Cytospora Species Associated with Walnut Canker Disease in China, with Description of a New Species C

fungal biology 119 (2015) 310e319

journal homepage: www.elsevier.com/locate/funbio

Cytospora species associated with walnut canker disease in China, with description of a new species C. gigalocus

Xinlei FANa, Kevin D. HYDEc,d, Min LIUa, Yingmei LIANGb, Chengming TIANa,* aThe Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, China bMuseum of Beijing Forestry University, Beijing 100083, China cInternational Fungal Research & Development Centre, The Research Institute of Resource Insects, Chinese Academy of Forestry, Bailongsi, Kunming 650224, China dSchool of Science, Mae Fah Luang University, Chaing Rai 57100, Thailand article info abstract

Article history: Cytospora species associated with canker disease are presently difficult to identify because Received 18 September 2014 of lack of ex-types cultures with molecular data, few distinguishable characters, and only Received in revised form Internal transcribed spacer (ITS) sequence data is available for most Cytospora strains in 16 December 2014 GenBank. We report on Cytospora species from the walnut tree in China. Collections Accepted 22 December 2014 were subjected to morphological and phylogenetic study. The relatedness of species asso- Available online 3 January 2015 ciated with walnut canker were established using combined ITS, nrLSU, b-tubulin, and ac- Corresponding Editor: tin gene sequence data. Cytospora atrocirrhata, Cytospora chrysosperma, Cytospora sacculus, Prof. Geoffrey Michael Gadd and a new species, Cytospora gigalocus, were identified causing canker disease of walnut. Cytospora gigalocus is formally described and compared with most similar species. Cytospora Keywords: chrysosperma and C. sacculus have previously been recorded from walnut, whereas C. atroc- Juglans regia irrhata is reported as associated with walnut canker for the first time. This is the first study Morphology that has established the Cytospora species causing walnut canker in China using a multi- Pathogen phasic approach. All species are recorded as being associated with walnut canker disease Phylogeny in China for the first time. Taxonomy ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction leading producer of commercial nuts (Savage 2001). China pro- duced 1.65 million metric tons of walnuts which accounted for The walnut tree is an important nut producing tree distributed 50 percent of the world production in 2011 (www.fao.org; in central-eastern Asia, Europe and the USA. Although all wal- accessed 1 Nov. 2013). The walnut tree is infected by a wide nut species can produce edible nuts, the English or Persian range of fungal pathogens which can seriously reduce walnut walnut (Juglans regia L.) is most widely cultivated and is the production. Cytospora is one of the most important pathogenic

* Corresponding author. Tel./fax: þ86 0 1062336196. E-mail address: [email protected] (C. Tian). http://dx.doi.org/10.1016/j.funbio.2014.12.011 1878-6146/ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Cytospora species associated with walnut canker disease 311

