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Research Article
Morphological and molecular identification of new species and records of Daldinia (Hypoxylaceae, Xylariales) from Guizhou Province, China
expand article infoYingying Wu‡, Shuji Li‡, Ning Jiang§, Chengming Tian‡
‡ Beijing Forestry University, Beijing, China
§ Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, China

Corresponding author: Chengming Tian ( chengmt@bjfu.edu.cn )

Academic editor: Samantha C. Karunarathna
© 2025 Yingying Wu, Shuji Li, Ning Jiang, Chengming Tian.
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: Wu Y, Li S, Jiang N, Tian C (2025) Morphological and molecular identification of new species and records of Daldinia (Hypoxylaceae, Xylariales) from Guizhou Province, China. MycoKeys 123: 253-269. https://doi.org/10.3897/mycokeys.123.160960
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Abstract

Members of Daldinia are widely distributed and commonly found on decaying wood, branches, and diseased leaves. In this study, four strains of Daldinia were isolated from diseased leaves of Indocalamus hirsutissimus and Rubus idaeus in Guizhou Province, China. Species identification was conducted using combined nuc rDNA ITS1-5.8S-ITS2 (ITS), partial sequences of the large subunit (28S), RNA polymerase II subunit 2 (rpb2), and beta-tubulin (tub2) sequence data, along with morphological comparisons. We introduce the new species D. rubi and D. eschscholtzii, accompanied by a new host record on Indocalamus hirsutissimus, supported by both morphological features and molecular evidence. In addition, a comprehensive analysis of D. eschscholtzii’s worldwide host distribution revealed that it spans 56 plant families across 31 countries. This study enhances our understanding of the species diversity of Daldinia and its broad host range, providing a new perspective for taxonomic research.

Key words:

Ascomycota, morphology, new taxon, phylogeny, taxonomy

Introduction

Daldinia was originally introduced by Cesati and De Notaris (Cesati and De Notaris 1863). In the early 20th century, Child (1932) conducted the first comprehensive global study on Daldinia, describing 13 species and introducing anamorphic features and physiological traits for classification purposes. However, her work contained inaccuracies in species descriptions, leading to confusion. Later, Ju et al. (1997) conducted the second global revision, clarifying some errors and providing detailed descriptions of D. bakeri, D. caldariorum, and D. loculata. Rogers et al. (1999) redefined the type species D. concentrica and held that it was actually D. childiae. In the early 21st century, the integration of molecular biology and chemotaxonomic techniques significantly advanced the taxonomic study of Daldinia. Johannesson et al. (2000) and Stadler et al. (2001a) constructed preliminary phylogenetic relationships using nuc rDNA ITS sequence analysis. The application of HPLC (high-performance liquid chromatography) and scanning electron microscopy (SEM) enabled the examination of chemical profiles and ultrastructures of historical specimens, revealing the diversity of secondary metabolites and their taxonomic significance in Daldinia (Stadler et al. 2001b, c, 2002; Bitzer et al. 2007). Subsequent ecological studies uncovered its endophytic lifestyle and rapid colonization ability following host damage (Rogers 2000; Stadler 2011). Stadler et al. (2014) integrated morphological, molecular phylogenetic, and chemotaxonomic approaches, analyzing data from thousands of specimens and cultures to elucidate the complex diversity of secondary metabolites and phylogenetic relationships in Daldinia. Overall, the taxonomy of Daldinia has evolved from a morphology-based approach to a multidisciplinary research framework, gradually resolving earlier taxonomic confusions and providing a foundation for understanding its ecological and evolutionary relationships.

In recent years, the classification framework of Daldinia has stabilized. Wendt et al. (2018) confirmed through multi-gene phylogenetic analysis (ITS, 28S, rpb2, and tub2) that Daldinia and its relatives form a distinct clade within Hypoxylaceae, clearly differentiated from Hypoxylon and Pyrenopolyporus. The application of a polyphasic approach has significantly increased species diversity, revealing multiple cryptic species. For instance, Stadler et al. (2004) described five new species from the Canary Islands, Channel Islands, and Sicily, while Wongkanoun et al. (2019) reported a new record and a new species from northern Thailand. Additionally, Pažoutová et al. (2013) identified D. hawksworthii, an insect-associated endophytic species, based on molecular data and conidial structures. Yin et al. (2024) isolated and identified seven new anamorphic species from diseased leaves in southern China, analyzing their geographical distribution and host specificity, which suggests that warm, humid, and vegetatively rich regions may harbor more Daldinia and fungal resources.

