonomy Several taxa collected in the present study were found to be morphologically and phylogenetically distinct from presently known species. The phylogenetic analyses based on the Bayesian analysis resolved a total of 15 Cercospora lineages from Thailand, with two clades representing undefined Cercospora species complexes (sensu Groenewald et al. 2013). The species representing novel taxa are treated below. Cercospora glycinicola Cheew., Crous & U. Braun, sp. nov. (Fig. 2). MycoBank MB812918 Type:—THAILAND. Chiang Mai: on Glycine max (Fabaceae), 29 Mar. 2013, S. Seekanha (holotype CBS H-22289, culture ex-type CPC 23911 = CBS 140164, CPC 23912). Leaf spots amphigenous, subcircular to irregular, pale brown, surrounded by a darker brown margin, 1−3 mm diam. Mycelium internal. Caespituli amphigenous, punctiform, brown. Stromata brown, intraepidermal or substomatal, 32.5−57.5 μm diam. Conidiophores in moderately dense fascicles (4−25), straight or sinuous to geniculate due to sympodial proliferation, unbranched, brown, paler toward the apex, 30−113 × 4−6 μm, 1−4-septate. Conidiogenous cells proliferating sympodially, integrated, terminal or conidiophores reduced to conidiogenous cells, 28−60 μm long; conidiogenous loci conspicuous, apical and formed on shoulders caused by geniculation, lateral, multi-local, loci distinctly thickened, darkened, 3–4 μm diam. Conidia solitary, obclavate-cylindrical, hyaline, obtuse at the apex, truncate to slightly obconically truncate at the base, 1−3-septate, 23−83 × 3−5 μm; hila thickened and darkened, 2.5–3 μm diam. Culture characteristics:—Colonies spreading, with moderate to dense, felty aerial mycelium, entire to undulate margin, reaching 42 mm diam after 2 wk. On MEA white, with somewhat greyish pink exudates. On PDA white to slightly grey, with diffuse red pigment in agar surrounding colony. On OA white to slightly grey, with diffuse red pigment in agar. Etymology:—Named after its Glycine-inhabiting habit. Notes:—Cercospora glycinicola is morphologically close to C. sojina (Shin & Kim 2001) but distinct in having shorter conidiophores (30–113 μm, versus 40–200 μm) and above all narrower conidia with few septa (3–5 μm wide, 1–3-septate, versus 4–8 μm wide, 3–7-septate). Phylogenetically it is also distinct from species presently known from DNA sequence data, including C. sojina. However, although these two isolates originate from the same lesion, they were morphologically different. Isolate CPC 23911 had more geniculate conidiophores, shorter conidiogenous cells TAXONOMY AND PHYLOGENY OF CERCOSPORA Phytotaxa 233 (1) © 2015 Magnolia Press • 39 (28−38 μm) and slightly shorter conidia (23−68 μm). In contrast, isolate CPC 23912 had straight conidiophores, longer conidiogenous cells (45−60 μm) and somewhat acicular conidia. However, phylogenetically the two isolates only differed via one nucleotide position in cmdA. FIGURE 2. Cercospora glycinicola (CBS H-22289). A. Leaf spot; B. Close-up of leaf spot; C, D. Conidiophores and conidiogenous cells; E−I. Conidia; J. Colony on MEA.—Scale bars: C−D = 40 μm; E−I = 50 μm. Cercospora cyperacearum Cheew., Crous & U. Braun, sp. nov. (Fig. 3). MycoBank MB812919 Type:—THAILAND. Chiang Mai: on leaves of Cyperus alternifolius (Cyperaceae), 12 May 2013, S. Seekanha (holotype CBS H-22290, culture ex-type CPC 23918 = CBS 140165). Other specimens examined:—THAILAND. Chiang Mai: on unknown monocot, 12 May 2013, S. Seekanha, CPC 24811; Lamphun on leaves of Solanum mammosum (Solanaceae), 9 Dec. 2010, J. Nguanhom, CPC 22014. Leaf spots amphigenous, pale brown to brown, margin indefinite, elongated to irregular. Caespituli amphigenous, punctiform, brown. Stromata substomatal to intraepidermal, brown, 38–63 μm high, 18–38 μm wide. Conidiophores fasciculate, pale olivaceous, paler and narrower towards the apex, unbranched, main portion straight, subcylindrical, only conidiogenous cells distinctly geniculate, 18−68 × 4−5 μm. Conidiogenous cells proliferating sympodially 5−9 times, integrated, terminal, 18–35 μm long, conidiogenous loci conspicuous, thickened and darkened, apical and lateral, circumspersed, 1−3 μm diam. Conidia solitary, hyaline, thin-walled, smooth, obclavate-cylindrical, subacute to acute at the apex, truncate at the base, 40−63 × 