New and Interesting Pine-Associated Hyphomycetes from China

Abstract

Pine trees play a crucial role in the forests of Sichuan Province, boasting rich species diversity and a lengthy evolutionary history. However, research and investigation on fungi associated with pine trees are insufficient. This study investigated the diversity of hyphomycetes fungi associated with pine trees in Sichuan Province, China. During the survey, we collected five specimens of hyphomycetes from branches and bark of species of Pinus. Five barcodes were selected for study and sequenced, including ITS, SSU, LSU, TEF1, and RPB2. Morphological examination and multi-locus phylogenetic analyses revealed three new species, viz. Catenulostroma pini sp. nov. within Teratosphaeriaceae, Kirschsteiniothelia longisporum sp. nov. within Kirschsteiniotheliaceae, Sporidesmiella sichuanensis sp. nov. within Junewangiaceae, and two known species, Paradictyoarthrinium diffractum and P. hydei within Paradictyoarthriniaceae, which are the new host records from Pinus species. Catenulostroma pini, distinguished from other species in the genus by its unique morphology, has three conidial morphologies: small terminal helicoconidia, scolecoconidia with many septa, and phragmoconidia conidia. Kirschsteiniothelia longisporum has longer spores when compared to the other species in the genus. According to phylogenetic analysis, Sporidesmiella sichuanensis formed an independent clade sister to S. aquatica and S. juncicola, distinguished by differences in conidial size.

1. Introduction

Hyphomycetes are a polyphyletic group of fungi that lack fruiting bodies (conidiomata), have hyphae that may be immersed in the substrate or not, and where sporulation mainly occurs on differentiated septate hyphae [1,2]. Hyphomycetes represent the asexual forms of many fungal species and are highly diverse, with more than 2265 genera and 13,800 species reported worldwide [2,3]. Hyphomycetes are widely distributed and can be saprophytic in freshwater, marine, and terrestrial ecosystems, or they can parasitize animals and plants as pathogens [4,5,6,7]. Many of the fungi in this group are aquatic and characterized by the production of conidia that are passively discharged from the hyphae, facilitating dispersal [8,9,10]. Since most conidia are characteristically shaped, species can often be identified by morphology, a common practice in the study of dematiaceous hyphomycetes [11,12,13,14]. However, since species may have undergone convergent evolution in morphology, phylogenetic analysis should also be incorporated into classification.

Catenulostroma (Teratosphaeriaceae, Dothideomycetes) was introduced by Crous et al., with the type C. protearum, which was previously placed in the genus Trimmatostroma [15]. Trimmatostroma and Catenulostroma are morphologically similar; however, phylogenetically, they appear as distinct genera, with the type species of Trimmatostroma belonging to the order Helotiales [15]. The characteristics of Catenulostroma are hypha-like conidiophores and conidia in basipetal chains [15,16]. Species of this genus include saprobic and pathogenic fungi that are occasionally isolated from opportunistic human diseases [15].

Kirschsteiniothelia (Kirschsteiniotheliaceae, Dothideomycetes) was established by Hawksworth to accommodate the type species K. aethiops [17]. Kirschsteiniothelia is a holomorphic genus with two types of asexual morphs: dendryphiopsis-like and sporidesmium-like [18]. Combining morphological and molecular evidence, Dendryphiopsis was confirmed to be an asexual of Kirschsteiniothelia [19,20]. Subsequently, Wijayawardene et al. recommended using Kirschsteiniothelia over Dendryphiopsis [21]. Members of this genus are primarily saprophytes found on dead or decaying wood in freshwater and terrestrial habitats and have occasionally been associated with mycoses [8,18,20,22,23,24,25,26,27]. Currently, there are 49 epithets are listed in Index Fungorum (http://www.indexfungorum.org; 7 July 2024).

Paradictyoarthrinium (Paradictyoarthriniaceae, Dothideomycetes) was introduced as a monotypic genus with P. diffractum as the type species [28]. The genus is characterized by gregarious, black, powdery colonies and macronematous conidiophores with asymmetrically and unevenly dictyoseptate, muriform, subglobose to ellipsoidal conidia [29,30]. Currently, there are only five species in the Paradictyoarthrinium, viz. P. aquatica, P. diffractum, P. hydei, P. salsipaludicola and P. tectonicola, of which P. aquatica, P. diffractum and P. hydei have records from China [28,29,30,31]. Paradictyoarthrinium species are primarily saprophytes found on decaying wood in terrestrial, freshwater, and marine environments [28,29,30,31].

Kirk erected Sporidesmiella (Junewangiaceae, Sordariomycetes) with the type S. claviformis and introduced two newly described species and four new combinations [32]. Sporidesmiella is mainly characterized by clavate or obovoid to cuneate, rounded or coronate at the apex, distoseptate conidia, seceding schizolytically from monoblastic, integrated, terminal, annellidic or rarely sympodially extending conidiogenous cells [32,33,34]. Species of Sporidesmiella are saprophytes found on leaves, submerged wood, or decaying wood in freshwater and terrestrial habitats [10,33,34,35,36,37]. To date, there are 54 epithets of Sporidesmiella (http://www.indexfungorum.org; 7 July 2024).

