Species of Dendrostoma (Erythrogloeaceae, Diaporthales) associated with chestnut and oak canker diseases in China

Abstract Dendrostoma was recently proposed in Erythrogloeaceae (Diaporthales, Sordariomycetes), with all known members recorded as being plant pathogenic on economically important tree hosts. During our collections of Dendrostoma species in China, mild to severe canker symptoms were observed on sweet chestnut (Castaneamollissima) and oak (Quercus spp.) trees. Dead and dying plant tissues exhibiting Dendrostoma canker symptoms were sampled for fungal isolation. A total of 37 Dendrostoma isolates were obtained and analysed using morphological characteristics and molecular data (ITS, LSU, RPB2, TEF1-α). Based on these data, 10 novel clades could be distinguished, which also proved to represent morphologically distinct species described here as Dendrostomaaurorae, D.castaneae, D.castaneicola, D.chinense, D.dispersum, D. parasiticum, D.qinlingense, D.quercus, D.shaanxiense and D.shandongense spp. nov. A key to species of the genus is also provided.

Erythrogloeum (Petrak 1953) is the type genus of Erythrogloeaceae and causes severe anthracnose on Hymenaea courbaril in South America (Ferreira et al. 1992). Chrysocrypta was first proposed in Cryphonectriaceae, being associated with leaf spots on Corymbia spp. in Australia (Crous et al. 2012a), but was subsequently transferred to Erythrogloeaceae, based on DNA sequence data (Senanayake et al. 2017). Disculoides was introduced with two initial species, D. eucalypti and D. eucalyptorum, discovered on diseased Eucalyptus leaves in Australia (Crous et al. 2012b). Two additional Disculoides species, D. calophyllae and D. corymbiae, were subsequently reported as foliar pathogens of Corymbia calophylla (Crous et al. 2016.
Dendrostoma (Erythrogloeaceae, Diaporthales) was recently introduced as a phytopathogenic fungal genus causing canker diseases on several economic hardwoods such as Malus spectabilis, Osmanthus fragrans and Quercus acutissima ). Subsequently, Dendrostoma leiphaemia on Quercus trees was transferred from Amphiporthe based on ITS and LSU sequences analysis (Senanayake et al. 2018). Dendrostoma represents one of four genera in the family, but is the only one known to have a sexual morph. Hence, Erythrogloeaceae can be distinguished from the other diaporthalean families by multiguttulate and bicellular ascospores that are constricted at the septum and acervular conidiomata, with subcylindrical to ampulliform conidiogenous cells and hyaline to olivaceous, aseptate conidia (Rossman et al. 2007, Voglmayr and Jaklitsch 2014, Senanayake et al. 2017, Voglmayr et al. 2017.
The Erythrogloeaceae, including Chrysocrypta, Dendrostoma, Disculoides and Erythrogloeum, represent a family of fungal pathogens occurring on several commercially important tree genera such as Corymbia, Eucalyptus, Hymenaea, Malus, Osmanthus and Quercus in Australia, Brazil, China and Costa Rica (Petrak 1953, Ferreira et al. 1992, Crous et al. 2012a, b, 2016. Considering the importance of these tree diseases and the lack of taxonomic information on Dendrostoma, we conducted several surveys for members of the genus in China. The aims of present study were (i) to describe the important Dendrostoma spp. associated with canker diseases on chestnut and oak trees in China and (ii) to provide a multi-gene phylogeny for the genus Dendrostoma based on a large set of freshly collected specimens in China. In agreement with previous taxonomic studies in Erythrogloeaceae, where different Disculoides spp. were discovered on Myrtaceae (Crous et al. 2012a(Crous et al. , b, 2016, several Dendrostoma spp. were found on Fagaceae (Castanea and Quercus), being associated with mild to severe canker diseases. The Dendrostoma species were subsequently classified based on morphological characteristics and phylogenetic data.

