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Research Article
Recommendations on approving the name “ Entomosporium”, with a new species, E. dichotomanthes from China (Leotiomycetes, Drepanopezizaceae)
expand article infoHong De Yang‡§, Ruvishika S. Jayawardena§, Xiang Yu Zeng|, Vinodhini Thiyagaraja‡, Qi Zhao‡, Kevin D. Hyde‡§
‡ Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
§ Mae Fah Luang University, Chiang Rai, Thailand
| Guizhou University, Guiyang, China

Corresponding author: Kevin D. Hyde ( kdhyde3@gmail.com )

Academic editor: Xinlei Fan
© 2024 Hong De Yang, Ruvishika S. Jayawardena, Xiang Yu Zeng, Vinodhini Thiyagaraja, Qi Zhao, Kevin D. Hyde.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Yang HD, Jayawardena RS, Zeng XY, Thiyagaraja V, Zhao Q, Hyde KD (2024) Recommendations on approving the name “Entomosporium”, with a new species, E. dichotomanthes from China (Leotiomycetes, Drepanopezizaceae). MycoKeys 107: 1-20. https://doi.org/10.3897/mycokeys.107.121962
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Abstract

The phytopathogenic genus, Entomosporium can cause serious leaf diseases worldwide. Entomosporium has long been regarded as a synonym of Diplocarpon. However, different morphologies between Entomosporium and Diplocarpon make this doubtful. Based on morpho-phylogenetic analyses, the placement of the genus was re-evaluated in this study. The combined the internal transcribed spacer gene region (ITS) and the 28S large subunit ribosomal RNA gene region (LSU) phylogenetic analysis shows that Entomosporium is an independent clade within Drepanopezizaceae and formed a sister clade to the generic type Diplocarpon. Moreover, Hymenula and Pseudopeziza do not cluster in Drepanopezizaceae. We propose to resurrect the name Entomosporium, and exclude Hymenula cerealis and Pseudopeziza medicaginis from Drepanopezizaceae and propose to treat them under Ploettnerulaceae. A new species, E. dichotomanthes is also introduced from China based on morpho-molecular analyses which is associated with Dichotomanthes tristaniicarpa.

Key words

Diplocarpon, Hymenula cerealis, plant pathogen, phylogeny, Pseudopeziza medicaginis

Introduction

Entomosporium Lév, a synonym of Diplocarpon F.A. Wolf, is a member of the strongly plant-pathogenic family Drepanopezizaceae (Holtslag et al. 2003; Nunes et al. 2016; Johnston et al. 2019; Wöhner and Emeriewen 2019). The Entomosporium species causes entomosporium leaf disease worldwide and frequently occurs as an epidemic (Bogo et al. 2018). As many species are described without molecular data, the relationship with Diplocarpon species remains unclear. Although Diplocarpon species are common and widespread, studies on Diplocarpon have predominantly focused on their phytopathology, with the taxonomy utilizing molecular markers being largely overlooked (Wijayawardene et al. 2017; Ekanayaka et al. 2019).

Historically, Diplocarpon has undergone several revisions. Diplocarpon was erected by Wolf (1912), with the type species D. rosae (syn. Asteroma rosae) which caused black spot disease on Rose. The sexual stage of Diplocarpon forms cup-like apothecia, in which the asci develop. The ascospores are hyaline, 2-celled, marssonina-like and oblong-elliptical (Wolf 1912; Stowell and Backus 1967). The asexual stage comprises acervuli that develop on leaf surfaces accompanied by typical black dot disease (Frick 1943). Conidia of D. rosae are hyaline, oblong-elliptical, 2-celled with one constricted septum. By the interpretations of the morphology of these taxa, a broad concept of species circumscription was employed. Some members of Bostrichonema, Entomosporium, Gloeosporium and Marssonina were treated as synonyms of Diplocarpon. For example, Ascochyta coronariae (= D. coronariae), Bostrichonema alpestre (= D. alpestre), Dothidea impressa (= D. impressa), Leptothyrium fragariae (= D. fragariae) and Phacidium saponariae (= D. saponariae) were transferred into Diplocarpon (Johnston et al. 2014; Braun 2018; Crous et al. 2020). In addition, all members in Entomopeziza, Entomosporium and Morthiera were regarded as congruent with D. mespili (Stowell and Backus 1966; Gamundí et al. 2004; Johnston et al. 2014).

