Accepted Manuscript Polyphasic characterization of four new plant pathogenic Phyllosticta species from China, Japan and the United States Nan Zhou, Qian Chen, George Carroll, Ning Zhang, Roger G. Shivas, Lei Cai PII: S1878-6146(14)00136-6 DOI: 10.1016/j.funbio.2014.08.006 Reference: FUNBIO 512 To appear in: Fungal Biology Received Date: 13 May 2014 Revised Date: 21 August 2014 Accepted Date: 25 August 2014 Please cite this article as: Zhou, N., Chen, Q., Carroll, G., Zhang, N., Shivas, R.G., Cai, L., Polyphasic characterization of four new plant pathogenic Phyllosticta species from China, Japan and the United States, Fungal Biology (2014), doi: 10.1016/j.funbio.2014.08.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 Polyphasic characterization of four new plant pathogenic 2 Phyllosticta species from China, Japan and the United States 3 4 5 6 7 8 9 10 11 12 13 14 15 Nan Zhou a,b, Qian Chen a, George Carroll c, Ning Zhang d, Roger G. Shivas e and Lei Cai a*. M AN US C RI PT State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P. R. China b University of Chinese Academy of Sciences, Shijingshan Rd, Shijingshan, Beijing 100049, P.R. China. c Department of Biology, University of Oregon, Eugene, Oregon. d Department of Plant Biology and Pathology, Department of Biochemistry and Microbiology, Rutgers University, 59 Dudley Road, Foran Hall 201, New Brunswick, New Jersey 08901, USA. e Department of Agriculture, Fisheries and Forestry, Plant Pathology Herbarium (BRIP), Plant Biosecurity Science, GPO Box 267, Brisbane, QLD 4001, Australia. Corresponding author: Lei Cai: mrcailei@gmail.com. EP TE D Abstract: The black rot disease of Vitis species and other host genera of Vitacease is caused by Phyllosticta ampelicida and allied taxa which is considered to be a species complex. In this paper, we introduce four new species of Phyllosticta, including two from the Phyllosticta ampelicida complex, based on a polyphasic characterization including disease symptoms and host association, morphology, and molecular phylogeny. The phylogenetic analysis was conducted based on the ribosomal internal transcribed spacer (ITS) region and a combined multi-locus alignment of the ITS, actin (ACT), partial translation elongation factor 1–alpha (TEF-1) and glyceraldehydes 3–phosphate dehydrogenase (GPDH) gene regions. Our study confirms the phylogenetic distinctions of the four new species, as well as their phenotypic differences with known species in the genus. Key words: Guignardia bidwellii, cryptic species, morphology, phylogeny, systematics. INTRODUCTION Phyllosticta is an important plant pathogenic genus known to cause leaf spots and fruit diseases worldwide on many important economic plants and ornamentals such as citrus, banana, apple, grapes, cranberry, orchids, Ophiopogon sp. and gugertree (Engelman 1861; Ellis 1880; McManus 1998; Baayen et al. 2002; Olatinwo et al. 2003; Paul et al. 2005; Wikee et al. 2011; Su et al. 2012; Wang et al. 2012; Wikee et al. 2012; Wong et al. 2012; Shivas et al. 2013; Zhang et al. 2013b). Several Phyllosticta species are also known as endophytes and saprobes, e.g., P. capitalensis and P. cocoicola, respectively (Punithalingam 1974; Baayen et al. 2002; Taylor and Hyde 2003; Glienke et al. 2011; Wikee et al. 2013a). Recently, some Phyllosticta species have been found to be able to produce novel bioactive metabolites such as phyllostine and phyllostoxin (Evident et al. 2008a, b; Yan et al. 2011; Wikee et al. 2011, 2013c), which offer promise in biocontrol. AC C 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 a ACCEPTED MANUSCRIPT EP TE D M AN US C RI PT Phyllosticta belongs to the Phyllostictaceae, with P. convallariae as the generic type (Persoon 1818; Desmazières 1847; Donk 1968; Seaver 1922; Wikee et al. 2013b). In 1973, van der Aa revised the genus and defined Phyllosticta as “pycnidia globose, pyriform or tympaniform, conidiogenous cells holoblastic, with percurrent proliferation, conidia hyaline, 1-celled, ovoid, obovate, ellipsoid, short cylindrical, or globose to subglobose, usually surrounded by a slime layer and bearing an apical extracellular appendage”, which has been widely accepted (van der Aa 1973). According to these criteria, van der Aa & Vanev (2002) reconsidered 2 936 names in Phyllosticta, accepting 141 species based on literature and a re-examination of herbarium specimens. Most other species were re-classified in Phoma, Asteromella and Diaporthe (syn. Phomopsis). Some Phyllosticta species have been named as Leptodothiorella for their spermatial state (van der Aa 1973). The genus Guignardia, which currently comprises 353 names in MycoBank (2014), is the sexual morph of Phyllosticta, but only a few anamorphic teleomorph connections were established (Wikee et al. 2011). Phyllosticta takes priority over Guignardia as it is the oldest name (Persoon 1818; Viala & Ravaz 1892), and further it has more species and is more firmly established in the plant pathology literature (Wikee et al. 2011, 2013b). In recent years, molecular phylogenetic tools have significantly improved our understanding of evolutionary relationships between species of Phyllosticta (Wulandari et al. 2009; Glienke et al. 2011; Wicht et al. 2012). For example, Baayen et al. (2002) studied isolates of P. citricarpa from Citrus and found two phylogenetically distinct groups. One group was slow-growing and pathogenic, while another was morphologically similar but fast-growing and nonpathogenic. The objective of this study was to name and characterize four novel Phyllosticta species from China, Japan and the USA, based on morphological characters and phylogenies derived from ITS and combined multi-locus sequences (ITS, ACT, TEF-1, GPDH). MATERIALS and METHODS Isolates Strains of Phyllosticta were collected from diseased leaves of ornamental or economic plant species from China and the USA, or requested from NBRC Japan. Tissue pieces (5 × 5 mm) were taken from the margin of leaf lesions and consecutively immersed in 75 % ethanol solution