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Article

Four New Endophytic Apiospora Species Isolated from Three Dicranopteris Species in Guizhou, China

by
Jing-Yi Zhang
1,2,3,
Meng-Lan Chen
1,
Saranyaphat Boonmee
2,3,
Yu-Xuan Wang
1 and
Yong-Zhong Lu
1,*
1
School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang 550003, China
2
School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
3
Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
*
Author to whom correspondence should be addressed.
J. Fungi 2023, 9(11), 1096; https://doi.org/10.3390/jof9111096
Submission received: 17 September 2023 / Revised: 2 November 2023 / Accepted: 9 November 2023 / Published: 10 November 2023
(This article belongs to the Section Fungal Evolution, Biodiversity and Systematics)
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Abstract

:
Endophytic fungi isolated from medicinal ferns serve as significant natural resources for drug precursors or bioactive metabolites. During our survey on the diversity of endophytic fungi from Dicranopteris species (a genus of medicinal ferns) in Guizhou, Apoiospora was observed as a dominant fungal group. In this study, seven Apiospora strains, representing four new species, were obtained from the healthy plant tissues of three Dicranopteris species—D. ampla, D. linearis, and D. pedata. The four new species, namely Apiospora aseptata, A. dematiacea, A. dicranopteridis, and A. globosa, were described in detail with color photographs and subjected to phylogenetic analyses using combined LSU, ITS, TEF1-α, and TUB2 sequence data. This study also documented three new hosts for Apiospora species.

1. Introduction

Apiosporaceae, typified by Apiospora Sacc., was established by Hyde et al. [1] to accommodate Arthrinium-like taxa characterized by a basauxic, arthrinium-like conidiogenesis producing apiospores [2,3]. Currently, only three genera, Apiospora, Arthrinium Kunze, and Nigrospora Zimm, are accepted in this family [4,5]. Apiospora Sacc. was introduced by Saccardo, P. A. [6] within the family Apiosporaceae (Amphisphaeriales, Sordariomycetes), with A. montagnei Sacc. as the type species. Due to the morphological similarities between the genera Apiospora and Arthrinium, they were long considered synonymous based on the one fungus one name principle [7,8,9,10,11]. It was not until the study by Pintos, Á. & P. Alvarado [12] that they were clarified as separate genera based on genetic, ecological, and morphological evidence. This delineation was confirmed and supported by subsequent studies [13,14,15,16,17,18,19,20]. Evidence of genome draft was employed within the fungal group Arthrinium/Apiospora for the first time, which also supported them into two separate genera [21]. Currently, 157 epithets under the genus Apiospora are listed in Index Fungorum (September 2023), while 116 epithets are listed in Species Fungorum. Out of these, molecular data have confirmed 91 Apiospora species [12,13,14,15,17,18,19,20,22].
The sexual morph of Apiospora is characterized by immersed to erumpent, multi-loculate, perithecial ascostromata, unitunicate, broadly clavate to cylindric–clavate asci, and hyaline, ellipsoidal, inequilaterally 2-celled ascospores with or without a gelatinous sheath [5,9,10,14,22,23]. The asexual morphs of Apiospora include hyphomycetes and coelomycetes. The hyphomycetous asexual morphs feature septate, subhyaline, or brown conidiophores emerging from basal cells or reduced to conidiogenous cells, basauxic conidiogenous cells, and typically globose to subglobose, aseptate conidia that appear lenticular or obovoid from side view [11,13,16,18,19,24]. The coelomycetous asexual morph of Apiospora is marked by its erumpent, pustulate, coriaceous conidiomata, hyphoid conidiophores, blastic, integrated, determinate, doliiform or cylindrical conidiogenous cells, and oval, brown conidia, which may have a truncate basal scar and a germ slit [2,9,20].
Endophytes are endosymbiotic flora, microorganisms that colonize the internal tissues of healthy plants without causing any direct, noticeable negative effects [25,26]. The taxonomic research of endophytic fungi has become a popular trend not only because of their beneficial effects on plants but also because of compounds including antibiotics and other compounds of therapeutic significance [26,27,28]. That is, endophytic fungi (especially medicinal plants) possess significant potential to discover or synthesize more bioactive compounds and mimic the structure and function of host compounds [25,28,29,30], which shows a new source of potentially useful pharmaceutical compounds [26,28,31].
Dicranopteris Bernh. is an ancient and widespread fern genus belonging to the family Gleicheniaceae (Filicopsida) found in tropical and subtropical ecosystems [32,33]. It belongs to a group of medically important ferns known for their significant pharmacological effects, including removing blood stasis, clearing heat and diuresis, anticancer, antinociception, and anti-inflammation [34,35,36,37,38]. Extracts from Dicranopteris are rich in bioactive compounds and have the potential to yield new structural compounds [34,39].
In this study, we examined endophytic fungi isolated from three Dicranopteris species (D. ampla, D. linearis, and D. pedate) in Guizhou, China, aiming to explore the diversity of fungi with research significance. We isolated nearly a thousand endophytic taxa from various parts of the three Dicranopteris species, belonging to 146 genera, based on NCBI searches of the ITS and LSU sequence data. Among these isolates, Apiospora emerged as a common genus. Within this collection, seven taxa were identified herein as four endophytic Apiospora species new to science, viz. A. aseptata, A. dematiacea, A. dicranopteridis, and A. globosa. To further determine the taxonomic placement of these four Apiospora species, we employed phylogenetic analyses using combined LSU, ITS, TEF1-α, and TUB2 sequence data, complemented by morphological features. A backbone tree of Apiosporaceae is provided in this study.

