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Canker Disease of Willow and Poplar Caused by Cryptosphaeria Pullmanensis Recorded in China

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Canker Disease of Willow and Poplar Caused by Cryptosphaeria Pullmanensis Recorded in China For. Path. 46 (2016) 327–335 doi: 10.1111/efp.12261 © 2016 Blackwell Verlag GmbH Canker disease of willow and poplar caused by Cryptosphaeria pullmanensis recorded in China By R. Ma1,2, Y.-F. Zhu3, X.-L. Fan1 and C.-M. Tian1,4 1The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, China; 2College of Forestry and Horticulture, Xinjiang Agricultural University, Urumqi, China; 3Forestry Research Institute, Xinjiang Agricultural University, Urumqi, China; 4E-mail: [email protected] (for correspondence) Summary A new canker disease of Salix alba and Populus alba has been observed in Xinjiang, China. Black circular spots on dead branches and stems are the symptoms of the disease. Sixty-seven isolates recovered from Salix matsudana, S. alba and Populus alba were identified as Cryp- tosphaeria pullmanensis based on morphological features and multigene phylogeny. Pathogenicity tests were performed on S. alba and P. alba stems using the C. pullmanensis isolates. Cankers on and Cryptosphaeria pullmanensis of C. pullmanensis from the stems fulfilled Koch’s postulates and confirmed C. pullmanensis as the causal agent of the canker disease. C. pullmanensis is characterized by its yellow stromatic tissue surrounded by a black conceptacle with regularly arranged multiple locules sharing common walls and hyaline, allantoid, aseptate conidia (mean size 7.42 9 1.72 lm). This is the first report of C. pullmanensis causing Cryptosphaeria canker in China, and S. alba and P. alba are new host records for C. pullmanensis. 1 Introduction Willow and poplar are fast-growing and easily propagated trees with a wide geographical range and are able to tolerate a wide range of environmental conditions. These trees are extensively used in forestry or integrated with agricultural sys- tems (Jensen et al. 2009; Madejona et al. 2013). In China, willows and poplars are ecologically and economically important, where they are used to form windbreaks and as a method of sand fixation, soil and water conservation and amelioration of saline–alkaline soils (Wang et al. 2015). Poplar has become the main tree planted in China to create shelter forest and for timber, and poplar plantations now cover more than 700 ha (Li 2010). Recently, a new canker disease associated with the occurrence of a Cryptosphaeria sp. on the stems and branches of willow and poplar has been observed in Xinjiang, China. A general survey of forest health revealed that many young trees had subsequently died 2–3 years after infection. Adult trees that had been infected were susceptible to being killed by the pathogen, other pathogens, or by abiotic stresses. In the survey area, approximately 10% of forest area is affected. If the disease continue to large area happened, most trees will die, where the disease is seriously affecting the health of poplars and willows in Xinjiang province. Cryptosphaeria (Diatrypaceae, Xylariales) is a genus of fungi that contain 63 species epithets listed in Index Fungorum (http://www.indexfungorum.org). Most of them without well clarification, however, were assigned to Eutypa and then Cryptosphaeria involved in urgent need to taxonomic revision (Rappaz 1987). Kirk et al. (2008) recorded eight species in the Dictionary of Fungi and Vasilyeva and Ma (2014) introduced Cryptosphaeria nigrescens in north-eastern China. Several species in this genus have been shown to cause plant disease. Cryptosphaeria populina (Pers.) Sacc. (syn Cryptosphaeria lig- niota (Fr.) Auersw.) is the best-known plant pathogen in this genus and is the causal agent of widespread canker through- out the range of aspen (Populus tremuloides Michx.), particular in the western states of the USA (Hinds 1972, 1981; Hinds and Laurent 1978; Juzwik and French 1990; Vasilyeva and Ma 2014). In addition, the occurrence of Cryptosphaeria euno- mia (Fr.) Fuckel on Fraxinus excelsior L., Cryptosphaeria mangrovei K.D. Hyde on mangrove (Hyde 1993; Venkateswara Sarma et al. 2001), Cryptosphaeria subcutanea (Wahlenb.) Rappaz on willow (Rappaz 1987) and Cryptosphaeria pullmanen- sis Glawe on aspen (Glawe 1984, 1989) has been reported in Switzerland, the Czech Republic, Argentina, Germany, Philip- pines, Estonia and the USA (Juzwik and French 1990; Carmaran et al. 2006). Until recently, our knowledge of Cryptosphaeria in China has been limited. C. populina has been previously recorded on Populus 9 berolinensis (K. Koch) Dippel in the north-east of China (Tai 1979; Wang et al. 2008), and C. populina and C. lig- niota have been recorded on Populus sp. in Jiangsu (Liu et al. 2013). Zhao et al. (2006) cited Deng (1963) in connection with the occurrence of C. populina and C.ligniota on Populus sp. in Jiangsu; however, there is no record of these findings in Deng (1963). The occurrence of Cryptosphaeria ligniota, C. exornata Lar.N. Vassiljeva, and C.venusta Lar.N. Vassiljeva in the Ussuri