for 1 min, 5 % sodium hypochlorite solution for 30 s, rinsed in sterile distilled water for 1 min. The pieces were blotted dry in sterile paper towels and incubated on 25% strength potato–dextrose agar (PDA) (Cai et al. 2009). Type specimens were deposited in Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS), with ex–type living cultures deposited in China General Microbiological Culture Collection Center (CGMCC). Morphology Obtained isolates were cultured on PDA media at room temperature (ca. 25 °C) for microscopic examination. Fungal structures (conidia, condiogenous cells) were mounted on glass slides in water for observation with a light microscope. Colony morphologies were examined after 14 d growth on PDA at room temperature in darkness, with colours assessed according to the colour charts of Rayner (1970). At AC C 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 ACCEPTED MANUSCRIPT EP TE D M AN US C RI PT least 30 pycnidia, conidiogenous cells or conidia were measured to calculate the mean sizes and standard deviations (SD) given in the formal descriptions. DNA extraction, PCR amplification and sequencing Mycelia were taken from 7-d-old cultures and grinded with organization disruptor FastPrep–24. Genomic DNA was exacted following the extraction protocol of Cubero et al. (1999). Quality and quantity of DNA were estimated visually by staining with GelRed after 1 % agarose gel electrophoresis. Internal transcribed spacer (ITS), Actin (ACT), translation elongation factor 1–alpha (TEF-1) and glyceraldehyde–3–phosphate dehydrogenase (GPDH) genes were amplified with primer pairs ITS1/ ITS4 (White et al. 1990), ACT512F/ACT783R (Carbone and Kohn 1999), EF1–728F/EF1–786R (Carbone and Kohn 1999) and GDF1/Gpd2–LM (Myllys et al. 2002, Guerber et al. 2003). Amplification mixtures and conditions were followed as described by Zhang et al. (2013b). DNA sequencing was performed at BGI, Beijing. Sequence alignment and phylogenetic analyses Sequences from forward and reverse primers were aligned to obtain a consensus sequence and deposited in the GenBank. Sequences of our isolates, together with reference sequences obtained from GenBank (Table 1), were assembled and aligned using MEGA v. 5.02 (Tamura et al. 2011) and MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html) (Katoh and Frith 2012). Novel sequence data were deposited in GenBank (Table 1), alignments in TreeBASE (www.treebase.org, study no.: 15802), and taxonomic novelties in MycoBank (Crous et al. 2004). Phylogenetic analyses were conducted using PAUP v. 4.0b10 (Swofford 2003) with Botryosphaeria obtusa as the outgroup. Ambiguously aligned regions were excluded from all analyses. An unweighted parsimony (UP) analysis was performed. Trees were inferred using the heuristic search option with TBR branch swapping and 1 000 random sequence additions, branches of zero length were collapsed and all equally most parsimonious trees were saved. Descriptive tree statistics such as tree length [TL], consistency index [CI], retention index [RI], rescaled consistency index [RC], and homoplasy index [HI], were calculated for trees generated. Clade stability was assessed in a bootstrap analysis with 1 000 replicates, each with 10 replicates of random stepwise addition of taxa. A Shimodaira–Hasegawa test (SH test) (Shimodaira & Hasegawa 1999) was performed in order to determine whether trees were significantly different. Trees were visualised in TreeView v. 1.6.6 (Page 1996). For the Bayesian analyses, the models of evolution were estimated by using MrModeltest v. 2.3 (Nylander 2004). Posterior probabilities (PP) (Rannala & Yang 1996, Zhaxybayeva & Gogarten 2002) were determined by Markov Chain Monte Carlo sampling (BMCMC) in MrBayes v. 3.0b4 (Huelsenbeck & Ronquist 2001), under the estimated model of evolution. Six simultaneous Markov chains were run for 1 000 000 generations and trees were sampled every 100th generation (resulting in 10 000 total trees). The first 2 000 trees, representing the burn–in phase of the analyses, were discarded and the remaining 8 000 trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree. AC C 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 ACCEPTED MANUSCRIPT 131 RESULTS Phylogenetic relationships were inferred using ITS, and the combined ACT, GPDH, ITS and TEF-1 dataset. The ITS dataset comprised 585 total characters including gaps, of which 303 characters were constant; 218 were parsimony informative; and 64 variable characters were parsimony–uninformative. Parsimony analysis generated 111 equally parsimonious trees, and the treewith shortest length (TL = 1147, CI = 0.424, RI = 0.771, RC = 0.327, HI = 0.576) was shown in Fig. 1. For the Bayesian analyses, model (GTR+I+G) was selected in MrModeltest 2.3. The branches with significant Bayesian posterior probability (≥ 95 %) were thickened in the phylogenetic tree. All four species described as new in this manuscript appear in distinct lineages and are well separated from other known species (Fig. 1). The combined dataset of ITS, ACT, TEF-1 and GPDH contained 52 combined sequences from 45 taxa and comprised 1777 total characters including gaps, of which 990 characters were constant; 576 were parsimony informative; 211 variable characters were parsimony-uninformative. The parsimony analysis generated one equally most parsimonious trees and the tree with shorter tree length (TL = 2568, CI = 0.475, RI = 0.708, RC = 0.337, HI = 0.525) was shown in Fig. 2. For the Bayesian analyses, the best-fit model (GTR+I+G) was selected in MrModeltest 2.3. The branches with significant Bayesian posterior probability (≥ 95 %) were thickened in the phylogenetic tree. Similarly all four novel species appear in different and distinct lineages (Fig. 2). 