Sample collections and fungal isolates
Surveys for Dendrostoma species were conducted in plantations, nurseries, parks, gardens, on mountains and natural reserves in Beijing, Hebei, Shaanxi, Shandong, Tian-jin and Zhejiang Provinces in China from 2017 to 2018. Typical canker symptoms were observed on stems, branches and twigs of different hosts, including Castanea mollissima, Quercus aliena, Q. aliena var. acuteserrata, Q. wutaishanica and other Quercus species (Fig. 1). Diseased samples were collected and placed in paper bags, then transferred to the laboratory for further study.
A total of 37 Dendrostoma isolates were established by removing a mucoid spore mass from sporulating ascomata and conidiomata produced on diseased bark, spreading the suspension on the surface of potato dextrose agar (PDA) plates and incubating the plates at 25 °C in the dark for up to 24 h. Single germinating spores were then transferred to clean plates under a dissecting microscope with a sterile needle. Specimens and isolates were deposited in the Museum of Beijing Forestry University (BJFC). Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Morphological analysis
The identification of Dendrostoma spp. was based on morphological features observed on the natural substrates. Cross-sections for ascomata and conidiomata from tree barks were prepared by hand using a double-edged blade under a dissecting microscope. At least 10 conidiomata/ascomata, 10 asci and 50 conidia/ascospores were measured to calculate the mean size and standard deviation. Measurements are reported as maxima and minima in parentheses and the range representing the mean plus and minus the standard deviation of the number of measurements is given in parentheses (Voglmayr et al. 2017). Microscopy photographs were captured with a Nikon Eclipse 80i compound microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast illumination. Nomenclatural novelties and descriptions were deposited in MycoBank (Crous et al. 2004). Cultural characteristics were recorded for isolates incubated on PDA in the dark at 25 °C.

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fungal colonies growing on PDA using a modified cetyl trimethyl ammonium bromide method (CTAB; Doyle andDoyle 1990, Zhang et al. 2010). The ITS region was amplified using the primers ITS1 and ITS4 (White et al. 1990), the LSU region with the primers LR0R and LR5 (Vilgalys and Hester 1990), the RPB2 region with primers fRPB2-5F and fRPB2-7cR (Liu et al. 1999) and the partial TEF1-α gene with the primers EF1-728F and EF1-986R (Carbone and Kohn 1999
A partition homogeneity test with heuristic search and 1000 replicates was performed using PAUP v. 4.0b10 to assess the discrepancy amongst the ITS, LSU, TEF1-α and RPB2 sequence datasets in reconstructing phylogenetic trees. MP analysis was run using a heuristic search option of 1000 search replicates with random-additions of sequences with a tree bisection and reconnection algorithm. Maxtrees were set to 5000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). ML analysis was performed using a GTR site substitution model including a gamma-distributed rate heterogeneity and a proportion of invariant sites (Guindon et al. 2010). The branch support was evaluated using a bootstrapping method of 1000 replicates (Hillis and Bull 1993). Phylograms were shown using FigTree v. 1.3.1 (Rambaut and Drummond 2010). Novel sequences generated in the current study were deposited in GenBank (Table 1) and the aligned matrices used for phylogenetic analyses in TreeBASE (accession number: S22929).