Although genera, such as Entomopeziza, Entomosporium and Morthiera have morphological similarities to Diplocarpon, it is perplexing that they are considered as synonyms. For example, 15 epithets of Entomosporium were regarded as D. mespili, as the sexual stage of Entomosporium morphologically resembles Diplocarpon (Naoui 2013; Johnston et al. 2014). However, Entomosporium produces cruciform, insect-like, 2–6-celled conidia, which is distinct from the conidia of Diplocarpon (Stowell and Backus 1966). Moreover, Entomosporium species are widely distributed in Argentina, Australia, Brazil, Canada, China, India, Israel, Italy, Japan, New Zealand, North America, Pakistan, and South Africa, on a wide host range of Rosaceae (Stowell and Backus 1966; Cariddi et al. 2009; Batool et al. 2014). Diplocarpon on the other hand, is mostly or specifically parasitic on herbaceous Rosaceae or low shrubs. The proposal to adopt Diplocarpon over Entomosporium is doubtful (Horie and Kobayashi 1980; Wijayawardene et al. 2021). The hypothesis that Diplocarpon mespili did not speciate with its worldwide spread should be re-evaluated (Chethana et al. 2021). An example of evidence is that Entomosporium sp. from Japan has more lateral cells (2–4) (Horie and Kobayashi 1979). Chen et al. (2022) introduced a new species with insect-like conidia but under the name “Diplocarpon”. In Index Fungorum (https://www.indexfungorum.org, 23 Nov 2023), 12 Diplocarpon species are recorded, namely D. alpestre, D. coronariae, D. earlianum, D. fragariae, D. hymenaeae, D. impressum, D. mali, D. mespili, D. mespilicola, D. polygoni, D. rosae and D. saponariae.

We are studying the pathogens of urban and forest tree species in Yunnan Province (Thiyagaraja et al. 2024) and in this study Entomosporium leaf disease was found in Dichotomanthes tristaniicarpa and has not been reported before. Dichotomanthes is endemic to Yunnan and Sichuan provinces in China (Zhou et al. 2000). It belongs to Rosaceae, with only one species D. tristaniicarpa which is a rare evergreen shrub tree, and is used as ornamental and medicinal plants (Tang et al. 2010; Yang et al. 2018). The ITS sequence blastn search of the newly generated sequences showed the close hits to Diplocarpon, and identified it as a new species based on the evidence from both morphology and phylogeny. Since the increasing number of members and updating molecular data of Diplocarpon, this study has provided an opportunity for a better understanding of the taxonomy of the genus. In this study, we interpret the relationship between Entomosporium and Diplocarpon, and further re-evaluate the taxonomy of Drepanopezizaceae.

Materials and methods

Sampling, isolation and morphological observations

Leaves with lesions of Dichotomanthes tristaniicarpa were collected from Yunnan Province. For single-spore isolation, the fruit bodies were transferred to sterilized water in a centrifuge tube using a syringe needle, then crushed into pieces using pipette tips. Subsequently, 200 μL of the spore suspension was transferred to potato dextrose agar (PDA) using a micropipette (Zhang et al. 2013). For tissue isolation, the leaves were washed with distilled water for 1 minute and then air-dried. The margins of the disease lesions were cut into fragments (0.5 × 0.5 cm) under aseptic conditions. These fragments were surface-sterilized with 75% ethanol for 30 seconds, followed by dipping in 1% sodium hypochlorite for 40 seconds. They were then rinsed three times in sterile demineralized distilled water before being transferred onto a PDA plate, with four fragments per plate (Senanayake et al. 2020). The Petri dishes were incubated in the dark at 25 °C. Specimens were deposited at the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS). Morphological observations were performed using Nikon SMZ745T dissecting microscope (DM) and Nikon Eclipse 80i compound microscope, equipped with IMG Camera SC2000C. Index Fungorum and Facesoffungi numbers were obtained as in Index Fungorum (https://www.indexfungorum.org/) and Jayasiri et al. (2015) and the details of the fungus were deposited in the Greater Mekong Subregion database (Chaiwan et al. 2021).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted by using Lysis Buffer for Microorganism to Direct PCR (Takara), following the user manual. PCR amplifications were performed in T100 Thermal Cycler (T100™, Bio-Rad, USA) with ingredients of 21 µL GoldenStar T6 Super PCR Mix (Tsingke), 1 µL (10 µM) of each primer and 2 µL DNA template. Amplification conditions include 3 min initial denaturation at 95 °C, followed by 35 cycles of 95 °C denaturation for 15 s, 53 °C ~ 56 °C annealing for 15 min, 72 °C extension for 20 s, followed by a final extension at 72 °C for 5 min. The primer set ITS5/ITS4 (White et al. 1990) was used to amplify the internal transcribed spacer gene region (ITS); and LROR/LR5 for the 28S large subunit ribosomal RNA gene region (LSU) (Vilgalys and Hester 1990; White et al. 1990) and 983F/2218R for translation elongation factor 1-alpha gene region (tef-α) (Rehner and Buckley 2005). PCR products were purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China.