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 TAXONOMY Phyllosticta carochlae N. Zhou & L. Cai, sp. nov. (Fig. 3) – MycoBank: MB809846 Etymology: named with the combination of the initial letters of the genus and species names of its host, Caryota ochlandra. Description: Leaf spots irregular, dark brown to black, surrounded by pale brown margins. Pycnidia on PDA brown to black, subglobose to globose, 70–300 (–400) m in diam. Conidiogenous cells 2–6.5 × 5.5–13 m ( x = 8.8 ± 1.8 × 4.1 ± 0.9, n = 50), holoblastic, hyaline, cylindrical, proliferating 1–2 times percurrently near apex. Conidia 6–8.5 × (9–) 10–12 (–13) m ( x = 7.1 ± 0.5 × 11.3 ± 0.8, n = 50), unicellular, ovoid, obovoid, ellipsoidal to subglobose, enclosed in a mucilaginous sheath, 2–5 m ( x = 3.5 ± 0.8, n = 50) thick, and bearing a hyaline, mucoid apical appendage, 6–16 m ( x = 10.6 ± 2.5, n = 50) long, straight to flexible, unbranched. Culture characteristics: Colonies on PDA flat, with irregular margins, olive green in obverse and reverse when young, becoming greenish black at maturity. Specimen examined: CHINA, Fujian, Zhangzhou, living leaves of Caryota ochlandra, 08 Nov. 2012, L. Cai, HMAS 245578 (holotype); ex–type culture CGMCC 3.17317; ibid LC2861, living culture CGMCC 3.17318. Notes: Phyllosticta carochlae was isolated from the common ornamental and medicinal plant Caryota ochlandra (Arecaceae). It is characterized by its thick mucilaginous conidial sheath (2–5 m) which is distinct from most other Phyllosticta AC C EP TE D M AN US C RI PT 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 ACCEPTED MANUSCRIPT M AN US C RI PT species. Many species of Phyllosticta have been reported on Arecaceae, including P. arecae, P. capitalensis, P. catechu, P. cearensis, P. caryotae, P. cocoicola, P. cocoina var. phoenicis, P. cocophila, P. cocos, P. elaeidis, P. guillielmicola, P. helleborella, P. hesperidearum, P. palmarum, P. palmetto, P. palmicola, P. palmivora, P. sabalicola and P. viniferae (Farr et al. 2014). Among these, only P. arecae and P. capitalensis were accepted as Phyllosticta species by van der Aa (van der Aa 1973, van der Aa and Vanev 2002). Although P. caryotae was associated with C. ochlandra by Shen (1932), it was re-identified as Phomopsis caryotae–urentis based on its morphological description (van der Aa and Vanev 2002). P. carochlae is phylogenetically most closely related to Guignardia mangiferae (97% ITS identity to JF261459 derived from authentic strain IMI260.576) and P. ardisiicola (96% ITS identity to AB454274 ex-type) (Fig. 1), and clustered in the same clade in the multi-locus tree with G. mangiferae (Fig. 2). In morphology, P. carochlae produce larger pycnidia (160–400 m vs. 71–96 × 74–108 m), wider conidiogenous cells (2–6.5 × 5.5–13 m vs. 5–12.5 × 1.2–2.5 m), longer apical appendages (6–16 m vs. 2.5–5 m) as well as thicker mucilaginous sheaths (2–5 m) than P. ardisiicola (Motohashi K et al. 2008). G. mangiferae has smaller conidia (8–12 × 5–7 m vs. 9–13 × 6–8.5 m), and slightly shorter apical appendages (7–13 m vs. 6–16 m) than P. carochlae (Glienke et al. 2011). EP TE D Phyllosticta partricuspidatae N. Zhou & L. Cai, sp. nov. (Fig. 4) – MycoBank: MB809850 Etymology: named with the combination of the initial letters of the genus and species names of its host, Parthenocissus tricuspidata. Description: Pycnidia on PDA black, aggregated or decentralized, subglobose to globose or irregular, (100–) 150–300 m in diam. Conidiogenous cells 2–6 × 3.5–12 m ( x = 7.0 ± 2.5 × 3.7 ± 0.9, n = 35), become smaller when ageing, holoblastic, hyaline, long cylindrical, subcylindrical to ampulliform, proliferating 1–2 times percurrently near apex. Conidia 5–8.5 × 8–12 m ( x = 6.6 ± 0.8 × 10.0 ± 1.0, n = 40), unicellular, thin– and smooth–walled, ampulliform, ovoid, obovoid, ellipsoidal to subglobose, become smaller when older, 3–4.5 × 3–5 m ( x = 3.5 ± 0.3 × 4.3 ± 0.3, n = 30) , enclosed initially in a mucilaginous sheath and bearing a hyaline, mucoid apical appendage, 4–11 m ( x = 7.0 ± 2.1, n = 20) long, straight to flexible, unbranched, deciduous. Spermatia aseptate, 3–5 (–7) × 1–2.5 m ( x = 1.6 ± 0.3 × 4.7 ± 0.7, n = 35), cylindrical to dumbbell-shaped, with one guttule at each end. Culture characteristics: Colonies on PDA flat, with irregular margins, greenish yellow in obverse and reverse when young, grey olivaceous at maturity. Pycndia visible after 15 days, black. Specimen examined: JAPAN, on Parthenocissus tricuspidata, HMAS 245577 (holotype); ex–type culture NBRC9466, LC2862; JAPAN, on Parthenocissus tricuspidata, living culture NBRC 9757, LC2863. Notes: The strain NBRC 9466 was obtained from NBCR as Guignardia bidwellii (syn. P. ampelicida according to van der Aa (1973)). According to our phylogenetic analyses, P. ampelicida sensu lato comprises three species, P. ampelicida sensu stricto AC C 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 ACCEPTED MANUSCRIPT (syn. G. bidwellii), which was neotypified by Zhang et al. (2013b), P. parthenocissi and P. partricuspidatae. P. partricuspidatae shares 96% and 95% ITS sequence identity with P. parthenocissi (CBS 111645, EU683672) and P. ampelicida (ATCC200578, KC193586), respectively. A comparison of their morphological characters is provided in Table 2. EP TE D M AN US C RI PT Phyllosticta schimicola N. Zhou & L. Cai, sp. nov. (Fig. 5) – MycoBank: MB809853 Etymology: named after the genus name of its host, Schima superba. Description: Leaf spots ellipsoid or circular to somewhat irregular, pale brown in center, surrounded by dark brown borders. Pycnidia on PDA black, subglobose to globose, 50–100 (–130) m in diam. Conidiogenous cells 1.5–4.5 × 5–12 (–16) m ( x = 8.2 ± 2.2 × 3.3 ± 0.6, n = 35), holoblastic, hyaline, long cylindrical, subcylindrical to ampulliform, proliferating 1–2 times percurrently near apex. Conidia 5–8 × 8–11 (–12) m ( x = 6.5 ± 0.5 × 10.0 ± 0.9, n = 40), unicellular, smooth–walled, ovoid to long ovoid, ampulliform, ellipsoidal to subglobose, truncate at the base when young, enclosed in a smooth and a mucilaginous sheath, 1–3.5 m ( x = 2.0 ± 0.6, n = 30) thick, and bearing a hyaline, mucoid apical appendage, 8–30 m ( x = 15.5 ± 5.4, n = 35) long, straight to flexible, unbranched. Culture characteristics: Colonies on PDA flat, with irregular