11 urn:lsid:arphahub.com:pub:C004A564-9D6A-5F9F-B058-6A3815DFE9C3 urn:lsid:zoobank.org:pub:A4FD9303-3F01-45D9-B438-FCCFE465D2F7 MycoKeys MC 1314-4057 1314-4049 Pensoft Publishers 10.3897/mycokeys.121.154055 154055 Research Article Ascomycota Phylogeny Taxonomy Asia The polyphasic approach reveals two new species and two new records of Nigrospora (Apiosporaceae, Amphisphaeriales) associated with Aquilaria sinensis from China Zhang Shiyu https://orcid.org/0009-0001-4174-6103 1 2 3 Li Junfu lijunfu@mail.kib.ac.cn 2 3 Jiang Hongbo https://orcid.org/0000-0001-9655-4662 2 3 Ye Shuang 4 Mapook Ausana 1 Xu Jianchu 2 3 Hyde Kevin D. https://orcid.org/0000-0002-2191-0762 1 Eungwanichayapant Prapassorn Damrongkool prapassorn@mfu.ac.th https://orcid.org/0000-0001-8005-4137 1 School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand Center for Mountain Futures, Kunming Institute of Botany, Kunming 650201, China CIFOR-ICRAF China Program, World Agroforestry (ICRAF), Kunming 650201, China Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand Tea Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650201, China

Corresponding authors: Junfu Li (lijunfu@mail.kib.ac.cn); Prapassorn Damrongkool Eungwanichayapant (prapassorn@mfu.ac.th)

Academic editor: Samantha C. Karunarathna

 2025 21 08 2025 121 1 20 6A1BFFBE-162F-566E-B7BB-10D52CBAB298 16931613 27 03 2025 10 06 2025 Shiyu Zhang, Junfu Li, Hongbo Jiang, Shuang Ye, Ausana Mapook, Jianchu Xu, Kevin D. Hyde, Prapassorn Damrongkool Eungwanichayapant 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.

This study isolated endophytic fungi of the genus Nigrospora from Aquilaria sinensis (Chinese agarwood) in Guangxi, China. Through rigorous morphological comparisons and molecular phylogenetic analyses, we have identified two new species (N. guangxiensis and N. pubeiensis) and two new host-recorded species (N. oryzae and N. camelliae-sinensis). The morphological analysis revealed that the new species align with the genus-level definition of Nigrospora in colony morphology, conidiophores, and conidial characteristics. However, they were distinguished from known species by finer features such as conidial dimensions and conidiogenous structures. For instance, conidiophores in Nigrospora are typically differentiated from hyphae, characterized by dark pigmentation, which is consistent with broader taxonomic descriptions. Molecular phylogeny, based on concatenated datasets of the ITS, TEF1-α, and TUB2 loci, showed that N. pubeiensis is most closely related to N. chinensis, while N. guangxiensis forms a distinct basal clade relative to these two groups. Additionally, N. oryzae and N. camelliae-sinensis were first recorded on A. sinensis, expanding their known host ranges. The study enriches the taxonomic framework of Nigrospora by providing novel data from China, emphasizing the importance of integrating morphological and molecular approaches in fungal systematics. It also underscores the ecological diversity of fungal-host interactions in agarwood-producing trees, a topic explored in related research on endophytic fungi. These findings have significant implications for our understanding of fungal diversity and their ecological roles in agarwood-producing trees, and they pave the way for further research in this area.

 2 new species asexual fungi endophyte Hyphomycetes multigene phylogeny Sordariomycetes Yunnan Department of Sciences and Technology of China（Grant No: 202302AE090023, 202303AP140001) The Yunnan Province Postdoctoral Orientation Training Fund (E33O386261) Citation

Zhang S, Li J, Jiang H, Ye S, Mapook A, Xu J, Hyde KD, Eungwanichayapant PD (2025) The polyphasic approach reveals two new species and two new records of Nigrospora (Apiosporaceae, Amphisphaeriales) associated with Aquilaria sinensis from China. MycoKeys 121: 1–20. https://doi.org/10.3897/mycokeys.121.154055

 Introduction

Species of Nigrospora Zimm. are pathogens, endophytes, or saprobes on various plant hosts in different habitats (Wang et al. 2017). This genus belongs to Apiosporaceae K.D. Hyde, J. Fröhl., Joanne E. Taylor & M.E. Barr (Hyde et al. 2024). Nigrospora was introduced by Zimmerman (1902) for N. panici Zimm., isolated from the dead leaves of Panicum amphibium Steud, in Indonesia. It was known from the asexual characters because of its black opaque conidial spores borne from grey conidiophores. Mason (1927) classified four species occurring on monocotyledons, viz., N. oryzae (Berk. & Broome) Petch, N. arundinacea (Cooke & Massee) Potl., N. sacchari (Speg.) E.W. Mason, and N. sphaerica (Sacc.) E.W. Mason, by their black-spored hyphomycetous characters and further indicated N. javanica Palm as a synonym of N. panici. Species of Nigrospora were delimited based on morphological characteristics until it was found that some key morphological characteristics overlapped between phylogenetically distinct species, such as conidial dimensions (Kirk et al. 2008). To address this issue, Wang et al. (2017) analyzed recent species in the genus Nigrospora and introduced twelve novel species based on morphological characters, host associations, and a multigene combined phylogeny of ITS, TEF1-α, and TUB2. They further placed this genus within the Apiosporaceae (Xylariales). Moreover, Hao et al. (2020) reported N. gorlenkoana Novobr., N. oryzae, N. osmanthi Mei Wang & L. Cai, N. rubi Mei Wang & L. Cai, and N. sphaerica with 13 novel host associations, including economically important crops from China. They confirmed the genus in the family Apiosporaceae based on multi-gene phylogenetic analyses. Liu et al. (2024) isolated N. anhuiensis Y. Liu & F. He as a pathogen from rice in Anhui Province, China. Zou et al. (2024) established a novel species, N. yunnanensis M.T. Zou & Yong Wang, from Juglans regia L. in Yunnan Province, China.