Phylogenetic analyses

Reverse and forward sequences were assembled using Chromas Pro (2.1.8) and initial identification was subjected to the NCBI (https://www.ncbi.nlm.nih.gov/) using BLAST search. Sequences of similar taxa were retrieved from the NCBI, and additional reference sequence selections based on Johnston et al. (2019) were downloaded from the DataStore (https://datastore.landcareresearch.co.nz/). The alignment was constructed with the online tool MAFFT v.7 (http://mafft.cbrc.jp/alignment/server) (Katoh and Standley 2013), and refined using BioEdit v. 7.7.1 (Hall 1999). The final combined data matrix was converted by the online tool ALTER (https://www.sing-group.org/ALTER/) (Glez-Peña et al. 2010). A quick Phylogenetic analysis was conducted using OFPT (Zeng et al. 2023) following its default protocol. The final Phylogenetic analyses were conducted on the CIPRES Science Gateway platform (https://www.phylo.org), using tools of RAxML-HPC v.8 on XSEDE (8.2.12) for maximum likelihood (ML) and MrBayes on XSEDE (3.2.7a) for Bayesian inference (BI). In the Bayesian inference, the best optimal substitution model was determined by using ModelFinder (Kalyaanamoorthy et al. 2017) under the Bayesian information criterion (BIC). The final phylogenetic tree was visualized with FigTree v. 1.4.4 and edited using Adobe Photoshop CS6 version 10.0. Sequences of the new strain generated in this study are deposited in GenBank (Table 1).

Table 1.
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GenBank accession numbers used in the phylogenetic analyses.

Species Strain Country GenBank accession number
ITS LSU
Acephala applanata CBS 109321T Switzerland NR_119482 KF951051
Blumeriella hiemalis CBS 146.35 USA MH855609 MH867119
B. kerriae JS20160615 United Kingdom KY929501 –
Cadophora fascicularis CBS 146382 Germany NR_170729 MN339414
Cheirospora botryospora MFLUCC 17-1399 China MN535816 MN535856
Collembolispora aristata CPC 21145T Czech Republic NR_111830 NG_042760
Cylindrosporium concentricum CBS:157.35 Australia MH855615 MH867125
Diplocarpon coronariae Satoko Kanematsu Dip-ap6-3 Japan AB609188 –
D. coronariae CS 01 Korea AB494960 AB494964
D. coronariae 5C11 USA MW364818 –
D. coronariae NL1 China KY672995 –
D. earlianum CBS 162.32 Unknown MH855259 MH866712
D. rosae CBS 163.31 Unknown MH855164 MH866612
D. rosae CBS 829.72 Netherlands – MH872311
D. rosae CFCC6814 Unknown KP099199 –
D. rosae DortE4T Germany Genome Genome
Drepanopeziza balsamiferae 14-19 USA MN315242 –
D. brunnea Marbr1 Unknown genome genome
D. ribis CBS:200.36 Netherlands MH855774 MH867284
D. salicis CBS:405.64 Switzerland MH858467 MH870102
D. tremulae CBS 408.64 Switzerland MH858468 MH870103
D. triandrae CBS 409.64 Switzerland MH858469 MH870104
Entomosporium dichotomanthes HKAS 131154 China PP333041 PP333042
E. mespili CBS 166.28 England – MH877689
E. mespili CBS 402.65 Unknown – MH870277
E. mespili KACC 42361 Korea EF600984 –
E. mespili KACC 42436 Korea EF600985 –
E. mespilicola CF 2 China OM237437 MW809414
E. mespilicola CF 3 China OM237438 MW809415
E. mespilicola CGMCC3 20492T China OM237436 MW809413
Hyaloscypha ericae UAMH 6735T Canada AF284122 MH018947
H. gabretae CBS 145341T Czech Republic MZ520780 NG_081311
H. gryndleri CBS 145337T Czech Republic MZ520785 MZ520774
H. minuta G.M.2015-04-06.2T Luxembourg KY769526 –
H. vitreola Huhtinen M220T Finland FJ477059 FJ477058
Hymenula cerealis CBS 132.34T Japan NR_171209 NG_070839
H. cerealis CBS 540.63 United Kingdom MH858350 MH869971
Leuconeurospora capsici CBS:176.44 Netherlands MH856125 MH867637
L. pulcherrima AFTOL-ID 1397 Unknown KF049206 FJ176884
Meliniomyces variabilis UAMH 8861T Canada NR_121313 NG_073616
Mycochaetophora gentianae MAFF 239231T Japan NR_121201 AB496937
Neospermospora avenae CBS 227.38T USA MW298276 NG_077377
Oculimacula yallundae CBS 110665T South Africa MW810278 MW715035
Pseudaegerita corticalis ICMP 15324T New Zealand EF029224 –
Pseudopeziza medicaginis CBS 283.55 USA MH857484 MH869025
Rhexocercosporidium carotae CBS 418.65T Norway NR_111086 MH870289
Rhynchosporium agropyri CBS:146762 Switzerland MW298346 MW298448
Thedgonia ligustrina CBS 132025 Korea GU269839 GU253856
T. ligustrina CBS:148.59T Netherlands NR_175086 NG_078647
T. ligustrina CPC 10530 Netherlands FJ839628 FJ839665
Vibrissea truncorum AFTOL-ID 1322T Canada EU434854 FJ176874
Ypsilina buttingtonensis CPC 39109T United Kingdom NR_170831 MT373355

Type strains are marked with “T”, and strains from the present study are in black bold.