Pine trees, the primary community-forming species of coniferous forests in the Northern Hemisphere, constitute the largest group of extant gymnosperms and have a long evolutionary history [38]. Pine trees are a crucial component of the forests in Sichuan Province. This Province has a diverse climate, complex terrain, and abundant pine species, providing a wide range of habitats conducive to the growth and diversification of fungi, resulting in vast fungal diversity. Despite numerous new taxa being reported in recent years, our understanding of them remains limited [39,40,41]. We regularly conduct fungal diversity surveys in Sichuan Province and investigate the taxonomy of fungi associated with Pinus spp. During the study, five hyphomycetous fungi were collected from Pinus spp. Through multi-locus phylogenetic analysis and morphological examination, we identified these five collections as three new species: Catenulostroma pini sp. nov., Kirschsteiniothelia longisporum sp. nov., Sporidesmiella sichuanensis sp. nov., and two new host records of Paradictyoarthrinium from Pinus spp.

2. Materials and Methods

2.1. Sample Collection, Morphological Examination and Isolation

We surveyed the fungal diversity of Hyphomycetes on Pinus spp. in Sichuan Province, China, from March to August 2023. The specimens were taken into the laboratory in paper envelopes for examination. Microscopic characters were observed and recorded using a Nikon SMZ800N stereo microscope equipped with a Nikon DS-Fi3 camera (Nikon Corporation, Tokyo, Japan) and a Nikon ECLIPSE Ni-U microscope (Nikon Corporation, Tokyo, Japan) fitted with a Nikon DS-Ri2 microscope camera (Nikon Corporation, Tokyo, Japan). Measurements were conducted using the Nikon NIS-Elements Documentation Imaging Version 5.21.00 (Nikon Corporation, Tokyo, Japan). All photographs were processed using Adobe Photoshop version 22.0 (Adobe Inc., San Jose, CA, USA). Single conidium isolation was made following the method described by Senanayake et al. [42]. Germinated conidia were individually transferred to potato dextrose agar (PDA) media plates and incubated in the dark at 25 °C. Culture characteristics were examined and recorded regularly after 1–3 weeks.

The holotype specimens were deposited in the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China, and all specimens were deposited in the Herbarium of the University of Electronic Science and Technology (HUEST), Chengdu, China. The living ex-type cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, and all living cultures were deposited in the University of Electronic Science and Technology Culture Collection (UESTCC), Chengdu, China. The taxonomic descriptions of the new taxa have been deposited in MycoBank.

2.2. DNA Extraction, PCR Amplification and Sequencing

Fungal genomic DNA was extracted from mycelia using the Trelief^TM Plant Genomic DNA Kit (TSINGKE Biotech, Shanghai, China) according to the manufacturer’s protocol. For Sporidesmiella sichuanensis specimens (HKAS 136267), obtaining a culture was not feasible, necessitating the direct extraction of DNA from fruiting structures using the method used by Wanasinghe et al. [43]. Five loci, the nuclear ribosomal internal transcribed spacer (ITS: ITS1-5.8S-ITS2), the nuclear ribosomal small subunit rRNA (SSU), the nuclear ribosomal large subunit rRNA (LSU), the partial translation elongation factor 1-alpha (TEF1), the partial second largest subunit of RNA polymerase II (RPB2), were selected for study and amplified by polymerase chain reaction (PCR). The corresponding primer pairs and PCR conditions are listed in

Table 1. Loci used in this study with the corresponding PCR primers and conditions.

Locus	PCR Primers	PCR: Thermal Cycles	References
ITS	ITS9mun or ITS5/ ITS4_KYO1 or ITS4	(94 °C: 30 s, 56 °C: 30 s, 72 °C: 30 s) × 35 cycles	[44,45]
LSU	LR0R/LR5	(94 °C: 30 s, 56 °C: 30 s, 72 °C: 1 min) × 35 cycles	[46,47]
SSU	PNS1/ NS41	(94 °C: 30 s, 56 °C: 30 s, 72 °C: 1 min) × 35 cycles	[48]
TEF1	EF1-983/ EF1-2218R	(94 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 35 cycles	[49,50]
RPB2	dRPB2-5f/dRPB2-7r	(94 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 35 cycles	[51]

. The final reaction volume of the PCR reagent was 25 µL, containing 2 µL of DNA template, 1 µL each of the forward and reverse primer, 8.5 µL of double-distilled water (ddH₂O), and 12.5 µL of 2×Flash PCR MasterMix (mixture of DNA Polymerase, dNTPs, Mg²⁺ and optimized buffer; CoWin Biosciences, Taizhou, China). The PCR products were visualized by 1% agarose gel electrophoresis. Sanger sequencing was conducted by Tsingke Biological Technology (Beijing, China). Newly generated sequences were deposited in GenBank, and the accession numbers are listed in

Table 2. Species details and their GenBank accession numbers used in phylogenetic analyses of Catenulostroma. Type strains are in bold, and newly generated sequences are in red.

Species	Culture/Specimen No.	GenBank Accession Numbers
		SSU	LSU	ITS
Catenulostroma chromoblastomycosum	CBS 597.97	GU214516	EU019251	AJ244260
C. corymbiae	CBS 133584	–	KC005805	KC005783
C. elginense	CBS 111030	GU214517	EU019252	–
C. hermanusense	CBS 128768	–	JF499853	JF499833
C. lignicola	CBS 130285	–	NG_059023	NR_154848
C. lignicola	FMR 11491	–	KY853489	KY853429
C. pini	UESTCC 24.0185	PQ046106	PQ038269	PQ038262
C. protearum	CBS 125421	–	KF902090	MH863677
Teratosphaeria fibrillosa	CPC 1876	–	GU214506	EU019282

,

Table 3. Species details and their GenBank accession numbers used in phylogenetic analyses of Kirschsteiniothelia. Type strains are in bold, and newly generated sequences are in red.

