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 bi- ological 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.