Results

A total of 50 ingroup taxa from Drepanopezizaceae, Hyaloscyphaceae, Ploettnerulaceae and Vibrisseaceae were used in the phylogenetic tree analysis, of which 20 species were from the type (Fig. 1). In total, 31 isolates contained all extant species that have available molecular data within Drepanopezizaceae. The combined LSU and ITS yield a 1409 bp alignment, with the best substitution models for each summarised as TIM2e+I+G4 and TIM2+F+R3, respectively.

Figure 1. 

Maximum likelihood phylogenetic tree inferred from combined LSU and ITS sequence data of Drepanopezizaceae and its closely related families. The tree is artificially rooted with Leuconeurospora capsici (CBS:176.44) and Leuconeurospora pulcherrima (AFTOL-ID 1397). Maximum likelihood bootstrap values ≥65% and Bayesian Posterior Probabilities (BYPP) ≥ 0.90 are given at the nodes. Novel taxon is in bold. Type sequences are labeled asterisk (*).

Phylogenetic analysis demonstrated that Diplocarpon divided into two phylogenetically close relative clades, Diplocarpon and Entomosporium. Diplocarpon clade is composed of D. coronariae (from China, Japan, Korea and the USA), D. earlianum (unknown country) and D. rosae (from China, Germany and an unknown country). Those three species have common characteristics of two-celled conidia. The new species Entomosporium dichotomanthes (from China), along with E. mespili (from England, Korea and an unknown country) and E. mespilicola (from China) consisted of clade Entomosporium, which showed insect-like conidia. Moreover, Hymenula cerealis and Pseudopeziza medicaginis were within Ploettnerulaceae.

Sequence comparison reveals the intergeneric and interspecific variation (Fig. 2). ITS sequence shows a high nucleotide variation within Diplocarpon, with an average of 58.1, compared to Diplocarpon, the Entomosporium, Blumeriella, Drepanopeziza and Thedgonia have an average of 62.6, 68, 71.3 and 76, respectively. The sequence comparison results align with the phylogenetic analysis, indicating that the closely related species exhibit less nucleotide variation. The LSU sequences have a lower variation. The Diplocarpon has an average of 28, and the Drepanopeziza, Blumeriella, Entomosporium and Thedgonia have an average of 35.5, 37, 40, and 44, respectively. The interspecific variation of Drepanopeziza and Entomosporium is 26.8 and 41.3.

Figure 2. 

Intergeneric and interspecific variation analysis A mean of ITS sequence variation within genera B means of LSU sequence variation within genera C ITS sequence variation of the query sequence and the subject, “S” is the subject, “x” is the mean value of nucleotide variation within species.

Taxonomy

Drepanopezizaceae Baral

MycoBank No: MycoBank No: 828889
Facesoffungi Number: FoF 05864
Fig. 3

Type

Drepanopeziza (Kleb.) Jaap 1914.

Description

Sexual morph : Ascomata small-sized, up to 2 mm in diameter, apothecial, cupulate, margin often protruding, with or without lobes, sessile and mostly immersed. Excipulum is composed of cells of textura angularis. Paraphyses hyaline, thin-walled, aseptate or septate, apically swollen. Asci 4–8-spored, clavate or cylindrical, apex obtuse to conical, with or without apical ring. Ascospores ellipsoid to fusoid, aseptate or 1–2-septate. Asexual morph: Conidiomata solitary to gregarious or confluent, mostly epiphyllous, acervulus. Conidiogenesis holoblastic. Conidia hyaline, thin-walled.

Figure 3. 

