Diaporthalean fungi associated with canker and dieback of trees from Mount Dongling in Beijing, China

Abstract Diaporthales is a fungal order comprising important plant pathogens, saprobes and endophytes on a wide range of woody hosts. It is often difficult to differentiate the pathogens in this order, since both the morphology and disease symptoms are similar among the various species. In the current study, we obtained 15 representative diaporthalean isolates from six tree hosts belonging to plant families Betulaceae, Fagaceae, Juglandaceae, Rosaceae, and Ulmaceae from Mount Dongling in China. Six species were identified residing in four families of Diaporthales (Diaporthaceae, Erythrogloeaceae, Juglanconidaceae and Melanconidaceae). Based on morphological comparison and the phylogenetic analyses of partial ITS, LSU, cal, his3, rpb2, tef1-α and tub2 gene sequences, we identified five known species (Diaporthe betulina, D. eres, D. rostrata, Juglamconis oblonga and Melanconis stilbostoma) and one novel species (Dendrostoma donglinensis). These results represent the first study of diaporthalean fungi associated with canker and dieback symptoms from Mount Dongling in Beijing, China.


Introduction
Diaporthales is an important order in class Sordariomycetes containing taxa that have broad host ranges and widely distributed as plant pathogens, endophytes or saprobes (Fan et al. 2018a. Most families of the Diaporthales are responsible for diseases on a wide range of host plants, some of which are economically important worldwide, causing anthracnose, blights, cankers, dieback, leaf spots and rots of root and fruit (Alvarez et al. 2016, Guarnaccia and Crous 2017, Voglmayr et al. 2017, Jiang et al. 2019a, Xavier et al. 2019, Fan et al. 2020). The order is characterized by perithecia often with elongate beaks, immersed in stromatic tissues, producing deliquescent paraphyses and unitunicate asci that generally deliquesce, become detached from the perithecial wall when mature, and have a characteristic refractive apical annulus in sexual morph; and acervuli, pycnidia or rarely synnemata, producing phialidic or annellidic conidiogenous cells with 0-1-septate conidia in asexual morph (Barr 1978, Rossman et al. 2007, Fan et al. 2020. The classification of Diaporthales has been confused over the past decades because of the wide variation in morphological characters. Several recent studies have helped to resolve taxonomic problems of Diaporthales by multigene phylogenetic analyses and accepted 30 families in the order (Senanayake et al. 2017, Braun et al. 2018, Fan et al. 2018a, Guterres et al. 2019, Xavier et al. 2019. Mount Dongling has a high diversity of plant species in western Beijing, which is considered as a biodiversity hotspot with more than 1000 plant species (Ma et al. 1995). As more plant species were recorded in this region, the exploration of fungal diversity gradually increased as most fungi are often linked to particular host plants as parasites or endophytes. Alternaria, Diaporthe, Leptostroma, Pestalotiopsis and Phoma were the most commonly isolated endophytic fungi from Pinus tabuliformis, and later additional 38 endophytic taxa were identified from Acer truncatum from the Mount Dongling (Guo et al. 2008, Sun et al. 2011. Further, pathogens of Botryosphaeriales have been identified from Mount Dongling, including species from the genera Aplosporella, Botryosphaeria and Phaeobotryon (Zhu et al. 2018).
During the trips to collect forest pathogens causing canker or dieback symptoms in Mount Dongling in Beijing, several specimens associated with typical diaporthalean symptoms were collected from various tree hosts, i.e. Betula dahurica (Betulaceae), Juglans regia, J. mandshurica (Juglandaceae), Prunus davidiana (Rosaceae) and Quercus mongolica (Fagaceae). As the higher-level phylogeny of many genera within the diaporthalean taxa remains largely unresolved in this region, the current study aims to clarify the systematics and taxonomy of these diaporthalean fungi with detailed descriptions.