genera causing walnut canker disease. The disease mainly im- Valsa ambiens (Spielman 1985; Adams et al. 2005). Cytospora pacts walnut branches and leads to large areas of dieback, juglandicola lacks conceptacles and has smaller spores thus reducing productivity of walnut orchards and inflicting (3.5e4 mm) than the generic type, Cytospora chrysosperma economic loss (Abbasi et al. 2013). (Pers.) Fr. (Saccardo 1884). The genus Cytospora (sexual state Valsa)wasintroduced In China, Cytospora species from popular and apple trees by Ehrenberg (1818). Cytospora species usually produce asex- have been well studied using morphology and phylogeny ual fruiting bodies that contain either a single or labyrin- (Wang et al. 2007, 2011; Zhang et al. 2007). Fan et al. (2014) clar- thine of locules, filamentous conidiophores (or asci), and ified and illustrated C. chrysosperma and Cytospora sophorae allantoid hyaline conidia (or allantoid hyaline ascospores) Bres. from the Sophora japonica L. and introduced a new spe- (Spielman 1983, 1985; Adams et al. 2005). In moist condi- cies, Cytospora sophoricola C.M. Tian & X.L. Fan using a multi- tions, the conidia emerge from the fruiting bodies in the phasic approach. form of yellow, orange to red gelatinous tendrils (Adams The aim of the present study was to (i) delimit the species et al. 2005, 2006). Species identification has generally been associated with walnut canker in China based on morpholog- established according to host affiliation, while morphologi- ical observation and phylogenetic analysis; and (ii) provide cal descriptions generally have lacked detail. However, a sin- a multi-locus phylogenetic data of ITS, nrLSU, TUB2, ACT gle species of Cytospora often occurs on taxonomically gene for several species of Cytospora. unrelated host plants, or a single host plant may be infected by different species (Adams et al. 2005; Wang et al. 2011). Kirk et al. (2008) estimated that there are 110 species of Cyto- Materials and methods spora, although 560 epithets are listed in Index Fungorum (2014). Isolation Synonymies and frequent name changes within Cytospora have caused much confusion for plant pathologists and my- Strains of Cytospora were isolated from diseased branches of cologists (Spielman 1983, 1985). Thus, a systematic account walnut trees during collecting trips to Gansu, Ningxia, Qing- of the genus Cytospora is needed which takes into account hai, and Shaanxi provinces in China. Isolations were made morphology and phylogenetic analysis (Adams et al. 2002; directly from conidiomata or ascomata (if sexual state was Fotouhifar et al. 2010; Hyde et al. 2010). The genus Cytospora present), and a direct morphological study was made of ma- has asexual morphs in Valsa, Leucostoma, Valsella, and Valseu- terial on the host, whenever possible. Part of hymenium was typella (Adams et al. 2005). More recently all sexual genera removed from 3 to 4 sporocarps of fresh material cut hori- were synonymized with Valsa either as a subgenus or species zontally with a sterile blade and mixed in a drop of sterile with no additional infrageneric rank (Adams et al. 2005). water on a glass slide. The contents were broken up further According to the International Code of Nomenclature for Algae, with the blade until a spore suspension was obtained. Half of Fungi, and Plants the suspension was spread over the surface of a Petri dish (ICN) in 2011, a single name is needed for a biological species and for genera. The principle of priority ap- with 1.8 % of potato dextrose agar (PDA) at 25 C. After incu- plies and the older name Cytospora has priority (Adams et al. bation for up to 24 h, single germinating conidia or asco- 2005; Fotouhifar et al. 2010). However, if another name is spores were transferred to fresh plates of PDA. The other more commonly used a case can be made for keeping this half of the suspension was used for further measurement name (Cai et al. 2009; Hyde et al. 2009; Udayanga et al. 2011; of spores. This method allowed unambiguous connection Wikee et al. 2011; Huang et al. 2013; Wei et al. 2013). Cytospora, to be established between the two morphs and correspond- as the older name (1818), is also more common in nature and ing pure cultures were used for phylogenic study. The taxon should have priority. names and accession numbers of GenBank sequences in the Several species of Cytospora associated with canker dis- current study are provided (Table 1). Specimens have been ease have been systematically studied using a geography- deposited at Museum of Beijing Forestry University (BJFC). centered or a host-centered strategy. Adams et al. (2005) com- Living cultures are deposited at China Forestry Culture Col- bined morphology and phylogeny using ITS sequence data to lection Center (CFCC) and China Center for Type Culture Col- describe 28 species of Cytospora from Eucalyptus, of which lection (CCTCC). eleven species were new to science. Adams et al. (2006) also described fourteen species from South Africa using the Morphological studies of Cytospora species same methodology. Fotouhifar et al. (2010) identified twelve species from Iran using morphology and phylogeny. Specimens were observed based on the morphological charac- Mehrabi et al. (2011) used morphology to identify six species teristics of their fruiting bodies on infected plant tissues, in- of Cytospora from apple trees in Iran and described their sex- cluding the structure and size of conidiomata; presence or ual states. Currently, several Cytospora species have been absence of a conceptacle; the colour, size, and shape of discs; recorded from walnut trees worldwide i.e., Cytospora cincta number and diameter of ostioles per disc; number and ar- Sacc., Cytospora juglandina Sacc., Cytospora juglandis Sacc., rangement type of locules; and the size and shape of conidio- Cytospora leucostoma Sacc., Cytospora leucosperma (Pers.) Fr., phores. Over 20 conidiomata were sectioned and 50 conidia and Cytospora juglandicola Sacc. (Tai 1979; Zhuang 2005; were selected randomly to measure their lengths and widths Fotouhifar et al. 2010). The first four species have multi- using a Leica light microscope (LM, DM 750). Cultural charac- locules with obvious black conceptacles (Fries 1823; teristics were recorded after 3, 7, and 30-days growth on Saccardo 1884). Cytospora leucosperma is the asexual state of PDA at 25 C in the dark. 312 X. Fan et al.