The secondary metabolites of Nigrospora species typically exhibit good biological activities and are thus regarded as an interesting source of natural products due to their potential application value. For example, Nigrospora sphaerica produces hydroanthraquinones, which exhibit anti-inflammatory activity (Kuang et al. 2022). The metabolites of this species have also shown prominent herbicidal activity in the treatment of intact, greenhouse-grown plants (Fukushima et al. 1998). Nigrospora sphaerica and Nigrospora camelliae-sinensis Mei Wang & L. Cai have also been proven to be a rich source of other secondary metabolites, such as hydroanthraquinone diketopiperazines, lactones, isochromene, and cyclopentanone derivatives (Metwaly et al. 2014; Zhang et al. 2015; Wu et al. 2018; Zhu et al. 2018; Huang et al. 2021; Kuang et al. 2022). Some have been used in the production of green tea, textile dyes, and food preservatives (Xu et al. 2022).

Aquilaria sinensis (Lour.) Spreng. (Thymelaeaceae) is an important economic and medicinal material in China. The high-value, fragrant, and dark resinous heartwood of A. sinensis is known by its popular name “agarwood” (Liu et al. 2017; Wang et al. 2018; Tan et al. 2019; Wang et al. 2019). The damage and infection of the white wood of A. sinensis stimulate the plant’s defense response, and subsequently, dark resin is produced to suppress further infection, resulting in the formation of agarwood (Chen et al. 2017; Chhipa et al. 2017; Faizal et al. 2020). Fungal inoculation is considered to be effective in the agarwood-producing industry, and most of the fungi used for inoculation are endophytes from agarwood-producing trees, including A. sinensis (Han et al. 2014; Azren et al. 2018; Subasinghe et al.2018; Du et al. 2024). Du et al. (2022b) reviewed the biological activity and agarwood induction potential of endophytic fungi; they indicated that Nigrospora oryzae isolated from A. sinensis produced biological activity metabolites such as β-phenylethyl alcohol, 11-hydroxycapitulatin B, and Capitulatin B (Li et al. 2014), showing that endophytic Nigrospora species from Aquilaria play a potential application value in the agarwood-producing industry.

During the survey of endophytic fungi, five strains of Nigrospora were isolated and examined from Aquilaria sinensis in Guangxi Zhuang Autonomous Region, China, for their morphological characteristics. Phylogenetic relationships based on DNA sequence analyses from nucleotides were investigated to evaluate the taxonomic and phylogenetic status of our strains and other Nigrospora species using combined ITS, TEF1-α, and TUB2 sequence data. Full descriptions, illustrations, and phylogenetic analysis results for the two new species and two new host records of Nigrospora are provided.

 Materials and methods Sampling and isolation of endophytic <italic><tp:taxon-name><tp:taxon-name-part taxon-name-part-type="genus" reg="Nigrospora">Nigrospora</tp:taxon-name-part></tp:taxon-name></italic> species

Healthy leaf samples of Aquilaria sinensis were collected in Qinzhou, Guangxi Zhuang Autonomous Region, China. The samples were transported to the laboratory in self-sealing plastic bags stored in insulated ice boxes(Rathnayaka et al. 2025). Leaf samples were rinsed with tap water to remove soil and debris, then aseptically cut into 0.5 cm × 0.5 cm pieces. Surface sterilization was carried out using 75% ethanol for 30 s, followed by 3% H2O2 for 3 min, and another 30 s treatment with 75% ethanol. The samples were then rinsed three times with sterile distilled water and dried on sterile tissue paper (Du et al. 2022a). After drying, four pieces of leaf tissue were placed on potato dextrose agar (PDA) plates and incubated at room temperature (25 °C). Fungal culture characteristics on PDA were observed after one month. Living cultures were deposited in the Kunming Institute of Botany Culture Collection, Kunming, China (KUNCC).

MycoBank numbers and Facesoffungi numbers were obtained as per the instructions in (https://www.mycobank.org/) and Jayasiri et al. (2015), respectively.

 DNA extraction, <abbrev xlink:title="polymerase chain reaction" id="ABBRID0EQPAE">PCR</abbrev> amplification, and sequencing

Fungal mycelia from 30-day-old PDA cultures were scraped off using a sterilized scalpel and transferred into 1.5 mL centrifuge tubes. Genomic DNA was extracted using the Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux®, P.R. China), following the manufacturer’s instructions.

DNA amplification was performed using polymerase chain reaction (PCR) targeting three loci: ITS, TEF1-α, and TUB2.