The stromata of Daldinia fungi are prominent and persistent, often forming dense clusters on woody plants, making them easily noticeable. Internally, the stromata exhibit horizontal zonation, a key feature distinguishing this genus from other pyrenomycete fungi (Stadler et al. 2014). Traditionally, Daldinia was considered a saprophyte, primarily causing white rot on dead angiosperm wood. However, recent studies suggest they may persist as endophytes within host tissues, forming stromata only when the host is damaged or stressed, leading to their previous misidentification as “rare” despite potentially widespread distribution (Stadler and Hellwig 2005). In this paper, during the identification of plant pathogenic fungi from diseased leaves in Guizhou Province, China, two Daldinia species were unexpectedly isolated from Indocalamus hirsutissimus and Rubus idaeus. Molecular and morphological analyses confirmed the establishment of a new species, D. rubi sp. nov., and reported a new host record species, D. eschscholtzii. This study provides detailed descriptions, illustrations, and DNA-based phylogenetic analyses to validate their taxonomic classification.

Materials and methods

Sample collection and fungal isolation

Diseased leaves of Indocalamus hirsutissimus and Rubus idaeus were sampled from Guizhou, China, and sampling information was recorded (Rathnayaka et al. 2025). For sampling positions on the leaf, the junction of diseased and healthy tissue was targeted. The diseased leaf tissues were cut into small pieces of approximately 5 mm × 5 mm using a sterile scalpel. These tissue pieces were then subjected to surface sterilization (rinsed in 75% ethanol for 30 s, followed by 2% sodium hypochlorite for 2 min, and finally rinsed three times in sterile distilled water). The surface-sterilized tissues were plated onto potato dextrose agar (PDA), which consisted of 20% diced potatoes, 2% agar, and 2% glucose. Petri dishes were incubated at 25 °C in the dark for 2–3 days. After colony formation, hyphal tips were carefully picked under a stereomicroscope and transferred to fresh PDA plates to obtain pure cultures, following the method described by Crous et al. (2019a). Type specimens of the new species were deposited in the Museum of Beijing Forestry University (BJFC), and ex-type living cultures were preserved at the China Forestry Culture Collection Center (CFCC), Beijing, China.

Morphological observation

Cultures were grown on PDA at 25 °C under a 12 h light/dark cycle (Crous et al. 2019b). After 14 days, colony measurements were taken, and characteristics such as color, shape, and aerial mycelium density were observed and recorded. Slide mounts were prepared in water from sporulating colonies on PDA. Observations were performed using a LEICA DM 2500 dissecting microscope (Wetzlar, Germany) and a NIKON ECLIPSE 80i compound microscope with differential interference contrast (DIC) illumination. Images were captured using a NIS DS-RI2 camera with Nikon NIS-Elements F4.30.01 software. Conidial length was measured from the base of the basal cell to the base of the apical appendage, and conidial width was measured at the widest point. A random selection of 50 conidia was used for measurements. Taxonomic novelties were deposited in MycoBank (Crous et al. 2004).

DNA extraction, PCR amplification, and sequencing

When mycelia spread fully on PDA, genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. PCR primers (forward and reverse) and amplification conditions are detailed in Table 1. PCR amplification was performed on a BIO-RAD PTC-200 thermal cycler. Each 20 μL reaction system contained 10 μL of Master Mix (Promega Corporation), 7 μL of double-deionized water, 1 μL each of forward and reverse primers, and 1 μL of DNA template. PCR products were analyzed by 2% agarose gel electrophoresis and sent to Tsingke Biotechnology Co., Ltd. (Beijing, China) for sequencing.

Table 1.
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Genes used in this study with PCR primers.

Locus PCR primers PCR: thermal cycles (annealing temp. in bold) References
ITS ITS1/ITS4 (95 °C: 30 s, 51 °C: 30 s, 72 °C: 1 min) × 35 cycles White et al. 1990
28S LROR/LR5 (95 °C: 45 s, 55 °C: 30 s, 72 °C: 1 min) × 35 cycles Vilgalys and Hester 1990
rpb2 fRPB2-5F/fRPB2-7cR (95 °C: 15 s, 55 °C: 30 s, 72 °C: 1 min) × 35 cycles Liu et al. 1999
tub2 T1/T22 (95 °C: 35 s, 52 °C: 55 s, 72 °C: 2 min) × 35 cycles O’Donnell and Cigelnik 1997