Phylogenetic analyses
The alignment based on the combined sequence dataset (ITS, LSU, TEF1-α and RPB2) included 46 ingroup taxa and three outgroup taxa, comprising 3536 characters in the aligned matrix. Of these, 2612 characters were constant, 175 variable characters were parsimony-uninformative and 749 characters were parsimony informative (101 from ITS, 21 from LSU, 389 from TEF1-α and 238 from RPB2). The MP analysis resulted in 108 equally most parsimonious trees (TL = 1590, CI = 0.744, RI = 0.897, RC = 0.668); the first tree is shown in Fig. 2. The phylogram based on the four gene sequences indicated 10 new species in Dendrostoma (Fig. 2), as described below.  Description. Sexual morph: Pseudostromata small to large, distinct, circular, erumpent, consisting of an inconspicuous ectostromatic disc, semi-immersed to superficial, causing a pustulate bark surface. Ectostromatic disc flat or concave, orange, surrounded by bark flaps. Central column beneath the disc more or less conical. Stromatic zones lacking. Ascomata perithecial, conspicuous, umber to fuscous black, embedded in orange to umber pseudostromatic tissue, regularly scattered, surrounding the ectostromatic disc, with small to long ostioles that emerge within the ectostromatic disc. Ostioles flat in the disc or sometimes slightly projecting, cylindrical, sometimes obscuring the disc, covered by an orange, umber to fuscous black crust. Paraphyses deliquescent. Asci fusoid, 8-spored, 2-3-seriate, with an apical ring, becoming detached from the perithecial wall. Ascospores hyaline, fusoid to cylindrical, symmetrical to asymmetrical, straight to curved, bicellular, with a median septum, constricted at the septum, smooth, multiguttulate. Asexual morph: Conidiomata pycnidial, spherical to conical to pulvinate, occurring separately, immersed to semi-immersed in bark; wall of several layers of yellow textura angularis. Central column beneath the disc conical or not. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of cavity, hyaline, smooth, subcylindrical to ampulliform. Conidia hyaline, aseptate, smooth, multiguttulate or not, thin-walled, ellipsoid to fusoid, straight to curved. Diagnosis. Dendrostoma aurorae differs from D. chinensis and D. shandongense by the existence of obvious central column.
Culture characters. On PDA, cultures are initially white, becoming isabelline after 2 weeks. The colonies are flat with irregular edge; texture uniform within 1 month at 25 °C in the dark.
Notes. Dendrostoma aurorae was discovered on stems of dying chestnut trees and appears morphologically similar to the chestnut blight pathogen, Cryphonectria parasitica. However, these two diaporthalean pathogens can be distinguished by the existence of a central column inside the conidiomata of Dendrostoma aurorae. In the genus Dendrostoma, D. aurorae differs from D. chinensis and D. shandongense by the existence of an obvious central column. Diagnosis. Dendrostoma castaneae is distinguished from the phylogenetically closely related species D. castaneicola by its narrower conidia.
Culture characters. On PDA, cultures are initially white, becoming black after 2 weeks. The colonies are flat with irregular edge; texture uniform, producing a circle with faint orange conidiomata distributed along the edge of the circle within 1 month at 25 °C in the dark. Notes. Dendrostoma castaneicola, D. castaneae and D. shaanxiense comprise three closely related pathogen species causing chestnut canker diseases in China, all three species occurring on Castanea mollissima. They differ with regard to conidiomatal characteristics, including conidial dimensions (Table 2)  Diagnosis. Dendrostoma chinense differs from D. shandongense by the appearance of conidiomata and is again similar to D. shandongense in its conidial characteristics.
Culture characters. On PDA, cultures are initially white, becoming dark orange after 2 weeks. The colonies are flat with irregular edge; texture uniform, producing concentric circles within 1 month at 25 °C in the dark. Notes. Dendrostoma parasiticum constitutes a widely distributed species occurring on several Fagaceae tree species including Castanea mollissima, Quercus aliena, Q. aliena var. acuteserrata and Q. wutaishansea. Dendrostoma parasiticum appears to be associated with tree dieback, canker and even tree death, although its pathogenicity remains unproven. Dendrostoma parasiticum is close to D. quercus in the phylogram (Fig. 2), but differs from D. quercus with shorter (9.3-11.7 μm in D. parasiticum vs. 13.3-16.1 μm in D. quercus) and narrower (2.8-3.3 μm in D. parasiticum vs. 3.5-4.2 μm in D. quercus) conidia.
Culture characters. On PDA, cultures are initially white, exhibiting light grey after 2 weeks. The colonies are flat with irregular edge; texture uniform, producing concentric circles with sparse conidiomata irregularly distributed on the centre of the plate within 1 month at 25 °C in the dark.

Dendrostoma quercus C.M. Tian & N. Jiang, sp. nov.
MycoBank: MB826824 Figure 10 Diagnosis. Dendrostoma quercus is recognised by the existence of dimorphic conidia, which is unique in the genus.
Notes. Dendrostoma shandongense and D. chinensis occasionally occur on the same branches. These species are best distinguished by the appearance of their conidiomata, which are black in Dendrostoma chinense and orange in D. shandongense.