The internal transcribed spacer (ITS) region was amplified using the primer pair ITS1 and ITS4 (White et al. 1990). The partial translation elongation factor 1-alpha (TEF1-α) region was amplified using EF1-728F and EF2 primers (Carbone and Kohn 1999; O’Donnell et al. 1998). The beta-tubulin (TUB2) fragment was amplified using Bt-2a and Bt-2b (Glass and Donaldson 1995). The PCR reaction volume was 25 µL, containing 1 µL forward primer, 1 µL reverse primer, 22 µL JPmix (Tsingke Biotech Co., Ltd.), and 1 µL of template DNA (48 ng/µL). PCR amplification conditions were as follows: initial denaturation at 98 °C for 2 min, followed by 32 cycles of denaturation at 98 °C for 15 s, annealing at 53 °C for 15 s, extension at 72 °C for 45 s, and a final extension at 72 °C for 5 min. Purification and sequencing of PCR products were carried out by Tsingke Biotech Co., Ltd. (Kunming, P.R. China). The DNA product was stored at -20 °C for long-term preservation.

 Phylogenetic analyses

Phylogenetic analyses were performed following the procedures described in Dissanayake et al. (2020). Sequence quality was assessed using SnapGene v6.1. A BLAST search based on ITS, TEF1-α, and TUB2 sequences was conducted to identify closely related taxa in GenBank (https://www.ncbi.nlm.nih.gov/genbank/), and corresponding sequences were downloaded. Apiospora obovata (LC4940) and Ap. malaysiana (CBS 102053) were selected as outgroup taxa (Table 1). Sequences were aligned in MEGA v7.0 using MUSCLE, with manual adjustments where necessary (Kumar et al. 2016). Phylogenetic trees were generated using both maximum likelihood (ML) and Bayesian inference (BI) methods via the CIPRES Science Gateway (Miller et al. 2010). The ML analysis was conducted using the GTRGAMMA model with 1000 bootstrap replicates. The BI analysis employed the Invgamma+5 model with 100,000 generations. Resulting trees were visualized in FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/), with bootstrap support values indicated at the nodes. The phylogram was rendered in TreeView (Page 1996), illustrated in Microsoft PowerPoint, and exported as a JPEG image using Adobe Photoshop CS5 (Adobe Systems Inc., United States). Newly generated sequences were deposited in GenBank (Table 1). Sequence alignments were submitted to TreeBASE under accession number S31751 (https://treebase.org/).

Names, culture collection numbers, hosts, and corresponding GenBank accession numbers of the taxa used in the phylogenetic analyses.