Species	Culture/Specimen No.	GenBank Accession Numbers
		SSU	LSU	ITS
Kirschsteiniothelia acutispora	MFLU 21-0127	ON980754	ON980758	OP120780
K. aquatica	MFLUCC 16-1685	MH182618	MH182594	MH182587
K. arasbaranica	IRAN 2509C	KX621988	KX621987	KX621986
K. arasbaranica	IRAN 2508C	KX621985	KX621984	KX621983
K. atra	CBS 109.53	AY016344	AY016361	–
K. atra	MFLUCC 16-1104	MH182615	MH182589	MH182583
K. atra	MFLUCC 15-0424	KU500585	KU500578	KU500571
K. cangshanensis	MFLUCC 16-1350	–	MH182592	MH182584
K. chiangmaiensis	MFLU 23-0358	OR575475	OR575474	OR575473
K. crustaceum	MFLU 21-0129	–	MW851854	MW851849
K. dushanensis	GZCC 19-0415	MW134610	MW133830	OP377845
K. ebriosa	CBS H-23379	–	LT985885	–
K. emarceis	MFLU 10-0037	–	NG_059454	NR_138375
K. extensum	MFLU 21-0130	–	MW851855	MW851850
K. esperanzae	T. Raymundo 6581	–	OQ880482	OQ877253
K. fluminicola	MFLUCC 16-1263	–	MH182588	MH182582
K. guangdongensis	MHZU 22-0137	–	OR164974	OR164946
K. inthanonensis	MFLUCC 23-0277	OR764784	OR762781	OR762773
K. longisporum	UESTCC 24.0190	PQ046108	PQ038273	PQ038266
K. lignicola	MFLUCC 10-0036	HQ441569	HQ441568	HQ441567
K. nabanheensis	HJAUP C2006	OQ023037	OQ023275	OQ023274
K. nabanheensis	HJAUP C2004	OQ023038	OQ023273	OQ023197
K. ramus	GZCC 23-0596	–	OR091333	NR_190260
K. phoenicis	MFLU 18-0153	–	NG_064508	NR_158532
K. phoenicis	MFLUCC 18-0216	MG859979	MG860484	MG859978
K. puerensis	ZHKUCC 22-0272	OP451021	OP451018	OP450978
K. puerensis	ZHKUCC 22-0271	OP451020	OP451017	OP450977
K. rostrata	MFLUCC 15-0619	KY697278	KY697276	KY697280
K. septemseptatum	MFLU 21-0126	ON980752	ON980757	OP120779
K. saprophytica	MFLUCC 23-0275	–	OR762783	OR762774
K. saprophytica	MFLUCC 23-0276	–	OR762782	OR762775
K. spatiosum	MFLU 21-0128	ON980753	–	NR_187065
K. submersa	S-481	MH182616	MH182591	–
K. submersa	S-601	–	MH182593	MH182585
K. submersa	MFLUCC 15-0427	KU500584	KU500577	KU500570
K. tectonae	MFLUCC 12-0050	–	KU764707	KU144916
K. tectonae	MFLUCC 13-0470	–	–	KU144924
K. tectonae	MFLUCC 23-0271	OR764782	OR762779	OR762771
K. tectonae	MFLUCC 23-0272	OR764783	OR762780	OR762772
K. thailandica	MFLUCC 20-0116	MT984280	MT984443	MT985633
K. thujina	JF13210	KM982717	KM982718	KM982716
K. vinigena	CBS H-23378	–	NG_075229	–
K. xishuangbannaensis	ZHKUCC 22-0221	OP289565	OP303182	OP289563
K. xishuangbannaensis	ZHKUCC 22-0220	OP289564	OP303181	OP289566
K. xishuangbannaensis	MFLUCC 23-0273	OR764781	OR762778	OR762770
K. xishuangbannaensis	MFLUCC 23-0274	OR764780	OR762777	OR762769
K. zizyphifolii	MFLUCC 23-0270	OR764779	OR762776	OR762768
Tenuitholiascus porinoides	HMAS-L0139638	MK352441	MK206259	–

,

Table 4. Species details and their GenBank accession numbers used in phylogenetic analyses of Paradictyoarthrinium. Type strains are in bold, and newly generated sequences are in red.

Species	Culture/Specimen No.	GenBank Accession Numbers
		LSU	ITS	RPB2
Nigrograna obliqua	CBS 141477	KX650560	KX650560	KX650580
Paradictyoarthrinium aquatica	MFLUCC 16-1116	MG747495	MG747496	MG780231
P. diffractum	MFLUCC 13-0466	KP744498	KP744455	KX437764
P. diffractum	MFLUCC 12-0557	KP744497	KP744454	KX437765
P. diffractum	KUMCC 19-0111	MN582756	MN582741	MN643158
P. diffractum	UESTCC 24.0187	PQ038271	PQ038264	PQ050360
P. hydei	KUNCC 10440	OQ146990	OQ135179	–
P. hydei	KUMCC 19-0185	–	MN582742	MN643159
P. hydei	MFLUCC 17-2512	MG747497	MG747498	MG780232
P. hydei	UESTCC 24.0188	PQ038272	PQ038265	PQ050361
P. salsipludicola	MFLUCC 22-0054	OR589801	OR589800	–
P. tectonicola	MFLUCC 13-0465	KP744500	KP744456	KX437763
P. tectonicola	MFLUCC 12-0556	KP744499	–	–

and

Table 5. Species details and their GenBank accession numbers used in phylogenetic analyses of Sporidesmiella. Type strains are in bold, and newly generated sequences are in red.

