High diversity of Diaporthe species associated with dieback diseases in China, with twelve new species described

Abstract Diaporthe species have often been reported as important plant pathogens, saprobes and endophytes on a wide range of plant hosts. Although several Diaporthe species have been recorded in China, little is known about species able to infect forest trees. Therefore, extensive surveys were recently conducted in Beijing, Heilongjiang, Jiangsu, Jiangxi, Shaanxi and Zhejiang Provinces. The current results emphasised on 15 species from 42 representative isolates involving 16 host genera using comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions, as well as their morphological features. Three known species, D.biguttulata, D.eres and D.unshiuensis, were identified. In addition, twelve novel taxa were collected and are described as D.acerigena, D.alangii, D.betulina, D.caryae, D.cercidis, D.chensiensis, D.cinnamomi, D.conica, D.fraxinicola, D.kadsurae, D.padina and D.ukurunduensis. The current study improves the understanding of species causing diebacks on ecological and economic forest trees and provides useful information for the effective disease management of these hosts in China.


Introduction
The genus Diaporthe Nitschke represents a cosmopolitan group of fungi occupying diverse ecological behaviour as plant pathogens, endophytes and saprobes (Muralli et al. 2006, Rossman et al. 2007, Garcia-Reyne et al. 2011, Udayanga et al. 2011, 2012a, b, 2014a, b, 2015, Gomes et al. 2013, Fan et al. 2015, Du et al. 2016, Dissanayake et al. 2017b, Guarnaccia and Crous 2017, Yang et al. 2017a, b, 2018, Marin-Felix et al. 2018. Diaporthe species are responsible for diseases on a wide range of plant hosts, including agricultural crops, forest trees and ornamentals, some of which are economically important. Several symptoms such as root and fruit rots, dieback, stem cankers, leaf spots, leaf and pod blights and seed decay are caused by Diaporthe spp. (Uecker 1988, Rehner and Uecker 1994, Mostert et al. 2001, Santos et al. 2011, Thompson et al. 2011, Udayanga et al. 2011. For example, D. ampelina, the causal agent of Phomopsis cane and leaf spot, is known as a severe pathogen of grapevines (Hewitt and Pearson 1988), infecting all green tissues and causing yield reductions of up to 30% in temperate regions (Erincik et al. 2001). Diaporthe citri is another well-known pathogen exclusively found on Citrus spp. causing melanose, stem-end rot and gummosis in all the citrus production areas except Europe (Mondal et al. 2007, Udayanga et al. 2014a, Guarnaccia and Crous 2017. Similarly, stem canker, attributed to several Diaporthe spp., is one of the most important diseases of sunflower (Helianthus annuus) worldwide (Muntañola-Cvetković et al. 1981, Thompson et al. 2011).
Several species of Diaporthe include a broad number of endophytes associated with hosts present in temperate and tropical regions (Udayanga et al. 2011). Gomes et al. (2013) considered that D. endophytica is a sterile endophyte on Schinus terebinthifolius and Maytenus ilicifolia based on molecular phylogeny. Huang et al. (2015) distinguished seven undescribed Diaporthe species associated with citrus in China. Moreover, some endophytes have been shown to act as opportunistic plant pathogens. For instance, D. foeniculina has been found as both endophyte and opportunistic pathogen on various herbaceous weeds, ornamentals and fruit trees (Udayanga et al. 2014a, Guarnaccia et al. 2016.