Taxa names Culture collection number Host ITS TUB2 TEF1-α
Apiospora obovata LC4940 – KY494696 KY705166 KY705095
Ap. malaysiana CBS 102053 – NR120273 KF144988 KF145030
Nigrospora aurantiaca (T) CGMCC3.18130 = LC7302 Nelumbo sp. (leaf) KX986064 KY019465 KY019295
N. aurantiaca LC7034 Musa paradisiaca KX986093 KY019598 KY019394
N. bambusae (T) CGMCC3.18327 = LC7114 Bamboo (leaf) KY385307 KY385319 KY385313
N. bambusae LC7244 Bamboo (leaf) KY385306 KY385320 KY385314
N. bambusae LC7245 Bamboo (leaf) KY385305 KY385321 KY385315
N. camelliae-sinensis LC2710 Castanopsis sp. KX985957 KY019484 KY019310
N. camelliae-sinensis LC3287 Camellia sinensis KX985975 KY019502 KY019323
N. camelliae-sinensis LC3496 Camellia sinensis KX985985 KY019510 KY019327
N. camelliae-sinensis (T) CGMCC3.18125 = LC3500 Camellia sinensis KX985986 KY019460 KY019293
N. camelliae-sinensis LC6684 Camellia sinensis KX986046 KY019570 KY019449
N. camelliae-sinensis KUNCC23-16748 Aquilaria sinensis PQ553689 PQ613611 PQ613606
N. chinensis LC2696 Lindera aggregata KX985947 KY019474 KY019424
N. chinensis LC3493 Camellia sinensis KX985984 KY019509 KY019434
N. chinensis LC4433 Castanopsis sp. KX986013 KY019536 KY019436
N. chinensis LC4558 Unknown host plant KX986020 KY019543 KY019441
N. chinensis (T) CGMCC3.18127 = LC4575 Machilus breviflora KX986023 KY019462 KY019422
N. chinensis LC4660 – – KY019548 KY019445
N. chinensis LC6631 Camellia sinensis KX986043 KY019569 KY019448
N. chinensis LC6851 Unknown host plant KX986049 KY019579 KY019450
N. gorlenkoana (T) CBS 480.73 Vitis vinifera KX986048 KY019456 KY019420
N. gorlenkoana JZB3230001 Cirsium setosum MN495939 MN549381 MN54464
N. guangxiensis KUNCC23-16747 Aquilaria sinensis PQ553688 PQ613610 PQ613605
N. guilinensis LC7301 Vitis vinifera KX986063 KY019608 KY019404
N. guilinensis (T) CGMCC3.18124 = LC3481 Nelumbo sp. (stem) KX985983 KY019459 KY019292
N. hainanensis (T) CGMCC3.18129 = LC7030 Musa paradisiaca (leaf) KX986091 KY019464 KY019415
N. hainanensis LC6979 Musa paradisiaca (leaf) KX986079 KY019586 KY019416
N. hainanensis LC7031 Musa paradisiaca KX986092 KY019597 KY019417
N. hainanensis LC7042 Musa paradisiaca (leaf) KX986094 KY019599 KY019418
N. lacticolonia (T) CGMCC3.18123 = LC3324 Camellia sinensis KX985978 KY019458 KY019291
N. lacticolonia LC7009 Musa paradisiaca (leaf) KX986087 KY019594 KY019454
N. musae (T) CBS 319.34 Musa paradisiaca (fruit) KX986076 KY019455 KY019419
N. musae LC6385 Camellia sinensis KX986042 KY019567 KY019371
N. oryzae LC6761 Oryza sativa KX986056 KY019574 KY019376
N. oryzae LC7297 Nelumbo sp. (leaf) KX985936 KY019605 KY019400
N. oryzae LC2693 Neolitsea sp. KX985944 KY019471 KY019299
N. oryzae LC2707 Rhododendron simiarum KX985954 KY019481 KY019307
N. oryzae LC4338 Camellia sp. KX986008 KY019532 KY019349
N. oryzae LC4961 Pittosporum illicioides KX986031 KY019553 KY019358
N. oryzae LC5243 Submerged wood KX986033 KY019555 KY019360
N. oryzae LC6923 Oryza sativa L. KX986051 KY019581 KY019383
N. oryzae JZB3230002 Phyllostachys nigra MN495940 – MN544639
N. oryzae JZB3230003 Rudbeckia hirta MN495941 – MN544640
N. oryzae JZB3230004 Scirpus sp. MN495942 MN549382 MN544641
N. oryzae KUNCC23-16746 Aquilaria sinensis PQ553687 PQ613609 PQ613604
N. osmanthi (T) CGMCC3.18126 = LC4350 Osmanthus sp. KX986010 KY019461 KY019421
N. osmanthi LC4487 Hedera nepalensis KX986017 KY019540 KY019438
N. osmanthi JZB3230005 Rosa chinensis MN495943 MN549383 MN508179
N. osmanthi JZB3230006 Rosa chinensis MN495944 MN549384 MN508180
N. osmanthi JZB3230007 Phragmites australis MN495945 MN549385 MN508181
N. osmanthi JZB3230008 Cirsium setosum MN495946 MN549386 MN508182
N. osmanthi JZB3230009 Phyllostachys nigra MN495947 MN549387 MN508183
N. osmanthi JZB3230010 Phyllostachys nigra MN495948 MN549388 MN508184
N. osmanthi JZB3230011 Rudbeckia hirta MN495949 MN549389 MN508185
N. pubeiensis KUNCC23-16745 Aquilaria sinensis PQ553686 PQ613608 PQ613603
N. pyriformis (T) CGMCC3.18122 = LC2045 Citrus sinensis KX985940 KY019457 KY019290
N. pyriformis LC2688 Lindera aggregata KX985941 KY019468 KY019297
N. pyriformis LC2694 Rubus reflexus KX985945 KY019472 KY019300
N. pyriformis LC3099 Camellia sinensis KX985971 KY019498 KY019322
N. pyriformis LC3292 Camellia sinensis KX985976 KY019503 KY019324
N. rubi (T) CGMCC3.18326 = LC2698 Rubus sp. KX985948 KY019475 KY019302
N. rubi JZB3230012 Fraxinus sp. MN495950 – MN54464
N. sphaerica LC7312 Nelumbo sp. (leaf) KX985935 KY019618 KY019414
N. sphaerica LC7298 Nelumbo sp. (leaf) KX985937 KY019606 KY019401
N. sphaerica LC2840 Harpullia longipetala KX985965 KY019492 KY019318
N. sphaerica LC3477 Camellia sinensis KX985982 KY019508 KY019326
N. sphaerica LC4264 Rhododendron arboretum KX985993 KY019517 KY019334
N. sphaerica LC4307 Rhododendron arboretum KX986005 KY019529 KY019346
N. sphaerica LC5901 Submerged wood KX986034 KY019556 KY019361
N. sphaerica LC6294 Camellia sinensis KX986044 KY019565 KY019369
N. sphaerica LC6996 Musa paradisiaca (leaf) KX986085 KY019592 KY019390
N. sphaerica JZB3230013 Cirsium setosum MN495951 MN549390 MN544642
N. sphaerica JZB3230014 Phragmites australis MN495952 MN549391 MN544643
N. sphaerica JZB3230015 Fraxinus sp. MN495953 MN549392 MN544644
Nigrospora sp. 1 LC2725 Symplocos zizyphoides KX985960 KY019487 KY019313
Nigrospora sp. 1 LC4566 Lithocarpus sp. KX986022 KY019545 KY019354
Nigrospora sp. 2 LC6704 Camellia sinensis KX986047 KY019571 KY019373
N. vesicularis LC0322 Unknown KX985939 KY019467 KY019296
N. vesicularis (T) CGMCC3.18128 = LC7010 Musa paradisiaca (leaf) KX986088 KY019463 KY019294
N. zimmermanii CBS 167.26 Unknown KY385308 KY385318 KY385312
N. zimmermanii (T) CBS 290.62 Saccharum officinarum (leaf) KY385309 KY385317 KY385311
N. zimmermanii CBS 984.69 Saccharum officinarum (leaf) KY385310 KY385322 KY385316
Nigrospora sp. YL-2024a – OP677969 PP103614 PP103590
N. shadeganensis SCUA-Saf-N27 – PP256499 PP263821 PP263812
N. coryli W18 Corylus heterophylla PP218065 PP218065 PP461302
N. humicola CFCC 56884 Soil ON555686 ON557392 ON557394
N. yunnanensis GUCC24-0008 – PP915796 PP947937 PP947933

– Refers to the data unavailability, (T)refers to the type specimens, and the taxa produced in this study are in bold.