borders, greenish grey in obverse and reverse, forming an arabesque crust, margin white when young, becoming olivaceous black to greenish black at maturity. Aerial mycelium loose, white. Pycndia black, visible after 15 days. Specimen examined: CHINA, Jingxi, Ganzhou, Chongyi, Yang Ling National Forest Park, living leaves of Schima superba, 24 Apr. 2013, Q. Chen, HMAS 245575 (holotype); ex–type culture CGMCC 3.17319 ; ibid LC 2865, living culture CGMCC 3.17320. Notes: Phyllosticta schimicola was isolated from Schima superba (Theaceae), which is one of the dominant tree species in the evergreen broad leaf subtropical forest in south and southeast China and has important economic, medical, and environmental value. P. schimae and two other unnamed Phyllosticta isolates have been reported from Schima (Kobayashi 2007, Su et al. 2012). P. schimicola produces smaller pycnidia (50–30 m vs. 150–200 m), shorter conidiogenous cells (1.5–4.5 × 5–16 m vs. 2–4 × 8–30 m), thicker mucilaginous sheaths (1–3.5 m), and much longer apical appendages (8–30 m vs. 4–10 m) than P. schimae (Su et al. 2012). P. schimicola appears closely related to P. schimae in the phylogenetic tree (Fig. 1-2), but significant genetic divergences were observed between the two species (e.g. 94% ITS identity to JN692534 ex-type). The GCPSR analysis also supports P. scimicola to be a distinct species from P. schimae. AC C 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 Phyllosticta vitis-rotundifoliae N. Zhou & L. Cai, sp. nov. (Fig. 6) – MycoBank: MB 809855 Etymology: named after its host, Vitis rotundifolia. Description: Pycnidia on PDA black, aggregated or decentralized, subglobose to ACCEPTED MANUSCRIPT TE D M AN US C RI PT globose or somewhat irregular, 100–300 m in diam. Conidiogenous cells (3–) 5–11 × 2–5.5 m ( x = 8.0 ± 1.7 × 3.4 ± 0.7, n = 35), holoblastic, hyaline, long cylindrical, subcylindrical to ampulliform, proliferating 1–2 times percurrently near apex. Conidia 6–9.5 × 9–13 m ( x = 7.4 ± 0.7 × 11.2 ± 0.9, n = 40), unicellular, thin– and smooth–walled, ampulliform, ovoid, obovoid, ellipsoidal to subglobose, truncate at the base, enclosed in a mucilaginous sheath and bearing a hyaline, short mucoid apical appendage, 1.5–6.5 (–7) m ( x = 3.7 ± 1.3, n = 30) long, straight to flexible, unbranched, deciduous. Culture characteristics: Colonies on PDA flat, greenish black in obverse and reverse. Sometimes aerial mycelium forms a white to grey olivaceous crust on the surface or the margins. Pycndia visible after 1–3 months, black. Specimen examined: United States, Florida, Living leaves of Vitis rotundifolia, 2003, James Kimborough, HMAS 245576 (holotype); ex–type culture CGMCC 3.17322, VITRO3-1; United States, Georgia, on Vitis rotundifolia, Fall 2003, R. Hanlin, living culture CGMCC 3.17321, VITRO2-4. Notes: Phyllosticta vitis-rotundifoliae was isolated from the native north American species Vitis rotundifolia (Vitaceae), which has commercial value as a table and wine grape (Sandhu & Gu 2010; Vislocky et al. 2013). Other Phyllosticta species reported from Vitis include P. ampelicida (van der Aa and Vanev 2002). Phyllosticta vitis-rotundifoliae differs from P. ampelicida in producing slightly larger conidia (6–9.5 × 9–13 m vs 5–8.5 × 8–12 m), shorter conidiogenous cells (3–11 m vs 9–15 m), and lacking spermatia (Zhang et al. 2013b). In the phylogenetic tree, P. vitis-rotundifoliae appears most closely related to P. ampelicida (93% ITS identity to KC193586 ex-type), Phyllosticta parthenocissi (93% ITS identity to EU683672 ex-type), and P. partricuspidatae (94% ITS identity to KJ847424 ex-type). A comparison of the morphological characters of Phyllosticta species on Vitaceae is provided in Table 2. EP DISCUSSION In this paper we describe four new Phyllosticta species including two from the P. ampelicida complex, based on a polyphasic approach employing disease symptoms and host association, morphology and molecular phylogeny. Each of these species presented typical morphological characteristics for Phyllosticta, i.e., conidia with mucilaginous sheaths and an apical appendage (Van der Aa 1973). Phyllosticta species haven been historically indentified by morphology and host association, with several taxonomic revisions of the genus (van der Aa 1973; van der Aa and Vanev 2002). The recent application of molecular phylogenetic analysis has resulted in a rapid increase in the number of Phyllosticta species (Motohashi et al. 2008; Wulandari et al. 2009, 2010; Glienke et al. 2011; Su et al. 2012; Wang et al. 2012; Wikee et al. 2012; Wong et al. 2012; Shivas et al. 2013, Wikee et al. 2013b; Zhang et al. 2013a, b), and the discovery of species complexes with morphologically similar but evolutionarily distinct taxa (Wulandari et al. 2009; Glienke et al. 2011; Wicht et al. 2012; Wikee et al. 2013b; Zhang et al. 2013b). Here, we described P. carochlae, P. partricuspidatae, P. schimicola and P. vitis-rotundifoliae as new species AC C 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 ACCEPTED MANUSCRIPT with highly supported independent lineages in the phylogenetic tree. These new species are also morphologically distinguished from currently known species. P. ampelicida was first described as Naemospora ampelicida by Engelman (1861) and neotypified by Zhang et al. (2013b). Guignardia bidwellii (Ellis) Viala & Ravaz was known as the sexual morph of P. ampeilcida (van der Aa 1973), which causes black rot of grapes in North America (Kuo & Hoch 1995, 1996; Shaw et al. 1998; Shaw & Hoch 1999). The connection between P. ampelicida and G. bidwellii was established on co-occurrence with the host (Ellis 1880). As P. ampelicida and G. bidwellii are species complexes (Witch et al. 2012, Zhang et al. 2013b), their synonymy is unproven. When establishing G. bidwellii, neither type specimen nor location of the specimen was designated (Ellis 1880). Although Viala & Ravaz mentioned that it was found in North America (Viala & Ravaz 1892), it is impossible to designate a neotype for G. bidwellii