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

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 ligniota (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 pullmanensis 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. ligniota 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. nigrescens 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 diseased 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 internal transcribed spacer (ITS) region and b-tubulin gene sequence in combination with biological and morphological characteristics 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 characteristics (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. Ma: Akesu, Xinjiang, China KM588259 KM593245 BJFC-S1010 2014-05-02 CFCC89937 Salix matsudana R. Ma: Kashi, Xinjiang, China KM588260 KM593246 BJFC-S1011 2013-08-12 CFCC899381 Salix alba R. Ma: Urumuqi, Xinjiang, China KM588261 KM593247 BJFC-S1012 2013-08-05 CFCC89939 Populus alba R. Ma: Hami, Xinjiang, China KM588262 KM593248 BJFC-S1013 2013-08-07 CFCC89940 Populus alba R. Ma: Qitai, Xinjiang, China KM588263 KM593249 BJFC-S1014 2014-05-03 CFCC89941 Salix matsudana R. Ma: Atushi, Xinjiang, China KM588264 KM593250 BJFC-S1015 2014-06-23 CFCC89942 Salix alba R. Ma: Urumuqi, Xinjiang, China KM588265 KM593251

1This strain has been used in the pathogenicity test.

To examine the culture morphology, seven isolates from different hosts and regions were first subcultured onto PDA plates at 28°C for 5 days. A 7-mm-diameter plug from the colony margin of each isolate was then placed in the centre of a 90-mm Petri dish containing PDA. To determine the effect of culture conditions on the growth of the fungal isolates, cultures of each isolate were grown on PDA at 11 different incubation temperatures ranging from 0 to 40°C (Chen et al. 2014), under different light and dark regimes, either 24-h light and 24-h dark, or 12-h light and 12-h dark, and under different pH conditions ranging from pH 1 to À11 for 7 days. Meanwhile, the growth speed of all isolates was tested on different media, either PDA (fresh potato 200 g, glucose 20 g, agar 20 g, ddH2O 1000 ml, natural pH), potato sucrose agar (PSA, fresh potato 200 g, sucrose 20 g, agar 20 g, ddH2O 1000 ml, natural pH), potato maltose agar (PMA, fresh potato 200 g, maltose 20 g, agar 20 g, ddH2O 1000 ml, natural pH), Czapek’s agar (NaNO3 3g,K2HPO3 1 g, MgSO4Á7H2O 0.5 g, KCl 0.5 g, FeSO4 0.01 g, agar 20 g ddH2O 1000 ml, natural pH), oats medium (oats 30 g, agar 20 g, ddH2O 1000 ml, natural pH) or beef extract peptone medium (beef extract 5 g, peptone 10 g, NaCl 5 g, agar 20 g, ddH2O 1000 ml, pH 7.0–7.2) (Zang et al. 2007). There were three replicate plates of each isolate. The colony colour, texture and mean growth rates were recorded (Fang 1998). Colony diameters were measured after 7 day using the crossing method (Niu et al. 2015). Data were analysed by analysis of variance (ANOVA) and Duncan’s multiple range tests (p < 0.05) using SPSS version 20.0 (IBM Corporation, Armonk, NY, USA).