The genus Diaporthe (syn. Phomopsis) was established by Nitschke (1870). Species identification criteria in Diaporthe were originally based on host association, morphology and culture characteristics (Mostert et al. 2001, Santos and Phillips 2009, Udayanga et al. 2012. As a consequence, a broad increase in the number of proposed Diaporthe species occurred. More than 1000 epithets for Diaporthe and 950 for Phomopsis were listed in Index Fungorum (2018) (http://www.indexfungorum. org/) (accessed 1 March 2018). The abolishment of the dual nomenclature system for pleomorphic fungi raised the question about which generic name to use. Given that both names are well known amongst plant pathologists and have been equally used, Rossman et al. (2015) proposed that the name Diaporthe (Nitschke 1870) has priority over Phomopsis (Saccardo and Roumeguère 1884) and has been adopted as the generic name in recent major studies (Gomes et al. 2013, Udayanga et al. 2014a, b, 2015, Fan et al. 2015, Huang et al. 2015, Du et al. 2016, Gao et al. 2017, Yang et al. 2017a, b, c, 2018. The sexual morph of Diaporthe is characterised by immersed ascomata and an erumpent pseudostroma with elongated perithecial necks. Asci are unitunicate, clavate to cylindrical. Ascospores are fusoid, ellipsoid to cylindrical, hyaline, biseriate to uniseriate in the ascus and sometimes with appendages (Udayanga et al. 2011). The asexual morph is characterised by ostiolate conidiomata, with cylindrical phialides producing three types of hyaline, aseptate conidia (Udayanga et al. 2011). Previously, species identification of Diaporthe was largely referred to the assumption of host-specificity, leading to the proliferation of names (Gomes et al. 2013). More than one species of Diaporthe can colonise a single host, while one species can be associated with different hosts (Santos and Phillips 2009, Diogo et al. 2010, Santos et al. 2011, Gomes et al. 2013). In addition, considerable variability of the phenotype characters is present within a species (Rehner and Uecker 1994, Mostert et al. 2001, Udayanga et al. 2011, 2012a. Species identification is essential for understanding the epidemiology and plant diseases management and to guide the implementation of phytosanitary measures (Santos and Phillips 2009, Udayanga et al. 2011, Santos et al. 2017. Thus, molecular data are necessary to resolve Diaporthe taxonomy and, during the recent years, many species have been described through a polyphasic approach together with morphology (Gomes et al. 2013, Udayanga et al. 2014a, b, 2015, Huang et al. 2015, Gao et al. 2017, Guarnaccia and Crous 2017. Santos et al. (2017) revealed that the use of a five-loci dataset (ITS-cal-his3-tef1-tub2) is the optimal combination for species delimitation, showing the ribosomal ITS locus as the least informative, which is contrary to the result of Santos et al. (2010).
Although the classification of Diaporthe has been on-going, species are currently being identified based on a combination of morphological, cultural, phytopathological and phylogenetical analyses (Gomes et al. 2013, Huang et al. 2013, Udayanga et al. 2014a, b, 2015, Fan et al. 2015, Du et al. 2016, Gao et al. 2016, Guarnaccia and Crous 2017, Perera et al. 2018a, b, Tibpromma et al. 2018. However, fungi isolated from forest trees in China were recorded in old fungal literature without any living culture and molecular data (Teng 1963, Tai 1979, Wei 1979. The current study aimed to investigate the major ecological or economic trees in China by large-scale sampling and to identify isolates via morphology and multi-locus phylogeny based on modern taxonomic concepts. From 2015 to 2017, several surveys were conducted in six Provinces representing 16 host genera. The objectives of the present study were (i) to provide a multi-gene phylogeny for the genus Diaporthe based on a large set of freshly collected specimens in China; (ii) to identify Diaporthe taxa associated with disease symptoms or non-symptomatic tissues of various host genera distributed over six Provinces in China; (iii) to define the species limits of D. eres and closely related species based on multi-gene genealogies.

Isolates
From 2015 to 2017, fresh specimens of Diaporthe were collected from symptomatic or non-symptomatic twigs or branches from Beijing, Heilongjiang, Jiangsu, Jiangxi, Shaanxi and Zhejiang Provinces in China (Table 1). A total of 105 isolates were obtained by removing a mucoid spore mass from conidiomata and spreading the suspension on the surface of 1.8% potato dextrose agar (PDA) in a Petri dish and incubating at 25 °C for up to 24 h. Single germinating conidia were transferred on to fresh PDA plates. Forty-two representative Diaporthe strains were selected based on cultural characteristics on PDA, conidia morphology and ITS sequence data. Specimens were deposited in the Museum of the Beijing Forestry University (BJFC). Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Morphological analysis