Phylogenetic analyses

The sequences obtained were assembled using SEQMAN software, and reference sequences from related publications (Yin et al. 2024; Liu et al. 2025) were retrieved from the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov). All sequences generated in this study were submitted to GenBank (Table 2). Sequences were aligned in MAFFT on the web server (https://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013; Katoh et al. 2019), and further adjustments and editing were made with MEGA (Tamura et al. 2013). The multigene sequence alignments and the resulting trees were deposited in TreeBASE (https://treebase.org; study ID S32158). Maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) were selected to construct phylogenetic trees using PAUP, PHYML, and MRBAYES (Huelsenbeck and Ronquist 2001; Swofford 2003; Silvestro and Michalak 2012). Phylograms were visualized with FIGTREE (http://tree.bio.ed.ac.uk/software/figtree/) and further edited with ADOBE ILLUSTRATOR CS (Adobe Systems Inc., USA). Maximum-parsimony bootstrap values (MPBP) and maximum-likelihood bootstrap values (MLBP) ≥ 50% and Bayesian posterior probabilities (BYPP) ≥ 0.90 were shown on the tree.

Table 2.
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Information on strains used in phylogenetic analysis of the genus Daldinia.

Species Strains Country GenBank Accession Numbers
ITS 28S rpb2 tub2
Annulohypoxylon annulatum CBS 140775 USA KY610418 N/A KY624263 KX376353
A. moriforme CBS 123579 France KX376321 KY610425 KY624289 KX271261
A. nitens MFLUCC 12.0823 Thailand KJ934991 KJ934992 KJ934994 KJ934993
A. truncatum CBS 140778 USA KY610419 N/A KY624277 KX376352
Daldinia analina CBS 114736T Ecuador AM749918 KY610430 KY624239 KC977259
D. bambusicola CBS 122872T Thailand KY610385 KY610431 KY624241 AY951688
D. bambusicola TBRC 8878 Thailand MH922869 MH922870 MK165431 MK165422
D. bambusicola TBRC 8879 Thailand MH922872 MH938543 MK165432 MK165423
D. bambusicola BCC33678 Thailand MN153860 MN153877 MN172218 N/A
D. brachysperma BCC33676 Thailand MN153854 MN153871 N/A MN172205
D. caldariorum MUCL 49211 France AM749934 KY610438 KY624242 KC977282
D. caldariorum CBS 122874 USA KU683756 KU683796 KU684289 KU684128
D. chiangdaoensis BCC88202T Thailand MN153850 MN153867 MN172208 MN172197
D. chiangdaoensis BCC88221 Thailand MN153851 MN153868 MN172209 MN172198
D. concentrica CBS 113277 Germany AY616683 KY610434 KY624243 KC977274
D. dennisi CBS 114741T Australia JX658477 KY610435 KY624244 KC977262
D. ehretiae SAUCC228302T China PP145319 PP198888 PP263613 PP277051
D. ehretiae SAUCC228303 China PP145320 PP198889 PP263614 PP277052
D. eschscholtzii CFCC 72597 China PV565503 PV548037 N/A PV649844
D. eschscholtzii CFCC 72598 China PV565504 PV548038 N/A PV649845
D. eschscholtzii MUCL 45435 Benin JX658484 KY610437 KY624246 KC977266
D. eschscholtzii TRRC 8876 Thailand MH938532 MH938541 MK165429 MK165420
D. eschscholtzii BCC28017 Thailand MN153862 MN153879 MN172215 N/A
D. eschscholtzii BCC62428 Thailand MN153863 MN153880 MN172216 N/A
D. flavogranulata BCC89363T Thailand MN153856 MN153873 MN172211 MN172200
D. flavogranulata BCC89365 Thailand MN153857 MN153874 MN172212 MN172201
D. flavogranulata BCC89376 Thailand MN153858 MN153875 MN172213 MN172202
D. guizhouensis GMB0719T China PQ884703 PQ885415 PQ893623 PQ893600
D. guizhouensis GMB5611 China PQ884704 PQ885416 PQ893624 PQ893601
D. jianfengensis SAUCC373804T China PP145325 PP198890 PP263615 PP277053
D. jianfengensis SAUCC373805 China PP145326 PP198891 PP263616 PP277054
D. korfii EBS067 Argentina KY204018 N/A N/A KY204014
D. korfii EBS473 Argentina KY204020 N/A N/A KY204016
D. kretschmaroides TBRC 8875 Thailand MH938531 MH938540 MK165425 MK165416
D. ledongensis SAUCC393602T China PP145327 PP198892 N/A PP277055