2. Materials and Methods

2.1. Collection and Isolation

Fresh, healthy plant tissues (leaves, rhizomes, roots, and stems) from three Dicranopteris species were collected along with relevant metadata (date, habitat, and locality). Samples were transported to the laboratory and processed for fungal isolation within 48 h. Healthy tissue pieces were first washed under running tap water. Surface sterilization of plant tissues followed the method described by Nontachaiyapoom et al. [40], with some modifications. To eliminate epiphytic microorganisms, the materials were surface-sterilized on a benchtop by immersing them in 75% (v/v) ethanol for 1–3 min (ca. 1 min for leaves and stems; ca. 3 min for rhizomes and roots). They were then rinsed with sterilized distilled water for 2 min, followed by a soak in 10% (v/v) NaClO for 0.5–2 min (ca. 0.5 min for leaves and stems; ca. 2 min for rhizomes and roots). The tissues were then rinsed with sterile distilled water three times in succession. After drying the sterilized plant tissues on sterilized filter paper, they were cut into approximately 2 mm2 pieces using a sterile blade. These small pieces were placed on fresh potato dextrose agar (PDA) containing antibiotics (50 μg/mL penicillin) and cultivated at 25 °C. Once fungal hyphae growth was observed emerging from the plant segments, the hyphae were picked from the edge of the colonies and transferred to fresh PDA media to obtain the pure cultures.

2.2. Morphological Study and Conservation

Isolates were grown on a PDA for one week, and cultural characteristics such as size, shape, color, and texture were recorded. These characteristics were further examined using a stereomicroscope (SMZ168-BL, Motic, Shanghai, China). Micro-morphological characteristics were described based on cultures that sporulated on either water agar (WA) or PDA [11,16,19,23,41,42]. These were photographed using an ECLIPSE Ni-U compound microscope (Nikon, Tokyo, Japan) equipped with an EOS 90D digital camera (Canon, Tokyo, Japan). Measurements of conidiophores, conidiogenous cells, conidia, and mycelia were conducted using the Tarosoft (R) Image Frame Work (version 0.9.7). Figures and the photoplates were processed with Adobe Illustrator CS6 v. 24.0.1 (Adobe Systems, San Jose, CA, USA). Dried materials were deposited in the Herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China, and the Herbarium of Guizhou Academy of Agricultural Sciences (GZAAS), Guiyang, China. Living cultures were deposited at the Kunming Institute of Botany, the Chinese Academy of Sciences (KUNCC), and the Guizhou Culture Collection (GZCC). Faces of Fungi and Index Fungorum numbers were registered in accordance with the guidelines presented in Jayasiri et al. [43] and Index Fungorum (http://www.indexfungorum.org/Names/Names.asp; accessed on 15 September 2023).

2.3. DNA Extraction, PCR Amplification and Sequencing

Genomic DNA was extracted from fresh fungal mycelia using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux®, Shanghai, China) according to the manufacturer’s instructions. Four primer pairs, namely LR0R and LR5 [44], ITS5 and ITS4 [45], EF2 and EF1-728f [46,47], and T1 and Bt2b [48,49], were employed to amplify the large subunit of the ribosomal DNA (LSU), the internal transcribed spacer (ITS), the elongation factor 1-alpha (TEF1-α), and the β-tubulin (TUB2) gene regions, respectively. The polymerase chain reaction (PCR) was carried out in a 50 μL reaction volume containing 2 μL of DNA template, 2 μL of each forward and reverse primer (10 μM), 25 μL of 2× Taq PCR Master Mix with blue dye (Sangon Biotech, China), and 19 μL of distilled–deionized water. The amplification conditions for LSU, ITS, TEF-1α, and TUB2 were based on the protocol described by Feng et al. [23]. Successful PCR products were sent to Sangon Biotech (Shanghai, China) for purification and sequencing. The sequences generated in this study have been deposited in NCBI GenBank (Table 1).

2.4. Alignments and Phylogenetic Analyses

The quality of the original sequences was checked using BioEdit v. 7.1.3.0 [50] and assembled with SeqMan v. 7.0.0 (DNASTAR, Madison, WI, USA). Consensus sequences underwent BLASTn analysis in the NCBI GenBank database for preliminary identification of similar sequences. Taxa (Table 2), including type and additional strains of Apiospora species and related genera (Nigrospora and Arthrinium) in Apiosporaceae, were selected for the phylogenetic analyses based on data obtained from Genbank and previous studies [8,11,12,14,19,20,23,41,51]. Sequence alignment was performed using MAFFT v.7.0 (https://mafft.cbrc.jp/alignment/server/; accessed on 20 August 2023) [52] and subsequently manually verified in BioEdit 7.1.3.0 [50]. The phylogenetic relationships, based on a combined LSU–ITS–TEF-1α–TUB2 dataset, were analyzed using both maximum likelihood (ML) and Bayesian inference (BI) criteria.
Maximum likelihood (ML) analysis was conducted on the CIPRES web portal (https://www.phylo.org/portal2/home.action; accessed on 20 August 2023) using the RAxML-HPC Blackbox (8.2.10) tool with rapid bootstrap analysis and 1000 bootstrap replicates [53,54]. The final tree was selected from the suboptimal trees of each run by comparing likelihood scores under the GTRGAMMA substitution model.
Posterior probabilities (PP) [55] were calculated using the Bayesian Markov Chain Monte Carlo (BMCMC) sampling method in MrBayes 3.2.7a via CIPRES [53]. The appropriate substitution model best fitting the DNA evolution model for the combined dataset was determined using MrModeltest v.2.3 [56]. For the LSU, ITS, and TUB2 datasets, GTR+I+G was selected, whereas HKY+I+G was selected for TEF1-α. Four simultaneous Markov chains run for 1 million generations, with trees sampled every 100 generations, yielding 10,000 trees. The first 2000 trees, representing the burn-in phase, were discarded, and the remaining 8000 trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree [57].
Phylogenetic trees were visualized using FigTree v. 1.4.4 [58] and adjusted using Adobe Illustrator CS6 (Adobe Systems, San Jose, CA, USA).