Agar plugs (6 mm diam.) were taken from the edge of actively growing cultures on PDA and transferred on to the centre of 9 cm diam Petri dishes containing 2% tap water agar supplemented with sterile pine needles (PNA; Smith et al. 1996) and potato dextrose agar (PDA) and incubated at 20-21 °C under a 12 h near-ultraviolet light/12 h dark cycle to induce sporulation as described in recent studies (Gomes et al. 2013, Lombard et al. 2014. Colony characters and pigment production on PNA and PDA were noted after 10 d. Colony colours were rated according to Rayner (1970). Cultures were examined periodically for the development of ascomata and conidiomata. The morphological characteristics were examined by mounting fungal structures in clear lactic acid and 30 measurements at 1000× magnification were determined for each isolate using a Leica compound microscope (DM 2500) with interference contrast (DIC) optics. Descriptions, nomenclature and illustrations of taxonomic novelties are deposited in MycoBank (www.MycoBank.org; Crous et al. 2004b).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from colonies grown on cellophane-covered PDA using a modified CTAB [cetyltrimethylammonium bromide] method (Doyle and Doyle 1990). DNA was estimated by electrophoresis in 1% agarose gel and the quality was measured using the NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), following the user manual (Desjardins et al. 2009). PCR amplifications were performed in a DNA Engine Peltier Thermal Cycler (PTC-200; Bio-Rad Laboratories, Hercules, CA, USA). The primer sets ITS1/ITS4 (White et al. 1990) were used to amplify the ITS region. The primer pair CAL228F/CAL737R (Carbone and Kohn 1999) were used to amplify the calmodulin gene (cal) and the primer pair CYLH4F (Crous et al. 2004a) and H3-1b (Glass and Donaldson 1995) were used to amplify part of the histone H3 (his3) gene. The primer pair EF1-728F/EF1-986R (Carbone and Kohn 1999) were used to amplify a partial fragment of the translation elongation factor 1-α gene (tef1). The primer sets T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) were used to amplify the beta-tubulin gene (tub2); the additional combination of Bt2a/Bt2b (Glass and Donaldson 1995) was used in case of amplification failure of the T1/Bt2b primer pair. Amplifications of different loci were performed under different conditions (Table 2). PCR amplification products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM® 3730XL DNA Analyser with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses
DNA generated sequences were used to obtain consensus sequences using SeqMan v.7.1.0 DNASTAR Lasergene Core Suite software programme (DNASTAR Inc., Madison, WI, USA). Sequences were aligned using MAFFT v.6 (Katoh and Toh 2010) and edited manually using MEGA6 (Tamura et al. 2013). Two different datasets were employed to estimate two phylogenetic analyses: one for Diaporthe species and one for Diaporthe eres complex. The first analysis was undertaken to infer the interspecific relationships in Diaporthe. All the Diaporthe isolates recovered from samples collected during this study and additional reference sequences of Diaporthe species were included in the dataset of combined ITS, cal, his3, tef1, and tub2 regions (Table 1), with Diaporthella corylina (CBS 121124) as outgroup. The second analysis focused on the Diaporthe eres complex based on cal, tef1 and tub2 loci (Table 3) according to recent publications (Gao et al. 2014, Udayanga et al. 2014b, Tanney et al. 2016, Fan et al. 2018, with Diaporthe citri (AR3405) as outgroup. Maximum Parsimony analysis was performed by a heuristic search option of 1000 random-addition sequences with a tree bisection and reconnection (TBR) 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). Maximum Likelihood analysis was performed with a GTR site substitution model (Guindon et al. 2010).
Branch support was evaluated with a bootstrapping (BS) method of 1000 replicates (Hillis and Bull 1993). Bayesian inference (BI) analysis, employing a Markov chain Monte Carlo (MCMC) algorithm, was performed (Rannala and Yang 1996). MrModeltest v. 2.3 was used to estimate the best-fit model of nucleotide substitution model settings for each gene (Posada and Crandall 1998). Two MCMC chains started from random trees for 1,000,000 generations and trees were sampled every 100 th generation, resulting in Newly sequenced material is indicated in bold type. a total of 10,000 trees. The first 25% of trees were discarded as the burn-in phase of each analysis. Branches with significant Bayesian posterior probabilities (BPP) were estimated in the remaining 7500 trees. Sequences data were deposited in GenBank (Table 1). The multilocus sequence alignments were deposited in TreeBASE (www.treebase.org) as accession S22702 and S22703. The taxonomic novelties were deposited in MycoBank (Crous et al. 2004b).

Collection of Diaporthe strains
Forty-two representative Diaporthe strains were isolated from 16 different host genera (Table 1) collected from six Provinces (Beijing, Heilongjiang, Jiangsu, Jiangxi, Shaanxi and Zhejiang) in China. All of these strains were isolated from symptomatic or nonsymptomatic branches or twigs and preserved in the China Forestry Culture Collection Centre (CFCC).