2−2.5 μm, indistinctly 1–6-septate, hila slightly thickened, darkened and refractive, 1−3 μm diam. Culture characteristics:—Colonies spreading, with dense aerial mycelium, reaching 40 mm diam after 2 wk. On MEA surface green-glaucous with pink pigment surrounding colony with undulate margins; reverse fucous-black. On PDA surface smoke grey with pink at the margin, entire margin; reverse fucous-black. On OA surface dense mycelium, with sparse entire margin, pure olivaceous-grey with diffuse livid red pigment surrounding colony; reverse dark vinaceous with lavender grey at the centre. Etymology:—Epithet derived from the host genus, Cyperus. 40 • Phytotaxa 233 (1) © 2015 Magnolia Press NGUANHOM ET AL. FIGURE 3. Cercospora cyperacearum (CBS H-22290). A. Leaf spot; B. Close-up of leaf spot; C, D. Conidiophores and conidiogenous cells; E−G. Conidia; H. Colony on MEA.—Scale bars: C−G = 40 μm. Notes:—Cercospora cyperacearum is undoubtedly plurivorous, as it is known from DNA sequences retrieved from unrelated hosts, including dicots and monocots, rendering a final conclusion impossible. The occurrence on additional hosts cannot be excluded and is probable, i.e., previously described Cercospora species might be involved. The Cercospora species described from Cyperus spp. are morphologically distinct (Braun et al. 2014). Cercospora cyperigena U. Braun & Crous has much shorter, 0–1-septate conidiophores, 5–20 × 2–5 μm, and C. cyperi Sawada has small stromata, 10–25 μm diam, smaller conidiogenous loci, (1–)1.5–2(–2.5) μm diam, and broader conidia, (2–)2.5– 5(–5.5) μm (Braun et al. 2014). Cercospora cyperacearum on Cyperus alternifolius in Thailand is characterised by forming large stromata and narrow conidia, and agrees well with the description of Cercospora ugandensis in Vasudeva (1963) based on Indian material on Cyperus sp., which is, however, not in agreement with the original description of this species that has been reduced to synonymy with C. cyperi in Braun et al. (2014). The characters of conidiophores and conidia of Cercospora spp. on Solanum are not in agreement with the material on Solanum mammosum. C. solanicola and C. melongenae are C. apii-like, i.e., with consistently acicular conidia, and were reduced to synonymy with C. physalidis s. lat. in Braun & Mel’nik (1997), which was considered to be part of the C. apii s. lat. complex in Crous & Braun (2003). These species are characterised by having long, pluriseptate conidiophores to 200 μm, and conidia to 300 × 2.5–5 μm. The conidiophores in C. solani agree well with those of C. cyperacearum but the lesions are indistinct and the acicular conidia are 3.5–6 μm wide (Chupp 1954; type material examined: Thüm., Mycoth. univ. 2070, HAL). C. solanigena (Bhartiya et al. 2000), described from India on Solanum melongena, resembles C. cyperacearum. However, the stromata are smaller, 10–30 μm diam, and the conidiophores are 16–100 × 3–5 μm, 1–6-septate, with conidia being cylindrical-obclavate to acicular (based on the original illustrations), 15–85 × 2–5 μm, 1–5-septate. TAXONOMY AND PHYLOGENY OF CERCOSPORA Phytotaxa 233 (1) © 2015 Magnolia Press • 41 Cercospora cyperina Nguanhom, Crous & U. Braun, sp. nov. (Fig. 4). MycoBank MB812920 Type:—THAILAND. Chiang Mai: on leaves of Cyperus alternifolius (Cyperaceae), 12 May 2013, S. Seekanha (holotype CBS H-22291, culture ex-type CPC 23919 = CBS 140166). FIGURE 4. Cercospora cyperina (CBS H-22291). A. Leaf spot; B. Close-up of leaf spot; C, D. Conidiophores and conidiogenous cells; E−H. Conidia; I. Colony on MEA.—Scale bars: C−D = 40 μm; E−H = 50 μm, and G applies to H. Leaf spots amphigenous, pale brown to brown, margin indefinite, elongated to irregular. Mycelium internal. Caespituli amphigenous, punctiform, brown. Stromata none or composed of 2−3 brown cells. Conidiophores loosely fasciculate, with fascicles of 2−9 conidiophores, brown, paler towards the apex, cylindrical to geniculate, somewhat constricted at septa, branched, 2−6-septate, 73−138 × 5−6 μm. Conidiogenous cells proliferating sympodially 1−6 times, integrated, terminal and intercalary, 20–38 μm long; conidiogenous loci conspicuous, thickened and darkened, multi-local, formed apical or on shoulders caused by proliferation, 2.5−4 μm diam. Conidia