River Valley has been recorded (Wang et al. 2008; Vasilyeva 2011). Recently, Vasilyeva and Ma (2014) described a new species, C. nigrescens, growing on dead branches of Populus davidiana Dode in north-eastern China (Jilin provinces). However, the occurrence of C. pullmanensis has not previously been recorded in China. During a general survey of forestry disease in Xinjiang, China, diseased trees showing signs of Cryptosphaeria infection that showed different disease characteristics to those observed when infected by C. ligniota, C. exornata, C. venusta or C. ni- grescens were observed in five regions. The disease characteristics of the Cryptosphaeria sp. observed on the host and the proliferation, the size and shape of the conidium growing on the isolated colonies were like those previously reported for C. pullmanensis (Glawe 1984, 1989). Cryptosphaeria pullmanensis was first reported on fallen branches of Populus Received: 5.7.2015; accepted: 16.12.2015; editor: T. Sieber http://wileyonlinelibrary.com/ 328 R. Ma, Y.-F. Zhu, X.-L. Fan and C.-M. Tian trichocarpa Torr. & A.Gray in Pullman, Whitman Co., WA, USA (Glawe 1984). The species has also been reported in dis- eased wood of grapevines in California (Trouillas et al. 2010; Andolfi et al. 2011) and Nevada (Trouillas et al. 2010; Urbez- Torres et al. 2012, 2013) and on Populus deltoides W.Bartram ex Humphry Marshall, Populus nigra L. (Trouillas et al. 2010) and Populus fremontii S. Watson (Trouillas et al. 2010) in California. C. pullmanensis has not previously been recorded in association with Populus alba L. or Salix alba L. Several species of Cryptosphaeria isolated from P. tremuloides and Vitis vinifera have previously been identified based on the morphological characteristics of the asexual state and sexual state on the host and the morphology of the anamorph in culture (Hinds 1981; Juzwik and French 1990; Romero and Carmaran 2003; Trouillas et al. 2010). More recently, the inter- nal transcribed spacer (ITS) region and b-tubulin gene sequence in combination with biological and morphological charac- teristics have been widely used for the discrimination of fungal species (Acero et al. 2004; Phillips et al. 2007; Trouillas et al. 2010). The aim of the present study was to identify the pathogenic fungi causing canker disease on willow and poplar in Xin- jiang, China, using Koch’s postulates, morphological features, and by phylogenetic comparison of the internal transcribed spacer (ITS) region and the b-tubulin gene sequence data with those of known sequences in GenBank. The biological char- acteristics (light, temperature, pH and medium) of Cryptosphaeria species isolated from Salix alba and Populus alba were also investigated. 2 Materials and methods 2.1 Sample collection, isolation and preservation Canker samples were collected from Populus alba, Salix matsudana and Salix alba in six regions of Xinjiang, China, between 2013 and 2014 (Fig. 1). To isolate the pathogen, samples were taken from five to 10 cankers of five trees per region. Sam- ples were taken from diseased and healthy tissues at the canker margins and cut into three pieces with a side length of 2–5 mm per canker (Carlucci et al. 2015), surface sterilized with 75% ethanol for 3–5 s and 0.1% mercuric chloride for 1 min, and then washed three times in sterile distilled water and blotted dry with sterilized filter paper. The sections of twig were placed on sterile plates of potato dextrose agar (PDA) and incubated at 28°C. The growing edges of hyphae were transferred onto a new PDA plate after 1–2 day. To obtain pure cultures of each isolate, a block of agar with a single hyphal tip was aseptically excised from the edge of the culture with the aid of a dissecting microscope and a sterile scalpel and transferred to a new PDA plate, which was then incubated at 28°C. All isolates were maintained on fresh PDA slants and stored at 4°C (Zhu et al. 2014). Seven diseased twigs from different areas of Xinjiang were deposited at the Museum of Beijing Forestry University (BJFC), and live cultures of the fungal isolates were deposited at the China Forestry Culture Collection Center (CFCC) (Table 1). 2.2 Morphological and culture characterization The morphological characteristics of the fruiting bodies were examined under the light microscope, including the location of the conidiomata, the size and shape of the conidiogenous cells and the conidia. The length and width of 50 conidia from randomly selected conidiomata of each successful isolate were measured under a Leica light microscope (DM 750). Sampling location City China Prefecture boundary Urumuqi Qitai Atushi Akesu Kashi Hami 0 120 240 480 720 Km Fig. 1. Map of Xinjiang, China, showing the locations of the Cryptosphaeria canker reported in this study. Canker caused by Cryptosphaeria pullmanensis 329 Table 1. Collection data relating to the strains used in this study that had been isolated from cankerous stems from Xinjiang. GenBank accession numbers Species number Date Isolate Host Collector and origin ITS b-tubulin BJFC-S1009 2013-07-11 CFCC89936 Salix alba R.
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