with confidence, especially as we did not observe a teleomorphic stage for P. ameplicida, P. parthenocissi, P. partricuspidatae and P. vitis-rotundifoliae. Wicht et al. (2012) found that the isolates of Phyllosticta from grapevine cultivars could be separated into two species by analyzing samples using a polyphasic approach employing morphological, molecular and proteomic data. Zhang et al. (2013b) described a new species, P. parthenocissi (from Parthenocissus quinquefolia), in the P. ampelicida complex. In this study, we introduce two additional new species, P. partricuspidatae, P. vitis-rotundifoliae in this group based on morphology and phylogenetic analyses. P. partricuspidatae is referred to as ‘clade B’ from Parthenocissus tricuspidata in Zhang et al. (2013b), and P. vitis-rotundifoliae is represented by two strains from Vitis rotundifolia in the USA. According to several recent studies of Phyllosticta on citrus, banana, Vitis, Vaccinium and other hosts (Glienke et al. 2011; Wang et al. 2012; Wong et al. 2012; Wicht et al. 2012; Zhang et al. 2013), the identification of species based on morphology and host association provides unsatisfactory species delimitation and consequently a polyphasic approach becomes imperative. Phylogenetic analysis has become a standard technique in fungal classification and has been well applied in several other ascomycete genera such as Colletotrichun (Cai et al. 2009; Crouch et al. 2009; Hyde et al. 2009) and Phoma (Aveskamp et al. 2008, 2010; de Gruyter et al. 2010). Our taxonomic understanding of Phyllosticta species is still poor due to the few living ex-type cultures in collections (Hyde et al. 2010; Zhang et al. 2013b). Undoubtedly many more species of Phyllosticta remain to be discovered and their agricultural and environmental importance determined. 343 Acknowledgments: This work was financially funded by CASKSCX2-YW-Z-1026 344 and NSFC (31110103906, and 31322001). Ms. Fang Liu is thanked for technical 345 assistance. 346 347 348 References AC C EP TE D M AN US C RI PT 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 Aveskamp M, De Gruyter J, Woudenberg J, Verkley G, Crous PW. 2010. Highlights of the ACCEPTED MANUSCRIPT EP TE D M AN US C RI PT Didymellaceae: a polyphasic approach to characterise Phoma and related pleosporalean genera. Stud Mycol 65(1): 1–60. Aveskamp MM, De Gruyter J, Crous PW. 2008. Biology and recent developments in the systematics of Phoma, a complex genus of major quarantine significance. Fungal Divers 31: 1–18. Baayen R, Bonants P, Verkley G, Carroll G, Van Der Aa H, et al. 2002. Nonpathogenic isolates of the citrus black spot fungus, Guignardia citricarpa, identified as a cosmopolitan endophyte of woody plants, G. mangiferae (Phyllosticta capitalensis). Phytopathology 92(5): 464–477. Cai L, Hyde KD, Taylor PWJ, Weir B, Waller J, et al. 2009. A polyphasic approach for studying Colletotrichum. Fungal Diversity 39: 183–204. Carbone I, Kohn LM. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553–556. Crouch JA, Clarke BB, Hillman BI. 2009. What is the value of ITS sequence data in Colletotrichum systematics and species diagnosis? A case study using the falcate−spored graminicolous Colletotrichum group. Mycologia 101(5): 648–656. Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G. 2004. MycoBank: an online initiative to launch mycology into the 21st century. Studies in Mycology 50: 19–22. Cubero OF, Crespo A, Fatehi J, Bridge PD. 1999. DNA extraction and PCR amplification method suitable for fresh, herbarium–stored, lichenized, and other fungi. Plant Syst Evol 216: 243–249. de Gruyter J, Woudenberg JH, Aveskamp MM, Verkley GJ, Groenewald JZ, et al. 2010. Systematic reappraisal of species in Phoma section Paraphoma, Pyrenochaeta and Pleurophoma. Mycologia 102(5): 1066–1081. Desmazieres MJBHJ. 1847. Quatorzieme notice sur les plantes cryptogames recemment decouvertes en france. Ann Sci Nat Bot Ser 3(8): 9–37. Donk MA. 1968. Report of the committee for Fungi and Lichen 1964–1968. Taxon 17: 578–581. Ellis, J.B. 1880. A new Sphaeria on grapes. Bulletin of the Torrey Botanical Club 7(8):90–91. Engelman. 1861. Trans. St Louis Acad. Sci. J Proc 2: 165. Evidente A, Cimmino A, Andolfi A, Vurro M, Zonno M, et al. 2008a. Phyllostoxin and phyllostin, bioactive metabolites produced by Phyllosticta cirsii, a potential mycoherbicide for Cirsium arvense biocontrol. J Agric Food Chem 56(3): 884–888. Evidente A, Cimmino A, Andolfi A, Vurro M, Zonno MC, et al. 2008b. Phyllostictines A–D, oxazatricycloalkenones produced by Phyllosticta cirsii, a potential mycoherbicide for Cirsium arvense biocontrol. Tetrahedron 64(8):1612–1619. Farr DF, Rossman AY. 2014. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved April 26, 2014, from http://nt.ars-grin.gov/fungaldatabases/ Glienke C, Pereira O, Stringari D, Fabris J, Kava–Cordeiro V, et al. 2011. Endophytic and pathogenic Phyllosticta species, with reference to those associated with Citrus Black Spot. Persoonia 26(1):47–56. Guerber JC, Liu B, Correll JC, Johnston PR. 2003. Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility. Mycologia 95: 872–895. Huelsenbeck JP, Ronquist FR. 2001. MrBayes: Bayesian inference of phylogenetic trees. Biometrics 17: 754–755. Hyde KD, Abd–Elsalam K, Cai L. 2010. Morphology: still essential in a molecular world. Mycotaxon 114(1): 439–451. AC C 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 ACCEPTED MANUSCRIPT EP TE D M AN US C RI PT Hyde KD, Cai L, Cannon PF, Crouch JA, Crous PW, et al. 2009. Colletotrichum—names in current use. Fungal Divers 39:147–182. Katoh K, Frith MC. 2012. Adding unaligned sequences into an existing alignment using MAFFT and LAST. Bioinformatics 28(23): 3144–3146. Kobayashi T. 2007. Index of fungi inhabiting woody plants in Japan. Host, distribution and literature. Zenkoku-Noson-KyoikuKyokai Publishing Co., Ltd. Kuo KC, Hoch H. 1995. Visualization of the extracellular matrix surrounding pycnidiospores, germlings and appressoria of Phyllosticta ampelicida. Mycologia 89: 