 Results Phylogenetic analyses

The combined ITS, TEF1-α, and TUB2 sequence dataset comprised 77 taxa, with Apiospora obovata (LC4940) and Ap. malaysiana (CBS 102053) selected as outgroup taxa.

Bayesian inference (BI) and maximum likelihood (ML) analyses of the combined dataset were conducted to determine the placement of our new taxa, infer relationships at the intrageneric level, and resolve phylogenetic relationships within Apiosporaceae. The phylogenetic trees derived from BI and ML analyses exhibited largely similar topologies and were consistent with previous studies based on ML analysis (Li et al. 2017; Su et al. 2018). The best-scoring RAxML tree is shown in Fig. 1, with a final ML optimization likelihood value of –10322.409322 (ln). The dataset consisted of 1,595 total characters, including gaps (ITS: 1–591 bp; TEF1-α: 592–1170 bp; TUB2: 1171–1595 bp). RAxML analysis yielded 1,595 distinct alignment patterns, with 33.97% of characters undetermined or containing gaps.

10.3897/mycokeys.121.154055.figure1 16931702 C1014B8E-90D2-50A4-A6B9-D2766215E48A

Tree generated by RAxML analysis showing the placement of Nigrospora species based on combined ITS, TEF1-α, and TUB2 sequence data. Bootstrap support values for ML (left) ≥ 75% and Bayesian posterior probabilities (PP, right) ≥ 0.9 are indicated at the nodes. The tree is rooted with Ap. obovata (LC4940) and Ap. malaysiana (CBS 102053). Strains generated in this study are shown in red and blue bold; red indicates new species, and blue indicates new host records.

https://binary.pensoft.net/fig/1398939 Fungi Amphisphaeriales Apiosporaceae BACB0194-7817-50D4-A2D7-8E81E28862B4 Nigrospora camelliae-sinensis 820731 Mei Wang & L. Cai, in Wang, Liu, Crous & Cai, Persoonia 39: 127 (2017) Fig. 2 Description.

Endophytic from healthy leaves of Aquilaria sinensis. Sexual morph: Undetermined. Asexual morph: Hyphae branched, septate, hyaline, or light grey, 2–4 µm diam. Conidiophores 3–6 µm diam. micronematous, mononematous, solitary, smooth, branched or not, 0–1 septate, hyaline to subhyaline, consisting of 1–2 cells or usually reduced to conidiogenous cells. Conidiogenous cells 5.5–7.2 × 6.3–9.4 µm diam. x̄ = 5.8 × 12.1 µm, n = 30), monoblastic, discrete, determinate, smooth, subglobose to ampulliform, subhyaline to light brown. Conidia 8–13 µm diam. x̄ = 11 µm, n = 30) solitary, smooth-walled, aseptate, spherical, light grey to black, globose or subglobose.

 Cultural characteristics.

Mycelium effusing on PDA within 12 hours from the edges of the surface sterilized leaf tissue piece. Colonies growing on PDA, hairy, black, reaching 9 cm in 7 days at 30 °C; mycelium partly superficial, partly immersed, slightly effuse, radially striate, with an irregular edge, black. Asexual conidia spores were formed after 25 days on PDA. Sexual spores were not formed within 60 days.

10.3897/mycokeys.121.154055.figure2 16931704 28EF6EB6-B1C2-5F74-8447-3DE326343A40

Nigrospora camelliae-sinensis (HKAS–134953, new host record). a. Culture on PDA for 30 days; b–d. Mass of conidia; e–j. Conidiogenous cells attached conidia; k. Conidiogenous cell arising from hyphae; l–o. Conidiophores bearing conidia; p–t. Conidia. Scale bars: 10 μm (b–t).

https://binary.pensoft.net/fig/1398940 Material examined.

China • Guangxi Zhuang Autonomous Region, Pubei City, in healthy living leaves of Aquilaria sinensis, September 25, 2020, Shiyu Zhang, GX9-3 (HKAS 134953, new host record) . Living culture KUNCC23-16748.

 Known distributions

(based on molecular data): China (Wang et al. 2017, this study).

 Known hosts

(based on molecular data): Aquilaria sinensis (this study), Camellia sinensis (Wang et al. 2017), Castanopsis sp. (Wang et al. 2017), Musa paradisiaca (Wang et al. 2017).

 Notes.

Nigrospora camelliae-sinensis has been reported to have a cosmopolitan distribution and a broad host range (Wang et al. 2017). In this study, our isolation (KUNCC23-16748), collected from Aquilaria sinensis in Guangxi Zhuang Autonomous Region, China, clustered in one single clade with N. camelliae-sinensis (94% ML, 1PP). Its morphological characteristics were in good agreement with those of N. camelliae-sinensis. Therefore, we regard this isolation (KUNCC23-16748) as N. camelliae-sinensis collected from Aquilaria sinensis for the first time. The study showed that N. camelliae-sinensis is also characterized by having determinate and micro- and mononematous, subcylindrical conidiophores.

 Fungi Amphisphaeriales Apiosporaceae 38F8999F-AAAB-59E3-8093-81A7A5415CC2 Nigrospora guangxiensis 859462 Facesoffungi Number: FoF17621 S.Y. Zhang, J.F. Li & K.D. Hyde sp. nov. Fig. 3 Etymology.