Sampling and isolation
Fresh specimens of diaporthalean fungi were collected from infected branches of six hosts from Mount Dongling in Beijing, China (Table 1), during the course of cognitive practice at the Beijing Forestry University (BJFU). Diaporthalean canker symptoms include elongated, slightly sunken and discolored areas in the bark, which often splits along the canker margin, forming several prominent dark sporocarps immersed and erumpent through the surface of the bark (Fig. 1). A total of 15 isolates were obtained by removing the mucoid spore mass from conidiomata or ascomata of fresh material, which was cut horizontally with a sterile blade and mixed in a drop of sterile water on a glass slide. The contents were broken up further with the blade until a spore suspension was obtained. The suspension was spread over the surface of 1.8 % potato dextrose agar (PDA). Single germinating spores were transferred on to fresh PDA plates. Specimens and isolates were deposited in the Key Laboratory for Silviculture and Conservation of the Ministry of Education in BJFU, and the working Collection of X.L. Fan (CF) housed at the BJFU. Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Morphology
Descriptions were performed based on morphological features of the ascomata or conidiomata from infected host materials. The macro-morphological photographs were captured using a Leica stereomicroscope (M205 FA) (structure and size of stromata, structure and size of ectostromatic disc and ostioles). Micro-morphological observations (shape and size of conidiophores, asci and conidia/ascospores) were determined under a Nikon Eclipse 80i microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast (DIC) illumination and the Nikon software NIS-Elements D Package v. 3.00. Adobe Bridge CS v. 6 and Adobe Photoshop CS v. 5 were used for the manual editing. Over 10 conidiomata/ ascomata, 10 asci and 30 conidia/ascospores were measured to calculate the mean size/ length and respective standard deviations (SD). Colony diameters were measured and the colony features were described using the color charts of Rayner (1970). Nomenclatural novelties and descriptions were deposited in MycoBank (Crous et al. 2004).

DNA isolation, amplification and sequencing
Genomic DNA was extracted from colonies grown on cellophane-covered PDA using a modified CTAB method (Doyle and Doyle 1990). The primers and PCR conditions are listed in Table 2. DNA sequencing was performed using an ABI PRISM 3730XL     (Rossman et al. 2007, Suetrong et al. 2015, Norphanphoun et al. 2016, Hongsanan et al. 2017, Senanayake et al. 2017, Voglmayr et al. 2017, Yang et al. 2018, Fan et al. 2018a, b, 2020 (Table 1). Subsequent alignments for each gene were generated using MAFFT v.7 (Katoh and Standley 2013) and manually improved where necessary using MEGA v. 6 (Tamura et al. 2013). Novel sequences generated in the current study were deposited in GenBank (Table 1, Suppl. materials 1-3: Tables S1-S3) and the aligned matrices used for phylogenetic analyses were submitted to TreeBASE (www.treebase.org; accession number: S24893).

Phylogenetic analyses
To infer the first phylogenetic relationships at the family level, an initial alignment combining the here generated and available ITS, LSU, rpb2 and tef1-α sequences was compiled following Fan et al. (2018a). This alignment was analyzed based on Maximum Parsimony (MP), Maximum Likelihood (ML), and Bayesian Inference (BI) methods. The MP analysis was conducted using a heuristic search (1,000 bootstrap) by PAUP v. 4.0b10 (Swofford 2003). The MP analysis was conducted with random sequence additions as option to stepwise-addition (1,000 bootstrap replicates and one tree held at each addition step), and maxtrees limited to 100 by replicate. The tree bisection and reconnection (TBR) was selected as option to the branch swapping algorithm (Swofford 2003). The branches of zero length were collapsed and all equally most parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). The ML analysis was performed using a GTR site substitution model, including a gamma-distributed rate heterogeneity and a proportion of invariant sites in PhyML v. 3.0 (Guindon et al. 2010). The BI analysis was conducted using the best-fit evolutionary models for each partitioned locus estimated in MrModeltest v. 2.3 (Posada and Crandall 1998) following the Akaike Information Criterion (AIC), with a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003). Two MCMC chains were run from random trees for 10 million generations and terminated when the average standard deviation of split frequencies dropped below 0.01. Trees were saved in each 1,000 generations. The first 25 % of trees were discarded at the burn-in phase of each analysis, and the Bayesian posterior probabilities (BPP) were calculated to assess the remaining trees (Rannala and Yang 1996). The MP bootstrap support (BS) equal to or above 50 were shown at the first and second position in branches. The branches with significant BPP equal to or above 0.95 were thickened in the phylogram.