Species	Culture/Specimen No.	GenBank Accession Numbers
		LSU	ITS	RPB2	TEF1
Junewangia thailandica	MFLU 15-2682	MW287762	–	–	–
Sporidesmiella aquatica	DLUCC 0777	MK849843	MK828692	–	MN194034
S. hyalosperma	DLUCC 1518	MK849842	MK828691	MN124523	MN194033
S. hyalosperma	KUMCC 15-0431	MK849841	MK828690	MN124522	MN194032
S. junci	CBS 149443	NG_229048	OP675893	OP676106	–
S. juncicola	CPC 41075	OK663757	OK664718	OK651165	–
S. juncicola	CPC 41109	OK663758	OK664719	OK651166	OK651188
S. lignicola	JAUCC 3436	OK091615	MZ613187	OK323222	OK323223
S. motuoensis	KUNCC 10425	OR229720	OP626348	–	–
S. motuoensis	KUNCC 10463	OR229719	OR286630	–	–
S. novae-zelandiae	DLUCC 0951	MK849847	MK828695	MN124526	MN194037
S. novae-zelandiae	DLUCC 1256	MK849845	MK828693	MN124525	MN194036
S. obovoidia	MFLUCC 17-2372	MW287766	MW286492	–	–
S. pini	CPC 40067	OK663786	OK664747	OK651177	–
S. sichuanensis	HKAS136267	PQ038270	PQ038263	PQ050359	PQ050356

.

2.3. Phylogenetic Analyses

According to the corresponding Sanger sequencing chromatograms, misleading data from the ends of raw sequencing fragments were manually trimmed and assembled into consensus sequences using SeqMan Pro version 7.1.0 (DNASTAR, Inc., Madison, WI, USA). Barcode sequences of all species (Table 2, Table 3, Table 4 and Table 5) were downloaded from the NCBI nucleotide database using the R package Analysis of Phylogenetics and Evolution 5.0 (APE, http://ape-package.ird.fr, 7 July 2024) [52].

The multiple sequence alignments were conducted using MAFFT version 7.310 [53] with options “--adjustdirection --auto”, and the alignment files were further trimmed using trimAl version 1.4 [54] with the option “-gapthreshold 0.5”, which only allows 50% of taxa with a gap in each site. The best-fit nucleotide substitution models for each locus were selected using ModelFinder version 2.1.1 [55] under the Corrected Akaike Information Criterion (AICC). All sequence alignments were combined using an in-house Python script.

Maximum Likelihood (ML) and Bayesian analysis (BI) were conducted based on individual and combined datasets. Four phylogenetic trees were constructed by multi-locus phylogenetic analyses. The first tree represents the phylogenetic analysis of Catenulostroma, the second tree represents the phylogenetic analysis of Kirschsteiniothelia, the third tree represents the phylogenetic analysis of Paradictyoarthrinium, and the fourth tree represents the phylogenetic analysis of Sporidesmiella within the Junewangiaceae. ML phylogenetic trees were obtained using the IQ-TREE version 2.0.3 [56], and the topology was evaluated using 1000 ultrafast bootstrap replicates. The BI was conducted using parallel MrBayes version 3.2.7a [57]. The ML trees were visualized using ggtree version 2.4.1 [58] and further edited in Adobe Illustrator version 16.0.0.

3. Results

3.1. Phylogenetic Analyses

Sequences of three loci were successfully obtained for the Catenulostroma pini (UESTCC 24.0185). Nine taxa were included in the combined ITS, LSU and SSU sequence data, with Teratosphaeria fibrillosa (CPC 1876) as the outgroup (Figure 1). The combined dataset (ITS: 1–644, LSU: 645–1871, SSU: 1872–3619) was composed of 273 distinct patterns, 136 parsimony-informative sites, 324 singleton sites and 3159 constant sites. The best-fit evolution models were K2P + G4 for the ITS partitions, K2P + I for the LSU partition, K2P for the SSU partition. The best-scoring ML tree (lnL = −7762.005) with support values from ML and Bayesian analysis at the node is shown in Figure 1.

According to the multi-locus phylogeny (Figure 1), our collection (UESTCC 24.0185) formed an independent clade sister to Catenulostroma hermanusense (CBS 128768) and C. protearum (CBS 125421) with 99% ML, 1.00 PP statistical support. Combining the morphological evidence with phylogeny, we propose a new species, C. pini, isolated from Pinus massoniana.

Sequences of three loci were successfully obtained for the Kirschsteiniothelia longisporum (UESTCC 24.0190). A phylogenetic tree of species in Kirschsteiniothelia was constructed (Figure 2), including 48 taxa, with Tenuitholiascus porinoides (HMAS-L0139638) as the outgroup. The combined dataset (ITS: 1–507, LSU: 508–1368, SSU: 1369–2392) was composed of 988 distinct patterns, 585 parsimony-informative sites, 252 singleton sites and 1555 constant sites. The best-fit evolution models were GTR + F + G4 for the ITS partitions, GTR + F + G4 for the LSU partition, and K2P + I + G4 for the SSU partition. The best-scoring ML tree (lnL = −14,653.774) with support values from ML and Bayesian analysis at the node is shown in Figure 2.