solitary, hyaline, thin-walled, smooth, acicular to somewhat obclavate, subobtuse at the apex, truncate to slightly obconically truncate at the base, 55−155 × 2.5−4 μm, indistinctly 15–20-septate, hila slightly thickened, darkened and refractive, 2.5–3 μm diam. Culture characteristics:—Colonies spreading, low convex, with sparse to moderate aerial mycelium, entire margin and folded surface, reaching 44 mm diam after 2 wk. On MEA surface whitish with patches of greyish rose; reverse olivaceous-black. On PDA surface whitish, pale grey at the centre with diffuse red pigment surrounding the colony; reverse olivaceous-black. On OA whitish with patches of pale grey, but red at the margin; reverse olivaceousgrey. Etymology:—Epithet derived from the host genus, Cyperus. Notes:—Cercospora cyperina is morphologically close to C. cyperi Sawada (Braun et al. 2014) but distinct by having longer, distinctly geniculate conidiophores with constrictions and much larger conidiogenous loci, 3–4 μm diam [conidiophores (10–)20–90 μm long, without constrictions, loci (1–)1.5–2(–2.5) μm diam in C. cyperi]. This taxon was also supported as a new species based on its distinct phylogenetic position. In the combined tree (Fig. 1), it is sister to C. cyperacearum and thus separate from other species occurring on Cyperus. 42 • Phytotaxa 233 (1) © 2015 Magnolia Press NGUANHOM ET AL. Cercospora musigena Nguanhom, Crous & U. Braun, sp. nov. (Fig. 5). MycoBank MB812921 Type:—THAILAND. Chiang Mai: on leaves of Musa sp. (Musaceae), 27 May 2013, S. Seekanha (holotype CBS H-22292, culture ex-type CPC 24809 = CBS 140167, CPC 24831). FIGURE 5. Cercospora musigena (CBS H-22292). A. Leaf spot; B, C. Conidiophores and conidiogenous cells; D−H. Conidia.—Scale bars: B−C = 50 μm; D−H = 30 μm. Leaf spots irregular, pale brown along the leaf margins, often surrounded by a yellow halo. Caespituli amphigenous, punctiform, brown. Stromata intraepidermal to subepidermal, brown, 30−45 μm diam. Conidiophores in moderately large fascicles (11−20 per fascicle), erumpent through the cuticle, brown, paler toward the apex, 3−5-septate, cylindrical, 1−3 times geniculate in upper part, tapering to flat-tipped loci, branched, 30−75 × 4−5 μm. Conidiogenous cells proliferating sympodially 1−4 times, integrated, terminal, rarely intercalary; conidiogenous loci distinct, apical or formed on shoulders due to sympodial proliferation, thickened and darkened, protruding, 2−3 μm diam. Conidia solitary, hyaline, straight to mildly curved, acicular, truncate at the base, obtuse at the apex, thin-walled, smooth, 15−130 × 3−5 μm, 2−20-septate, hila thickened, darkened, 2–2.5 μm diam. Culture characteristics:—Colonies spreading, flat, with sparse to dense aerial mycelium, even margin, reaching 48 mm diam after 2 wk. On MEA surface pale purplish grey, with rosy buff outer region; reverse sepia. On PDA surface vinaceous-buff, with red diffuse pigment surrounding culture; reverse bay. On OA surface whitish, with patches of grey; reverse chestnut, with rust in outer region. Etymology:—Named after the host from which it was isolated, Musa sp. Notes:—Cercospora musigena is similar to C. hayi Calp. by its acicular conidia with truncate bases, being part of the C. apii complex (Braun et al. 2014). In C. hayi, however, there are fewer conidiogenous loci per conidiogenous cell, and the conidial tips are acute to subacute (Calpouzos 1955). C. apii s. lat. (including C. hayi) on Musa spp. is genetically heterogeneous. Sequences retrieved from C. apii-like cultures isolated from banana clustered in three TAXONOMY AND PHYLOGENY OF CERCOSPORA Phytotaxa 233 (1) © 2015 Magnolia Press • 43 different clades (Groenewald et al. 2013). However, the identity of the name C. hayi is still unresolved and will need to be clarified by means of epitypification (Braun et al. 2014). Cercospora sp. (Fig. 6). Specimen examined:—THAILAND. Chiang Mai: on leaves of Crassocephalum crepidioides (Asteraceae), 29 Mar. 2013, J. Nguanhom (specimen CBS H-22293, culture CPC 23905, CPC 23906 = CBS 140168). FIGURE 6. Cercospora sp. (CBS H-22293). A. Leaf spot; B. Close-up of leaf spot; C, D. Conidiophores and conidiogenous cells; E−H. Conidia; I = colony on MEA.