In addition to the above analyses, we provided separate phylogenetic trees for two additional genera (Dendrostoma and Diaporthe) in Diaporthales, based on various gene regions (see below) including the same parameters as in the analyses described above. The branch support from MP and ML analyses was evaluated with a bootstrap support (BS) method of 1,000 replicates (Hillis and Bull 1993). Phylograms were plotted in Figtree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and edited in Adobe Illustrator CS6 v.16.0.0 (https://www.adobe.com/cn/products/illustrator.html).

Phylogenetic analysis
The combined matrix (ITS, LSU, rpb2 and tef1-α) of Diaporthales included 198 ingroup accessions (15 from the current study and 183 retrieved from GenBank) and two outgroup taxa. The aligned matrix comprised 4,047 characters including gaps (773 characters for ITS, 1,190 for LSU, 1,114 for rpb2 and 970 for tef1-α), of which 2,002 characters were constant, 158 variable characters were parsimony-uninformative and 1,887 characters were variable and parsimony-informative. MP analyses generated 100 parsimonious trees of which the first tree is presented in Fig. 2 (TL = 12,631, CI = 0.313, RI = 0.792, RC = 0.248). The tree topologies of ML and BI analyses were mostly similar to the generated MP tree. The 15 isolates obtained in this study were clustered within the families Diaporthaceae, Erythrogloeaceae, Juglanconidaceae and Melanconidaceae in Diaporthales (Fig. 2). To delimitate to the species level, phylogenetic trees for Dendrostoma and Diaporthe were constructed separately based on different DNA datasets.
For the genus Diaporthe (Diaporthaceae), a concatenated ITS, cal, his3, tef1-α and tub2 matrix was produced with 201 ingroup accessions (6 from this study and 195 retrieved from GenBank). The combined matrix comprised 3,237 characters including gaps (544 characters for ITS, 593 for cal, 587 for his3, 645 for tef1-α and 868 for tub2) of which 1,330 characters were constant, 442 variable characters parsimony-uninformative and 1,465 characters variable and parsimony-informative. The MP analysis generated 100 parsimonious trees and the first tree is presented in Fig. 3 (TL = 12,978, CI = 0.280, RI = 0.712, RC = 0.199). The isolates of Diaporthe clustered in three different clades, corresponding to the three known species in this genus. The second combined matrix (cal, tef1-α and tub2) focusing on the Diaporthe eres complex included 56 ingroup accessions (4 from this study and 52 retrieved from GenBank). The concatenated matrix comprised 1,198 characters including gaps (405 for cal, 363 for tef1-α and 430 for tub2) of which 933 characters were constant, 112 variable characters parsimony-uninformative and 153 characters variable and parsimony-informative. The MP analysis generated 100 parsimonious trees of which the first is presented in Fig. 4 (TL = 415, CI = 0.701, RI = 0.882, RC = 0.618). The tree topologies of the ML and BI analyses were almost similar to the MP tree.  For the genus Dendrostoma (Erythrogloeaceae), ITS, rpb2 and tef1-α alignments were concatenated, including 42 ingroup accessions (three from this study and 39 retrieved from GenBank) was produced. The full matrix comprised 2,400 characters including gaps (561 characters for ITS, 1,078 for rpb2 and 761 for tef1-α), of which 1,486 characters are constant, 231 variable characters are parsimony-uninformative and 683 characters are variable and parsimony-informative. The only parsimonious tree generated in MP analyses is presented in Fig. 5   Notes. Diaporthaceae was introduced by von Höhnel (1917) and subsequently involved in confusing the taxonomy due to many genera with wide variation of morphological characters and the majority without culture or DNA phylogeny. Senanayake et al. (2017Senanayake et al. ( , 2018 accepted 14 genera in Diaporthaceae, including Allantoporthe,       (1870). The identification of Diaporthe was confused due to the historical species recognition criteria based on overlapped morphology, culture characteristics and host affiliation (Dissanayake et al. 2017). The phylogenetic analysis recommended to delimitate taxa to the species level was first proposed by Udayanga et al. (2012) and later modified to include concatenated alignments of ITS, cal1, his3, tef1-α, tub2 (Gomes et al. 2013  Notes. Yang et al. (2018) described Diaporthe betulina from cankers of Betula spp. in Heilongjiang Province. The only strain CFCC 53144 representing D. betulina clusters in a well-supported clade and appear most closely related to D. betulae, which was also isolated from Betula platyphylla in Sichuan Province . Diaporthe betulina (strain CFCC 52562) differs from D. betulae by its slender alpha conidia (2.5-3 vs. 3-4 μm) , and 13 bp for ITS, 7 bp for cal, 19 bp for his, 12 bp for tef and 6 bp for tub2 based on alignment of the concatenated five-gene deposited in TreeBASE (S24893). Both morphology and sequence data confirmed that our isolates belong to this species. Fig. 6 Description. Sexual morph: not observed. Asexual morph: Pycnidial stromata immersed in bark, scattered, slightly erumpent through the bark surface, unilocular, with a conspicuous central column. Central column beneath the disc more or less conical, pale grey with yellow. Ectostromatic disc orange, elliptical, 160-300 μm in diam., with one ostiole per disc. Ostiole dark brown to black, at the same level as or slightly above the disc surface, 70-80 μm in diam. Locule single, 210-260 μm in diam. Conidiophores cylindrical, hyaline, unbranched, straight or slightly curved, tapering towards the apex, 12-13.5 × 2-3 μm. Conidiogenous cells enteroblastic, phialidic. Alpha conidia hyaline, aseptate, smooth, ellipsoidal, biguttulate, rounded at both ends, 6.5-8.5 × 2.5-3 (av. = 7.3± 0.5 × 2.8 ± 0.3, n = 30) μm. Beta conidia were not observed.

Diaporthe eres Nitschke, Pyrenomyc. Germ. 2: 245 (1870)
Culture characteristics. Cultures on PDA are initially white, growing up to 4 cm in diam. after 3 days, and becoming yellow green to brown after 7-10 days. Colonies are flat felty with a thick texture at the marginal area, with a thin texture at the center, abundant aerial mycelium, sterile. Notes. Diaporthe eres is the type species of Diaporthe, and is also the most common species causing canker disease on a wide range of hosts (Gomes et al. 2013, Udayanga et al. 2014, Dissanayake et al. 2017, Yang et al. 2018. Our isolates are associated with canker disease of Prunus davidiana in China, which belong to the Diaporthe eres species complex (Fig. 4). Fan et al. (2018c) treated many Diaporthe species as D. eres, and showed the combined cal, tef1-α and tub2 genes provide a better topology than the combined five-gene phylogeny for the D. eres complex. Both sequence data and morphology confirm that our isolates belong to this species (Fig. 4). Notes. Fan et al. (2015) introduced Diaporthe rostrata from Juglans mandshurica causing walnut dieback in China. Yang et al. (2017) introduced D. juglandicola as a sister clade with D. rostrata, but it has no conspicuous rostrate necks on the bark. However, we recommend to treat D. juglandicola as a synonym of D. rostrate, based on the same host species, and lacking of phylogenetic support to separate them after involving our current materials (CF 2019807 and CF 2019910) with conspicuous rostrate necks. Description. Sexual morph: not observed. Asexual morph: Pycnidial stromata immersed in the bark, scattered, erumpent through the surface of bark, unilocular, with a conspicuous central column. Central column beneath the disc more or less conical, yellow. Conceptacle absent. Ectostromatic disc hyaline, circular to ovoid, 750-1190 μm in diam., with a single ostiole per disc. Ostiole grey to black, at the same level as the disc surface, 240-270 μm in diam. Locule single, circular to irregular, undivided, 550-750 μm in diam. Conidiophores hyaline, unbranched, approximately cylindrical. Conidiogenous cells enteroblastic, phialidic. Conidia hyaline, fusoid, acute at each end, smooth or occasional not smooth, aseptate, 16.5-20.5 × 2-3.5 (av. = 18 ± 1.1 × 3 ± 0.3, n = 30) μm.