759–771. Kuo KC, Hoch H. 1996. The parasitic relationship between Phyllosticta ampelicida and Vitis vinifera. Mycologia 88: 626–634. McManus PS. 1998. First report of early rot of cranberry caused by Phyllosticta vaccinii in Wisconsin. Plant Dis 82(3): 350–350. Motohashi K, Araki I, Nakashima C. 2008. Four new species of Phyllosticta, one new species of Pseudocercospora, and one new combination in Passalora from Japan. Mycoscience 49(2): 138–146. Myllys L, Stenroos S, Thell A. 2002. New genes for phylogenetic studies of lichenized fungi: glyceraldehyde–3–phosphate dehydrogenase and betatubulin genes. Lichenologist 34: 237–246. Nylander JAA. 2004. MrModeltest v. 2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University. Olatinwo RO, Hanson EJ, Schilder AMC. 2003. A first assessment of the cranberry fruit rot complex in Michigan. Plant Dis 87: 550–556. Page RDM, 1996. TreeView: An application to display phylogenetic trees on personal computers. Computer Applications Biosciences 12: 357–358. Paul I, Van Jaarsveld A, Korsten L, Hattingh V. 2005. The potential global geographical distribution of Citrus Black Spot caused by Guignardia citricarpa (Kiely): likelihood of disease establishment in the European Union. Crop Prot 24(4): 297–308. Persoon CH. 1818. Traité sur les Champignons Comestibles, Contenantl’ Indication des Espèces Nuisibles Précédéd’une Introduction à l’Histoire des Champignons. Paris, Belin-Leprieur. Punithalingam E. 1974. Studies on Spheropsidales in culture II. Mycol Pap 136: 1–63. Rannala B, Yang Z. 1996. Probability distribution of molecular evolutionary trees: a new method of phylogenetic inference. Journal of Molecular Evolution 43: 304–311. Rayner RW. 1970. A mycological colour chart. Common wealth Mycological Institute. Kew, Surrey, UK. Sandhu AK, Gu LW. 2010. Antioxidant Capacity, Phenolic Content, and Profiling of Phenolic Compounds in the Seeds, Skin, and Pulp of Vitis rotundifolia (Muscadine Grapes) As Determined by HPLC-DAD-ESI-MSn. J. Agric. Food Chem. 58 (8): 4681–4692. Seaver FJ. 1922. Phyllostictaceae. North American Flora 6: 3–84. Shaw B, Hoch H. 1999. The pycnidiospore of Phyllosticta ampelicida: surface properties involved in substratum attachment and germination. Mycol Res 103: 915–924. Shaw B, Kuo KC, Hoch H. 1998. Germination and appressorium development of Phyllosticta ampelicida pycnidiospores. Mycologia 90: 258–268. Shen CI. 1932. Fungi of Nanking. IV. Contributions from the Biological Laboratory of the Science Society of China 8(2): 153-161. Shimodaira H, Hasegawa M. 1999. Multiple comparisons of log–likelihoods with applications to AC C 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 ACCEPTED MANUSCRIPT EP TE D M AN US C RI PT phylogenetic inference. Molecular Biology and Evolution 16: 1114–1116. Shivas RG, Tan YP, Grice KRE. 2013. Phyllosticta species on orchids (Orchidaceae), introducing Phyllosticta speewahensis sp. nov. (Phyllostictaceae, Ascomycota) from northern Australia. Sydowia 65(1): 139–146. Su YY, Cai L. 2012. Polyphasic characterisation of three new Phyllosticta spp. Persoonia 28: 76–84. Swofford DL. 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts, USA. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739. Taylor JE, Hyde KD. 2003. Microfungi of tropical and temperate palms. Fungal Divers Res Ser 12: 1–459. van der Aa HA, Vanev S. 2002. A revision of the species described in Phyllosticta. CBS, Utrecht. van der Aa HA. 1973. Studies in Phyllosticta I. Stud Mycol 5: 1–110. Viala P, Ravaz L. 1892. Sur la dénomination botanique (Guignardia bidwellii) du black–rot. Bulletin de la Société Mycologique de France 8: 63–63. Vislocky LM, Fernandez ML. 2013. Grapes and Grape Products: Their Role in Health. Nutrition Today 48(1): 47–51. Wang X, Chen G, Huang F, Zhang J, Hyde KD, Li H. 2012. Phyllosticta species associated with Citrus diseases in China. Fungal Diversity 52: 209–224. White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR Protocols: A guide to methods and applications: 315–322. Academic Press, San Diego, USA. Wicht B, Petrini o, Jermini M, Gessler C, Broggini GAL. 2012. Molecular, proteomic and morphological characterization of the ascomycete Guignardia bidwellii, agent of grape black rot: a polyphasic approach to fungal identification. Mycologia 104(5): 1036–1045. Wikee S, Lombard L, Crous PW, Nakashima C, Motohashi K, et al. 2013a. Phyllosticta capitalensis, a widespread endophyte of plants. Fungal Diversity 60: 91–105. Wikee S, Lombard L, Nakashima C, Motohashi K, Chukeatirote E et al. 2013b. A phylogenetic re–evaluation of Phyllosticta (Botryosphaeriales). Studies in Mycology 76: 1–29. Wikee S, Udayanga D, Crous PW, Chukeatirote E, McKenzie EHC et al. 2011. Phyllosticta—an overview of current status of species recognition. Fungal Diversity 51: 43–61. Wikee S, Wulandari NF, McKenzie EHC, Hyde KD. 2012. Phyllosticta ophiopogonis sp. nov. from Ophiopogon japonicus (Liliaceae). Saudi Journal of Biological Sciences 19: 13–16. Wikee S, Jaidee P, Wongkam S, Mckenzie EHC, Hyde KD, Chukeatirote E. 2013c. Antimicrobial activity of crude extracts of Phyllosticta spp. Mycology 4(2): 112–117. Wong MH, Crous PW, Henderson J, Groenewald JZ, Drenth A. 2012. Phyllosticta species associated with freckle disease of banana. Fungal Diversity 56: 173–187. Wulandari N, To-Anun C, Hyde KD, Duong L, De Gruyter J, et al. 2009. Phyllosticta citriasiana sp. nov., the cause of Citrus tan spot of Citrus maxima in Asia. Fungal Divers 34: 23–39. Wulandari NF, To-Anun C, Lei C, Abd-Elsalam KA, Hyde KD. 2010. Guignardia/Phyllosticta species on banana. Cryptogam Mycol 31 (4): 403–418. Yan X, Sikora RA, Zheng J. 2011. Potential use of cucumber (Cucumis sativus L.) endophytic AC C 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 ACCEPTED MANUSCRIPT fungi as seed treatment agents against root.knot nematode Meloidogyne incognita. J Zhejiang Univ Sci B 12(3): 219–225. Zhang K, Su YY, Cai L. 2013a. Morphological and phylogenetic characterisation of two new species of Phyllosticta from China. Mycol Progress 12: 547–556. Zhang K, Zhang N, Cai L. 2013b. Typification and phylogenetic study of Phyllosticta ampelicida and P. vaccinii. Mycologia 105: 1030–1042. Zhaxybayeva O, Gogarten JP. 2002. Bootstrap, Bayesian probability and maximum likelihood mapping: exploring new tools for comparative genome analyses. Genomics 3: 1–15. RI PT 481 482 483 484 485 486 487 488 489 490 Table 1 Sources of isolates and GenBank accession number used in this study. 