Named after the type location, Guangxi Zhuang Autonomous Region, China.

 Holotype.

HKAS 134952.

 Description.

Endophytic from healthy leaves of Aquilaria sinensis. Sexual morph: Undetermined. Asexual morph: Hyphae branched, septate, hyaline to pale brown, 1–3 µm diam. Conidiophores usually reduced to conidiogenous cells, which are dispersed on hyphae. Conidiogenous cells 4.5–6 × 5.3–7.3 µm diam. x̄ = 5 × 6.8 µm, n = 30), discrete, solitary, monoblastic, determinate, subglobose, pale brown to brown colored. Conidia 8–14 μm diam. x̄ = 10.5 µm, n = 30), solitary, discrete, dark brown to black, globose or subglobose.

 Cultural characteristics.

Mycelium effusing on PDA within 12 hours from the edges of the surface sterilized leaf tissue piece. Colonies growing on PDA, hairy, reaching 9 cm in 7 days at 30 °C; mycelium partly superficial, partly immersed, slightly effuse, radially striate, with irregular edge, initially greyish-brown, becoming black colored with age; asexual spores were formed after 25 days on PDA, and sexual spores not formed within 60 days on PDA.

10.3897/mycokeys.121.154055.figure3 16931706 94908DD8-56B1-5953-9D92-9D2F91D8840A

Nigrospora guangxiensis (HKAS–134952, holotype). a. Culture on PDA for 30 days; b, c. Mass of conidia; d–g. Conidiogenous cells attached conidia; h, i. Conidiophores; j–s. Conidia. Scale bars: 10 μm (b–s).

https://binary.pensoft.net/fig/1398941 Known distribution

(based on molecular data): China (this study).

 Known hosts.

Aquilaria sinensis (this study).

 Material examined.

China • Guangxi Zhuang Autonomous Region, Pubei city, on healthy living leaves of Aquilaria sinensis, September 25, 2020, Shiyu Zhang, GX4-1 (HKAS 134952, holotype) ; extype living culture, KUNCC23-16747.

 Notes.

In the multi-locus phylogenetic analyses, our strain Nigrospora guangxiensis (KUNCC23-16747) formed a separate branch basal (97% ML, 1PP) to N. pubeiensis (KUNCC23-16745) and N. chinensis (eight strains). Nigrospora guangxiensis differs from N. pubeiensis in having smaller conidiogenous cells (5 × 6.8 µm vs. 5.8 × 9.4 µm) and shorter conidia (10.5 µm vs. 11.3 µm). Additionally, its initial culture on PDA medium is greyish-brown. Nigrospora guangxiensis is distinct from N. chinensis in having smaller conidia (10.5 µm vs. 12.97 ± 1.07 µm), pale brown to brown-colored conidiogenous cells, and sterile cells, which are absent in N. guangxiensis (Wang et al. 2017). A nucleotide base comparison of these species is shown in the notes of N. pubeiensis and Table 2.

Base pair comparison in ITS, TEF1-α, and TUB2 sequences of N. chinensis, N. pubeiensis, and N. guangxiensis.

Species Base pair comparison in ITS, TUB2, and TEF1-α
ITS TUB2 TEF1-α
N. chinensis (CGMCC-38127) vs. N. pubeiensis (KUNCC23-16745) 3/492(0.6%) 26/365(7.1%) 0
N. chinensis (CGMCC-38127) vs. N. guangxiensis (KUNCC23-16747) 10/492(2.0%) 26/373(7.0%) 62/425(14.5%)
N. guangxiensis (KUNCC23-16747) vs. N. pubeiensis (KUNCC23-16745) 9/511(1.8%) 0 62/425(14.5%)
Fungi Amphisphaeriales Apiosporaceae CB306075-7C85-5702-9640-4036E33016A2 Nigrospora oryzae 253729 Facesoffungi Number: FoF06596 (Berk. & Broome) Petch, J. Indian bot. Soc. 4: 24 (1924) Fig. 4 Monotospora oryzae Berk. & Broome, J. Linn. Soc., Bot. 14: 99. 1873. Basionym. Khuskia oryzae H.J. Huds., Trans. Brit. Mycol. Soc. 46: 358. 1963. Synonym. Description.

Endophytic from healthy leaves of Aquilaria sinensis. Sexual state: Undetermined. Asexual state: Hyphomycetes. Hyphae 1–3.5 µm diam. branched, smooth, septate, hyaline, or subhyaline. Conidiophores 2–5 µm diam. micronematous, solitary in sporodochia, subcylindrical, pale grey, smooth, 0–2-septate, branched or not, usually reduced to conidiogenous cells. Conidiogenous cells 4.7–5.2 × 6.3–7.4 µm diam. x̄ = 5.0 × 6.9 µm, n = 50), monoblastic, solitary, determined, hyaline to subhyaline, ampliform to subspherical, discrete, smooth-walled. Conidia 8–13.5 µm diam x̄ = 11.3 µm, n = 50) solitary, globose or subglobose, black, shiny, smooth, aseptate.

 Cultural characteristics.

Mycelium effusing on PDA within 12 hours from the edges of the surface-sterilized leaf tissue piece. Colonies growing on PDA, hairy, black, reaching 5 cm in 7 days at 30 °C; mycelium partly superficial, partly immersed, slightly effuse, radially striate, with an irregular edge, dark brown to black colored; asexual spores were formed after 25 days on PDA, and sexual spores were not formed within 60 days on PDA.

