Unveiling Sordariomycetes taxa associated with woody litter in Northern Thailand

Chayanard Phukhamsakda; Kevin D. Hyde; Milan C. Samarakoon; Johnny Louangphan; Kedsara Navasit; Fatimah Al-Otibi; Chitrabhanu S. Bhunjun

doi:10.3897/mycokeys.115.145330

Research Article

Unveiling Sordariomycetes taxa associated with woody litter in Northern Thailand

Chayanard Phukhamsakda^‡, Kevin D. Hyde^§|‡, Milan C. Samarakoon^¶, Johnny Louangphan^§‡, Kedsara Navasit^‡, Fatimah Al-Otibi^|, Chitrabhanu S. Bhunjun^‡

‡ Mae Fah Luang University, Chiang Rai, Thailand

§ Kunming Institute of Botany, Chinese Academy of Science, Kunming, China

| King Saud University, Riyadh, Saudi Arabia

¶ Chiang Mai University, Chiang Mai, Thailand

Corresponding author: Chitrabhanu S. Bhunjun ( chitrabhanu.bhu@mfu.ac.th )

Academic editor: Danushka Sandaruwan Tennakoon

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Phukhamsakda C, Hyde KD, Samarakoon MC, Louangphan J, Navasit K, Al-Otibi F, Bhunjun CS (2025) Unveiling Sordariomycetes taxa associated with woody litter in Northern Thailand. MycoKeys 115: 155-185. https://doi.org/10.3897/mycokeys.115.145330

Abstract

Sordariomycetes species are abundant in woody litter samples. In this study, we introduce two novel species, Diaporthe thailandica (Diaporthaceae) and Occultitheca chiangraiensis (Xylariaceae), from woody litter materials. We also describe a new host record of D. tulliensis and a new geographical record for D. melonis. All collections were identified based on morphology and phylogenetic analyses of combined datasets. The morphologies of the taxa fit the generic concepts of Diaporthe and Occultitheca, respectively. Diaporthe thailandica formed a sister clade with D. raonikayaporum but differs from D. raonikayaporum in the sizes of conidiomata, conidiogenous cells, and beta conidia. Diaporthe thailandica also differs from D. raonikayaporum by the absence of gamma conidia. Occultitheca chiangraiensis differs from the sister taxon O. rosae in having smaller ascomata and a thicker mucilaginous sheath. We also provide a synopsis of Occultitheca species with details on their morphology, host, and country. These findings provide valuable insights into the diversity and ecological roles of Sordariomycetes, emphasising the need for continued exploration of fungal biodiversity in various environments.

Key words:

2 new records, 2 new taxa, anthostomella-like taxa, phylogeny, saprobes, taxonomy

Introduction

Plant litter plays an important role in shaping ecological processes and supporting biodiversity, which represents a major source of organic carbon and nutrient cycling (Zhang et al. 2023). As natural decomposers, fungi hold a significant role in the breakdown of woody litter by degrading complex organic compounds efficiently (Dashtban et al. 2010; Hyde et al. 2019; Mapook et al. 2022). Although woody litter harbours various groups of fungi, comprehensive studies are limited (Hyde et al. 2020a; Phukhamsakda et al. 2022; Wanasinghe and Mortimer 2022; Xu et al. 2022; Madagammana et al. 2023).

Diaporthe (Diaporthaceae, Diaporthales, Sordariomycetes, Ascomycota) was established by Fuckel (1867) with D. alnea as the type species. Diaporthe is an important plant pathogen that also comprises endophytes and saprobes on a wide range of hosts (Dissanayake et al. 2017; Hyde et al. 2020b; Phukhamsakda et al. 2020; Bhunjun et al. 2022). As pathogens, Diaporthe cause diebacks, cankers, leaf spots, blights, melanoses, stem-end rots, and gummosis on economically and ecologically important plants (Gomes et al. 2013; Manawasinghe et al. 2021; Bhunjun et al. 2024a). The sexual morph of Diaporthe is characterised by immersed ascomata, erumpent pseudostroma with elongated perithecial necks, and unitunicate asci that produce hyaline ascospores (Udayanga et al. 2011; Hongsanan et al. 2023). The asexual morph has ostiolate conidiomata, cylindrical phialides, and aseptate and hyaline conidia (alpha, beta, and gamma) (Udayanga et al. 2011; Gomes et al. 2013). Molecular approaches are essential for accurate identification due to overlapping morphological characters among distinct species (Bhunjun et al. 2022; Norphanphoun et al. 2022). The taxonomy of Diaporthe species has been the subject of several studies (Gomes et al. 2013; Udayanga et al. 2014; Gao et al. 2017; Sun et al. 2021; Norphanphoun et al. 2022). The genus has recently been revised, and 31 species were synonymised based on multi-gene phylogeny, GCPSR (Genealogical Concordance Phylogenetic Species Recognition), as well as coalescence-based analyses of ITS, tef1, tub2, cal, and his3 sequences (Dissanayake et al. 2024). Pereira and Phillips (2024) further reduced 53 species to synonymy and introduced a new species, D. pygmaeae, based on several molecular approaches.