Discussion
In this study, we reviewed the taxonomic circumscription of Dendrostoma using molecular and morphological data. This is the first study that presents a robust phylogeny using a number of Dendrostoma isolates from different geographic origins. The results revealed up to 14 species in Dendrostoma based on the observation of type specimens and ex-type cultures (D. leiphaemia was not observed), of which 10 species were shown to represent new species, namely D. aurorae, D. castaneae, D. castaneicola, D. chinense, D. dispersum, D. parasiticum, D. qinlingense, D. quercus, D. shaanxiense and D. shandongense.
The 13 type specimens in Dendrostoma (except D. leiphaemia) were examined to establish robust morphological characteristics amongst specific ranks. Amongst these, 3 species, Dendrostoma mali, D. osmanthi and D. quercinum, were discovered to only have a sexual morph on natural hosts; 9 species, D. aurorae, D. castaneae, D. castaneicola, D. chinense, D. dispersum, D. parasiticum, D. qinlingense, D. shaanxiense and D. shandongense, were observed with only an asexual morph and only one species, D. quercus, was represented by both asexual and sexual morphs. Hence, morphological differences amongst Dendrostoma species were mainly established based on conidiomata produced on diseased host tissues, including colours of conidiomata, culture characteristics (Fig. 13), existence or non-existence of a central column, conidial shape and dimensions.
Dendrostoma shandongense and D. chinense are similar in conidial shape and size, but differ markedly from the other species. Additionally, Dendrostoma shandongense and D. chinense comprise the only two species in the genus with conidiomata lacking a central column structure, although they differ considerably with regard to in conidiomatal appearance (Figs. 6, 13). The remaining eight species differ by the existence of a central column inside the conidiomata and can be further distinguished by their conidial characteristics, namely length, width and l/w ratio. Additionally, a key to the 14 Dendrostoma species is provided below. The genus Dendrostoma was initially proposed to include three presumed plant pathogens causing canker diseases on hardwood trees, namely D. mali on Malus spectabilis, D. osmanthi on Osmanthus fragrans and D. quercinum on Quercus acutissima . Consistent with the previous study, the newly described 10 species were all isolated from fruiting structures associated with typical canker symptoms on several hardwood tree species, namely Castanea mollissima and Quercus spp.

Key to
The tree genera Castanea and Quercus in Fagaceae contain numerous important and common tree species in China, including C. mollissima, C. crenata, C. henryi, C. seguinii, Q. acutissima, Q. aliena, Q. dentata, Q. mongolica and Q. wutaishanica (Flora of China website: http://frps.eflora.cn/). Castanea mollissima constitutes one the most important crop tree species widely cultivated in 26 provinces in China. However, many plantations and nurseries planting Chinese chestnut suffer from fungal diseases that cause high production losses (Jiang et al. 2018). In particular, chestnut blight caused by Cryphonectria parasitica represents the most serious fungal disease, reducing host vitality and potentially killing the host (Jiang et al. 2018, Rigling andProspero 2018).
In the present study, seven Dendrostoma species were observed on the host Castanea mollissima including D. aurorae, D. castaneae, D. castaneicola, D. chinense, D. parasiticum, D. shaanxiense and D. shandongense, causing chestnut canker diseases, termed Dendrostoma canker herein. Dendrostoma canker constitutes a newly discovered disease that has been observed in chestnut plantations and nurseries. Species of Dendrostoma usually infect host branches and stems, with occasional infection of twigs. Maturation of the fruiting structures from June to July resulted in death of the infected branches. Notably, no sexual fruiting structures were discovered during our investigations on chestnut trees.
Accurate recognition and identification of plant diseases are essential as fungal pathogens are constantly evolving and traditional control methods are frequently insufficient for disease control. In comparison, in the present study, Dendrostoma canker is considered to be caused by up to eight different species of Dendrostoma. Further studies are, however, required to confirm their pathogenicity and fully resolve their ecology.