Diaporthe rostrata
Culture characteristics. Cultures on PDA are initially white, growing slowly to 2 cm in diam. after 3 days and 4 cm after 14 days, becoming salmon in the center after 7-10 days. Growth stops when colony reaches 8 cm and cultures becoming salmon to honey after the 30 days. Colonies are felty with a uniform texture; sterile. Notes. Dendrostoma donglinensis is associated with canker disease of Quercus mongolica in China. It can be distinguished from its closest relative D. parasiticum by its fusoid, acute at each end and larger conidia (16.5-20.5 × 2-3.5 vs. 9.3-11.7 ×2.8-3.3 μm). The isolates are phylogenetically distinct from all other available strains of Dendrostoma included in this study and we therefore describe this species as new, based on DNA sequence data and morphology. Notes. Juglanconis was introduced by Voglmayr et al. (2017) to accommodate previous Melanconium juglandinum, M. oblongum and M. pterocaryae based on morphology and DNA data of type materials. The genus is restricted to one host in Juglandaceae, which is identified by having perithecial ascomata, 8-spored asci with an apical ring, hyaline, bicelled ascospores in the sexual morph; and acervular conidiomata, brown conidia with gelatinous sheaths in asexual morph (Voglmayr et al. 2017). Notes. Juglanconis oblonga (previous Melanconium oblongum) is associated with canker disease of Juglandaceae hosts in North America and Southeast Asia (Graves 1923, Voglmayr et al. 2017, Fan et al. 2018b. This species is similar to J. juglandina in disease symptoms but can be distinguished by its longer conidia (22 × 12.5 compared to 20 × 13 μm) and DNA sequence data (Fan et al. 2018b). This species is a common pathogen causing walnut canker in China (Fan et al. 2018b). Notes. Melanconidaceae was introduced by Winter (1886) and has been subject to some confusion due to the overlap in morphological characters between genera and the absence of DNA sequence data supporting the family concept (Barr 1978). Castlebury et al. (2002) and Rossman et al. (2007) restricted this family to a single genus Melanconis based on LSU rDNA sequences, which was adapted by recent studies (Senanayake et al. 2017, Fan et al. 2018b (Zhuang 2005, 2018b.
The classification of Diaporthales presented here follows the previous studies (Castlebury et al. 2002, Rossman et al. 2007) and discoveries of new taxa from many other works (Suetrong et al. 2015, Dissanayake et al. 2017, Voglmayr et al. 2017, Senanayake et al. 2017. We performed frequently and used four genes (ITS, LSU, rpb2 and tef1-α) to evaluate the 30 families in this order, but it was found to be confusing in some taxa such as Apoharknessia and Lasmenia in Apoharknessiaceae (Fig. 2). It suggests that more studies using a multiphasic approach are still needed to clarify some issues in this order. Diaporthales includes many phytopathogenic genera such as Dendrostoma, Diaporthe, Melanconis and Juglanconis, which have been reported causing canker disease of tree hosts in China , 2018b, Yang et al. 2018, Jiang et al. 2019b. The current study focuses on diaporthalean fungi in Mount Dongling of Beijing, which is considered as a biodiversity hotspot with a high diversity for fungal species and (Guo et al. 2008, Zhu et al. 2018. We hope that the descriptions and molecular data of diaporthalean fungi in this study could provide a resource for future studies in this region.