1 EP TE D M AN US C ATCC: American Type Culture Collection, Virginia, USA; BRIP: Plant Pathology Herbarium, Biosecurity Queensland, Dutton Park, Queensland, Australia; CBS: CBS–KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands; CGMCC: China General Microbial Culture Collection; CPC: Culture collection of P. W. Crous, housed at CBS; IMI International Mycological Institute, CABI–Bioscience, Egham, Bakeham Lane, U.K.; LGMF: Culture collection of Laboratory of Genetics of Microorganisms, Federal University of Parana, Curitiba, Brazil; MAFF: the Microbiological Genebank, National Institute of Agrobiological Sciences, Japan; MUCC: Culture Collection, Laboratory of Plant Pathology, Mie University, Tsu, Mie prefecture, Japan; NBRC: Biological Resource Center, the National Institute of Technology and Evaluation, Japan; VIC: Culture collection of Federal University of Viçosa, Viçosa, Brazil; Lab. of Plant Pathology, Mie University, Japan; ZJUCC: Zhejiang University Culture Collection, China. * indicates the ex–type cultures 2 ITS: Internal transcribed spacers 1 and 2 together with 5.8S nrDNA; TEF-1: partial translation elongation factor 1-alpha gene; ACT: actin gene; GAPDH: glyceraldehyde–3–phosphate dehydrogenase gene. Table 2 Morphological characters of related Phyllosticta species from Vitaceae plants. AC C 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 Fig. 1 Maximum parsimony tree based on the ITS nrDNA sequence alignment. Values above the branches represent parsimony bootstrap support values (> 50 %). Thickened branches represent significant Bayesian posterior probability values (≥ 95 %). Novel sequences are printed in bold and the scale bar indicates 10 changes. The tree is rooted to Botryosphaeria obtusa. An asterisk (*) indicates the ex-type strains. Fig. 2 Maximum parsimony tree based on combined ITS, ACT, TEF-1 and GAPDH sequence sequence alignment, showing the phylogenetic relationships of the four new species. Values above the branches represent parsimony bootstrap support values (> 50 %). Thickened branches represent significant Bayesian posterior probability (≥ 95 %). Novel sequences are printed in bold and the scale bar indicates 10 changes. The tree is rooted to Phyllosticta owaniana. An asterisk (*) indicates the ex-type ACCEPTED MANUSCRIPT strains. Fig. 3 Phyllosticta carochlae (from holotype). a. diseased leaves of Caryota ochlandra. b. Colony on PDA (front). c. Colony on PDA (reverse). d. Pycnidia on colonies. e–f. Conidiogenous cells. g–i. Condia. Bars: d = 100 m, e–i = 10 m. RI PT Fig. 4 Phyllosticta partricuspidatae (from holotype). a. Young colony on PDA (front). b. Young colony on PDA (reverse). c. Mature colony on PDA (front). d. Mature colony on PDA (reverse). e–f. Pycnidia on colonies. g–h. Young conidiogenous cells. i. Old conidiogenous cells. j–k. Young condia on colonies. l–m. Condia. n. Spermatia. Bars: e = 300 m, f = 100 m, g–n= 10 m. M AN US C Fig. 5 Phyllosticta schimicola (from holotype). a–b. deseased leaves of Schima superba. c. Colony on PDA (front). d. Colony on PDA (reverse). e–f. Pycnidia on colonies. g–h. Conidiogenous cells. i–l. Condia. Bars: e–f = 100 m, g–l = 10 m. EP TE D Fig. 6 Phyllosticta vitis-rotundifoliae (from holotype). a. Colony on PDA (front). b. Colony on PDA (reverse). c. Pycnidia on colonies. d–e. Conidiogenous cells. f–h. Condia. Bars: d–h = 10 m. AC C 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 ACCEPTED MANUSCRIPT Table 1 Sources of isolates and GenBank accession number used in this study. Strain no.1 Species Host Locality GenBank Accession number2 ITS ACT TEF-1 GPDH Botryosphaeria obtusa CMW8232 Conifers South Africa AY972105 AY972111 DQ280419 – Guignardia alliacea MUCC0014* Allium Japan AB454263 – – – USA JN692543 JN692519 JN692531 JN692508 G. gaultheriae CBS447.70* Gaultheria humifusa RI PT fistulosum G. mangiferae IMI260.576* Manifera indica India JF261459 JF343641 JF261501 JF343748 G. philoprina CBS447.68* Taxus baccata Netherlands AF312014 – – – G. vaccinii CBS 126.22* Oxycoccus USA FJ538353 – – – Canada KF170306 KF289238 – – Phyllosticta abieticola CBS112067* Abies concolor P. aloeicola CPC21020* Aloe ferox P. ampelicida ATCC200578* Vitis riparia Isolate Sb22.6 Vitis vinifera MUCC0031 Ardisia crenata P. ardisiicola South Africa KF154280 KF289311 KF289193 KF289124 USA KC193586 KC193581 – KC193584 France HM008728 – – – Japan AB454274 – – – Japan AB454260 – – – Australia AY042927 KF306232 KF289170 KF289074 Brazil JF343565 JF343649 JF343586 JF343744 Brazil JF343572 JF343656 JF343593 JF343758 Stanhopea sp. Brazil JF261465 JF343647 JF261507 JF343776 CPC16592 Citrus limon Argentina KF206187 KF289273 KF289178 KF289092 CGMCC 3.17317* Caryota China KJ847422 KJ847430 KJ847444 KJ847438 China KJ847423 KJ847431 KJ847445 KJ847439 Taiwan JQ743562 – – – =MAFF240060 =NBRC102261* P. aspidistricola MUCC0010 Aspidistra =MAFF240040 elatior =NBRC102244* P. brazilianiae Muehlenbekia = IMI 298910 * adpressa VIC30556 Bifrenaria =CBS128855* harrisoniae LGMF330 =CBS126270* CPC18848 EP P. capitalensis D P. bifrenariae CBS535.87 TE P. beaumarisii M AN US C macrocarpos Mangifera indica =CBS128856* AC C P. carochlae CGMCC 3.17318 P. cavendishii BRIP554196* ochlandra Caryota ochlandra Musa cv. Formosana P. citriasiana CBS 120486* Citrus maxima Thailand FJ538360 FJ538476 FJ538418 JF343686 P. citribraziliensis CBS 100098* Citrus limon Brazil FJ538352 FJ538468 FJ538410 JF343691 P. citricarpa CBS127454* Citrus limon Australia JF343583 JF343667 JF343604 JF343771 CPC16603 Citrus limon Uruguay KF170295 KF289274 KF289213 KF289147 P. citrichinaensis