Morphology of genera in Drepanopezizaceae. Diplocarpon: a ascomata, b asci, paraphyses and ascospore (a, b D. rosae, redraw from Wolf 1912) c acervulus and conidia (D. rosae, redraw from Lee and Shin 2000), Entomosporium d ascomata e asci and paraphyses (d, e E. maculatum, redraw from Stowell and Backus 1967) f acervulus g conidia (f, g E. mespilicola, redraw from Chen et al. 2022), Drepanopeziza h ascomata (Dr. populorum, redraw from Spiers and Hopcroft 1998) i asci, paraphyses and ascospore j conidiogenous cells and conidia (i, j Dr. ribis, redraw from https://www.centrodeestudiosmicologicosasturianos.org), Blumeriella k ascomata l asci, paraphyses and ascospore (k, l B. haddenii, redraw from Williamson and Bernard 1988) m acervulus n conidia (m, n B. jaapii, redraw from https://www.forestryimages.org), Thedgonia o acervulus and conidia (T. ligustrina, redraw from Crous et al. 2009) p Hymenula: conidiogenous cells and conidia (H. gramineum redraw from Wiese and Ravenscroft 1978), Pseudopeziza q ascomata (P. trifolii, redraw from Kirchner and Boltshauser 1987) r asci, paraphyses and ascospore (P. ribis, redraw from https://www.pesticidy.ru/pathogens_genus/Pseudopeziza).

Notes

Drepanopezizaceae was described with sexual and asexual morphs. Both life morphs were found as parasitic on leaves of various dicotyledons, and rarely on herbaceous (Johnston et al. 2019). The sexual morph is recognized by the cupulate, apothecial ascomata, and the paraphyses with swollen apical (Harada et al. 1974; Williamson and Bernard 1988; Spiers and Hopcroft 1998). The asexual morph is acervular but varies in conidial shape among genera (Crous et al. 2009; Khodadadi et al. 2022). The family name was first time used by Batista and Maia (1960), but was invalid because of unavailable diagnosis or description (Johnston et al. 2019). It was difficult to trace back the history of members accommodated in the family until Johnston et al. (2019), validated the family name based on the phylogenetic analysis (Table 2).

Table 2.
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Main versions of classification of Drepanopezizaceae and its accepted genera.

Batista and Maia (1960) Wijayawardene et al. (2017) Johnston et al. (2019) Ekanayaka et al. (2019) Wijayawardene et al. (2022) Zhu et al. (2023) This study
The family name was used Blumeriella Erected the family Blumeriella Blumeriella Blumeriella Blumeriella Blumeriella
Diplocarpon Diplocarpon Diplocarpon Diplocarpon Diplocarpon Diplocarpon
Drepanopeziza Drepanopeziza Drepanopeziza Drepanopeziza Drepanopeziza Drepanopeziza
Felisbertia Felisbertia Felisbertia Felisbertia Felisbertia Entomosporium
Leptotrochila Leptotrochila Leptotrochila Leptotrochila Hymenula Felisbertia
Pseudopezicula Pseudopeziza Marssonina Pseudopeziza Leptotrochila Leptotrochila
Pseudopeziza Spilopodia Pseudopezicula Spilopodia Pseudopeziza Spilopodia
Spilopodia Spilopodiella Spilopodiella Spilopodiella Spilopodia Spilopodiella
Spilopodiella Thedgonia Spilopodia Spilopodiella Thedgonia
Thedgonia Thedgonia

The taxa used in the phylogenetic analysis are labeled in bold.

Entomosporium Lév. 1857

MycoBank No: MycoBank No: 8180
Facesoffungi Number: FoF 15505

Type

Entomosporium maculatum Lév. 1856.

Description

Sexual morph : Ascomata small-sized, apothecial, cupulate, epiphyllous. Excipulum composed of cells of textura angularis. Paraphyses numerous, hyaline, thin-walled, septate, apically swollen, simple or branched, longer than aci. Asci 8-spored, bitunicate to uniseriate, thick-walled, clavate, short pedicel, apex obtuse, amyloid, with apical ring. Ascospores 2-celled, ellipsoidal, smooth, hyaline, thick-walled, unequal, the upper cell slightly lager. Asexual morph: Conidiomata solitary to gregarious or confluent, mostly epiphyllous, acervulus. Conidiogenesis hyaline, cylindrical, holoblastic. Conidia 2–6-celled, hyaline, thin-walled, cruciform or insect-like, basal cell developed from the conidiogenous cell, cylindrical, globose to obovate, and other cells attached basal cell in both upper sides and apex, apical cell larger, globose to subglobose, lateral cells globose to ellipsoidal, smaller than the apical and basal cells, the apical and basal cells with a tubular appendage.

Notes

Entomosporium was erected by Leveille in 1856, based on E. maculatum from leaves of Pyrus communis (Rosaceae), and was characterized by 4-celled, cross-like conidia (Stowell and Backus 1966; Horie and Kobayashi 1980). Historically, Entomosporium is composed of multiple morphologically indistinguishable species. Sivanesan and Gibson combined all species to E. mespili, but they did not mention their taxonomic basis (Horie and Kobayashi 1980). Atkinson recorded the process by which ascospores from a cupulate fungi formed the conidia of E. maculatum, and named the species as Fabraea maculata, while he later proposed that F. maculata may be identical to E. mespili (Atkinson 1897, 1909). Taxonomic status changed for the morphologically similar taxa, viz. Diplocarpon, Entomopeziza, Fabraea and Marssonina (Stowell and Backus 1967; Johnston et al. 2014). After versions, Jørstad (1945) combined Diplocarpon and Entomosporium, and recognized Atkinson’s collection as the type. This opinion was also discussed by Stowell and Backus (1967), but they failed the verification through experiments. The mystery of Entomosporium associated with sexual morph is still not confirmed by molecular data, since no new collection was found in sexual stage in recent decades.