D. ledongensis SAUCC393603 China PP145328 PP198893 N/A PP277056
D. loculatoides CBS 113279 UK AF176982 KY610438 KY624247 N/A
D. macaronesica CBS 113040 Spain KY610398 KY610477 KY624294 N/A
D. menghaiensis SAUCC242404T China PP145323 PP198894 PP263617 PP277057
D. menghaiensis SAUCC242405 China PP145324 PP198895 PP263618 PP277058
D. petriniae MUCL 49014 Austria AM749937 KY610439 KY624248 N/A
D. phadaengensis BCC89349T Thailand MN153852 MN153869 MN172206 MN172195
D. phadaengensis BCC89359 Thailand MN153853 MN153870 MN172207 MN172196
D. pyrenaica MUCL 53969 France KY610413 N/A KY624274 N/A
D. rhododendri SAUCC460001T China PP145330 PP198896 N/A PP277059
D. rhododendri SAUCC460002 China PP145329 PP198897 N/A PP277060
D. rubi CFCC 72599T China PV565505 PV548039 N/A PV649846
D. rubi CFCC 72600 China PV565506 PV548040 N/A PV649847
D. spatholobi SAUCC203501T China PP145318 PP198898 N/A PP277061
D. spatholobi SAUCC203502 China PP145317 PP198899 N/A PP277062
D. steglicilii MUCL 43512 Papua New KY610399 KY610479 KY624250 N/A
D. subvernicosa TBRC 8877T Thailand MH938533 MH938542 MK165430 MK165421
D. theissenii CBS 113044 Argentina KY610388 KY610441 KY624251 N/A
D. thunbergiae SAUCC228601T China PP145322 PP198900 N/A PP277063
D. thunbergiae SAUCC228602 China PP145321 PP198901 N/A PP277064
D. vernicosa CBS 119316 Germany KY610395 KY610442 KY624252 N/A
Hypomontagnella monticulosa MUCL 54604 French Guiana KY610404 KY610487 KY624305 KX271273
H. submonticulosa CBS 115280 France KC968923 KY610457 KY624226 KC977267
Hypoxylon fragiforme MUCL 51264 Germany KC477229 KM186295 KM186296 KX271282
Hy. fuscum CBS 113049 France KY610401 KY610482 KY624299 KX271271
Hy. haematostroma MUCL 53301 Martinique KC968911 KY610484 KY624301 KC977291
Jackrogersella cohaerens CBS 119126 Germany KY610396 KY610497 KY624270 KY624314
J. minutella CBS 119015 Portugal KY610381 KY610424 KY624235 KX271240
Pyrenopolyporus hunteri MUCL 52673T Ivory Coast KY610421 KY610472 KY624309 KU159530
P. laminosus TBRC 8871 Thailand MH938527 MH938536 MK165424 MK165415
P. laminosus MUCL 53305T Martinique KC968934 KY610485 KY624303 KC977292
P. nicaraguensis CBS 117739T Burkina Faso AM749922 KY610489 KY624307 KC977272
Rostrohypoxylon terebratum CBS 119137T Thailand DQ631943 DQ840069 DQ631954 DQ840097
Xylaria arbuscula CBS 126415 Germany KY610394 KY610463 KY624287 KX271257
X. brunneovinosa HAST 720 Martinique EU179862 N/A GQ853023 GQ502706

Notes: Strains in this study are marked in bold. “T”: ex-type strains. N/A = not available.

Maximum parsimony (MP) analysis used the tree bisection and reconnection (TBR) branch-swapping algorithm with 1,000 random-addition sequences in a heuristic search (Swofford 2003). The maximum number of trees was set at 5,000 branches of zero length, and all parsimonious trees were saved. Tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency index (RC) were calculated (Swofford 2003). For maximum likelihood (ML) analysis, the GTR GAMMA model of site substitution was applied, estimating gamma-distributed rate heterogeneity and the proportion of invariant sites (Guindon et al. 2010). Branch support in MP and ML was evaluated using a 1,000-replicate bootstrap (BS) method (Hillis and Bull 1993). Bayesian inference (BI) analysis, using a Markov chain Monte Carlo (MCMC) algorithm, was employed to calculate Bayesian posterior probabilities (Rannala and Yang 1996). MRMODELTEST (Posada and Crandall 1998) was used to estimate the nucleotide substitution model for weighted Bayesian analysis. Two MCMC chains, starting from random trees, were run for 1,000,000 generations until the average standard deviation of split frequencies dropped below 0.01, sampling trees every 100th generation. The first 25% of trees were discarded as burn-in, and Bayesian posterior probabilities (BPP) were calculated from the remaining 7,500 trees.