According to the multi-locus phylogeny (Figure 2), our collection (UESTCC 24.0190) formed a branch sister to Kirschsteiniothelia aquatica (MFLUCC 16–1685). Based on the morphological evidence and phylogeny, we propose a new species, K. longisporum, isolated from Pinus taeda.

Sequences of three loci were successfully obtained for the Paradictyoarthrinium diffractum (UESTCC 24.0187) and Paradictyoarthrinium hydei (UESTCC 24.0188). A phylogenetic tree of species in Paradictyoarthrinium was constructed (Figure 3), including 13 taxa, with Nigrograna obliqua (CBS 141477) as the outgroup. The combined dataset (ITS: 1–518, LSU: 519–1364, SSU: 1365–2418) was composed of 314 distinct patterns, 73 parsimony-informative sites, 347 singleton sites and 1998 constant sites. The best-fit evolution models were K2P + I for the ITS partitions, K2P + I for the LSU partition, and K2P + I for the RPB2 partition. The best-scoring ML tree (lnL = −5397.698) with support values from ML and Bayesian analysis at the node is shown in Figure 3.

According to the multi-locus phylogeny (Figure 3), our collection (UESTCC 24.0188) nest with P. hydei strains with 99% ML, 1.00 PP statistical support and our collection (UESTCC 24.0187) nest with P. diffractum strains. Based on the morphological evidence and phylogeny, we report our collections (UESTCC 24.0188 and UESTCC 24.0187) as new host records of P. hydei and P. diffractum from Pinus spp.

Sequences of four loci were successfully obtained for the Sporidesmiella sichuanensis (HKAS 136267). A phylogenetic tree of species in Sporidesmiella within the Junewangiaceae was constructed (Figure 4), including 15 taxa, with Junewangia thailandica (MFLU 15–2682) as the outgroup. The combined dataset (ITS: 1–520, LSU: 521–1326, RPB2: 1327–2323, TEF1: 2324–3181) was composed of 576 distinct patterns, 375 parsimony-informative sites, 398 singleton sites and 2408 constant sites. The best-fit evolution models were GTR + F + G4 for the ITS partitions, K2P + I for the LSU partition, HKY + F + I for the RPB2 partition, GTR + F + G4 for the TEF1 partition. The best-scoring ML tree (lnL = −9362.397) with support values from ML and Bayesian analysis at the node is shown in Figure 4.

According to the multi-locus phylogeny (Figure 4), our collection (HKAS 136267) formed an independent clade sister to Sporidesmiella aquatica (DLUCC 0777) and Sporidesmiella juncicola strains. Based on the morphological evidence and phylogeny, we identified S. sichuanensis as a novel species from Pinus taeda.

3.2. Taxonomy

Catenulostroma pini W.H. Tian & Maharachch., sp. nov. (Figure 5).

MycoBank: MB 854981

Etymology: Named after the host genus where the fungus was collected.

Saprobic on dead bark of Pinus massoniana in terrestrial habitats. Asexual morph: Hyphomycetes. Colonies on the natural substratum effuse, scattered, gregarious, superficial, dark brown, powdery. Conidiophores mononematous, micronematous, branched, flexuous, septate, cylindrical, smooth, brown, reduced to conidiogenous cells. Conidiogenous cells 1.4–3.3 μm ($\overline{\mathrm{X}}$ = 2.2 μm, n = 20) width, integrated, holoblastic–thalloblastic, terminal or conidiophores reduced to conidiogenous cells, cylindrical, brown. Conidia three types: Helicoconidia 7–10 μm ($\overline{\mathrm{X}}$ = 8.5 μm, n = 35) diam., helicospores, solitary, acrogenous, terminal, euseptate, constricted at the septate, circinate, spherical, rounded at apex, thick-walled, brown to dark brown, verruculose. Scolecoconidia occasionally formed, 20–42 × 2.6–4.2 μm ($\overline{\mathrm{X}}$ = 30 × 3.4 μm, n = 25), occurring in branched chains, scolecospores, catenate, 5–15-septate, cylindrical, straight to slightly curved, pale brown to brown, smooth to finely verruculose. Phragmoconidia 12.5–44 × 3–5.5 μm ($\overline{\mathrm{X}}$ = 25 × 4 μm, n = 20), in simple or branched chains, cylindric–obclavate, straight to flexuous, septate, constricted at the septate, thick-walled, hyaline to pale brown, verruculose. Sexual morph: Undetermined.

Culture characteristics: Colonies on PDA reaching 25 mm diam. after 40 days at 25 °C, slow growing, colonies from above: irregularly circular, gray-brown, submerged margins, erumpent, with folded surface, and unevenly lobed; reverse: black.

Material examined: CHINA: Sichuan Province, Luzhou City, Daolingou, 29°15′1″ N, 105°42′1″ E, elevation 405 m, 31 March 2023, within dead bark of Pinus massoniana, W.H. Tian DLG24 (HKAS 136266, holotype), ex-type culture permanently preserved in a metabolically inactive state, UESTCC 24.0185.