3. Results

3.1. Phylogenetic Analysis

Seven endophytic taxa with asexual morphs, isolated from three Dicranopteris species, were identified as A. aseptata, A. dematiacea, A. dicranopteridis, and A. globosa spp. nov. within the genus Apiospora (Apiosporaceae, Amphisphaeriales) (Table 1). The combined LSU (840 bp), ITS (623 bp), TEF1-α (534 bp), and TUB2 (207 bp) sequence alignment comprised 135 taxa, with Seiridium phylicae (CPC 19962 and CPC 19965) serving as the outgroup taxa.
The dataset contained 2204 characters after alignment. The matrix presented 1181 distinct alignment patterns, with 25.29% being completely undetermined characters or gaps. Base frequencies and rates were A = 0.235259, C = 0.247975, G = 0.262058, and T = 0.254709; substitution rates were AC = 1.153887, AG = 2.614757, AT = 1.016680, CG = 0.924945, CT = 4.569867, and GT = 1.000000, with a tree length of 4.582782. The distribution shape parameter α equaled 0.227323. The tree topologies generated from both RAxML and Bayesian analyses were similar, showing no significant conflicts. The best-scoring RAxML tree is shown in Figure 1, with a final likelihood value of −25,698.717456. This phylogenetic tree revealed that the new species Apiospora aseptata (KUNCC 23-14169) clusters with two unidentified Apiospora taxa (SAUCC 1429 and SAUCC 1430), albeit with weak support. Apiospora dematiacea is closely related to the species, which includes four taxa of Apiospora hydei (LC7105, LC7103, CBS 114990, and SICAUCC 22-0032). Four isolates representing the new species, Apiospor dicranopteris (GZCC 23-0708, GZCC 23-0712, KUNCC 23-14177, and KUNCC 23-14171), form a distinct clade, which is basal to A. koreana (KUC21332) and A. qinlingensis (CFCC 52303). Lastly, Apiospor globosa forms its own clade, being a sister to A. neosubglobosa (KUMCC 16-0203 and JHB 006).