2.3 Molecular characterization and multilocus phylogenetic analysis For DNA extraction, cultures were grown on PDA for 5–7 days at 28°C to obtain pure single-genotype cultures. The mycelium was then scraped off the medium using a sterile scalpel (Acero et al. 2004), and the total genomic DNA was extracted from the mycelia using a modified CTAB method (Doyle 1990). Extracted DNA was electrophoresed on 1% agarose gel to check genomic DNA quality. The ITS region was amplified using the primers ITS1 and ITS4 (White et al. 1990). A portion of the b-tubulin gene was amplified using the primers Bt2A and Bt2B (Glass and Donaldson 1995; Urbez-Torres et al. 2014). Amplification was performed in a 20 ll volume comprising 1 ll (20 ng) DNA template, 1.6 ll dNTP (2.5 mmol/L), 2 ll109 PCR buffer (15 mM/ml MgCl2), 1 ll of each primer (10 ll mol/l), 0.1 ll Taq DNA polymerase (5 U/ll) and 14.3 ll ddH2O. PCR products were electrophoresed on 1% agarose gel (Fan et al. 2014). DNA sequencing was performed using an ABI PRISMâ 3730XL DNA Analyser with BigDyeâ Terminator Kit v.3.1 (Invitrogen) at the Shanghai Invitrogen Bio- logical Technology Company Limited (Beijing, China). The sequences of the fungal isolates were assembled using Seqman v.7.1.0 in the DNASTAR lasergene core suite software (DNASTAR Inc., Madison, WI, USA). Subsequently, the sequences were aligned and edited manually using MEGA 5 (Tamura et al. 2011; Adesemoye et al. 2014). Phylogenetic analysis was performed using PAUP v.4.0b10 for maximum parsimony (MP) analysis (Swofford 2003) and MrBayes v.3.1.2 was used for Bayesian analysis (Ronquist and Huelsenbeck 2003). The ITS and b-tubulin DNA sequences of the fungal isolates were compared with known sequences in GenBank by performing a BLAST search. Strains of Xylaria hypoxylon (L.) Grev. were included in the analysis as an outgroup (Table 2). The phylograms were constructed using FIGTREE v.1.3.1 (Rambaut and Drummond 2010). A maximum parsimony analysis was run using a heuristic search option of 1000 random-addition sequences with a tree bisection and reconnection (TBR) branch-swapping algorithm. The branches of zero length were collapsed, and all equally parsimonious trees were saved. Clade stability was assessed with a bootstrap analysis of 1000 replicates. The other parsimony scores calculated were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). Branch support was evaluated by bootstrapping with 1000 replicates. Bayesian analysis was performed using a Markov Chain Monte Carlo (MCMC) algorithm with Bayesian posterior probabilities (Rannala and Yang 1996). Two Markov chains were run from random trees for 1 000 000 generations, and trees were sampled every hundredth generation, resulting in 10 000 total trees. The first 25% of trees were discarded as the burn-in phase of each analysis, and the posterior probabilities (PP) were calculated using the remaining 7500 trees. All sequence data in our research were deposited in GenBank (Table 1). 330 R. Ma, Y.-F. Zhu, X.-L. Fan and C.-M. Tian

Table 2. Sequence and description of genera in the Diatrypaceae obtained from GenBank and used in the phylogenetic analyses.

GenBank accession number

Species Strain No. Host Origin Collector/Isolator ITS b-tubulin

Cryptosphaeria DWIN100 Populus nigra Yolo Co., CA, USA F.P. Trouillas GQ293930 GQ294015 pullmanensis C. pullmanensis UCD2371NV Vitis vinifera NV, USA J.R. Urbez-Torres GQ293966 GQ294011 C. ligniota C2C Populus emula Switzerland J.F. Acero AJ302418 - C. subcutanea C4C Salix borealis Norway J.F. Acero AJ302420 - Cryptovalsa AD100 Vitis vinifera South Australia F.P. Trouillas HQ692551 HQ692468 ampelina C. ampelina SAPN03 Populus nigra McLaren Flat, F.P. Trouillas HQ692555 HQ692461 South Australia Diatrypella HVGRF03 Citrus paradisi Hunter Valley, F.P. Trouillas/W.M. Pitt HQ692590 HQ692502 vulgaris New South Wales, Australia D. vulgaris HVPT01 Schinus molle Hunter Valley, F.P. Trouillas/W.M. HQ692594 HQ692506 New South Wales, Australia Eutypa lata SAPN01 Populus nigra McLaren Flat, F.P. Trouillas HQ692616 HQ692500 South Australia E. lata EP18 Vitis vinifera Tumbarumba, W.M. Pitt HQ692611 HQ692501 New South Wales, Australia E. leptoplaca SAPA01 Populus alba Adelaide, F.P. Trouillas HQ692599 HQ692488 South Australia E. leptoplaca TUQU01 Quercus sp. Tumbarumba, F.P. Trouillas HQ692598 HQ692491 New South Wales, Australia Eutypella HVGRF01 Citrus paradise Hunter Valley, F.P. Trouillas/W.M. HQ692589 HQ692521 citricola New South Wales, Australia Xylaria - - - - GU300095 GQ487703 hypoxylon