Table 1 e Species from walnut used in molecular phylogenetic analysis in this study (ex-type strain is bolded). Species Isolate Origin GenBank accession numbers

ITS nrLSU TUB2 ACT

Cytospora atrocirrhata CFCC 89615a Xining, Qinghai KF225610 KF225624 KF498688 KF498673 CFCC 89616 Xining, Qinghai KF225611 KF225625 KF498689 KF498674 Cytospora chrysosperma CFCC 89617 Haidong, Qinghai KF225605 KF225619 KF498683 KF498668 CFCC 89618 Xining, Qinghai KF225612 KF225626 KF498690 KF498675 CFCC 89619 Yinchuan, Ningxia KF225614 KF225628 KF498692 KF498677 CFCC 89628 Xining, Qinghai KF225613 KF225627 KF498691 KF498676 Cytospora gigalocus CFCC 89620 Xining, Qinghai KF225608 KF225622 KF498686 KF498671 CFCC 89621 Xining, Qinghai KF225609 KF225623 KF498687 KF498672 Cytospora sacculus CFCC 89624a Gannan, Gansu KF225615 KM401886 KM401890 KM401888 CFCC 89625 Gannan, Gansu KF225616 KM401887 KM401891 KM401889 CFCC 89626 Xianyang, Shaanxi KF225617 KF225631 KF498695 KF498681 CFCC 89627 Xianyang, Shaanxi KF225618 KF225632 KF498696 KF498682

a Isolates designated as epitype in this study.

DNA extraction in the phylogenetic analysis. The ITS sequence alignment file and the multi-locus sequences alignment file are depos- Mycelium for DNA extraction was grown on PDA with cello- ited in TreeBASE (www.treebase.org) as accession S14606 phane for 3 d and obtained from the surface of cellophane and S16280. Phomopsis vaccinii Shear was selected as out- by scrapping. Genomic DNA was extracted using the modified group in this analysis (Adams et al. 2005). CTAB method (Doyle & Doyle 1990). DNA concentrations were Maximum parsimony (MP) analysis was run by heuristic estimated visually by electrophoresis in 1 % agarose gels by search option of 1000 random-addition sequences with tree comparing band intensity with a DNA maker 1 kbp (Takara bisection and reconnection (TBR) as the branch-swapping Biotech). algorithm. The branches were collapsed if maximum branch length was zero and all equally parsimonious trees were saved. To estimate confidence of the clades in the resulting PCR amplifications, sequencing, and phylogenetic analysis trees, MP bootstrap values were generated from 1000 replicates. Other measures calculated were tree length (TL), DNA was amplified from the complete ITS regions, nrLSU, b- consistency index (CI), retention index (RI) and tubulin (TUB2), and actin (ACT) genes of each Cytospora iso- rescaled consistency (RC). The branch support (BS) was eval- late. A 580 bp fragment of ITS gene was amplified with the uated with bootstrap method of 1000 replicates (Hillis & Bull primer pair ITS1 and ITS4 (White et al. 1990). A 700 bp frag- 1993). ment of nrLSU region was amplified with the primer pair Maximum likelihood (ML) analysis was also performed us- NL1 and NL4 (O’Donnell 1993). A 550 bp fragment of TUB2 ing RAxML v.7.2.8 using a GTR model of site substitution in- was amplified with the primer pair Bt2a and Bt2b (Glass & cluding estimation of Gamma-distributed rate heterogeneity Donaldson 1995). A 300 bp fragment of ACT was amplified and a proportion of invariant sites (Stamatakis 2006). The with the primer pair ACT512F and ACT783R (Carbone & branch support (BS) was evaluated with bootstrap method of Kohn 1999). The PCR amplification products were separated 100 replicates (Hillis & Bull 1993). by electrophoresis in 2 % agarose gels with a DNA maker Bayesian analysis was performed by a Markov Chain 2000 bp (Takara Biotech). DNA sequencing was carried out Monte Carlo (MCMC) method with an invgamma distribution using an ABI PRISM 3730XL DNA Analyzer with BigDye and six rate categories (GTRþGþG). Two MCMC chains were Terminater Kit v.3.1 (Invitrogen) at the Shanghai Invitrogen run with starting from random trees for 1 000 000 genera- Biological Technology Company (Beijing, China). The first tions and trees were sampled every 100th generation, result- analysis of sequences from our isolates were based on a com- ing in 10 000 total trees. The first 2500 trees were discarded bined four concatenated sequences (ITS, nrLSU, TUB2, and as the burn-in phase of the analysis and the Bayesian poste- ACT). Sequences were aligned by Clustal W as implemented rior probabilities (BPP) were calculated using the remaining in MEGA v.5 (Adams et al. 2005; Fotouhifar et al. 2010; Tamura 7500 trees. et al. 2011). Ambiguously aligned sequences were excluded from analysis. Phylogenetic analysis were carried out by PAUP v.4.0b10 for maximum parsimony (MP) analysis Results and discussion (Swofford 2003), MrBayes v.3.1.2 for Bayesian analysis (Ronquist & Huelsenbeck 2003), and RAxML v.7.2.8 for maxi- Phylogeny mum likelihood (ML) analysis (Stamatakis 2006). In addition, a second analysis was performed to confirm the relationship Identification of Cytospora strains to species is complicated of available Cytospora species. Sequences of our isolates and by the lack of distinct morphological differences, lack of epi- representative ITS sequences of Cytospora, including ex-type typified names or names with authentic sequence data and strains obtained from previous systematic studies were used also by the fact that most species (which are putatively Cytospora species associated with walnut canker disease 313