Result

Phylogenetic analyses

The BLAST results indicated that the four isolates belong to Daldinia. In this genus, the combined ITS, 28S, rpb2, and tub2 dataset consisted of 3,145 characters, including alignment gaps (408 for ITS, 801 for 28S, 830 for rpb2, and 1,106 for tub2), of which 2,006 were constant and 228 were variable but parsimony-uninformative characters. MP analysis with the remaining 911 parsimony-informative characters resulted in one equally parsimonious tree: tree length (TL) = 4,936; consistency index (CI) = 0.369; retention index (RI) = 0.682; and rescaled consistency index (RC) = 0.251. In ML analysis based on the combined gene dataset, the matrix had 239 distinct alignment patterns. Estimated base frequencies were as follows: A = 0.238203, C = 0.267126, G = 0.257477, T = 0.237194, AC = 0.732392, AG = 5.679375, AT = 0.806583, CG = 0.787827, CT = 4.670947, GT = 1.000000, gamma distribution shape parameter α = 0.178803, and likelihood value ln = −26014.402666. The phylogenetic analysis revealed that the four isolates (CFCC 72597, CFCC 72598, CFCC 72599, CFCC 72600) were categorized into two clades, representing one new species, D. rubi, and one known species, D. eschscholtzii (Fig. 1). The single-gene trees for ITS, 28S, and tub2 of Daldinia are shown in Suppl. material 1.

Figure 1. 

Phylogram generated from RAxML analysis based on ITS, 28S, rpb2, and tub2 sequence data of Daldinia isolates. The tree was rooted with Xylaria brunneovinosa (HAST 720) and X. arbuscula (CBS 126415). MP, ML (≥ 50%), and BI (≥ 0.9) bootstrap supports are shown near the nodes. Isolates from this study are marked in red, and ex-type strains are marked in bold.

Taxonomy

Daldinia eschscholtzii (Ehrenb.: Fr.) Rehm, Annls mycol. 2(2): 175. 1904.

MycoBank No: 858704
Fig. 2

Description.

Sexual morph: not observed. Asexual morph: Conidiophores with Virgariella-like to Nodulisporium-like branching, mononematous or dichotomous, bearing 1–4 conidiogenous cells per terminus, smooth to finely roughened, hyaline, aseptate, 23.5–28.5 × 2.5–3 (av. ± S.D.= 26 ± 1.9 × 3 ± 0.2 µm, n = 30) μm. Conidiogenous cells terminal or lateral, cylindrical to phialidic, smooth-finely roughened, hyaline, aseptate, apical, 9–14.5 × 2–3 μm (av. ± S.D.= 11.5 ± 1.5 × 2.5 ± 0.3 µm, n = 30). Conidia ellipsoid, cylindrical, oval in shape, smooth, hyaline to pale yellow, aseptate, solitary, holoblastic-sympodial, 4.5–6.5 × 2–4 μm (av. ± S.D.= 5.5 ± 0.5 × 3.0 ± 0.4 µm, n = 50).

Culture characters.

Colonies were dense and uniform, with entire margins and velvety texture, fluffy, surface grayish-white, slightly darker centrally, reverse pale yellow to buff, dark brown at the center. Colonies developed abundant aerial hyphae and reached 60 mm in diameter after 7 days on PDA at 25 °C.

Figure 2. 

Daldinia eschscholtzii (BJFC-S2541) A, D. Leaf of host Rubus idaeus; B. Front colony morphology on PDA at 14 days; C. Reverse colony morphology on PDA at 14 days; E–I. Conidiogenous cells and conidia; J, K. Conidia. Scale Bars: 500 μm (D); 10 μm (E–K).

Materials examined.

China • Guizhou Province, Xingyi City, Maling River Canyon Scenic Area, 25°7'49"N, 104°57'19"E, on the leaf spots of Indocalamus hirsutissimus, 26 Jul 2024, C.M. Tian, N. Jiang, S.J. Li and Y.Y. Wu, BJFC-S2541, living culture CFCC 72597; ibid. BJFC-S2542, living culture CFCC 72598.