3.2. Taxonomy

Apiospora aseptata, J.Y. Zhang and Y.Z. Lu, sp. nov. (Figure 2)
Index Fungorum number: IF901115; Facesoffungi number: FoF14873.
Etymology: referring to the aseptate conidia.
Culture characteristics: Colonies on PDA are medium circular, spread, flat with an entire edge, with thin aerial hyphae, reaching ca. 50 mm diam after 10 d at 25 °C, grey-brown from above, yellow–brown in reverse. Mycelium consists of septate, branched, hyaline to brown hyphae.
Description: Endophytic in the healthy roots of Dicranopteris pedata. Sexual morph: Undetermined. Asexual morph: Conidiophores are cylindrical, septate, branched, rough-walled, flexuous, and often reduced to conidiogenous cells. Conidiogenous cells ca. 3.5 µm wide, aggregated in clusters on hyphae, solitary, mono-polyblastic, cylindrical to subglobose, hyaline to brown. Conidia amerospores, aseptate, globose or sub globose, 7–9.5 (–13) µm diam. ( x ¯ = 8 µm, n = 40) in surface view, subglobose; 6–8.5 × 5–7 µm ( x ¯ = 7 × 6 µm, n = 20) from side view, lenticular with a pale longitudinal germ slit, smooth to finely roughened, occasionally micro guttules, pale brown to brown.
Material examined: China, Guizhou Province, Qianxinan Buyi, and Miao Autonomous Prefecture, Chengheng County, isolated from the healthy leaf of Dicranopteris pedata near the roadside, 16 March 2022, J.Y. Zhang, 138-3 (HKAS 129875, holotype); ex-type living cultures, KUNCC 23-14169.
GenBank accession numbers: (LSU) OR590335, (ITS) OR590341, (TEF1-α) OR634949, and (TUB) OR634943.
Notes: Apiospora aseptata aligns well with the characteristics of the genus Apiospora and is most similar to A. pseudoparenchymaticum in the shape of its conidiogenous cells and conidia. However, they differ in conidial size [11]. Apiospora aseptata has noticeably smaller conidia than A. pseudoparenchymaticum (7–9.5 (–13) µm diam. vs. 13.5–27 × 12–23.5 µm). In phylogenetic analysis, Apiospora aseptata clusters with Apiospora sp. strains (SAUCC 1429 and SAUCC 1430) and forms a sister relationship with the clade that includes A. arctoscopi (KUC21331), A. jiangxiensis (LC 4577), and A. obovata (LC 4940). Unfortunately, we could not compare the morphological characteristics of the Apiospora sp. strains (SAUCC 1429 and SAUCC 1430), as the morphology of these strains has not been reported. Based on both phylogeny and morphology, we introduce Apiospora aseptate as a new species.
Apiospora dematiacea J. Y. Zhang and Y. Z. Lu, sp. Nov. (Figure 3)
Index Fungorum number: IF901116; Facesoffungi number: FoF14874.
Holotype: HKAS 129910
Etymology: referring to its dematiaceous spore.
Culture characteristics: Colonies on PDA medium circular, cottony, edge entire, flat, spreading, with abundant aerial mycelia, zonate with one concentric circle, reaching 47 mm diam after 10 d at 25 °C, white from above, yellowish white to grey to light yellow from center to edge in reverse. Vegetative hypha septate, branched, hyaline to light brown.
Description: Endophytic in the stems of Dicranopteris ampla. Sexual morph: Undetermined. Asexual morph: Conidiophores reduced to conidiogenous cells, cylindrical, septate, hyaline. Conidiogenous cells are cylindrical to subglobose, aggregated in clusters on hyphae, smooth, hyphae-like, and hyaline to brown. Conidia aseptate, globose to ellipsoid in surface view, 14.5–18(–20) µm diam. ( x ¯ = 16.5 µm, n = 30), lenticular to lageniform from side view, 18.5–23(–25) × 10–13 µm diam. ( x ¯ = 21.5 × 11.5 µm, n = 30), with longitudinal, pale germ slit. Sterile cells up to 32 µm long, 9–12(–16) µm ( x ¯ = 10.5 µm, n = 20) wide, elongated, mixed among conidia brown, rarely truncate, and have a darkened scar at the base.
Material examined: CHINA, Guizhou Province, Qianxinan Buyi and Miao Autonomous Prefecture, Ceheng County (24°59′44″ N 105°50′16″ E), isolated from the healthy stem of Dicranopteris ampla near the roadside, 16 March 2022, J.Y. Zhang, 307-1 (HKAS 129910, Holotype), ex-living cultures, KUNCC 23-14202.
GenBank accession numbers: (LSU) OR590339, (ITS) OR590346, (TEF1-α) OR634953, and (TUB) OR634948.
Note: Apiospora dematiacea morphologically resembles A. hydei, characterized by conidiogenous cells aggregated in clusters on hyphae and globose conidia in surface view, with a pale equatorial slit when viewed from the side [8]. However, A. dematiacea is distinguishable from A. hydei due to its hyphae-like conidiogenous cells and more varied conidial shapes that include sterile cells. From a phylogenetic perspective, while Apiospora dematiacea shares a sister relationship with A. hydei, it constitutes a distinct lineage. A comparison of nucleotide base pairs between the ex-type strain of A. hydei (CBS 114990) and our newly isolated strain of Apiospora dematiacea (KUNCC 23-14202) reveals differences of 1183/1185 bp (99%), 578/579 (99%, including 1 gap), 411/432 bp (95%, including 12 bp gaps), and 778/794 bp (98%, including 2 bp gaps) in the LSU, ITS, TEF1-α, and TUB2 sequences, respectively. This confirms that they are distinct species.