named) in GenBank only have ITS sequence data (Hyde & addition, the new isolates were all clearly distinguished Zhang 2008). In this study, we therefore suggest authentic from other species, which supported the morphological strains for some relevant species names as well as multi- results. locus gene data for the strains collected. Multi-locus analysis (ITS, nlLSU, TUB2, ACT) of twelve strains isolated from Taxonomy Juglans regia is given in Fig 1. There are 2002 characters of the combined matrix, 1520 characters are constant, 312 vari- Cytospora gigalocus C.M. Tian, X.L. Fan & K.D. Hyde, sp. nov. able characters are parsimony-uninformative and 170 are Fig 3. parsimony informative. MP analysis indicated one parsimo- MycoBank MB805397; Facesoffungi number: FOF000306. nious tree as shown in Fig 1 (TL ¼ 644, CI ¼ 0.891, Cytospora gigalocus is characterized by large multi-locules RI ¼ 0.921, RC ¼ 0.821). ML method and Bayesian analysis (1.63e2.18 mm) with the absence of a conceptacle, multi- was similar with the MP tree. The tree showed four ostioles, and hyaline, elongate-allantoid, eguttulate, aseptate individual clades of Cytospora species causing walnut canker conidia (4.6e5.6 0.8e1.3 mm). (Fig 1). Holotype: BJFC S647. Phylogenetic analysis of ITS sequence data (Fig 2) includes Etymology: gigalocus (Lat.), referring to the large size of the the strains from this study and representative sequences of locules. Cytospora from GenBank including sixteen ex-type strains. Habitat/Distribution: Known from Juglans regia in China. The alignment comprises 88 taxa (including one outgroup Pathogen on twigs and branches of Juglans regia. Sexual state: taxon). The dataset for Cytospora comprises a total of 591 unknown. Asexual state: Stromata immersed in bark. Conidio- base pairs used in analysis, of which 353 are constant, 65 vari- mata erumpent in a large circular area, discoid, with large able characters are parsimony-uninformative and 173 are par- multi-locules. Disc light brown, triangular, circular to ovoid, simony informative. A heuristic search generated 844 0.33e0.62 mm (average ¼ 0.52, n ¼ 20) in diameter, with one parsimonious trees, one of which is presented in Fig 2 to five ostioles per disc. Ostiole in the disc arranged in circles, (TL ¼ 835, CI ¼ 0.536, RI ¼ 0.844, RC ¼ 0.452). ML and Bayesian 0.13e0.19 mm (average ¼ 0.16, n ¼ 20) in diameter. Multi- analyses were similar to the MP tree. The ITS phylogenetic locules arrange in circles or ellipse with common walls, tree resolved 45 Cytospora clades. Cytospora gigalocus repre- 1.63e2.18 mm (average ¼ 1.91, n ¼ 20) in diameter. Conidio- sented the new species and formed a monophyletic clade phores hyaline, unbranched