ZJUCC200956* Citrus reticulata China JN791620 JN791533 JN791459 – P. citrimaxima CPC20276 Citrus maxima Thailand KF170304 KF289300 KF289222 KF289157 =CBS136059* ACCEPTED MANUSCRIPT P. concentrica CBS 937.70* Hedera helix Italy FJ538350 KF289257 FJ538408 JF411745 P. cordylinophila CPC20261* Cordyline Thailand KF170287 KF289295 KF289172 KF289076 fruticosa P. cornicola CBS111639 Cornus florida USA KF170307 KF289234 – – P. cussoniae CPC14873 Cussonia sp. South Africa JF343578 JF343662 JF343599 JF343764 Erica gracilis South Africa KF206170 KF289291 KF289227 KF289162 MUCC0113 Fallopia Japan AB454307 – =NBRC102266* japonica P. foliorum CBS 447.68* Taxus baccata Netherlands KF170309 P. hamamelidis MUCC149 Hamamelis Japan KF170289 =CBS136060* P. ericarum CBS132534 P. fallopiae – – KF289247 KF289201 KF289132 KF289309 – – CGMCC3.14355* Hosta P. hubeiensis CGMCC3.14986* Viburnum plantaginea odoratissimim P. hymenocallidicola CBS 131309* Hymenocallis littoralis P. hypoglossi CBS 434.92* Ruscus aculeatus P. ilicis–aquifolii CGMCC3.14358* Ilex aquifolium P. kerriae MAFF240047 Kerria japonica P. ligustricola MUCC0553 Leucothoe =CBS136073* catesbaei MUCC0024* TE P. leucothoicola D =NBRC102251* M AN US C japonica P. hostae Ligustrum RI PT =CPC19744* China JN692535 JN692511 JN692523 JN692503 China JX025037 JX025032 JX025042 JX025027 Australia JQ044423 KF289242 KF289211 KF289142 Italy FJ538367 FJ538483 FJ538425 JF343695 China JN692538 JN692514 JN692526 – Japan AB454266 – – – Japan AB454370 KF289310 – – Japan AB454269 AB704212 – – Australia JQ743570 – – – Thailand KF170305 KF289296 KF289190 KF289121 obtusifolium CPC20264* AC C P. mangifera–indica CPC18347* EP P. maculata Musa cv.Goly–goly pot–pot Mangifera indica P. minima CBS 585.84* Acer rubrum USA KF206176 KF289249 KF289204 KF289135 P. musarum BRIP55434* Hill banana India JQ743584 – – – P. musicola CBS 123405* Musa Thailand FJ538334 FJ538450 FJ538392 Japan AB454318 AB704233 – – South Africa FJ538368 KF289254 FJ538426 JF343767 Japan AB454317 AB704232 – – USA EU683672 JN692518 JN692530 – P. neopyrolae P. owaniana acuminata MUCC0125 Pyrola =CPC21879* asarifolia CBS 776.97* Brabejum stellatifolium P. pachysandricola MUCC0124* Pachysandra terminalis P. parthenocissi CBS 111645* Parthenocissus ACCEPTED MANUSCRIPT quinquefolia P. partricuspidatae Parthenocissus NBRC9466* Japan KJ847424 KJ847432 KJ847446 KJ847440 Japan KJ847425 KJ847433 KJ847447 KJ847441 Japan AB454268 – – – Japan AB454276 – – – USA KF206172 KF289239 KF289209 KF289140 tricuspidata Parthenocissus NBRC9757 tricuspidata MUCC0019 Parthenocissus MUCC0037 Parthenocissus RI PT tricuspidata tricuspidata P. paxistimae CBS 112527* Paxistima mysinites CBS616.72 Ilex aquifolium Germany KF154279 KF289251 KF289205 KF289136 P. podocarpi CBS111647 Podocarpus South Africa KF154276 KF289235 KF289232 KF289168 lanceolata P. podocarpicola CBS 728.79* Podocarpus maki P. pseudotsugae CBS111649 Pseudotsuga menziesii P. rhaphiolepidis MUCC0432* Rhaphiolepis indica CGMCC3.14354* Schima superba P. schimicola CGMCC 3.17319* Schima superba CGMCC 3.17320 Schima superba P. speewahensis BRIP 58044 Orchids P. spinarum CBS292.90 Chamaecyparis TE D P. schimae M AN US C P. philoprina USA KF206173 KF289252 KF289203 KF289134 USA KF154277 KF289236 KF289231 KF289167 Japan AB454349 AB704242 – – China JN692534 JN692510 JN692522 JN692506 China KJ847426 KJ847434 KJ847448 KJ854895 China KJ847427 KJ847435 KJ847449 KJ854896 northern KF017269 – KF017268 – France JF343585 JF343669 JF343606 JF343773 China JX025040 JX025035 JX025045 JX025030 China JX025041 JX025036 JX025046 JX025031 Tasmania KF206205 KF289255 KF289210 KF289141 USA KC193585 KC193580 KC193582 KC193583 USA KF170312 KF289287 KF289229 KF289165 USA KJ847429 KJ847437 KJ847451 KJ847443 USA KJ847428 KJ847436 KJ847450 KJ847442 New Zealand JN692541 JN692517 JN692529 JN692507 Australia pisifera P. styracicola CGMCC3.14985* Styrax EP grandiflorus CGMCC3.14989 CBS 777.97* AC C P. telopeae Styrax grandiflorus Telopea speciosissima P. vaccinii ATCC 46255* Vaccinium P. vacciniicola CPC18590* Vaccinium P. vitis-rotundifoliae CGMCC 3.17321 macrocarpon macrocarpum Vitis rotundifolia CGMCC 3.17322* Vitis rotundifolia P. yuccae CBS117136 Yucca elephantipes ACCEPTED MANUSCRIPT Table 2 Morphological characters of related Phyllosticta species from Vitaceae plants. brown to black; P. ampelicida globose or subglobose; to black; P. parthenocissi globose Conidia ( m) cells ( m) cylindrical to conical; 9–13(–15) × 3–4.5 150–300 brown Conidiogenous or subglobose; cylindrical to conical; 7.5–12.5 × black; Appendage ( m) Zhang et al. to dumbbell 2013 ellipsoidal, shaped; 5–7 × 8.5–12.5× 1.3–2.2 Zhang et al. globose to subglobose; 2013 – 7.5–10 × 6–9 subglobose to long cylindrical, ovoid, cylindrical to P. globose subcylindrical to obovoid, dumbbell-sha partricuspidatae irregular; ampulliform; ellipsoidal to ped; 3–5(–7) (100–) 3.5–12 × 2–6 subglobose; × 1–2.5 subglobose to vitis-rotundifolia somewhat irregular; subcylindrical to ampulliform; (3–) 2–5.5 5–11 AC C EP TE 100–300 or long cylindrical, × D black; globose 4–7 Present study 4–11 8–12 × 5–8.5 150–300 P. 2–7 ampulliform, or References size ( m) broadly ovoid 6–7.5 2.5–3.5 150–300 Spermatia RI PT species Pycnidia ( m) M AN US C Phyllosticta ampulliform, Present study ovoid, obovoid, ellipsoidal to subglobose; 6–9.5 × 9–13 – 1.5–6.5 (–7) AC C EP TE D M AN US C RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN US C RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN US C RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN US C RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN US C RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN US C RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Highlights: Four new Phyllosticta species are introduced based on a polyphasic characterization. Distinctions between four new species and other Phyllosticta species are presented. AC C EP TE D M AN US C RI PT Host association is proved to be unreliable in Phyllosticta species delimitation.