Apiospora dicranopteridis J.Y. Zhang and Y.Z. Lu, sp. nov. (Figure 4)
Index Fungorum number: IF901117; Facesoffungi number: FoF14875
Etymology: referring to the fungal host genus, Dicranopteris.
Holotype: HKAS 129877
Culture characteristics: Colonies on PDA medium circular, edge entire, floccose at the surface with dense, white aerial mycelia, growly fast, reaching 55 mm diam after 10 d at 25 °C, cottony, velvety, loose, white from above, yellow to yellowish whites in reverse. Vegetative hypha: septate, branched, sometimes coiled, guttulate, hyaline to pale brown.
Description: Endophytic in the stems of Dicranopteris pedata. Sexual morph: Undetermined. Asexual morph: Conidiophores are cylindrical, septate, branched, smooth-walled, and often reduced to conidiogenous cells. Conidiogenous cells 6–15 × 3.5–10 µm ( x ¯ = 10 × 6 µm, n = 25) µm, solitary to aggregated in clusters arising from dense aerial hyphae, mono- to polybasic, sympodial, sub-globose to doliiform to cylindrical, smooth, subhyaline. Conidia amerospores, aseptate, globose or sub globose, 10.5–13 µm diam. ( x ¯ = 11.5 µm, n = 15), cylindrical to broadly clavate 14–17(–22) × (6–)8–10.5 µm ( x ¯ = 16 × 9 µm, n = 8), with rounded at the apex and a slightly narrower and truncate base, smooth to finely roughened guttules, without an equatorial germ slit, hyaline to pale brown.
Material examined: China, Guizhou Province, Qianxinan Buyi and Miao Autonomous Prefecture, Ceheng County (24°59′44″ N 105°50′16″ E), isolated from the healthy stems of Dicranopteris pedata nearby the roadside, 16 March 2022, J.Y. Zhang, 139-2 (HKAS 129877, holotype), ex-type living cultures, KUNCC23-14171; Ibid., isolated from the root of D. pedate, 16 March 2022, J.Y. Zhang, 170-4 (GZAAS 23-0780, paratype), living cultures, KUNCC 23-14177; Ibid, Anlong County, Jia Jia Ya Kou (24°59′23″ N; 105°35′20″ E), isolated from the healthy leaf of D. pedate, 16 March 2022, J.Y. Zhang, 223-4 (HKAS 129895, paratype), living cultures, GZCC 23-0712; Ibid, isolated from the healthy rhizome of D. ampla, 16 March 2022, J.Y. Zhang, 225-1 (HKAS 129898, paratype), living cultures, GZCC 23-0708.
GenBank accession numbers: KUNCC23-14171: (LSU) OR590336, (ITS) OR590342, (TEF1-a) OR634950, (TUB2) OR634944; KUNCC 23-14177: (LSU) OR590337, (ITS) OR590343, (TEF1-a) OR634951, (TUB2) OR634945; GZCC 23-0712: (LSU) OR590338, (ITS) OR590345, (TEF1-a) OR634952, (TUB2) OR634947; GZCC 23-0708: (ITS) OR590344, (TUB2) OR634946.
Notes: Apiospora dicranopteridis is morphologically distinct from other Apiospora species by its mono- or polyblastic, elongated cylindrical conidiogenous cells and globose to cylindrical to broadly clavate, hyaline to pale brown conidia. Phylogenetically, four strains (GZCC 23-0708, GZCC 23-0712, KUNCC23-14177, and KUNCC23-14171) representing Apiospora dicranopteridis sp. nov. formed a distinct clade. They share a sister relationship with A. koreana (KUC21332) and A. qinlingensis (CFCC 52303), reinforcing the notion that they are separate species.
Apiospora globosa J.Y. Zhang and Y.Z. Lu, sp. nov. (Figure 5)
Index Fungorum number: IF901402; Facesoffungi number: FoF14658.
Etymology: referring to the globose to subglobose conidia
Holotype: HKAS 129921
Culture characteristics: Colonies on WA medium irregulate, with several dark spots, flat with undulate edge, reaching 33 mm diam after 15 d at 25 °C, hyaline to light brown. Vegetative hyphae are thin, sparse, septate, branched, guttulate, and hyaline, some curled in a ring structure.
Description: Endophytic in the stems of Dicranopteris linearis. Sexual morph: Undetermined. Asexual morph: Conidiophores undistinguishable, hyphae-like. Conidiogenous cells are undistinguishable and hyphae-like. Conidia produced directly from vegetative hypha inside the WA culture, 4.5–8.5 µm diam ( x ¯ = 6 µm, n = 20), aseptate, globose to subglobose, smooth to finely roughened, light yellow to gold to black.
Material examined: China, Guizhou Province, Anshun City, Ziyun Miao Buyi Autonomous County, Getu River Scenic Spot (25°48′26″ N 106°4′24″ E), isolated from the healthy stem of Dicranopteris linearis in a disturbed forest, 2 August 2022, J.Y. Zhang, S4-1 (dry WA culture, HKAS 129921, holotype; dry culture of WA-carrot mixture, GZAAS 23-0790), ex-type living cultures, KUNCC 23-14210.
GenBank accession numbers: (LSU) OR590340, (ITS) OR590347, and (TEF1-α) OR634954.
Notes: Phylogenetically, Apiospora globosa forms a distinct clade that is sister to the species Apiospora neosubglobosa (KUMCC 16-0203 and JHB 006). While Apiospora neosubglobosa has been described with only a sexual morph [9,22], our new species produces an asexual morph in culture. Morphologically, Apiospora globosa resembles A. xenocordella in conidial shape but has notably different conidiogenous cells and conidial size (4.5–8.5 µm diam. vs. 9–10 µm diam.). Apiospora globosa possesses indistinct, hyphae-like conidiogenous cells, whereas A. xenocordella features globose to clavate to doliiform conidiogenous cells [8].