2.4 Pathogenicity tests In vitro inoculation tests were performed to test pathogenicity of the isolates. Five mycelial plugs 5 mm in diameter were excised from each of the 7-day-old cultures of three strains that had been isolated from Salix alba, S. matsudana Koidz. and Populus alba. Branches of 5-year-old S.alba and P. alba were cut into 30-cm-long segments, surface-disinfected with 75% alcohol for 3–5 s and sealed with wax at the top. The bottom of the branches was standing in a pot of water. Then, the bark was removed with a 5-mm-diameter puncher in the upper part of the branch, 5-mm-diameter mycelial plug placed into the wounded tissue and the wound sealed with Parafilm. There were five replicates of each Cryptosphaeria isolate on each of the two hosts (S. alba and P. alba). Mock-inoculated stems were inoculated with sterile PDA plugs (Adesemoye et al. 2014; Carlucci et al. 2015). Both inoculated and mock-inoculated plants were kept in a moist chamber maintained at 28°C for 48 h. Any changes observed in the willow and poplar stems in the region around the inoculation site were recorded. Re-isolates were made from inoculated willow and poplar tissues that showed the same symptoms of disease as those observed in the diseased specimens collected from forest sites. The isolates were identified as described previously (Choi et al. 2014; Park et al. 2014).

3 Results

3.1 Taxonomy Black circular or oval stromata were observed on diseased stems (Fig. 2: a, c). The diameter of the stromata ranged from 1.35 to 3.12 mm (interquartile range 1.41–2.98 mm), with a mean diameter of 2.09 9 2.27 mm. Orange, ﬁliform curly conidial masses oozing from the conidiomata were observed 15 days after inoculation (Fig. 2b). Conidiomata were immersed in the bark and surrounded by a black conceptacle ﬁlled with yellow stromatic tissue. The multiple locules were regularly arranged, sharing common walls (Fig. 2: d–g). Palisades of conidiophores lined the conid- iomatal chambers. Conidiogenous cells were cylindrical, scarred in a sympodial manner, with cells infrequently bearing both annellations and sympodial scars (Fig. 2i indicate with arrows). Conidia were hyaline, allantoid and aseptate. The length of the conidia ranged from 5.63 to 10.07 lm (interquartile range 6.04–8.80 lm) and the width from 1.05 to 2.45 lm (interquartile range 1.23–2.20 lm), with a mean size of 7.42 9 1.7 lm. The isolates recovered from cankers on Salix alba and Populus alba had a similar morphology as those previously reported for isolates of C. pullmanensis. Canker caused by Cryptosphaeria pullmanensis 331

(a) (b)

(c)(d) (e)

(f) (g) (h)

(i) (j)

Fig. 2. Morphological characteristics of Cryptosphaeria pullmanensis on Salix alba; a and c, Symptoms seen on willow in the field. b, Symptoms on willow after inoculation with C. pullmanensis. d and f, Transverse sections through conidiomata. e and g, Longitudinal sections through conidiomata. h, Colony after 15 days on PDA incubated at 28°C: an aerial view of the colony is shown on the left and the underside of the colony is shown on the right; i, conidiophore; j, conidia. Bars in a and b represent 1 cm; bars in c represent 2 mm; bars in d–g represent 1 mm; bars in i and j represent 7 lm. The colonies of all the cultured isolates initially appeared white, but became pale yellow in the centre, fading to white at the margin when grown under conditions of 24-h light, whereas cultures grown in the dark which were white become gray. The colonies appeared rather felty in the centre and were thinner towards the margin. The colonies typically had sec- tors of both sparse and dense mycelium, the dense areas were white to gray, and the sparse areas were yellow. The central of the reverse of the colonies was ash black, and the margin was yellow (Fig. 2h). The colonies covered the plates in 7– 10 days and produced conidia after 10–14 days. The teleomorph was not found. The isolates showed little growth when incubated at temperatures between 0 and 15°C and between 37 and 40°C. The optimum conditions for colony growth were 28–31°C in darkness. The most suitable culture medium for colony growth was oats followed by PDA. All the strains showed significantly more growth on media with a pH of 5–7 than on media with different pH (p ≤ 0.05). However, the strains showed significantly more growth under alkaline conditions than under acid conditions (pH 1–4) (Fig. 3).