Notes: Multi-locus phylogenetic analysis indicated that our isolate (UESTCC 24.0185) constitutes an independent clade sister to Catenulostroma hermanusense (CBS 128768) and Catenulostroma protearum (CBS 125421) (Figure 1). In the NCBI BLASTn search, comparing the ITS and LSU sequence of our isolate (UESTCC 24.0185) and C. hermanusense (CBS 128768) revealed 92.42% (549/594 bp, gaps: 17/594 bp), 96.65% (663/686 bp, without gaps) similarity, respectively. Comparing the ITS and LSU sequence of our isolate (UESTCC 24.0185) and C. protearum (CBS 125421) revealed 90.82% (475/523 bp, gaps: 17/513 bp), 96.35% (659/684 bp, without gaps) similarity, respectively. Morphologically, our isolate (UESTCC 24.0185) differs from C. hermanusense (CBS 128768) by the shape of conidia (helicoconidia, scolecoconidia, phragmoconidia vs. subcylindrical to ellipsoid conidia) and longer and narrower conidia (12.5–44 × 3–5.5 μm vs. 10–25 × 5–10 μm) [59], our isolate (UESTCC 24.0185) differs from C. protearum by the shape of conidia (helicoconidia, scolecoconidia, phragmoconidia vs. variable muriform to transversely septate conidia) [60] and relatively narrower conidia (7–10 μm vs. 7–25 μm) [15]. Therefore, based on morphological characteristics and phylogenetic analysis results, we identified C. pini as a novel species from Pinus massoniana in China.

Kirschsteiniothelia longisporum W.H. Tian & Maharachch., sp. nov. (Figure 6).

MycoBank: MB 854982

Etymology: The epithet refers to the long spores.

Saprobic on a dead branch of Pinus taeda in terrestrial habitats. Asexual morph: Hyphomycetes. Colonies on the natural substratum effuse, hairy, black. Mycelium superficial, hairy, scattered, dark brown to black. Conidiophores 115–285 × 6.5–14 μm ($\overline{\mathrm{X}}$ = 215 × 9 μm, n = 30), macronematous, mononematous, sometimes branched, solitary or fasciculate, erect, straight or slightly flexuous, cylindrical, septate, verruculose, dark brown to black. Conidiogenous cells holoblastic, integrated, terminal and intercalary, cylindrical, obtuse at apex, dark brown, verruculose. Conidia 35–130 × 8.5–15 μm ($\overline{\mathrm{X}}$ = 65 × 11 μm, n = 35), tapering to 2–4.5 μm ($\overline{\mathrm{X}}$ = 3, n = 35) at the distal end, with a blackish-brown 3–7 μm wide ($\overline{\mathrm{X}}$ = 4.5, n = 35) scar at the base, phragmoconidia, solitary, acrogenous, 3–15-distoseptate, cylindric–obclavate, elongated, wide at the middle and lower part, straight or flexuous, uneven width, slender and rounded at apex, truncate at base, brown, thick-walled, verruculose, secession schizolytic. Sexual morph: Undetermined.

Culture characteristics—Colony on PDA reaching 11 mm diam. in 8 days at 25 °C in the dark, colonies from above: irregular circular, grey, uneven entire, raised in centre, with denser mycelium at the centre; reverse: black, cream at the margin, margin undulated.

Material examined: CHINA: Sichuan Province, Chengdu City, Jiudaoguai, 30°30′21″ N, 103°53′47″ E, elevation 502 m, 19 October 2023, within dead branches of Pinus taeda, W.H. Tian JDG36 (HKAS 136268, holotype), ex-type culture permanently preserved in a metabolically inactive state, CGMCC 3.27599 = UESTCC 24.0190.

Notes: Phylogenetic analysis based on the combined dataset of ITS, LSU and SSU loci revealed that our collection (UESTCC 24.0190) forms a branch sister to Kirschsteiniothelia aquatica (MFLUCC 16–1685) and K. cangshanensis (MFLUCC 16–1350) (Figure 2). Comparing the ITS sequence of our collection (UESTCC 24.0190) with K. aquatica (MFLUCC 16–1685) revealed 94.67% (515/544 bp, gaps: 4/544 bp) similarity, and with k. cangshanensis (MFLUCC 16–1350) revealed 91.91% (477/519 bp, gaps: 4/519 bp) similarity. Morphologically, our collection differs from the K. aquatica (MFLUCC 16–1685) and K. cangshanensis (MFLUCC 16–1350) by larger conidiophores (115–285 × 6.5–14 μm vs. 114–151 × 7–8 μm vs. 105–135 × 6–8 μm) and bigger conidia (35–130 × 8.5–15 μm vs. 35–46 × 7.5–8.8 μm vs. 33–43 × 7.5–8.5 μm) [61]. Our collection (UESTCC 24.0190) shares similar characteristics with K. fluminicola (MFLUCC 16–1263) in having slender conidia rounded at apex and multi-septate at maturity [61]. However, the conidiophores of our collection (UESTCC 24.0190) are wider than K. fluminicola (MFLUCC 16–1263) (6.5–14 μm vs. 7–9 μm), and the conidia of our collection (UESTCC 24.0190) is relatively bigger than K. fluminicola (MFLUCC 16–1263) (35–130 × 8.5–15 μm vs. 47.5–86.5 × 8–10 μm) [61]. Thus, our collection UESTCC 24.0190 is described as a new species based on morphological observation and phylogenetic evidence.