Occultitheca (Xylariaceae, Xylariales) is characterised by immersed ascomata, short pedicellate asci with J+ apical ring, brown ascospores with hyaline dwarf cells, and a straight germ slit (Rogers and Ju 2003; Samarakoon et al. 2022). The genus is notable for having the uppermost ascospore distant from the ascus apex (Rogers and Ju 2003; Samarakoon et al. 2022). The second species, O. rosae, was described almost two decades later from a dead branch of Rosa species in Guizhou, China (Samarakoon et al. 2022). Tian et al. (2024) introduced O. ananasi from dead pineapple leaves from Chiang Rai, Thailand, based on morphological and molecular analyses.

Chiang Rai and Chiang Mai are located in the northern part of Thailand and are considered biodiversity hotspots (Hyde et al. 2018). During the study of woody litter microfungi in northern Thailand, two novel species and two new records were discovered based on morphology and multigene phylogeny. This underscores a critical gap in our understanding of fungal biodiversity and its ecological significance in the area, thus highlighting the need for further research (Hyde et al. 2024).

Materials and methods

Fungal collection, isolation, and observation

Woody litter samples were collected from forest areas in Chiang Mai and Chiang Rai Provinces, Thailand. The area is covered with a canopy of tall trees, such as dipterocarp species and Bambusa species. The specimens were maintained in paper bags for transport to the laboratory. The fungal structures were observed using a Leica EZ4 stereomicroscope (Leica Microsystems (SEA) Pte Ltd, Singapore). and photographed using a Nikon ECLIPSE Ni compound microscope (Nikon, Japan) equipped with a Nikon DS-Ri2 camera. Tarosoft (R) Image Frame Work version 3.9.3.74 was used for measurements, and Adobe Photoshop CS6 software was used for the photo plates.

Single spore isolation was conducted to obtain pure culture on potato dextrose agar (PDA) as described in Senanayake et al. (2020). The culture plates were incubated at room temperature (25 ± 5 °C) for 4 weeks. Herbarium materials were deposited in the Mae Fah Luang University Fungarium (MFLU), and ex-type living cultures were deposited in the Mae Fah Luang University Culture Collection (MFLUCC) in Chiang Rai, Thailand. Index Fungorum (Index Fungorum 2024) and Facesoffungi numbers (FoF) (Jayasiri et al. 2015) were obtained. The data of the novel species will also be uploaded to the Fungalpedia website (Hyde et al. 2023).

DNA extraction, amplification, and sequencing

Fresh mycelium was scraped from a 4-week-old culture on PDA, and DNA was extracted using the E.Z.N.A. Forensic DNA Kit (BIO-TEK) according to the manufacturer’s instructions. The polymerase chain reaction (PCR) was used to amplify the ITS region (ITS5/ITS4) (White et al. 1990), cal (CAL228F/CAL737R) (Carbone and Kohn 1999), his3 (CYLH3F/H3-1b) (Glass and Donaldson 1995), LSU (LR0R/LR5) (Vilgalys and Hester 1990), rpb2 (fRPB25f/fRPB2-7cR) (Liu et al. 1999), tef1 (EF-728F/EF-986R) (Carbone and Kohn 1999), and tub2 (T1/T22 and BT2a/BT2b) genes (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997). The PCR conditions for each primer were set up following Samarakoon et al. (2022) and Dissanayake et al. (2024). A PCR reaction was carried out in a 25 μL reaction volume containing 12.5 µL of 2 × PCR MasterMix, 9.5 µL of double-distilled water, 1 µL of 20 µmol for each forward and reverse primer, and 1 µL of 30 ng of DNA template. The PCR products were visualised on 1% agarose gels with 6 μl of 4S green dye per 100 mL. Successful PCR products were purified and sequenced by Biogenomed Co., Ltd., South Korea. The newly generated sequences were deposited in GenBank (Tables 1, 2).

Table 1.

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GenBank accession numbers of the taxa used in the phylogenetic analyses of Diaporthe section sojae.