—Scale bars: C−D = 40 μm; E−H = 50 μm, G applies to H. Leaf spots amphigenous, circular to irregular, dark brown with pale brown centre, 3−5 mm diam. Caespituli amphigenous, punctiform, brown. Stromata medium in size, substomatal or intraepidermal, brown, 27.5–37.5 μm diam. Conidiophores in moderately large fascicles, arising from stromata, through stomata or erumpent, cylindrical to strongly geniculate, brown, paler towards the apex, unbranched, rarely constricted near the apex, 50−133 × 5−6 μm. Conidiogenous cells proliferating sympodially 2−12 times, integrated, terminal, 20−75 μm long; loci conspicuous, apical or on shoulders formed by geniculation, thickened and darkened, 2–3 μm diam. Conidia solitary, acicular, shorter ones subcylindrical, hyaline, smooth, thin-walled, straight to curved, apices subacute to obtuse, base truncate, indistinctly 3−12-septate, 27.5−180 × 2−5 μm, hila thickened and darkened, 2–3 μm wide; microcyclic conidiation observed. Culture characteristics:—Colonies spreading, flat, with sparse to moderate aerial mycelium, folded surface and even margins, reaching 45 mm after 2 wk. On MEA pale grey, with white centres, whitish at the margin; olivaceousgrey in reverse. On PDA whitish grey; reverse olivaceous-grey. On OA whitish grey (due to aerial mycelium); reverse blackish. Notes:—The genus Crassicephalum is close to Senecio and allied genera and in subtribe Senecioninae in tribe Senecioneae. The cercosporoid fungus on Crassocephalum crepidioides is morphologically indistinguishable from Cercospora apii s. lat., by having acicular conidia with truncate bases. C. senecionis Ellis & Everh. is morphologically very close, except for somewhat wider conidiophores, 4–8 μm, and conidia, 3–6 μm (Braun & Mel’nik 1997). C. 44 • Phytotaxa 233 (1) © 2015 Magnolia Press NGUANHOM ET AL. senecionicola Davis differs in having uniformly short, non-geniculate conidiophores, 15–55 × 4–5 μm, and acicular to obclavate conidia, 2–3.5 μm wide (Chupp 1954). The Indian C. senecionis-grahamii Thirum. & Govindu (Thirumalachar & Govindu 1962) is morphologically barely distinguishable from material on Crassocephalum crepidioides. Erechtites is another genus belonging in subtribe Senecininae and confusable with Crassocephalum. C. erechtitis G.F. Atk. is a widespread species, also known from Asia (Hsieh & Goh 1990), and is morphologically also similar to material on Crassocephalum, being C. apii-like in morphology. Sequence data of the species on hosts belonging to the Senecioninae, which are morphologically involved in this complex, are not yet available for comparison. Furthermore, Crassocephalum crepidioides is an African species not native in Thailand. Therefore, an infection of this host by another Cercospora species, native or exotic, cannot be excluded. Hence, the present data do not allow a final conclusion and taxonomic treatment of the Cercospora sp. occurring on Crassocephalum to be made until such time as the phylogeny of related taxa has been clarified. Discussion Approximately 500 cercosporoid species have been reported from Thailand, including 300 species of Cercospora. To date these taxa have primarily been identified based on their morphology, and only a few have been studied phylogenetically (To-anun et al. 2010, 2011). In several phylogenetic studies, multi-gene DNA sequence data have proven highly effective to distinguish among species of Cercospora (Groenewald et al. 2005, 2006a, b, 2010, 2013, Bakhshi et al. 2015a, b). The same approach was followed in this study, leading to the conclusion that morphological characters and molecular techniques are complementary, and both necessary to underpin novel species of Cercospora from Thailand. The results obtained here provide strong support for the distinction of several Cercospora species based on an analysis of ITS and cmdA DNA sequence data. Four new species of Cercospora were recognized in this study. Cercospora glycinicola is morphologically similar to C. sojina, which also occurs on Glycine max, but is distinct in that it has shorter conidiophores and narrower conidia. Two species were described from Cyperus, namely C. cyperacearum and C. cyperina. Based on a range of characters related to conidiophore length, septation, stromatal size, and conidium morphology, these species appear distinct from the taxa