or branched at the base, (marked in blue in Fig 2). It also showed that Cytospora chryso- 16.1e23.6 mm (average ¼ 17.4, n ¼ 20). Conidia hyaline, sperma in current study clustered with strain 334 and CFCC elongate-allantoid, eguttulate, aseptate, 4.6e5.6 0.8e1.3 89600 from previous studies in China and Iran (Fotouhifar mm (average ¼ 4.8 1.1, n ¼ 50). et al. 2010; Fan et al. 2014); Cytospora atrocirrhata clustered Cultures: colony white, flat, and thin, with uniform texture. with strain 22 from Salix excelsa in Iran (Fotouhifar et al. On PDA, the conidiomata distributed irregularly on the me- 2010); and Cytospora sacculus was clustered with strain CBS dium surface. 192.42 and CBS 116.21 from study of Adams et al. (2005).In Material examined: CHINA, QINGHAI PROVINCE, Xining, Xishan Botanical Garden, 3637025.8900 N, 10144048.9200 E, 2330 m asl., on stems of Juglans regia, collected by X.L. Fan, 19 August 2012 (BJFC S647, holotype), living culture, CFCC 89620, CCTCC AF2013017; Xishan Botanical Garden, 3637014.3200 N, 10144046.5800 E, 2321 m asl., on stems of J. regia, collected by X.L. Fan, 19 August 2012 (BJFC S648, paratype), living culture, CFCC 89621, CCTCC AF2013019. Notes: Cytospora gigalocus has multi-loculate conidiomata with one to five ostioles and shares common walls, and is morphologically similar to Cytospora schulzeri from Malus spp. and Cytospora carbonacea on Ulmus spp. (Fotouhifar et al. 2007; Mehrabi et al. 2011). Cytospora gigalocus has larger locules (1.6e2.2 mm) diam. with a hollow centre (Fig 3E,F) as compared with C. schulzeri (1.4e1.5 mm), and it has smaller conidia than C. carbonacea (mean 4.84 1.07 vs. 11.73 1.65) (Adams et al. 2006; Fotouhifar et al. 2007; Zhang et al. 2014). In the phylogenetic tree, C. gigalocus clusters in a separate clade (Figs 1 Fig 1 e Phylogram based on combined four genes (ITS, and 2) with high support values (MP BS/ML BS/BPPs ¼ 100/ nrLSU, TUB2, ACT) of Cytospora. Values above the branches 100/1). indicate maximum parsimony and maximum likelihood Cytospora atrocirrhata Gvritishvili, Mikol Fitopatol 7 (6): 547 bootstrap support (BS ‡ 50 %). Thickened branches repre- (1973). Fig 4. sent posterior probabilities (BPP ‡ 0.95) from Bayesian in- Habitat/Distribution: Known from Juglans regia and Salica- ference. The tree is rooted with Phomopsis vaccinii. Scale ceae in China; Salix in USSR; and Salix excelsa and Populus nigra bar [ 30 nucleotide substitutions. Data from the ex-type in Iran. strain of the new species is in bold. The isolates designated Pathogen on twigs and branches of Juglans regia. Sexual state: as epitype are shown with an asterisk. unknown. Asexual state: Stromata immersed in the bark. 314 X. Fan et al.