Notes.

Based on multi-locus phylogenetic analysis, two strains (CFCC 72597 and CFCC 72598) formed a highly supported clade with Daldinia eschscholtzii (100% MP/100% ML/0.99 BYPP). D. eschscholtzii is a multifunctional wood-inhabiting fungus exhibiting endophytic, saprophytic, and pathogenic traits (Stadler et al. 2014). Daldinia eschscholtzii exhibits endophytic, saprophytic, and pathogenic traits (Stadler et al. 2014) and has a broad host range spanning 56 plant families across 31 countries (Suppl. material 2), mainly colonizing decaying dicotyledonous wood and occasionally occurring on marine algae (Karnchanatat et al. 2007; Zhang et al. 2008; Tarman et al. 2012). Its human pathogenic potential is also confirmed (Ng et al. 2012, 2016; Yew et al. 2014; Chan et al. 2015). This study represents the first documented record of D. eschscholtzii on Indocalamus, supported by phylogenetic congruence with known D. eschscholtzii and alignment with its generalist ecology of colonizing lignocellulosic substrates, like Indocalamus.

Daldinia rubi Y.Y. Wu & C.M. Tian, sp. nov.

MycoBank No: 858703
Fig. 3

Type.

China • Guizhou Province, Guiyang City, Yunyan District, Qianlingshan Forest Park, 26°36'06"N, 106°41'42"E, on the leaf spots of Rubus idaeus, 26 Jul 2024, C.M. Tian, N. Jiang, S.J. Li, and Y.Y. Wu (holotype BJFC-S2543). Ex-type culture CFCC 72599.

Etymology.

Named after the host genus, Rubus.

Description.

Sexual morph: not observed. Asexual morph: Conidiophores are mononematous or dichotomously branched, displaying a Virgariella-like to Nodulisporium-like branching pattern. Conidiophores smooth to finely roughened, hyaline, aseptate, with 2–3 conidiogenous cells at each terminus, measuring (14–)16–36(–39) × 2.5–4(–7) μm (av. ± S.D.= 27 ± 8.4 × 3.5 ± 1.2 µm, n = 30). Conidiogenous cells terminal or lateral, cylindrical, hyaline to pale yellow, smooth to finely roughened, with flattened base, producing conidia apically, measuring (11.5–)13–20.5 × 2–3.5 μm (av. ± S.D.= 16 ± 2.5 × 3 ± 0.4 µm, n = 30). Conidia ellipsoid to dacryoid, hyaline, aseptate, smooth to finely roughened, solitary, mostly flat-based, holoblastic-sympodial, measuring 4.5–8 × 3–4.5 μm (av. ± S.D.= 7 ± 0.8 × 4 ± 0.3 µm, n = 50).

Figure 3. 

Daldinia rubi (BJFC-S2543) A, D. Leaf of host Rubus idaeus; B. Front colony morphology on PDA at 14 days; C. Reverse colony morphology on PDA at 14 days; E. Conidiomata development on medium; F–J. Conidiogenous cells and conidia; K. Conidia. Scale Bars: 500 μm (D); 200 μm (E); 10 μm (F–K).

Culture characters.

Colonies showed sparse, cobweb-like mycelium, appearing semi-transparent, pale gray, with small brown central structures. The reverse was pale grayish-brown with scattered black conidial masses. Aerial hyphae were sparse, growing to 60 mm on PDA in 7 days at 25 °C.

Other material examined.

China • Guizhou Province, Guiyang City, Yunyan District, Qianlingshan Forest Park, 26°36'06"N, 106°41'42"E, on the leaf spots of Rubus idaeus, 26 Jul 2024, C.M. Tian, N. Jiang, S.J. Li and Y.Y. Wu, BJFC-S2544, living cultures CFCC 72600.

Notes.