Phylogenetic analyses
The first sequences dataset for the ITS, cal, his3, tef1, and tub2 was analysed in combination to infer the interspecific relationships within Diaporthe. The combined species phylogeny of the Diaporthe isolates consisted of 236 sequences, including the outgroup sequences of Diaporthella corylina (culture CBS 121124). A total of 2948 characters including gaps (516 for ITS, 568 for cal, 520 for his3, 486 for tef1 and 858 for tub2) were included in the phylogenetic analysis. The maximum likelihood tree, conducted by the GTR model, confirmed the tree topology and posterior probabilities of the Bayesian consensus tree. For the Bayesian analyses, MrModeltest suggested that all partitions should be analysed with dirichlet state frequency distributions. The following models were recommended by MrModeltest and used: GTR+I+G for ITS, cal and his3, HKY+I+G for tef1 and tub2. The topology and branching order of ML were similar to BI analyses (Fig. 1). Based on the multi-locus phylogeny and morphology, 42 strains were assigned to 15 species, including 12 taxa which we describe here as new (Fig. 1).
The second dataset with cal, tef1 and tub2 sequences were analysed to focus on the Diaporthe eres complex. The alignment included 86 taxa, including the outgroup sequences of Diaporthe citri (Table 3). The aligned three-locus datasets included 1148 characters. Of these, 881 characters were constant, 105 variable characters were parsimony-uninformative and 162 characters were parsimony informative. The heuristic search using maximum parsimony (MP) generated 105 parsimonious trees (TL = 438, CI = 0.669, RI = 0.883, RC = 0.591), from which one was selected (Fig. 2). Based on the multi-locus phylogeny and morphology, seven strains were identified as D. eres, seven strains formed three distinct clades embedded in the D. eres complex, i.e. D. betulina, D. chensiensis and D. padina. MP and ML bootstrap support values above 50% are shown as first and second position, respectively. The branches with significant Bayesian posterior probability (≥ 0.70) in Bayesian analyses were thickened in the phylogenetic tree. The current results, based on the three genes (cal, tef1 and tub2), suggest that D. eres clade could be separated from other species in this complex (      Diagnosis. Diaporthe acerigena can be distinguished from the phylogenetically closely related species D. oraccinii in larger alpha conidia. Holotype. CHINA. Shaanxi Province: Qinling Mountain, on symptomatic twigs of Acer tataricum, 27 June 2017, N. Jiang (holotype: BJFC-S1466; ex-type culture: CFCC 52554).
Culture characters. Cultures incubated on PDA at 25 °C in darkness. Colony at first white, becoming dark brown in the centre with age. Aerial mycelium white, dense, fluffy, with cream conidial drops exuding from the ostioles.

Diaporthe alangii C.M. Tian & Q. Yang, sp. nov.
MycoBank: MB824704 Figure 4 Diagnosis. Diaporthe alangii can be distinguished from the phylogenetically closely related species D. tectonae and D. tulliensis by the size of conidiophores and alpha conidia.
Culture characters. Cultures incubated on PDA at 25 °C in darkness. Colony initially white, producing beige pigment after 7-10 d. The colony is flat, felty with a thick texture at the centre and marginal area, with thin texture in the middle, lacking aerial mycelium, conidiomata absent.
Culture characters. Cultures incubated on PDA at 25 °C in darkness. Colony flat with white felty aerial mycelium, turning white to dark brown aerial mycelium, conidiomata irregularly distributed on the agar surface.

Diaporthe biguttulata F. Huang, K.D. Hyde & H.Y. Li, 2015
Culture characters. Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming pale grey, with dense aerial mycelium in the centre and sparse aerial mycelium at the marginal area, conidiomata absent.
Specimens examined. CHINA. Zhejiang Province: Tianmu Mountain, on symptomatic branches of Juglans regia, 20 Apr. 2017, Q. Yang, living culture CFCC 52584 and CFCC 52585 (BJFC-S1504). Notes. Diaporthe biguttulata was originally described from a healthy branch of Citrus limon in Yunnan Province, China (Huang et al. 2015). In the present study, two isolates (CFCC 52584 and CFCC 52585) from symptomatic branches of Juglans regia were congruent with D. biguttulata based on morphology and DNA sequences data (Fig. 1). We therefore describe D. biguttulata as a known species for this clade.
Culture characters. Cultures incubated on PDA at 25 °C in darkness. Colony at first flat with white felty mycelium, becoming black in the centre and black at the marginal area with age, conidiomata not observed.
Culture characters. Cultures incubated on PDA at 25 °C in darkness showed colony at first white, becoming pale brown with yellowish dots with age, flat, with dense and felted mycelium, with visible solitary or aggregated conidiomata at maturity.
Culture characters. Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white felted aerial mycelium, becoming light brown mycelium due to pigment formation, conidiomata irregularly distributed over agar surface, with yellowish conidial drops exuding from the ostioles.