Fig 2 e Phylogram based on ITS sequence data for Cytospora species and closest relatives. Values above the branches indicate maximum parsimony and maximum likelihood bootstrap support (BS ‡ 50 %). Thickened branches represent posterior probabilities (BPP ‡ 0.95) from Bayesian inference. The tree is rooted with Phomopsis vaccinii. Scale bar [ 20 nucleotide substitutions. The taxa resulting from the current study are shown in blue. Ex-type taxa are in bold. The isolates designated as epitype are shown with an asterisk.

Conidiomata, erumpent, discoid, conceptacle dark, with a sin- reached 6e7 cm, with a regular edge, sparse sporulation in gle locule. Disc grey to black, nearly ﬂat, circular to ovoid, culture. 0.21e0.32 mm (average ¼ 0.25, n ¼ 20) in diameter, with one Material examined: CHINA, QINGHAI PROVINCE, Xining, ostiole per disc. Ostioles medium sometimes gray to black, Xishan Botanical Garden, 3637025.8900 N, 10144048.9200 E, prominent, 98e147 mm (average ¼ 126, n ¼ 20) in diameter. 2330 m asl., on stems of Juglans regia, collected by X.L. Fan, 19 Locule undivided, circular to ovoid, occasionally wrinkled, August 2012 (BJFC S649, reference specimen designated here), 1.13e1.42 mm (average ¼ 1.31, n ¼ 20) in diameter. Conidio- living culture, CFCC 89615, CCTCC AF2013020. Xining, Xishan phores hyaline, unbranched, or occasionally branched at the Botanical Garden, 3637048.2700 N, 10144045.3300 E, 2331 m asl., base, 15.3e20.4 mm (average ¼ 17.1, n ¼ 20). Conidia hyaline, on stems of Juglans regia, collected by X.L. Fan, 19 August 2012 eguttulate, elongate-allantoid, aseptate, 4.5e5.39 1e1.3 mm (BJFC S650), living culture, CFCC 89616, CCTCC AF2013021. (average ¼ 4.9 1.1, n ¼ 50). Notes: Juglans regia is reported here as a new host for Cyto- Cultures: colony white, producing red pigment after spora atrocirrhata, which has previously been reported from Pop- 7e10 d, ﬂat, felty, ceasing growth when colony diameter ulus and Salix in USSR and Iran (Fotouhifar et al. 2007, 2010). The Cytospora species associated with walnut canker disease 315

Fig 2 e (Continued)

first report of this species in China was from Populus and Salix Pathogen on twigs and branches of J. regia. Sexual state: (Wang et al. 2013). This species was first described from Salix V. ceratosperma (not found in current study). Asexual state: Stro- by Gvritishvili with black conceptacle and single locule and mata immersed in bark. Conidiomata, erumpent, discoid to then re-studied by Fotouhifar et al. (2007, 2010). In the current conical, with multi-locules. Disc light brown to gray, circular study, isolates were shown to be C. atrocirrhata based on unique to ovoid, 0.17e0.24 mm (average ¼ 0.22, n ¼ 20) in diameter, morphological characters of a well-developed conceptacle and with one ostiole per disc. Ostiole gray to black, in the centre a single locule (Fig 5BeF) from previous descriptions, as well of the disc, 79e103 mm (average ¼ 91, n ¼ 20) in diameter. as phylogenetic analysis from Fotouhifar et al. (2010). Multi-locules arrange in circles or ellipse with independent Cytospora atrocirrhata has not been epitypified and there- walls, 0.51e0.96 mm (average ¼ 0.78, n ¼ 20) in diameter. Co- fore we designate BJFC S649 as a reference specimen nidiophores hyaline, unbranched, or occasionally branched at (Ariyawansa et al. 2014). Fresh collections of C. atrocirrhata the bases, 15.3e22.5 mm (average ¼ 18.5, n ¼ 20). Conidia hya- are needed from the type location and host for epitypification. line, elongate-allantoid, eguttulate, aseptate, Cytospora sacculus (Schwein.) Gvrit., Mikol Fitopatol 3: 207 3.6e5.2 0.9e1.2 mm (average ¼ 4.2 1.0, n ¼ 50). (1969). Fig 5 Cultures: colony originally white becoming light grayish- Synonyms: Sphaeria sacculus Schwein., Schr. naturf. Ges. brown after 7 d and finally grayishyellow-brown at 30 d, flat, Leipzig 1: 26 (1822) with a uniform texture, conidiomata irregular on medium Valsa ceratosperma (Tode) Maire, Publ. Inst. Bot. Barcelona 3 surface. (no. 4): 20 (1937) Material examined: CHINA, GANSU PROVINCE, Gannan, Habitat/Distribution: Known from Fagus sylvatica in Nether- Diebu, Luoda, Mogou, 3356046.7700 N, 10352018.1600 E, 1702 m lands; Jacaranda acutifolia in South Africa; J. regia in China; asl., on stems of J. regia, collected by X.L. Fan, 11 August 2012 and Taxus baccata in Switzerland. (BJFC S774, reference specimen designated here), living 316 X. Fan et al.