4. Discussion

The genus Apiospora is relatively well studied, with species distributed across tropical, subtropical, temperate, and cold climates globally [8,12,14,41,51]. Members of the Apiospora species can function as endophytes [16,19,59,60], pathogens [8,19,61], or saprobes [5,10,13,23], found on various hosts, including various plants, air, water, soil debris, home dust, food, and the gut of insects [8,11,12,14,59,61]. They do not exhibit a clear lifestyle preference or pronounced sensitivity to environmental change. A fungus-host distribution of Arthrinium species (most of which have been synonymized under Apiospora) was provided by Wang et al. [11]. The data showed that Poaceae and Cyperaceae are the dominant host plant families, especially the former [10,12,14,17,20,22,42,62,63]. There have been no previous reports of Apiospora species from ferns, likely due to the neglect of fungi on ferns [64,65]. In this study, four new species (Apiospora aseptata, A. dematiacea, A. dicranopteridis, and A. globosa) were reported as endophytes isolated from three medicinal ferns—Dicranopteris ampla, D. linearis, and D. pedate—based on evidence from morphology and phylogenetic analyses of a concatenated dataset of LSU, ITS, TEF1-α, and TUB2 sequences. This study represents the first report of Apiospora species from Dicranopteris species, expanding the host diversity knowledge of Apiospora species.
The Apiospora-Arthrinium group has made certain achievements in bioactive secondary metabolites, with a high interest in agriculture, food, and the pharmaceutical industry [66,67,68,69]. The evaluation of the biological activities of Apiospora-Arthrinium spp. revealed this group has relatively biological activities for antifungal, antioxidant, and cellulolytic activity, especially Apiospora saccharicola [66]. A quick guide to secondary metabolites from the Apiospora–Arthrinium group was provided by Overgaard et al. [69], including the knowledge of 269 secondary metabolites and emphasizing some of the known biological or toxic compounds. For example, several species, including Apiospora arundinis, A. aurea, A. phaeosperma, A. sacchari, A. saccharicola, A. serenensis, A. terminalis, and Apiospora/Arthrinium spp., can produce 3-nitropropionic acid, which is associated with the food safety problem of poisonings or even deaths [70,71,72]. The study results of the genome sequence provided by Sørensen et al. [21] revealed that the Apiospora-Arthrinium group holds a high number of secondary metabolite gene clusters, which has attracted more attention to the compounds of this group [21,69]. In the current research context, we newly obtained seven endophytic taxa from the medicinal ferns of Dicranopteris rich in bioactive compounds and identified them as four new species (A. aseptata, A. dematiacea, A. dicranopteridis, and A. globosa) in the genus Apiospora, a fungal group that is currently attracting attention. These isolates are valuable fungi that are expected to explore secondary metabolites, which will also be the future research direction of our research.
All strains were initially cultured on a PDA medium. Most Apiospora strains sporulate on PDA substrate naturally under conventional conditions (room temperature 25–28°, natural light), which is consistent with many studies [8,11,16,19,23,41]. However, Apiospora globosa (KUNCC 23-14210) failed to sporulate on the PDA medium. Sporulation was later induced on various media, including CMM (Corn Meal Medium), MEA (malt extract agar), OA (oatmeal agar), SNA (synthrtic nutrient-poor agar), and WA (water agar). Eventually, sporulation of Apiospora globosa (KUNCC 23-14210) was successfully induced on WA and a WA-carrot mixture. During investigations of endophytic fungal diversity isolated from Dicranopteris in Guizhou, China, hundreds of isolates were selected from nearly a thousand endophytic strains for sporulation induction across multiple media (CMM, MEA, OA, PDA, SNA, and WA). Results showed that the sporulation rate on the WA medium exceeded that of other media. Therefore, WA is prioritized for inducing sporulation in plant endophytic fungi when the sporulation mechanism for this fungal group is not documented in previous publications.

Author Contributions

Conceptualization, J.-Y.Z.; data curation, J.-Y.Z. and M.-L.C.; formal analysis, J.-Y.Z.; investigation, J.-Y.Z., M.-L.C. and Y.-X.W.; methodology, J.-Y.Z.; project administration, Y.-Z.L.; software, J.-Y.Z.; supervision, S.B. and Y.-Z.L.; writing—original draft preparation, J.-Y.Z.; writing—review and editing, S.B. and Y.-Z.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (NSFC 32060013), the Youth Science and Technology Talent Development Project from the Guizhou Provincial Department of Education (QJHKYZ [2021]263) and Youth Science and Technology Talent Development Project from Guizhou Provincial Department of Education (QJHKYZ [2022]345).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All sequences generated in this study were submitted to GenBank.