Entomosporium dichotomanthes H.D. Yang, Jayaward & K.D. Hyde, sp. nov.

Index Fungorum: IF901675
Facesoffungi Number: FoF 15506
Fig. 4

Etymology

The species epithet ‘dichotomanthes’ refers to the host Dichotomanthes tristaniicarpa in which the holotype was collected.

Figure 4. 

Entomosporium dichotomanthes (HKAS 131154, holotype) a, b disease symptoms on the leaves, c–e conidiomata f, g conidiogenous cells and conidia,h–k conidia. Scale bars: 50 µm (e); 10 µm (f–k).

Holotype

HKAS 131154.

Description

Parasitic on leaf of Dichotomanthes tristaniicarpa in terrestrial habitat. Leaf spots: appear as tiny black spots or irregular black stripes on the upper side of the mature leaf when young, without injured disease symptoms. Later the spot enlarged to circular lesions or large dead areas with black edege. The area around the black spots remains green. Sexual morph: Not determined. Asexual morph: Conidiomata dark brown to black, stromatic, acervular, epiphyllous, solitary to gregarious or confluent, subcuticular to rounded or irregular in outline, rugose, erumpent through the cuticle. Conidiomatal wall mixed with host plant tissue, of several layers loose textura angularis cell. Conidiophores hyaline to pale brown, cylindrical, branched. Conidiogenous cells 5.0–8.4 × 2.8–4.4 µm, hyaline, cylindrical, holoblastic. Conidia hyaline, 3–4-celled, cruciform, the basal cell developed from the conidiogenous cell, and other cells attached to basal cell in both upper sides and apex. Basal cells 6.7–12.1 × 4.5–8.6 (avg. = 9.9 × 7.1, n = 30) µm, cylindrical, globose to obovate. Apical cell 5.8–13.0 × 5.1–10.7 (avg. = 9.9 × 8.0, n = 30) µm, globose to subglobose, the end with a tubular appendage. Lateral cells 3.5–7.5 × 2.5–4.1 (avg. = 5.7 × 4.1, n = 20) µm, subglobose to ellipsoidal, the end with a tubular appendage.

Material examined

China, Yunnan Province, Kunming City, Longchuanqiao park, 25°8'15.65"N, 102°47'13.70"E, on living leaf of Dichotomanthes tristaniicarpa, 14 December 2021, YHD 239-5 (HKAS 131154); YHD 202.

Notes

Entomosporium dichotomanthes is characterized by having three to four cells of conidia. Its morphology resembles D. mespili and D. mespilicola, but has different host plants association and distribution. E. dichotomanthes is easily detectable on the host substrate in the mountains around the lake of Longchuanqiao Park. However, we couldn’t find this fungus on nearby plants of the host, or on other plants in the mountains. We also failed to isolate the culture by using both single spore isolation and tissue isolation methods which indicates E. dichotomanthes strictly rely on D. tristaniicarpa.

Discussion

The taxonomic status and the phylogenetic relationship of Diplocarpon and Entomosporium in Drepanopezizaceae were assessed in this study. We included all extant species with molecular data in Drepanopezizaceae, as well as most genera of its sister family Ploettnerulaceae for the first time. Upon molecular phylogenetic analysis, Diplocarpon divided into two distinct clades representing Entomosporium and Diplocarpon. Sequence comparison reveals the average nucleotide variation of Blumeriella, Drepanopeziza, Entomosporium, and Thedgonia is higher than Diplocarpon which means intergeneric variation is greater than interspecific variation. Moreover, Diplocarpon and Entomosporium have a high nucleotide variation compared to the more speciose genus Drepanopeziza. Consequently, Entomosporium recovered separately from Diplocarpon and should not assign all species to E. mespili. On the plant host (Table 3), D. rosae is commonly reported on Roses, D. earlianum on strawberries and D. coronaria on apple trees. However, Entomosporium has a wide host range of woody plants like shrubs and trees, such as apple, hawthorn, saskatoon and pear (Holtslag et al. 2004; Nunes et al. 2016; Thurn et al. 2019). Likewise, the morphology features of conidia sustained the difference, Entomosporium displays insect-like conidia while Diplocarpon produces 2-celled conidia. Johnston et al. (2014) stated that Entomosporium and Diplocarpon are conspecific due to the linked asexual and sexual morphs, but this was not confirmed by molecular data. Conclusively, our study based on morphology coupled with molecular data supported the division of these genera. Entomosporium leaf disease is mainly associated with Entomosporium species (Holtslag et al. 2003; Seo et al. 2010; Nunes et al. 2016). Hence, the classification system of Diplocarpon was revised. We propose to recover the validity of the genus name “Entomosporium”, to accommodate species that have insect-like conidia species in Drepanopezizaceae. Furthermore, we introduced a new species E. dichotomanthes from China. Its taxonomical placement is basal in the “Entomosporium” clade supported by high bootstrap. The disease symptom appeared as black spots or irregular black stripes on the upper side of the mature leaf of Dichotomanthes tristaniicarpa, which was easily recognizable. We also generated the first sequence of the tef1-α gene for Entomosporium, from E. dichotomanthes. However, we used only LSU and ITS sequences data in our study, since scant tef1-α sequences data are available for reference taxa that cannot be used in this phylogenetic analysis. The blast against NCBI shows the tef1-α sequences have highest similarity with D. coronariae (MT674914) and Hyaloscypha fuckelii (MT254572), gained the value of 884/948(93%) and 860/948(91%), respectively.