Fig 3 e Morphology of Cytospora gigalocus from Juglans regia (BJFC S647, holotype). (A, D) Habit of conidiomata on twig. (B, C) Longitudinal sections through conidiomata. (E, F) Transverse sections through conidiomata. (G) Conidiophores. (H) Conidia. (I) Colony on PDA at 3-day. Bars: AeF [ 1 mm, G [ 20 mm, H [ 5 mm.

culture, CFCC 89624, CCTCC AF2013023. ibid., (BJFC S775), liv- the C. sacculus clade to be identical to results in Adams et al. ing culture, CFCC 89625, CCTCC AF2013024. SHAANXI PROV- (2005) and C. sacculus can be distinguished from other Cyto- INCE, Xianyang, Yangling, 3414006.0800 N, 10800048.3000 E, spora species (Figs 1e2). Cytospora sacculus has also been 534 m asl., on stems of J. regia, collected by X.L. Fan, 30 May recorded from Malus, Pyrus, and Ziziphus in China and Iran 2012 (BJFC S567), living culture, CFCC 89626, CCTCC (Wang et al. 2007; Fotouhifar et al. 2010; Du & Zhao 2013). AF2013013. ibid., (BJFC S568), living culture, CFCC 89627, The current study represents the first record of walnut canker CCTCC AF2013014. caused by C. sacculus in China. Notes: Cytospora sacculus has been recorded from a range of Cytospora sacculus has not been epitypified and therefore host plants. Adams et al. (2005) listed the sexual state as V. we treat BJFC S774 as a reference specimen (Ariyawansa ceratosperma and narrowed this morphological species con- et al. 2014). Fresh collections of C. sacculus from the type loca- cept. In the current study, isolates were shown to be C. saccu- tion and host are needed for epitypification. lus based on previous described morphological characters and Cytospora chrysosperma (Pers.) Fr., Sylv. mycol. berol. (Ber- phylogeny (Saccardo 1884; Adams et al. 2005). lin): 28 (1818). Cytospora sacculus has many taxonomic synonyms and Synonyms: Naemaspora chrysosperma Pers., Syn. meth. confusion in both morphology and molecular data. Adams fung. (Gottingen)€ 1: 108 (1801). et al. (2005) considered it as a distinct species with multi- Valsa sordida Nitschke, Pyrenomycetes Germanici 2: 203 ostioles per disc, independent locules walls and grayish to (1870). yellow-brown and brownish gray colonies, and provided the Habitat/Distribution: Known from Armeniaca vulgaris, Cratae- first ITS sequence data. ITS sequence analysis (Fig 2) show gus azarolus, Ficus carica, Ligustrum latifolium, Malus pumila, Cytospora species associated with walnut canker disease 317

Fig 4 e Morphology of Cytospora atrocirrhata from Juglans regia (BJFC S649). (A, D) Habit of conidiomata on twig. (B, C) Lon- gitudinal sections through conidiomata. (E, F) Transverse sections through conidiomata. (G) Conidiophores. (H) Conidia. (I) Colonies on PDA at 3-day and 30-day. Bars: A [ 1 mm, BeF [ 0.5 mm, G [ 20 mm, H [ 5 mm.