Notes. Diaporthe chensiensis occurs in an independent clade (Fig. 1) and is phylogenetically distinct from D. vaccinii. Diaporhe chensiensis can be distinguished from D. vaccinii by 57 nucleotides in concatenated alignment, in which 14 were distinct in the ITS region, 13 in the cal region, 10 in the his3 region, 15 in the tef1 region and 15 in the tub2 region. Although this species belongs to the D. eres complex, it is, however, distinct from the known species within the complex (Fig. 2).
Culture characters. Cultures incubated on PDA at 25 °C in darkness showed colony originally flat with white felty mycelium, developing petaloid mycelium after 7-10 d and turning yellowish at the centre and brownish at the marginal area after 15 d. Conidiomata erumpent at maturity.
Culture characters. Cultures incubated on PDA at 25 °C in darkness. Colony white to yellowish, with dense and felted mycelium, lacking aerial mycelium, with maize-coloured conidial drops exuding from the ostioles.
Notes. Diaporthe eres, the type species of the genus, was described by Nitschke (1870) on Ulmus sp. collected in Germany, which has a widespread distribution and a broad host range as a pathogen, endophyte or saprobe causing leaf spots, stem cankers and diseases of woody plants (Udayanga et al. 2014b). Fan et al. (2018) indicated that D. biguttusis, D. ellipicola, D. longicicola and D. mahothocarpus should be treated as synonyms of D. eres using cal, tef1 and tub2 gene regions. In this study, we extended the work presented in Fan et al. (2018) and found seven additional strains belonging to D. eres. Additionally, the phylogenetic tree demonstrated that D. camptothecicola and D. momicola should also be treated as synonyms of D. eres (Fig. 2). Diaporthe camptothecicola from Camptotheca acuminate and D. momicola from Prunus persica are described and illustrated based on the combined ITS, cal, his3, tef1 and tub2 regions (Dissanayake et al. 2017a, Yang et al. 2017c. Both of the two species are embedded in the D. eres complex. However, ITS analysis resulted in an unresolved phylogenetic tree without definitive bootstrap at the internodes, highly discordant to the trees resulting from the other four genes (Udayanga et al. 2014b). Therefore, the ITS region was not used in the combined analysis in the current study. To further investigate this complex, a second set of four (cal, his3, tef1 and tub2), three (cal, tef1 and tub2), two (tef1 and tub2) and one (tef1) data matrices were performed following Santos et al. (2017) and Fan et al. (2018). The results showed that the three genes analyses (cal, tef1 and tub2) appeared to be a better species recognition (Fig. 2). When it comes to this species complex, sequences supported by Udayanga et al. (2014b) are necessary to perform a more robust phylogenetic tree, clarifying the real species boundaries in this group in the future work.
Culture characters. Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming yellowish, dense and felted aerial mycelium with age, with visible solitary or aggregated conidiomata at maturity. Additional material examined. CHINA. Shaanxi Province: Zhashui city, Niubeiliang Reserve, on symptomatic twigs of Fraxinus chinensis, 7 July 2017, Q. Yang, living culture CFCC 52583 (BJFC-S1496).
Culture characters. Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming dense and felted aerial mycelium in the centre and grey to black mycelium at the marginal area with solitary conidiomata at maturity. Notes. This new species is introduced as molecular data show it to be a distinct clade with high support (ML/BI=100/1) and it appears most closely related to D. fusi- cola and D. ovoicicola. Diaporthe kadsurae can be distinguished from D. fusicola by 11 nucleotides in concatenated alignment, in which 4 were distinct in the ITS region and 7 in the cal region; from D. ovoicicola by 25 nucleotides in concatenated alignment, in which 12 were distinct in the ITS region, 6 in the cal region and 7 in the tef1 region. Morphologically, D. kadsurae differs from D. fusicola and D. ovoicicola in shorter co-nidiophores (7-11 μm in D. kadsurae vs. 11-24.1 μm in D. fusicola; 7-11 μm in D. kadsurae vs. 14.2-23.6 μm in D. ovoicicola) (Gao et al. 2014). Diagnosis. Diaporthe padina can be distinguished from the phylogenetically closely related species D. betulae in smaller conidiomata and alpha conidia.
Culture characters. Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming grey to brown in the centre, with pale grey, felted, valviform mycelium at the marginal area and aggregated conidiomata at maturity.

Culture characters.
Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming brown to pale black in the centre, dense, felted, conidiomata not observed.