Based on multi-locus phylogenetic analysis, the two isolates (CFCC 72599, CFCC 72600) formed an independent clade with 100% MP, 100% ML, and 1.00 BYPP values, clearly distinct from Daldinia ehretiae in the multi-locus analyses (Fig. 1). To further substantiate the recognition of D. rubi as a new species, a comprehensive comparison of asexual morphological traits within the Daldinia genus in China was conducted (Table 3). Morphologically, D. rubi can be readily distinguished from D. ehretiae by multi-trait divergence: as shown in Table 3, D. rubi produces larger conidia (ellipsoid to dacryoid, 4.5–8 × 3–4.5 μm) compared to D. ehretiae (ellipsoid or cylindrical, 4.2–6.6 × 1.7–2.8 μm). For conidiophores, D. rubi has shorter and narrower structures (mononematous or dichotomously branched, 16–36 × 2.5–4 μm) with more conidiogenous cells per terminus (2–3 cells) than D. ehretiae (mononematous or dichotomously branched, 100–210 × 3.1–4.3 μm, 1–2 cells per terminus). Conidiogenous cells of D. rubi are shorter in length (cylindrical or laterally cylindrical, (11.5–)13–20.5 × 2–3.5 μm) versus D. ehretiae (cylindrical, 16.8–24.5 × 2.7–4.1 μm). Molecularly, D. rubi also shows clear divergence from D. ehretiae. There is a 12 bp difference in ITS sequences (376 characters, 96.8% similarity, including one gap) and a 26 bp difference in tub2 sequences (745 characters, 96.5% similarity, no gaps). Collectively, the independent phylogenetic position, distinct morphological traits (as detailed in Table 3 for a comparison of asexual characteristics among Daldinia species in China), and molecular divergence confirm that D. rubi represents a new species.

Table 3.
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Comparative analysis of asexual morphological traits among Daldinia species.

Species Conidiophores (Branching/Conidiogenous Cells/Size) Conidiogenous Cells (Shape/Size) Conidia (Shape/Size) References
D. bambusicola Dichotomously or trichotomously branched, 2–3 conidiogenous cells per terminus, 110–160 × 2.1–2.7 μm Cylindrical, 10.1–15.3 × 2.5–3.1 μm Subglobose or ellipsoid, 3.4–4.5 × 2.5–3.1 μm Yin et al. (2024)
D. childiae Dichotomously or trichotomously branched, 2–3 conidiogenous cells per terminus, 150–220 × 2.5–3 μm Clavate (apically enlarged), 14.9–32.7 × 2.8–4.4 μm Subglobose or ellipsoid, 5.8–9.1 × 4.1–5.8 μm Yin et al. (2024)
D. ehretiae Mononematous or dichotomously branched, 1–2 conidiogenous cells per terminus, 100–210 × 3.1–4.3 μm Cylindrical, 16.8–24.5 × 2.7–4.1 μm Ellipsoid or cylindrical, 4.2–6.6 × 1.7–2.8 μm Yin et al. (2024)
D. eschscholtzii Mononematous, dichotomously or trichotomously branched, 2–3 conidiogenous cells per terminus, 120–214 × 2.3–4.1 μm Cylindrical, 14.9–22.7 × 2.1–3.6 μm Ellipsoid or dacryoid, 4.9–6.8 × 2.3–3.5 μm Yin et al. (2024)
D. jianfengensis Mononematous, dichotomously or trichotomously branched, 1–4 conidiogenous cells per terminus, 70–120 × 2.9–4.4 μm Cylindrical, 12.1–16.9 × 2.6–3.6 μm Subglobose or ellipsoid, 3.2–5.5 × 2.6–3.7 μm Yin et al. (2024)
D. ledongensis Rarely mononematous or dichotomously branched, 1 conidiogenous cell per terminus, 120–200 × 1.7–2.1 μm Clavate, 8.6–15.1 × 1.2–3.4 μm Ellipsoid or fusiform, 3.2–4.0 × 1.4–2.0 μm Yin et al. (2024)
D. menghaiensis Dichotomously or trichotomously branched (occasionally), 1–2 conidiogenous cells per terminus, 80–150 × 1.9–3.4 μm Cylindrical or clavate, 16.9–23.5 × 2.0–3.5 μm Ellipsoid, subglobose or dacryoid, 4.7–8.2 × 3.1–4.0 μm Yin et al. (2024)
D. rhododendri Rarely mononematous or dichotomously branched, conidiogenous cell number unclear, 40–90 × 1.4–2.0 μm Cylindrical or ampulliform, 5.9–11.6 × 1.1–2.9 μm Ellipsoid, cylindrical or banana - shaped, 3.2–5.1 × 1.1–2.3 μm Yin et al. (2024)
D. rubi Mononematous or dichotomously branched, 2–3 conidiogenous cells per terminus, 16–36 × 2.5–4 μm Cylindrical or laterally cylindrical, (11.5–)13–20.5 × 2–3.5 μm Ellipsoid to dacryoid, 4.5–8 × 3–4.5 μm This study
D. thunbergiae Mononematous, dichotomously or trichotomously branched, 1–4 conidiogenous cells per terminus, 70–220 × 1.8–4.1 μm Cylindrical or clavate, 6.7–17.3 × 1.9–2.5 μm Ellipsoid or teardrop - shaped, 3.2–5.0 × 2.2–3.1 μm Yin et al. (2024)