Acknowledgments

Jing-Yi Zhang would like to thank Shaun Pennycook (Manaaki Whenua Landcare Research, New Zealand) for advising on the fungal names and Ning-Guo Liu and Chuan-Gen Lin for the guidance. Jing-Yi Zhang would also like to thank Mae Fah Luang University for granting me a tuition scholarship for my Ph.D. studies.

Conflicts of Interest

The authors declare no conflict of interest.

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Jof 09 01096 g001aJof 09 01096 g001b
Figure 1. The phylogenetic tree generated from ML analysis is based on a concatenated LSU–ITS–TEF1α–TUB2 dataset for the family Apiosporaceae. Bootstrap support values for ML greater than 60% and Bayesian posterior probabilities (PPs) greater than 0.95 were indicated above or below the nodes as ML/PP. Seiridium phylicae (CPC 19962 and CPC 19965) were selected as the outgroup taxa. The newly generated sequences are shown in red.
Figure 1. The phylogenetic tree generated from ML analysis is based on a concatenated LSU–ITS–TEF1α–TUB2 dataset for the family Apiosporaceae. Bootstrap support values for ML greater than 60% and Bayesian posterior probabilities (PPs) greater than 0.95 were indicated above or below the nodes as ML/PP. Seiridium phylicae (CPC 19962 and CPC 19965) were selected as the outgroup taxa. The newly generated sequences are shown in red.
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Figure 2. Apiospora aseptata (HKAS 129875, holotype). (a,b) Cultures on PDA from above and below; (c) colonies on PDA; (d–g) conidiogenous cells with conidia; (h–j) conidia. Scale bars: (d) 20 μm; (e–j) 10 μm.
Figure 2. Apiospora aseptata (HKAS 129875, holotype). (a,b) Cultures on PDA from above and below; (c) colonies on PDA; (d–g) conidiogenous cells with conidia; (h–j) conidia. Scale bars: (d) 20 μm; (e–j) 10 μm.
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Figure 3. Apiospora dematiacea (HKAS 129910, Holotype). (a,b) Cultures on PDA from above and below; (c) colonies on PDA; (d–i) conidiogenous cells with conidia; (j–p) conidia. Scale bars: (d) 50 μm; (e–l) 20 μm; (m–p) 10 μm.
Figure 3. Apiospora dematiacea (HKAS 129910, Holotype). (a,b) Cultures on PDA from above and below; (c) colonies on PDA; (d–i) conidiogenous cells with conidia; (j–p) conidia. Scale bars: (d) 50 μm; (e–l) 20 μm; (m–p) 10 μm.
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Figure 4. Apiospora dicranopteridis (HKAS 129877, holotype). (a,b) Cultures on PDA from above and below; (c,d) colonies on PDA; (d–l) conidiophores, conidiogenous cells with conidia. Scale bars: (e,f) 50 μm; (g–l) 20 μm.
Figure 4. Apiospora dicranopteridis (HKAS 129877, holotype). (a,b) Cultures on PDA from above and below; (c,d) colonies on PDA; (d–l) conidiophores, conidiogenous cells with conidia. Scale bars: (e,f) 50 μm; (g–l) 20 μm.
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Figure 5. Apiospora globosa (HKAS 129921, holotype). (a,b) Cultures on WA from above and below; (c) colonies on WA; (d–f) conidiophores with conidia; (g–i) conidia. Scale bars: (d–f) 20 μm; (g–i) 10 μm.
Figure 5. Apiospora globosa (HKAS 129921, holotype). (a,b) Cultures on WA from above and below; (c) colonies on WA; (d–f) conidiophores with conidia; (g–i) conidia. Scale bars: (d–f) 20 μm; (g–i) 10 μm.
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Table 1. Apiospora endophytic isolates were used in this study.
Table 1. Apiospora endophytic isolates were used in this study.
Taxon Strain CodeSpecimenStatusHostTissuesSubstrate (Sproluration)
A. aseptataKUNCC 23-14169HKAS 129875HD. pedataLeafPDA
A. dematiaceaKUNCC 23-14202HKAS 129910HD. amplaStemPDA
A. dicranopterisGZCC 23-0708HKAS 129898PD. amplaRhizomePDA
A. dicranopterisGZCC 23-0712HKAS 129895PD. pedataLeafPDA
A. dicranopterisKUNCC23-14177GZAAS 23-0780PD. pedataRootPDA, WA
A. dicranopterisKUNCC23-14171HKAS 129877HD. pedataStemPDA
A. globosaKUNCC 23-14210HKAS 129921HD. linearisStemWA
Note: status: H denotes holotype; P denotes paratype.
Table 2. Taxa used in this study and their GenBank accession numbers.
Table 2. Taxa used in this study and their GenBank accession numbers.
Taxa NamesStrainsGenBank Accessions
LSUITSTEF1-αTUB2
Apiospora acutiapicumKUMCC 20-0210 TMT946339MT946343MT947360MT947366
Apiospora agariKUC21333 TMH498440MH498520MH544663MH498478
Apiospora aquaticumS 642 TMK835806MK828608N/AN/A
Apiospora arctoscopiKUC21331 TMH498449MH498529MN868918MH498487
Apiospora arundinisCBS 449 92KF144931KF144887KF145019KF144977
Apiospora arundinisGZCC 20-0116 TMW478899MW481720MW522952MW522968
Apiospora aureaCBS 244.83 TKF144935AB220251KF145023KF144981
Apiospora balearicaCBS 145129 TMK014836MK014869N/AMK017975
Apiospora bambusaeALV17304MK014841MK014874MK017951MK017980
Apiospora bambusicolaMFLUCC 20-0144 TMW173087MW173030MW183262N/A
Apiospora biserialisCGMCC 3.20135 TMW478885MW481708MW522938MW522955
Apiospora camelliae sinensisLC 5007 TKY494780KY494704KY705103KY705173
Apiospora chiangraienseMFLUCC 21-0053 TMZ542524MZ542520N/AMZ546409
Apiospora chromolaenaeMFLUCC 17-1505 TMT214436MT214342N/AN/A
Apiospora cordylinaeGUCC 10027 TN/AMT040106MT040127MT040148
Apiospora cyclobalanopsidisCGMCC 3.20136 TMW478892MW481713MW522945MW522962
Apiospora descalsiiCBS 145130 TMK014837MK014870 MK017976
Apiospora dichotomanthiLC 4950 TKY494773KY494697KY705096KY705167
Apiospora dongyingensisSAUCC 0302 TOP572424OP563375OP573264OP573270
Apiospora esporlensisCBS 145136 TMK014845MK014878 MK017983
Apiospora euphorbiaeIMI 285638bAB220335AB220241NAAB220288
Apiospora fermentiKUC21289 TMF615213MF615226MH544667MF615231
Apiospora gaoyouensisCFCC 52301 TN/AMH197124MH236793MH236789
Apiospora garethjonesiiKUMCC 16-0202 TKY356091KY356086N/AN/A
Apiospora gelatinosaKHAS 11962 TMW478888MW481706MW522941MW522958
Apiospora guiyangensisHKAS 102403 TMW240577MW240647N/AMW775604
Apiospora guizhouensisLC 5322 TKY494785KY494709KY705108KY705178
Apiospora hainanensisSAUCC 1681 TOP572422OP563373OP573262OP573268
Apiospora hispanicaIMI 326877 TAB220336AB220242N/AAB220289
Apiospora hydeiCBS 114990 TKF144936KF144890KF145024KF144982
Apiospora hydeiLC7103KY494791KY494715KY705114KY705183
Apiospora hydeiLC7105KY494793KY494717KY705116KY705185
Apiospora hydeiSICAUCC 22-0032ON185553ON183998ON221312ON221313
Apiospora hyphopodiiMFLUCC 15-0003 TN/AKR069110N/AN/A
Apiospora hyphopodiiKUMCC 16-0201KY356093KY356088N/AN/A
Apiospora hysterinaICPM 6889 TMK014841MK014874MK017951MK017980
Apiospora ibericaCBS 145137 TMK014846MK014879N/AMK017984
Apiospora intestiniCBS 135835 TKR149063KR011352KR011351KR011350