3.2 Phylogenetic analyses BLAST analyses showed that the ITS and b-tubulin DNA sequences of the seven isolates recovered from willow and poplar cankers in Xinjiang shared 99% sequence homology with the North American C. pullmanensis isolates in GenBank. To eval- uate the phylogenetic relationships among the seven diﬀerent Chinese Cryptosphaeria isolates (Table 1), their ITS and b- tubulin sequences were analysed using BLAST and compared with 13 closely related GenBank reference sequences belong- ing to genera in the family Diatrypaceae that had been reported by other studies (Trouillas et al. 2010, 2011). 332 R. Ma, Y.-F. Zhu, X.-L. Fan and C.-M. Tian

Fig. 3. Effect of light, temperature, medium and pH on colony diameters (mm) of C. pullmanensis after 7 days. (The bars indicate the mean Æ SE, and the letters indicate significant difference at p < 0.05 level by Duncan’s new multiple range test).

A total of 67 strains associated with Cryptosphaeria canker disease were isolated from specimens of S. alba, S. matsudana and P. alba growing in six counties of Xinjiang, of which seven strains representing the species were used in the phylogeny (Table 1). The sequence data sets for the ITS and b-tubulin were analysed in combination. Tree topologies computed from the MP and Bayesian analyses were similar for the individual gene regions and in the combined analysis. The multilocus analysis includes 932 characters, of which 570 characters are constant, 103 variable characters are parsimony uninformative, and 259 are parsimony informative. A heuristic search generated one parsimonious tree (TL = 645, CI = 0.767, RI = 0.827, RC = 0.634) (Fig. 4). The phylogram constructed using Bayesian analysis is in agreement and not signiﬁcantly diﬀerent from the MP tree. MP bootstrap support (MP-BS) was equal to or above 50%.

3.3 Pathogenicity tests The pathogenicity tests were performed on willows and poplars. They exhibit the same symptoms after inoculation. The bark of the inoculated S. alba and P. alba stems initially showed a light brown to orange discoloration after 2–3 days, which then became black with small, scattered, lens-shaped areas after 7–10 days, similar to those seen on naturally infected stems (Fig. 2b). The fungus produced fruiting bodies and viable spores on the dead bark after 10–15 days and caused extensive discoloration of the heartwood and sapwood, as seen on the infected hosts from which the pathogen was isolated. The mock-inoculated stems remained healthy. The morphology of the pure culture that was re-isolated from symptomatic tissues of willow and poplar was the same as that of the isolate used as the inoculum, thus fulﬁlling Koch’s postulates, and conﬁrming C. pullmanensis as a pathogenic fungus for willow and poplar.

4 Discussion This is the first report of C. pullmanensis causing Cryptosphaeria canker in China and the first record of C. pullmanensis causing Cryptosphaeria canker on Salix alba and Populus alba in the world. The isolates recovered from the cankerous tissues of willow and poplar and reisolated from the willow stem cankers in the pathogenicity test consistently showed the same cultural morphology, indicating that they were the causal agent of the canker by fulfilling Koch’s postulates. The morphological features of the anamorph observed on colonies of the isolates cultured on PDA conformed with previous descriptions of C. pullmanensis (Glawe 1984) and phylogenetic inferences based on the DNA sequence data from the ITS regions and b-tubulin gene. Several other Cryptosphaeria species are morphologically similar to C. pullmanensis. However, C. ligniota, C. exornata, C. sulcata and C. nigrescens have larger, darker, ascospores and shorter conidia than C. pullmanensis (Glawe 1984; Vasilyeva and Ma 2014). C. vicinula (Nyl.) P. Karst. is closely related to C. pullmanensis and has reduced stromata, dark ascospores, and a salica- ceous host. C. vicinula differs from C. pullmanensis because it lacks the unusual thickenings at the ends of the ascospores, has well-developed, pulvinate stromata as opposed to the poorly developed stromata observed in C. pullmanensis, and has significantly longer conidia than C. pullmanensis (Saccardo 1882; Glawe and Rogers 1986). Canker caused by Cryptosphaeria pullmanensis 333

Fig. 4. Phylogenetic tree generated using maximum parsimony and maximum-likelihood analyses based on combined ITS and b-tubulin genes for various species in the family Diatrypaceae. Values indicate maximum-likelihood bootstrap values (BT ≥50%) and the Bayesian posterior probabilities (BPP ≥0.9) before and after the slash, respectively. Xylaria hypoxylon served as outgroup taxon. The isolates recovered from willow and poplar cankers in this study are shown in bold.