Morus alba, Olea sativa, Persica vulgaris, Prunus domestica, Robinia Notes: Cytospora chrysosperma is the type species of Cyto- pseudoacacia, and Thuja orientalis in Iran; Fraxinus in Europe and spora, and occurs worldwide on a wide range of hosts. Accord- Iran; Juglans regia in China and Iran; Salicaceae in China, Iran, ing to earlier records, five plant genera are hosts to this species Netherland, South Africa, Switzerland, UK, and USA; Sophora in China, i.e., Castanea, Morus, Populus, Salix, and Ulmus (Deng japonica in China; Triticum in Germany; and Ulmus in USA; 1963; Tai 1979; Wei 1979; Chen 2002; Zhuang 2005). Infection of Material examined: CHINA, QINGHAI PROVINCE, Haidong, J. regia by this species has only been reported in Iran Ledu, Gaomiao, 3627030.1000 N, 10230014.2200 E, 1935 m asl., (Fotouhifar et al. 2010), thus this is the first report of C. chryso- on stems of Juglans regia, collected by X.L. Fan, 14 August sperma causing walnut canker in China. Isolates were identi- 2012 (BJFC S644), living culture, CFCC 89617, CCTCC fied as C. chrysosperma based on morphological characters, AF2013015. Xining, Xishan Botanical Garden, 3637026.1200 N, host, and phylogeny (Tai 1979; Saccardo 1884; Adams et al. 10144050.7500 E, 2298 m asl., on stems of J. regia, collected by 2005; Fotouhifar et al. 2010) and the taxon is described in Fan X.L. Fan, 19 August 2012 (BJFC S651), living culture, CFCC et al. (2014). Valsa sordida has been reported to be the sexual 89618, CCTCC AF2013022. Xining, Xishan Botanical Garden, state of C. chrysosperma but the Valsa sexual state is difficult 3637026.1200 N, 10144050.7500 E, 2298 m asl., on stems of J. regia, to collect and Cytospora is considered an older name. Thus collected by X.L. Fan, 19 August 2012 (BJFC S652), living culture, the name C. chrysosperma is used in this study. CFCC 89628, CCTCC AF2013011. NINGXIA PROVINCE, Yin- Cytospora chrysosperma has not been epitypified and there- chuan, Helan, 3831050.1300 N, 10617053.0100 E, 1233 m asl., on fore we use BJFC CGHs10 as a reference specimen (Ariyawansa stems of J. regia, collected by X.L. Fan, 5 Feb 2012 (BJFC S510), et al. 2014; Fan et al. 2014). Fresh collections of C. chrysosperma living culture, CFCC 89619, CCTCC AF2013012. from the type location and host are needed for epitypification. 318 X. Fan et al.

Fig 5 e Morphology of Cytospora sacculus from Juglans regia (BJFC S774). (A, D) Habit of conidiomata on twig. (B, C) Longitudinal sections through conidiomata. (E, F) Transverse sections through conidiomata. (G) Conidiophores. (H) Conidia. (I) Colonies on PDA at 3-day and 30-day. Bars: A [ 1 mm, BeF [ 0.5 mm, G [ 20 mm, H [ 5 mm.

obtained from Iranian walnut trees using molecular markers. Acknowledgements African Journal of Biotechnology 10: 15710e15716. Adams GC, Surve-Iyer RS, Iezzoni AF, 2002. Ribosomal DNA sequence divergence and group I introns within the Leucostoma This study was financed by National Natural Science Founda- species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., tion of China (Project No.: 31170603), Fundamental Research ascomycetes that cause Cytospora canker of fruit trees. Myco- Funds for the Central Universities (Project No.: BLYJ201404) logia 94: 947e967. and Programs for Undergraduate of Beijing Forestry University Adams GC, Roux J, Wingfield MJ, 2006. Cytospora species (Asco- Scientific and Technological Innovation and Training (Project mycota, Diaporthales, Valsaceae), introduced and native No.: X201410022002). Thanks are due to Zhongcheng Xi (Forest pathogens of trees in South Africa. Australasian Plant Pathology 35 e Seed and Seedling Management Station of Qingyang, Gansu, : 521 548. Adams GC, Roux J, Wingfield MJ, Common R, 2005. Phyloge- China) and Huaizhang Yao (Environment Protection Bureau netic relationships and morphology of Cytospora of Yulin Economic Development Area, Shaanxi, China) for col- species and related teleomorphs (Ascomycota, Diapor- lection of specimens. thales, Valsaceae) from Eucalyptus. Studies in Mycology 52: 1e144. references Ariyawansa HA, Hawksworth DL, Hyde KD, et al., 2014. Epitypification and neotypification: guidelines with appro- priate and inappropriate examples. Fungal Diversity 69: 1e35. Abbasi K, Abbasi S, Fotouhifar KB, Zebarjadi AR, Cheghamirza K, Cai L, Hyde KD, Taylor PWJ, et al., 2009. A polyphasic approach for 2013. Genetic diversity of Cytospora chrysosperma isolates studying Colletotrichum. Fungal Diversity 39: 183e204. Cytospora species associated with walnut canker disease 319

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