Paradictyoarthrinium diffractum Matsush., Matsush. Mycol. Mem. 9: 18 (1996) (Figure 7).

Saprobic on a dead branch of Pinus taeda in terrestrial habitats. Asexual morph: Hyphomycetes. Colonies on the natural substrate scattered, gregarious, superficial, black, powdery. Conidiophores thick-walled, black, macronematous, sometimes micronematous, erect to slightly curved, short, branched or unbranched, arising from hyphae, slightly constricted at the septa. Conidiogenous cells 3.8–8.3 × 2.5–7 μm ($\overline{\mathrm{X}}$ = 5.3 × 4 μm, n = 25), blastic, mostly terminal, determinate, peacock green. Conidia 14–28 × 10–20 μm ($\overline{\mathrm{X}}$ = 20 × 15 μm, n = 35), dictyoconidia, solitary or formed in chains, unevenly dictyoseptate, subglobose to ellipsoidal, verrucose, dark green when immature and dark when mature. Sexual morph: Undetermined.

Culture characteristics: Colonies on PDA reaching 20 mm diam. after 10 days at 25 °C, colonies from above: grey at the centre, creamy white until margin, hyaline mycelia at the entire edge, dense, fluffy, and circular, reverse: dark-olivaceous brown at the centre, and creamy white towards the edge.

Material examined: China, Sichuan Province, Chengdu City, Jiudaoguai, 30°30′21″ N, 103°53′47″ E, elevation 502 m, 19 October 2023, within dead branches of Pinus taeda, W.H. Tian JDG37 (HUEST 24.0204), living culture permanently preserved in a metabolically inactive state, UESTCC 24.0187.

Notes: According to the multi-locus phylogeny, our collection (UESTCC 24.0187) is nested with P. diffractum strains (Figure 3). Based on the BLASTn NCBI GenBank database search of ITS, LSU and RPB2 sequences, our collection (UESTCC 24.0187) is 99% similar to P. diffractum. In addition, the morphological characteristics of our collection (UESTCC 24.0187) overlap with the P. diffractum in having powdery colonies, lacking conidiophores, and solitary or formed in chains, pleomorphic conidia [62]. Thus, based on morphological comparison and phylogenetic analyses, we report our collections (UESTCC 24.0187) as a new host record of P. diffractum from Pinus taeda.

Paradictyoarthrinium hydei N.G. Liu & J.K. Liu, in Liu, Luo, Liu, Cheewangkoon & Chaiwat, Phytotaxa 338(3): 290 (2018) (Figure 8).

Saprobic on a dead branch of Pinus sp. in terrestrial habitats. Asexual morph: Hyphomycetes. Colonies on the natural substrate, scattered, gregarious, superficial, black, powdery. Conidiophores macronematous, rarely micronematous, thick-walled, black, erect to slightly curved, short, branched or unbranched, arising from hyphae, slightly constricted at the septa. Conidiogenous cells 4.3–5.8 × 2.7–5 μm ($\overline{\mathrm{X}}$ = 5 × 4 μm, n = 25), monoblastic, terminal, determinate, integrated, dark green. Conidia 18–40 × 9–35 μm ($\overline{\mathrm{X}}$ = 28 × 22 μm, n = 35), dictyoconidia, solitary or formed in chains, unevenly dictyoseptate, subglobose to ellipsoidal, constricted at the septa, verrucose, dark green when immature and dark when mature. Sexual morph: Undetermined.

Culture characteristics: Colonies on PDA reaching 34 mm diam. after 20 days at 25 °C, colonies from above: circular, divergent at the margin, dense, slightly raised, white at the margin, grey at the centre, reverse: dark-olivaceous brown at the centre, and creamy white towards the edge.

Material examined: China, Sichuan Province, Neijiang City, Songlin Village, 29°32′19″ N, 105°9′28″ E, elevation 373 m, 1 April 2023, decaying branches of Pinus sp., W.H. Tian SLC10 (HUEST 24.0205), living culture permanently preserved in a metabolically inactive state, UESTCC 24.0188.

Notes: Multi-locus phylogeny indicates that our collection’s (UESTCC 24.0188) sister is the Paradictyoarthrinium hydei group with 99% ML, 1.00 PP statistical support (Figure 3). In the NCBI BLASTn search, the ITS, LSU and RPB2 sequences of our collection (UESTCC 24.0188) are 99% similar to P. hydei. In addition, as morphological characteristics examined largely overlapped with the type strain of P. hydei [29], we report our collections (UESTCC 24.0188) as a new host record of P. hydei from Pinus sp.

Sporidesmiella sichuanensis W.H. Tian & Maharachch., sp. nov. (Figure 9).

MycoBank: MB 854983

Etymology: Named after the Sichuan province, China, where the holotype was collected.