presently known to occur on Cyperus (Braun et al. 2014). However, Cercospora cyperacearum is plurivorous, and also occurs on Solanum mammosum, although it is morphologically distinct from the species known from Solanum (Braun & Mel’nik 1997, Bhartiya et al. 2000, Crous & Braun 2003). Further collections in the region are required to determine if C. cyperacearum could also be found on additional hosts, but ultimately cross-inoculation experiments would be required to determine if the different hosts are only chance occurrences, or if this is a truly plurivorous species. Cercospora collections occurring on Musa have always been assigned to C. hayi, which was originally described from banana leaves collected in Cuba (Calpouzos 1955). However, in a recent phylogenetic study, Groenewald et al. (2013) showed that sequences retrieved from C. apii-like cultures isolated from banana leaves collected in different countries clustered in three different clades. It is therefore not surprising that the collection obtained from Thailand is distinct from these unnamed taxa, and from C. apii s. str. Unfortunately, the three taxa referred to by Groenewald et al. (2013) are sterile, and thus further collections would be called for to try and elucidate the Cercospora complex occurring on banana, which is a host that appears to harbour a range of unique cercosporoid fungi (Arzanlou et al. 2008). In addition to these novel taxa, three Cercospora species were found on new hosts (based on the clades phylogenetically defined by Groenewald et al. 2013): C. cf. citrullina on Cyathula prostrata (Amaranthaceae); C. cf. malloti on Abelmoschus esculentus (Malvaceae), Asystasia salicifolia (Acanthaceae), Brassica alboglabra (Brassicaceae), Codiaeum variegatum (Euphorbiaceae), Eupatorium odoratum (Asteraceae), Jatropha integerrima (Euphorbiaceae), Melampodium divaricatum (Asteraceae), Musa sapientum (Musaceae), Nicotiana tabacum (Solanaceae), Phlox drummondii (Polemoniaceae), Physalis peruviana (Solanaceae), and Plantago major (Plantaginaceae); and C. cf. nicotianae on Houttuynia cordata (Saururaceae). Results obtained in this study showed that the most common Cercospora sp. found in Thailand was C. cf. malloti, which occurred on a wide host range. The collected isolates of C. cf. malloti shared similar conidiophore characteristics, being thick-walled, with distinct loci formed at the apex and on the shoulders caused by conidiophore geniculation (Groenewald et al. 2013). However, there were also some variable characters, namely differences in conidiophore geniculation and conidium length, suggesting that either the DNA loci currently used are not sensitive enough to TAXONOMY AND PHYLOGENY OF CERCOSPORA Phytotaxa 233 (1) © 2015 Magnolia Press • 45 distinguish all species, or that different environmental conditions and hosts to some degree influence the observed Cercospora phenotype. As most isolates sporulate poorly in culture (if at all), comparisons were always done on material in vivo. Cercospora malloti was originally described from Mallotus (Euphorbiaceae) collected in the USA. Fresh material would thus need to be recollected from this host in the USA to resolve the phylogenetic relationships of this taxon. Other than these wide host range species, some taxa also appeared to be host specific, namely C. capsici on Capsicum spp. (Solanaceae), C. cf. mikaniicola on Mikania cordata (Asteraceae) and C. cf. zinnia on Zinnia elegans (Asteraceae). To fully resolve the taxonomy of the Cercospora spp. occurring in Thailand, however, a global initiative is called for, as the phylogenetic position of many “common” species remains unknown, and these species, like C. malloti, will have to be recollected on their original hosts from their respective countries of origin. Further global studies are presently underway to try and establish a phylogenetic reference tree, collection and database for the genus Cercospora. Acknowledgements This work was financially supported by the Royal Golden Jubilee Ph.D. Program (PHD/0061/2551) and the Thailand Research Fund (DBG5380011 and MRG5580163). J.N. also thanks the technical staff from the CBS-KNAW Fungal Biodiversity Centre for their invaluable assistance. 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