Morphological features were not sufficient to identify the isolates recovered in the present study to species level. There- fore, the identity was confirmed by performing DNA sequence comparisons of the ITS and b-tubulin gene regions, which has previously been shown to be an effective method of identifying species of Diatrypaceae (Trouillas et al. 2010, 2011). The results of the phylogenetic analyses were consistent with each other as well as with the combined dataset. Phyloge- netic analyses performed by previous studies have reported that genera in the Diatrypaceae occur in more than one clade, suggesting that genera in the Diatrypaceae have polyphyletic origins (Trouillas et al. 2010). The phylogenetic analysis also showed that C. pullmanensis formed a unique lineage that differed from other members of the Diatrypaceae genus (Crypto- valsa, Eutypella, Eutypa and Diatrypella) that were used in the phylogenetic analyses. C. pullmanensis and Eutypa lata have a similar stromatal morphology. The stromata of these two species can be distinguished by the degree of immersion of the stromata in the host tissue, which is cortical in Cryptosphaeria, whereas in Eutypa it develops in the wood (Rappaz 1987). The conidia of C. pullmanensis are significantly shorter than the conidia of C. subcutanea (Romero and Carmaran 2003) and phylogenetic analysis of this clade grouped these two species into sepa- rate subgroups. Specimens of Cryptosphaeria canker were accidently collected as part of a general survey of forest health that involved collecting specimens of Cytospora canker. The symptoms of both these diseases are similar but can be distinguished by a careful observation of the symptoms. Cytospora canker frequently produces fruiting bodies along the Cryptosphaeria canker perimeter. Because Cytospora fruiting bodies are very conspicuous on the dead tissue, it is often erroneously assumed to be the primary causal agent of the Cryptosphaeria canker. Cytospora canker usually has an irregular outline, with sunken, orange-discoloured perimeters. Cryptosphaeria canker has dead, black, stringy, sootlike bark that adheres tightly to the sapwood and contains small, scattered, lens-shaped, light-coloured areas (Hinds 1972, 1981, 1985; Hinds and Laurent 1978). The anamorph characteristics of C. pullmanensis on the host are morphologically similar to the multichambered conidiomata of Cytospora, which also have allantoid conidia (Sutton 1980). However, C. pullmanensis can be distinguished from Cytospora on the basis that it forms annellides and sympodulae rather than phialides. In this study, the C. pullmanensis isolates recovered from S. alba and P. alba showed most growth when plated on oats, PDA or beef extract media with a pH of 5–7 and incubated in the dark at 28–31°C. These optimum culture conditions for C. pullmanensis differ from those previously reported by Glawe (1984), who maintained cultures on PDA in an incubator operating on a cycle of 10 h of fluorescent light at 20°C followed by 14 h of darkness at 15°C. Accurate identification and diagnostics of fungal pathogens are important for determining the disease cycle and route of transmission. To date, C. pullmanensis has only been recorded on willow and poplar in six regions of Xinjiang. It is not known how or when C. pullmanensis was introduced into China or how widespread the disease is within China. A disease census will be carried out in other parts of China to determine the distribution and host range of C. pullmanensis in China. 334 R. Ma, Y.-F. Zhu, X.-L. Fan and C.-M. Tian Research is also needed to determine the route of transmission and to develop strategies to prevent the introduction of the disease in previously uninfected areas and to control the spread of the disease in areas that are already infected with C. pullmanensis in order to sustain the development of willow and poplar in China. No direct control measures are known. The prevention of trunk wounds and the pruning of dead, dying or diseased branches should aid in reducing the incidence of disease (Hinds 1981; Juzwik et al. 1978). Further investigations will be undertaken to determine the occurrence, epidemic, and impact of this new canker.

Acknowledgements This study was funded by the National Forestry Industry Research Special Funds for Public Welfare Projects (201204501), the National Natural Science Foundation of China (grant number: 31460198) and the Natural Science Foundation of Xin- jiang Uygur Autonomous Region (grant number: 2014211B019). We thank Meng Chen (Xinjiang Uygur Autonomous Region Forestry Bureau pest quarantine) for supporting this research, Ling Ren (Kashi Prefecture Bureau of Forestry pest quarantine) for assistance with collecting specimens and Jingf Sun, Xiaol Liu and Yingm Liu for assistance in the laboratory.

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