Table 3.
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Diplocarpon species documented from different countries and plant host.

Current name Original name Host Disease Symptom Location Reference
D. coronaria Diplocarpon mali Malus Rosaceae Apple blotch disease premature leaf fall of Apple India Goyal et al. (2018)
D. coronaria Diplocarpon mali Malus Rosaceae Apple blotch disease dark brown and irregularly shaped blotches or lesions China Lian et al. (2021)
D. coronaria Marssonina coronaria Malus Rosaceae Apple blotch disease tiny yellow spots at first, become grayish brown circular lesions Korea Lee et al. (2011)
D. coronaria Marssonina coronaria Malus baccata Rosaceae Leaf spot disease initially light brown to brown lesions without a distinctive margin, later reddish black to purple, finally appearing as a yellow blotch with green islands Korea Lee and Shin (2000)
D. coronaria Diplocarpon coronaria Malus Rosaceae Apple blotch disease dark brown and irregularly shaped blotches or lesions Germany Wöhner et al. (2021)
D. coronaria Diplocarpon mali Malus Rosaceae Apple blotch disease – Japan Tanaka et al. (2000)
D. coronariae Diplocarpon coronariae Malus spp. Rosaceae Apple blotch disease brown to black spots, with frond-like edges or surrounded by a yellow halo America Khodadadi et al. (2022)
D. earlianum Diplocarpon earlianum Strawberry Rosaceae Leaf scorch reddish-purple lesions Canada Dhanvantari (1967)
D. earlianum Diplocarpon earlianum Strawberry Rosaceae Leaf scorch disease – México Garay-Serrano et al. (2021)
D. earlianum Diplocarpon earlianum Fragaria x ananassa Rosaceae Leaf scorch disease – America Xue et al. (1996)
D. fragariae Diplocarpon fragariae Strawberry Rosaceae Leaf scorch disease – México Garay-Serrano et al. (2021)
D. fragariae Marssonina fragariae Duchesnea chrysantha, Fragaria × ananassa, Potentilla fragarioides, Potentilla feyniana Rosaceae Leaf spot disease initially reddish to brown, later dark brown, central area surrounded by yellowish halo Korea Lee and Shin (2000)
D. mespili Diplocarpon mespili Pyrus communis Rosaceae Entomosporium leaf disease reddish, purple, to dark brown spots Southern Brazil Nunes et al. (2016)
D. mespili Diplocarpon mespili Eriobotrya japonica Rosaceae Entomosporium leaf disease circular, bright red spots on young leaves, turned to purple blotches with ash brown grey centers, and coalesced to form large dead areas on leaf surfaces Pakistan Batool et al. (2014)
D. mespili Entomosporium maculatum Cydonia oblonga Rosaceae Entomosporium leaf disease – Southwestern Romania Borcean and Imbrea (2021)
D. mespili Entomosporium mespili Photinia x fraseri Rosaceae Entomosporium leaf disease reddish-colored lesions Georgia Mims et al. (2000)
D. mespili Entomosporium mespili Pyrus communis Rosaceae Entomosporium leaf disease – Southern Brazil Bogo et al. (2018)
D. mespili Entomosporium mespili Amelanchier alnifolia Rosaceae Entomosporium leaf and berry spot disease – Canada Holtslag et al. (2003)
D. mespili Entomosporium mespili Photinia glabra Rosaceae Entomosporium leaf disease Initially appeared as minute circular spots, later several small spots coalesced to make large necrotic blotches Korea Seo et al. (2010)
D. mespili Entomosporium mespili Cydonia oblonga Rosaceae Entomosporium leaf disease circular reddish-brown spots at first, coalesced producing large necrotic areas, the leaves turned yellow or reddish and fell prematurely Southern Italy Cariddi et al. (2009)
D. mespili Entomosporium sp. Amelanchier asiatica Rosaceae Entomosporium leaf disease black shiny pustule spots Japan Horie and Kobayashi (1979)
D. mespili Entomosporium sp. Eriobotrya japonica Rosaceae Entomosporium leaf disease yellowish or reddish spots with a greenish halo around Japan Horie and Kobayashi (1979)
D. mespili Entomosporium sp. Pyrus communis Rosaceae Entomosporium leaf and fruit spot disease reddish to purple at the beginning, later irregular dark brown to black necrotic patches on the leaf, sunken irregular black spot on the fruit India Altaf et al. (2019)
D. mespili Entomosporium mespili Crataegus Rosaceae small, irregularly shaped spots or larger lesions – America Thurn et al. (2019)
D. mespili Diplocarpon mespili Pyrus pyraster Rosaceae – Bulgaria Velinova and Tashev (2017)
D. mespilicola Diplocarpon mespilicola Crataegus pinnatifida Rosaceae Entomosporium leaf disease brown spots China Chen et al. (2022)
D. rosae Diplocarpon rosae Rose Rosaceae Black spot disease black spots United Kingdom Knight and Wheeler (1977)
D. rosae Diplocarpon rosae Rosa multiflora Rosaceae Black spot disease black spots Germany Von Malek and Debener (1998)
D. rosae Diplocarpon rosae Rose Rosaceae Black spot disease black spots North America Whitaker et al. (2007)
D. rosae Diplocarpon rosae Rose Rosaceae Black spot disease – Belgium Leus et al. (2002)
D. rosae Diplocarpon rosae Rosa rugosa Rosaceae Black spot disease black spot lesions Canada Bolton and Svejda (1979)
D. saponariae Diplocarpon saponariae Silene latifolia Caryophyllaceae Leaf spot disease pale yellow to pale brown spots, sometimes purple-bordered, regular or irregularly rounded, sometimes elongated Turkey Erdoĝdu and Hüseyin (2009)