Apiospora italicaCBS 145138 TMK014847MK014880MK017956MK017985
Apiospora jatrophaeAMH 9557 TN/AJQ246355N/AN/A
Apiospora jiangxiensisLC 4577 TKY494769KY494693KY705092KY705163
Apiospora kogelbergensisCBS 113333 KKF144938KF144892KF145026KF144984
Apiospora koreanaKUC21332 TMH498444MH498524MH544664MH498482
Apiospora locuta pollinisLC 11683 TN/AMF939595MF939616MF939622
Apiospora longistromaMFLUCC 11-0481 TKU863129KU940141N/AN/A
Apiospora malaysianaCBS 102053TKF144942KF144896KF145030KF144988
Apiospora marianiaeAP18219 TON692422ON692406N/AON677186
Apiospora mariiCBS 497.90 TKF144947AB220252KF145035KF144993
Apiospora marinaKUC21328 TMH498458MH498538MH544669MH498496
Apiospora mediterraneaIMI 326875AB220337AB220243N/AAB220290
Apiospora minutispora17E-042 TN/ALC517882LC518889LC518888
Apiospora montagneiAP301120 TON692424ON692408ON677182ON677188
Apiospora moriMFLU 18-2514 TMW114393MW114313N/AN/A
Apiospora multiloculataMFLUCC 21-0023 TOL873138OL873137N/AOL874718
Apiospora mytilomorphaDAOM 214595 TN/AKY494685N/AN/A
Apiospora neobambusaeLC 7106 TKY494794KY494718KY806204KY705186
Apiospora neochinenseCFCC 53036 TN/AMK819291MK818545MK818547
Apiospora neogarethjonesiiHKAS 102408 TMK070898MK070897N/AN/A
Apiospora neosubglobosaJHB 006KY356094KY356089N/AN/A
Apiospora neosubglobosaKUMCC 16-0203 TKY356095KY356090N/AN/A
Apiospora obovataLC 4940 TKY494772KY494696KY705095KY705166
Apiospora ovataCBS 115042 TKF144950KF144903KF145037KF144995
Apiospora paraphaeospermaMFLUCC 13-0644 TKX822124KX822128N/AN/A
Apiospora phragmitisCPC 18900KF144956KF144909KF145043KF145001
Apiospora phyllostachydisMFLUCC 18-1101 TMH368077MK351842MK340918MK291949
Apiospora piptatheriCBS 145149 TMK014860MK014893N/AN/A
Apiospora pseudomariiGUCC 10228 TN/AMT040124MT040145MT040166
Apiospora pseudoparenchymaticaLC7234 TKY494819KY494743KY705139KY705211
Apiospora pseudorasikravindraeKUMCC 20-0208 TN/AMT946344MT947361MT947367
Apiospora pseudosinensisCPC 21546 TKF144957KF144910KF145044N/A
Apiospora pseudospegazziniiCBS 102052 TKF144958KF144911KF145045KF145002
Apiospora pterospermaCPC 20193 TKF144960KF144913KF145046KF145004
Apiospora pusillispermaKUC21321 TMH498453MH498533MN868930MH498491
Apiospora qinlingensisCFCC 52303 TN/AMH197120MH236795MH236791
Apiospora rasikravindraeNFCCI 2144N/AJF326454N/AN/A
Apiospora rasikravindraeLC5449KY494789KY494713KY705112KY705182
Apiospora sacchariCBS 372.67KF144964KF144918KF145049KF145007
Apiospora saccharicolaCBS 831.71KF144969KF144922KF145054KF145012
Apiospora sargassiKUC21228 TKT207696KT207746MH544677KT207644
Apiospora sasaeCBS 146808 TMW883797MW883402N/AMW890120
Apiospora septataCGMCC 3.20134 TMW478890MW481711MW522943MW522960
Apiospora serenensisIMI 326869 TAB220344AB220250N/AAB220297
Apiospora setariaeMT492005N/AMT492005MW118457MT497467
Apiospora setostromaKUMCC 19-0217MN528011MN528012MN527357N/A
Apiospora sichuanensisHKAS 107008 TMW240578MW240648N/AMW775605
Apiospora sorghiURM 93000 TN/AMK371706N/AMK348526
Apiospora sp.SAUCC 1429OQ615287OQ592558N/AN/A
Apiospora sp.SAUCC 1430OQ615286OQ592557N/AN/A
Apiospora sphaerospermaCBS114314KF144951KF144904KF145038KF144996
Apiospora stipaeCBS 146804 TMW883798MW883403MW890082MW890121
Apiospora subglobosaMFLUCC 11-0397 TKR069113KR069112N/AN/A
Apiospora subroseaLC 7292 TKY494828KY494752KY705148KY705220
Apiospora taeanensisKUC21322TN/AMH498515MH544662MH498473
Apiospora thailandicaMFLUCC 15-0202 TKU863133KU940145N/AN/A
Apiospora tropicaMFLUCC 21 0056 TOK491653OK491657N/AN/A
Apiospora vietnamensisIMI 99670 TKX986111KX986096N/AKY019466
Apiospora xenocordellaCBS 478 86 TKF144970KF144925KF145055KF145013
Apiospora yunnanaMFLUCC 15 1002 TKU863135KU940147N/AN/A
Arthrinium austriacumGZU 345006MW208860MW208929N/AN/A
Arthrinium caricicolaCBS 145127MK014838MK014871N/AMK017977
Arthrinium cf. sporophleoidesGZU 345102MW208866MW208944N/AN/A
Arthrinium crenatumAG 19066 TMW208861MW208931N/AN/A
Arthrinium japonicumIFO 31098AB220358AB220264N/AAB220311
Arthrinium luzulaeAP7619 3 TMW208863MW208937N/AN/A
Arthrinium curvatum var. minusCBS 145131MK014839MK014872N/AMK017978
Arthrinium morthieriGZU 345043MW208864MW208938MW221920MW221926
Arthrinium puccinioidesCBS 145150MK014861MK014894N/AMK017998
Arthrinium sphaerospermumAP25619MW208865MW208943N/AN/A
Arthrinium sporophleumCBS 145154MK014865MK014898N/AMK018001
Nigrospora aurantiacaCGMCC 3.18130 TKX986098KX986064KY019295KY019465
Nigrospora bambusaeCGMCC 3.18327 TNG 069455KY385307KY385313KY385319
Nigrospora camelliae sinensisCGMCC 3.18125 TKX986103KX985986KY019293KY019460
Nigrospora chinensisCGMCC 3.18127 TKX986107KX986023KY019422KY019462
Nigrospora gorlenkoanaCBS 480.73 TKX986109KX986048KY019420KY019456
Nigrospora guilinensisCGMCC 3.18124 TKX986113KX985983KY019292KY019459
Nigrospora hainanensisCGMCC 3.18129 TKX986112KX986091KY019415KY019464
Nigrospora lacticoloniaCGMCC 3.18123 TKX986105KX985978KY019291KY019458
Nigrospora musaeCBS 319.34 TKX986110MH855545KY019419KY019455
Nigrospora oryzaeLC2693 KX986101KX985944KY019299KY019471
Nigrospora osmanthiCGMCC 3.18126 TKX986106KX986010KY019421KY019461
Nigrospora pyriformisCGMCC 3.18122 TKX986100KX985940KY019290KY019457
Nigrospora rubiLC2698 T KX986102KX985948KY019302KY019475
Nigrospora saccharicolaCGMCC 3.19362 TN/AMN215788MN264027MN329951
Nigrospora sphaericaLC7298KX986097KX985937KY019401KY019606
Nigrospora vesicularisCGMCC 3.18128 TKX986099KX986088KY019294KY019463
Nigrospora zimmermaniiCBS 290.62 TKY806276KY385309KY385311KY385317
Seiridium phylicaeCPC 19962 TNG 042759LT853092LT853189LT853239
Seiridium phylicaeCPC 19965KC005809LT853093LT853190LT853240
Note: T denotes type strains; “N/A” indicates no data are available in GenBank.
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MDPI and ACS Style

Zhang, J.-Y.; Chen, M.-L.; Boonmee, S.; Wang, Y.-X.; Lu, Y.-Z. Four New Endophytic Apiospora Species Isolated from Three Dicranopteris Species in Guizhou, China. J. Fungi 2023, 9, 1096. https://doi.org/10.3390/jof9111096

AMA Style

Zhang J-Y, Chen M-L, Boonmee S, Wang Y-X, Lu Y-Z. Four New Endophytic Apiospora Species Isolated from Three Dicranopteris Species in Guizhou, China. Journal of Fungi. 2023; 9(11):1096. https://doi.org/10.3390/jof9111096

Chicago/Turabian Style

Zhang, Jing-Yi, Meng-Lan Chen, Saranyaphat Boonmee, Yu-Xuan Wang, and Yong-Zhong Lu. 2023. "Four New Endophytic Apiospora Species Isolated from Three Dicranopteris Species in Guizhou, China" Journal of Fungi 9, no. 11: 1096. https://doi.org/10.3390/jof9111096

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