Discussion

In this study, two Daldinia species, D. eschscholtzii and D. rubi, were discovered on leaf spot samples of Indocalamus hispidus and Rubus idaeus in Guizhou Province, China. This represents the first record of Daldinia on host plants belonging to Rubus (Rosaceae) and Indocalamus (Poaceae). Globally, Daldinia exhibits an exceptionally broad host range, with the USDA database documenting over 600 host species (https://fungi.ars.usda.gov/). Within China, previously reported Daldinia species have primarily been found on woody plants of Fagaceae, Lauraceae, Sapindaceae, Moraceae, and Betulaceae (https://fungi.ars.usda.gov/). By contrast, records on Rosaceae and Poaceae hosts remain scarce. The present study significantly expands the known host range of Daldinia in China.

Notably, Daldinia exhibits distinctive biological characteristics in its sexual morph, most remarkably through the formation of large stromata on woody branches. These structures characteristically display concentric zonation patterns internally and produce ellipsoidal, brownish to dark brown ascospores (Stadler et al. 2001a; Stadler et al. 2004; Stadler et al. 2014; Liu et al. 2025). However, our investigations, consistent with previous studies, have failed to observe a sexual stage in Daldinia specimens collected from diseased foliage in Southwest China (Yin et al. 2024). This phenomenon suggests that the life cycle transition in Daldinia may be influenced by an intricate interplay of environmental conditions, climatic factors, and host-specific characteristics. Moreover, Daldinia may initially colonize leaves as endophytes or latent pathogens. When host vigor declines or environmental conditions become favorable, these fungi may exhibit pathogenicity. Subsequent studies should conduct artificial inoculation experiments to confirm Daldinia’s pathogenicity and determine how environmental factors and host conditions affect its virulence.

This study systematically compiled the global host range of D. eschscholtzii, revealing its ability to parasitize over 50 plant families while demonstrating distinct host preferences (Suppl. material 2). These fungi primarily colonize dicotyledonous plants, with a particular affinity for the Rosales and Fabales orders, showing the highest frequency on the Fabaceae, Lauraceae, and Moraceae families. Notably, D. eschscholtzii exhibits cross-clade infectivity, capable of rarely colonizing gymnosperms (Pinaceae) and ferns. As shown in Suppl. material 2, tropical-affiliated families (e.g., Lauraceae and Myrtaceae) are disproportionately represented among hosts, many of which include economically significant crops such as citrus and soybean. These characteristics reflect an evolutionary balance between broad-spectrum infectivity and specialized host adaptation in D. eschscholtzii.

Current studies reveal a wide distribution of Daldinia fungi around the world, demonstrating their strong adaptability to diverse ecological environments (Chen 2002; Stadler et al. 2014; Yin et al. 2024; Liu et al. 2025). This suggests potential undiscovered Daldinia species across various ecosystems, warranting more systematic investigations. Targeted sampling in special habitats (e.g., karst formations) and non-conventional hosts would particularly advance our understanding of the biodiversity and ecological functions of this genus.

Acknowledgments

We are grateful to Chungen Piao and Minwei Guo (China Forestry Culture Collection Center [CFCC], Chinese Academy of Forestry, Beijing) for support of strain preservation during this study.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Use of AI

No use of AI was reported.

Funding

This study was financed by the National Natural Science Foundation of China (Project No. 32371887).

Author contributions

All authors have contributed equally.

Author ORCIDs

Yingying Wu https://orcid.org/0009-0007-5095-2738

Shuji Li https://orcid.org/0009-0006-4734-8399

Ning Jiang https://orcid.org/0000-0002-9656-8500

Chengming Tian https://orcid.org/0000-0002-3352-7664

Data availability

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

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Supplementary materials

Supplementary material 1 

Supplementary image

Yingying Wu, Shuji Li, Ning Jiang, Chengming Tian

Data type: pdf

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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Supplementary material 2 

Supplementary table

Yingying Wu, Shuji Li, Ning Jiang, Chengming Tian

Data type: docx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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