Correspondingly, Hymenula cerealis and Pseudopeziza medicaginis were not clustered in Drepanopezizaceae in our phylogenetic tree. However, Hymenula was recovered within Drepanopezizaceae in Zhu et al. (2023). Further, the type material of H. cerealis (= Cephalosporium gramineum, CBS 132.34) was used in their study and obtained a good statistical support (MLBP/BIPP = 96%/100%), in the phylogenetic analyses conducted based on the combined five-gene data set. However, only H. cerealis as well as a small group of taxa from both Drepanopezizaceae and Ploettnerulaceae were applied in their phylogenetic analysis. The disease caused by Hymenula is cephalosporium stripe on herbaceous plants that is different from Drepanopezizaceae (Wiese and Ravenscroft 1978; Zhu et al. 2023). Similar situations with Hymenula, Pseudopeziza cause black spot disease mostly found on Alfalfa and Red clover (Fabaceae), not on Rosaceae plants (Jones 1919; Meyer and Luttrell 1986; Yuan et al. 2007). The taxonomy of Pseudopeziza is confusing (Meyer and Luttrell 1986). There are 135 species epithets that have been linked to Pseudopeziza in Index Fungorum (https://www.indexfungorum.org), of which many names have been transferred to other families, such as Diaporthaceae, Ploettnerulaceae and Rhytismataceae. Only three sequences labeled as Pseudopeziza were accessible in the GenBank, and P. medicaginis (CBS 283.55) was used in this study.

Morphologically, Hymenula was only found in the asexual stage. Meanwhile, the asexual morph of Drepanopezizaceae does not share highly persuasive common morphological characteristics for delimiting its generic members. The morphology of P. medicaginis fits Drepanopezizaceae (Jones 1919), but differs in having indistinctive swollen apical paraphyses (Meyer and Luttrell 1986). Thus, we propose to exclude H. cerealis and P. medicaginis from Drepanopezizaceae and to treat them under Ploettnerulaceae.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by the Chinese Research Fund (project no E1644111K1) entitled “Flexible introduction of high-level expert program, Kunming Institute of botany, Chinese Academy of Sciences’’. Vinodhini Thiyagaraja would like to thank Flexible Talent Introduction Program - Grant Number E16441 and post-doctoral grant program.

Author contributions

All authors have contributed equally.

Author ORCIDs

Ruvishika S. Jayawardena https://orcid.org/0000-0001-7702-4885

Vinodhini Thiyagaraja https://orcid.org/0000-0002-8091-4579

Qi Zhao https://orcid.org/0000-0001-8169-0573

Kevin D. Hyde https://orcid.org/0000-0002-2191-0762

Data availability

All of the data that support the findings of this study are available in the main text.

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