Culture characters. Cultures incubated on PNA at 25 °C in darkness. Colony entirely white at surface, reverse with pale brown pigmentation, white, fluffy aerial mycelium.
Notes. Diaporthe unshiuensis was originally described from twigs of non-symptomatic Fortunella margarita in Zhejiang Province, China (Huang et al. 2015). In the present study, two isolates from twigs of asymptomatic Carya illinoensis were congruent with D. unshiuensis based on morphology and DNA sequences data (Fig. 1). We therefore describe D. unshiuensis as a known species for this clade.

Discussion
The current study described 15 Diaporthe species from 42 strains based on a large set of freshly collected specimens. It includes 12 new species and 3 known species, which were sampled from 16 host genera distributed over six Provinces of China (Table 1). In this study, 194 reference sequences (including outgroup) were selected based on BLAST searches of NCBIs GenBank nucleotide database and included in the phylogenetic analyses (Table 1). Phylogenetic analyses based on five combined loci (ITS, cal, his3, tef1 and tub2), as well as morphological characters, revealed the diversity of Diaporthe species in China, mainly focusing on diebacks from major ecological or economic forest trees.
Several studies have been conducted associated with various hosts in China. For instance, the research conducted by Huang et al. (2015) revealed seven apparently undescribed endophytic Diaporthe species on Citrus. Gao et al. (2016) demonstrated that Diaporthe isolates, associated with Camellia spp., could be assigned to seven species and two species complexes. Recently, Diaporthe has been revealed as paraphyletic by Gao et al. (2017), showing that Ophiodiaporthe, Pustulomyces, Phaeocytostroma and Stenocarpella embed in Diaporthe s. lat. and eight new species of Diaporthe were introduced from leaves of several hosts. However, the identification of Diaporthe species associated with dieback of forest trees has rarely been studied, thus a large-scale investigation of Diaporthe spp. was conducted from 2015 to 2017. This study provides the first molecular phylogenetic frame of Diaporthe diversity associated with dieback in China, combined with morphological descriptions.
Diaporthe eres, the type species of the genus, was initially described by Nitschke (1870), from Ulmus sp. collected in Germany. The major problem with this generic type was the lack of an ex-type culture or ex-epitype culture, although a broad species concept has historically been associated with D. eres (Udayanga et al. 2014b). Udayanga et al. (2014b) designed strain AR5193 as the epitype of D. eres and provided the phylogram of this complex using seven loci (ITS, act, Apn2, cal, his3, FG1093, tef1 and tub2), amongst which the tef1, Apn2 and his3 genes were recognised as the best markers for defining species in the D. eres complex. Moreover, they showed that poorly supported non-monophyletic grouping was observed when ITS sequences were included in the combined analysis. In this study, although we conducted phylogenetic analysis as performed in previous studies on Diaporthe species (Santos et al. 2017), much confusion has, however, occurred in species separation of the D. eres complex (Fig. 1). Especially, the ITS region could lead to a confused taxonomic situation within this species complex. We found the three-gene analysis, excluding the ITS and his3 regions, resulted in a more robust tree congruent with Udayanga et al. (2014b) and resolved the species boundaries within the D. eres species complex. The isolates, clustering with D. eres in this study, occur on multiple hosts from many different geographic locations. This study revealed three new species belonging to the D. eres complex, i.e. D. betulina, D. chensiensis and D. padina. It also shows D. biguttusis, D. camptothecicola, D. ellipicola, D. longicicola, D. mahothocarpus and D. momicola were clustered in D. eres and should be treated as synonyms of D. eres, which is in conformity with Fan et al. (2018).
The initial species concept of Diaporthe, based on the assumption of host-specificity, resulted in the introduction of more than 1000 taxa (http://www.indexfungorum. org/). Thus, during the past decade, a polyphasic approach, employing multi-locus DNA data together with morphology and ecology, has been employed for species boundaries in the genus (Crous et al. 2012, Udayanga et al. 2014a, b, Huang et al. 2015, Gao et al. 2016, Guarnaccia and Crous 2017, Yang et al. 2017a, b, 2018, Perera et al. 2018a, b, Tibpromma et al. 2018.
Further studies are required in order to conduct an extensive collection of Diaporthe isolates, to resolve taxonomic questions and to redefine species boundaries. Multiple strains from different locations should also be subjected to multi-gene phylogenetic analysis to determine intraspecific variation. The descriptions and molecular data of Diaporthe species provided in this study represent a resource for plant pathologists, plant quarantine officials and taxonomists for identification of Diaporthe.