Saprobic on a dead branch of Pinus taeda in terrestrial habitats. Asexual morph: Hyphomycetes. Colonies on the natural substratum effuse, hairy, black. Mycelium superficial, hairy, yellow-brown to brown, scattered. Conidiophores 108–178 × 3.2–5.7 μm ($\overline{\mathrm{X}}$ = 137 × 4 μm, n = 20), macronematous, mononematous, solitary, unbranched, erect, straight or slightly flexuous, cylindrical, 6–12-septate, smooth, yellow-brown, paler towards the apex. Conidiogenous cells holoblastic, polyblastic, integrated, sympodial, terminal, cylindrical, subhyaline to pale brown, smooth, percurrently extended, with the extensions towards the apex. Conidia 20–27 × 9.5–12 μm ($\overline{\mathrm{X}}$ = 24 × 10 μm, n = 35), phragmoconidia, solitary, acrogenous, 3–4-distoseptate, cylindric–obclavate, widened and rounded at the apex, uneven width, truncate at the base, subhyaline to pale brown, thick-walled, smooth. Sexual morph: Undetermined.

Material examined: CHINA: Sichuan Province, Chengdu City, Jiudaoguai, 30°30′21″ N, 103°53′47″ E, elevation 502 m, 19 October 2023, within dead branches of Pinus taeda, W.H. Tian JDG40_1 (HKAS 136267, holotype).

Notes: Phylogenetic analyses of combined ITS, LSU, RPB2 and TEF1 sequence data showed that our collection (HKAS 136267) forms a separate branch sister to Sporidesmiella aquatica and Sporidesmiella juncicola (Figure 4). The closest match to our collection (HKAS 136267) is S. aquatica (DLUCC 0777) (Figure 4). Sequence comparison for the ITS, LSU and TEF1 region between our collection (HKAS 136267) and the type strain of S. aquatica showed 87.74% (315/359 bp, gaps: 14/359 bp), 96.95% (699/721 bp, gaps: 5/721 bp) and 95.25% (802/842 bp, without gaps) base pair similarity. However, morphologically, our collection (HKAS 136267) differs from the S. aquatica (DLUCC 0777) by smaller conidiophores (108–178 × 3.2–5.7 μm vs. 178–228 × 8–10 μm) and smaller conidia (20–27 × 9.5–12 μm vs. 51–59 × 18–22 μm) [10]. Therefore, based on morphology and phylogenetic analyses, we introduce S. sichuanensis as a novel species from Pinus taeda in China.

4. Discussion

In this study, two rarely seen species from Pinus spp. were reported. Catenulostroma pini sp. nov., isolated from the dead bark of Pinus massoniana, is the seventh species of the genus and the first time that the genus Catenulostroma has been recorded in China [15,16,59]. Sporidesmiella sichuanensis sp. nov., isolated from a dead branch of Pinus taeda, is the eleventh species in the genus to have sequence data. Sporidesmiella is an old genus with origins dating back to the 18th century (previously as Sporidesmium); hence, molecular data are unavailable for most species [32]. Sporidesmiella pini, was isolated from needles of Pinus sylvestris in the Netherlands [63]. This suggests that many unique and rare taxa on Pinus spp. may still await discovery and exploration.

Catenulostroma pini sp. nov. is an interesting species in which three conidial morphologies were observed. The first type is a helicoconidia small terminal conidia, characterized by helicospores that are solitary, acrogenous, euseptate, constricted at the septa, circinate, and spherical. This morphology is slightly similar to that of C. protearum obtained from OA, as reported by Crous et al. [60]. However, the conidia in our collection are circinate and spherical, whereas the conidia of C. protearum are variably muriform to transversely septate [60]. The transversely septate conidia of C. protearum are very similar to the second conidial morphology in our collection [60]. They all occur in branched chains, forming chain-like, septate, and scolecospores, which are also similar to the conidia of C. chromoblastomycosum [15,60]. After germination and sporulation of helicoconidia on PDA, we observed a third morphology of conidia characterized by simple or branched chains, cylindric–obclavate, straight to flexuous, hyaline to pale brown, septate, constricted at the septate. Similar to C. hermanusense, conidia are in simple or branched chains but with different morphologies: cylindric–obclavate and constricted at the septa, compared to subcylindrical to ellipsoid [59]. Besides the unique and diverse morphology of the genus Catenulostroma, it is noteworthy that C. chromoblastomycosum was described as a case of human chromoblastomycosis [15]. Currently, no other species of this genus have been found capable of infecting humans and causing disease.

In this study, four hyphomycetes fungi collected from Pinus spp. were isolated and identified: Catenulostroma pini sp. nov. (Teratosphaeriaceae), Kirschsteiniothelia longisporum sp. nov. (Kirschsteiniotheliaceae), Paradictyoarthrinium diffractum and P. hydei of Paradictyoarthriniaceae, all within Dothideomycetes. Dothideomycetes represent the largest and most diverse class of ascomycete fungi [6,25,64,65]. This class includes over 25 orders, 110 families and more than 19,000 species [66]. Their representatives have an incredibly diverse lifestyle and can be associated with various hosts and substrates [6]. Another collection was identified as Sporidesmiella sichuanensis sp. nov., a saprophyte in the Junewangiaceae family within Sordariomycetes. Sporidesmiella is a polyphyletic genus. Based on phylogenetic analysis of the LSU and RPB2 datasets, Shenoy et al. [67] classified S. fusiformis into the family Melanommataceae within Dothideomycetes. Subsequently, using combined ITS, LSU, RPB2, and TEF1 sequence data, many species in the genus were reassigned to Junewangiaceae within Sordariomycetes [10,33,34,35,36]. The diversity of fungal groups in Sordariomycetes is high, and most species are saprophytic fungi that can degrade organic matter in nature and promote the material cycle of the ecosystem [68,69,70].