10.3897/mycokeys.121.154055.figure4 16931708 51B851C5-8787-5EA0-B19F-F9E098530A07

Nigrospora oryzae (HKAS—134951), new host and geographical record. a. Culture on PDA for 30 days; b, c. Mass of conidia; d, e. Conidiogenous cells attached conidia; f–h. Conidiophores bearing conidia; i–r. Conidia. Scale bars: 10 μm (a–r).

https://binary.pensoft.net/fig/1398942 Material examined.

China • Guangxi Zhuang Autonomous Region, Pubei city, on healthy living leaves of Aquilaria sinensis, September 25, 2020, Shiyu Zhang, GX2-3 (HKAS 134951, new host record) . Living culture KUNCC23-16746.

 Known distribution

(based on molecular data): Australia (Barkat et al. 2016), Bangladesh (Begum et al. 2018), China (Sun et al. 2011; Chen and Kirschner2018; He et al. 2019; Zhang et al. 2019, this study), Iran (Hashemian et al. 2014), Italy (Lorenzini et al. 2016), Kazakhstan (Eken et al. 2016), Pakistan (Alam et al. 2017), Sri Lanka (Wang et al. 2017), and Thailand (Hyde et al. 2020).

 Known hosts

(based on molecular data): Acer truncatum (Sun et al. 2011), Actinidia sp. (Li et al. 2018), Aloe vera (Alam et al. 2017), Aquilaria sinensis (This study), Castanopsis sp. (Wang et al. 2017), Cephalotaxus sinensis (Wang et al. 2017), Citrullus lanatus (Chen and Kirschner 2018), Citrus reticulata (Wang et al. 2017), Cleyera japonica (Wang et al. 2017), Daphniphyllum macropodum (Wang et al. 2017), Daphniphyllum oldhamii (Wang et al. 2017), Gossypium hirsutum (Zhang et al. 2019), Hamamelis mollis (Wang et al. 2017), Nelumbo nucifera (Chen and Kirschner 2018), Nelumbo sp. (Wang et al. 2017), Neolitsea sp. (Wang et al. 2017), Oryza sativa (Wang et al. 2017), Osmanthus fragrans (Wang et al. 2017), Osmanthus sp. (Wang et al. 2017), Pennisetum americanum (Hashemian et al. 2014), Pentactina rupicola (Wang et al. 2017), Photinia serrulata (He et al. 2019), Phyllostachys heterocycla (Hyde et al. 2020), Rhododendron simiarum (Wang et al. 2017), Rhododendron sp. (Wang et al. 2017), Rubus reflexus (Wang et al. 2017), Rubus sp. (Wang et al. 2017), Symplocos zizyphoides (Wang et al. 2017), Ternstroemia sp. (Wang et al. 2017), Triticum aestivum (Eken et al. 2016; Barkat et al. 2016), Tutcheria microcarpa (Wang et al. 2017), Vaccinium corymbosum (Zhang et al. 2019), Vitis vinifera (Lorenzini et al. 2016).

 Notes.

Nigrospora oryzae has been reported to have a cosmopolitan distribution and a broad host range (Wang et al. 2017). In this study, our isolate (KUNCC23-16746) clustered in the same clade (100% ML, 1 PP) as N. oryzae, and its morphological characteristics were consistent with those of N. oryzae (Wang et al. 2017). Therefore, we regard this isolate (KUNCC23-16746) as N. oryzae, which was collected from Aquilaria sinensis in the Guangxi Zhuang Autonomous Region, China, for the first time.

 Fungi Amphisphaeriales Apiosporaceae 3676BF35-A7EA-5015-A981-698FE3D645F7 Nigrospora pubeiensis 859460 Facesoffungi Number: FoF17622 S.Y. Zhang, J.F. Li & K.D. Hyde sp. nov. Fig. 5 Etymology.

Named after the location from which it was collected, Pubei, Guangxi Zhuang Autonomous Region, China.

 Holotype.

HKAS 134950.

 Description.

Endophytic from healthy leaves of Aquilaria sinensis. Sexual morph: Undetermined. Asexual morph: Hyphae 3–6 µm diam. branched, smooth, septate, hyaline, or subhyaline. Conidiophores 1–4 µm diam, micronematous, solitary, subcylindrical, hyaline to pale grey, smooth, 0–2-septate, branched or not, usually reduced to conidiogenous cells. Conidiogenous cells 5–6.3 × 6.3–11.3 µm diam. (x̄ = 5.8 × 9.4 µm, n = 20), discrete, solitary, monoblastic, determinate, subglobose, straight, smooth, hyaline or dark brown. Conidia 6–14 μm diam (x̄ = 11.3 µm, n = 20), solitary, granular, dark brown to black, globose, or subglobose.

 Cultural characteristics.

Mycelium effusing on PDA within 12 hours from the edges of the surface-sterilized leaf tissue piece. Colonies growing on PDA, hairy, black, reaching 9 cm in 7 days at 30 °C; mycelium partly superficial, partly immersed, slightly effuse, radially striate, with irregular edge, initially grayish white, becoming black colored with age; asexual spores were formed after 25 days on PDA, and sexual spores not formed within 60 days on PDA.

 Known distribution

(based on molecular data): China (this study).

 Known hosts.

Aquilaria sinensis (this study).

 Material examined.

China • Guangxi Zhuang Autonomous Region, Pubei City, in healthy living leaves of Aquilaria sinensis, September 25, 2020, Shiyu Zhang, GX2-2 (HKAS 134950, holotype). Ex-type living culture at KUNCC23-16745.

 Notes.

Nigrospora pubeiensis is described herein as a new species, which resembles other species in the genus in having granular, dark brown to black, globose, or subglobose conidia, but differs in its short conidiophores, sometimes reduced to conidiogenous cells. The strain representing N. pubeiensis (KUNCC23-16745) clustered in a supported clade (100% ML, 1 PP) and is closely related to N. guangxiensis (KUNCC23-16747) and N. chinensis (CGMCC-38127). However, N. pubeiensis is distinct in nucleotide base pair comparison with N. guangxiensis (KUNCC23-16747) across the ITS gene region (9/511 bp, 1.8% difference, no gap) and the TEF1-α gene region (62/425 bp, 14.5% difference, no gap), TUB2 (0); it differs with N. chinensis (CGMCC-38127) across the ITS gene region (3/492 bp, 0.6% difference, no gap) and the TUB2 gene region (26/365 bp, 7.1% difference, no gap), TEF1-α (0). They also have different conidiophore structures. Compared with the photoplate from Wang et al. (2017), the conidiophore of N. chinensis has an ampulliform structure, but N. pubeiensis does not. Based on distinct morphological characteristics and phylogenetic support, we regard this isolation (KUNCC23-16745) as a new species, N. pubeiensis, which was collected from Aquilaria sinensis in Guangxi Province, China.

10.3897/mycokeys.121.154055.figure5 16931710 712BB29E-E065-56B3-AB93-C0DA91DEE41D

Nigrospora pubeiensis (HKAS–134950, holotype). a. Culture on PDA for 30 days; b. Mass of conidia; c, d. Conidiogenous cells attached conidia; e–i. Conidiophores; j. Conidiophores bearing conidia; k–t. Conidia. Scale bars: 10 μm (b–t).

https://binary.pensoft.net/fig/1398943 Discussion

The discovery of two novel Nigrospora species (N. pubeiensis and N. guangxiensis), along with new host records for N. oryzae and N. camelliae-sinensis, highlights the adaptability of Nigrospora to diverse ecological niches. The broad host range of Nigrospora on Aquilaria spp. may be linked to its ability to exploit host-specific chemical environments. For instance, endophytic fungi in A. sinensis often interact with host-derived secondary metabolites, such as sesquiterpenes and chromones, which are abundant in agarwood resin (Li et al. 2025). These compounds may create selective pressures that favor fungal lineages capable of tolerating or metabolizing them (Li et al. 2025). Evidence from other studies shows that Nigrospora species produce bioactive metabolites (e.g., antimicrobial compounds), which could confer competitive advantages in colonizing new hosts or resisting host defenses (Metwaly et al. 2014; Kuang et al. 2022).

Ecologically, the occurrence of Nigrospora on A. sinensis aligns with observations that endophytic fungal communities shift dynamically during agarwood formation. For example, fungal diversity decreases as resinous compounds accumulate, suggesting a trade-off between host defense and fungal survival (Li et al. 2025). Similar patterns have been observed in Fusarium-induced agarwood, where dominant fungi such as Colletotrichum thrive in resin-rich tissues (Fang et al. 2024). This implies that Nigrospora may occupy niches where host metabolites are less inhibitory or where their secondary metabolites counteract host defenses.

Evolutionarily, the divergence of Nigrospora lineages may reflect co-adaptation with Aquilaria hosts. Multi-gene phylogenies (e.g., ITS, TEF1, and TUB2) reveal that fungal speciation often correlates with host specificity (Tibpromma et al. 2021). For instance, Nemania species isolated from A. sinensis produce sesquiterpenoids identical to those found in agarwood, suggesting a symbiotic relationship in which the fungi contribute to resin biosynthesis (Tibpromma et al. 2021). Analogously, Nigrospora may have evolved metabolic pathways that enable coexistence with Aquilaria’s chemical defenses, thus facilitating colonization across diverse hosts.

 Acknowledgments

Li Junfu thanks the Yunnan Provincial Department of Human Resources and Social Security and the Yunnan Provincial Postdoctoral Orienteering Training and Funding Program (E33O386261), entitled “Investigation of Fungal Diversity and Fungal Resources in the Dry Valley Area of Honghe, Yunnan Province.” We thank two anonymous reviewers and Prof. Saowaluck Tibpromma for their valuable suggestions to improve the manuscript.

 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

The research is supported by the Yunnan Department of Science and Technology of China (Grant Nos: 202302AE090023 and 202303AP140001).

 Author contributions

Shiyu Zhang, Prapassorn Damrongkool Eungwanichayapant and Junfu Li conceived the study. Shiyu Zhang, Junfu Li and Shuang Ye performed the experiments. Shiyu Zhang, Hongbo Jiang and Junfu Li analyzed the data. Shiyu Zhang and Junfu Li drafted the manuscript. Ausana Mapook, Jianchu Xu, Kevin D. Hyde and Prapassorn Damrongkool Eungwanichayapant reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.

 Author ORCIDs

Shiyu Zhang https://orcid.org/0009-0001-4174-6103

Hongbo Jiang https://orcid.org/0000-0001-9655-4662

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

Prapassorn Damrongkool Eungwanichayapant https://orcid.org/0000-0001-8005-4137

 Data availability

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

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