Species	Strain	ITS	tef1	tub2	cal	his3
Diaporthe acaciarum	CBS 138862^T	KP004460	N/A	KP004509	N/A	KP004504
D. amaranthophila	MAFF 246900^T	LC459575	LC459577	LC459579	LC459583	LC459581
D. amaranthophila	MAFF 246901	LC459576	LC459578	LC459580	LC459584	LC459582
D. ambigua	CBS 114015^T	MH862953	KC343736	KC343978	KC343252	KC343494
D. ambigua	CBS 117167	KC343011	KC343737	KC343979	KC343253	KC343495
D. angelicae	CBS 111592^T	KC343027	KC343753	KC343995	KC343269	KC343511
D. angelicae	CBS 100871	KC343025	KC343751	KC343993	KC343267	KC343509
D. arctii	CBS 139280^T	KJ590736	KJ590776	KJ610891	KJ612133	KJ659218
D. arezzoensis	MFLU 19-2880^T	MT185503	MT454019	MT454055	N/A	N/A
D. batatas	CBS 122.21^T	KC343040	KC343766	KC344008	KC343282	KC343524
D. beilharziae	BRIP 54792^T	JX862529	JX862535	KF170921	N/A	N/A
D. biguttulata	CFCC 52584^T	MH121519	MH121561	MH121598	MH121437	MH121477
D. biguttulata	CFCC 52585	MH121520	MH121562	MH121599	MH121438	MH121478
D. brasiliensis	CBS 133183^T	KC343042	KC343768	KC344010	KC343284	KC343526
D. brasiliensis	LGMF926	KC343043	KC343769	KC344011	KC343285	KC343527
D. breyniae	CBS 148910^T	ON400846	ON409188	ON409186	ON409189	ON409187
D. caatingaensis	URM7485^T	KY085927	KY115604	KY115601	KY115598	KY115605
D. caatingaensis	URM7484	KY085928	N/A	KY115602	KY115599	KY115606
D. caryae	CFCC 52563^T	MH121498	MH121540	MH121580	MH121422	MH121458
D. caryae	CFCC 52564	MH121499	MH121541	MH121581	MH121423	MH121459
D. chimonanthi	SCHM 3614^T	AY622993	N/A	N/A	N/A	N/A
D. chimonanthi	SCHM 3603	AY620820	N/A	N/A	N/A	N/A
D. cichorii	MFLUCC 17-1023^T	KY964220	KY964176	KY964104	KY964133	N/A
D. cinnamomi	CFCC 52569^T	MH121504	MH121546	MH121586	N/A	MH121464
D. cinnamomi	CFCC 52570	MH121505	MH121547	MH121587	N/A	MH121465
D. citriasiana	ZJUD30^T	JQ954645	JQ954663	KC357459	KC357491	N/A
D. citriasiana	ZJUD81	KJ490616	KJ490495	KJ490437	N/A	KJ490558
D. convolvuli	CBS 124654^T	KC343054	KC343780	KC344022	KC343296	KC343538
D. convolvuli	FAU649	KJ590721	KJ590765	N/A	KJ612130	KJ659210
D. coracoralinae	URM 8912^T	PP192078	PP430449	PP402241	PP408214	PP421133
D. coracoralinae	FCCUFG 39	PP192079	N/A	PP402242	PP408215	PP421134
D. cucurbitae	DAOM 42078^T	KM453210	KM453211	KP118848	N/A	KM453212
D. cucurbitae	CBS 136.25	KC343031	KC343757	KC343999	KC343273	KC343515
D. cuppatea	CBS 117499^T	KC343057	KC343783	KC344025	KC343299	KC343541
D. cyatheae	YMJ-1364^T	JX570889	KC465406	KC465403	KC465410	N/A
D. discoidispora	ZJUD89^T	KJ490624	KJ490503	KJ490445	N/A	KJ490566
D. discoidispora	GZCC 22-0065	OP056659	OP150498	OP150576	OP150655	OP150730
D. eleutharrhenae	01^T*	OK017069	OK017070	OK017071	N/A	N/A
D. eleutharrhenae	02*	OK648457	OK648458	OK648459	N/A	N/A
D. fici-septicae	NCYUCC 19-0108^T	MW114349	MW192212	MW148269	N/A	N/A
D. fici-septicae	MFLU 20-20178	MW114348	MW192211	MW148268	N/A	N/A
D. foliorum	CMRP 1321^T	MT576688	MT584310	MT584327	MT584341	MT584338
D. foliorum	CMRP 1330	MT576671	MT584309	MT584328	MT584342	MT584340
D. ganjae	CBS 180.91^T	KC343112	KC343838	KC344080	KC343354	KC343596
D. ganjae	PSCG489	MK626955	MK654897	MK691287	MK691202	MK726204
D. goulteri	BRIP 55657a^T	KJ197290	KJ197252	KJ197270	N/A	N/A
D. gulyae	BRIP 54025^T	JF431299	JN645803	N/A	N/A	N/A
D. gulyae	BRIP 53158	JF431284	JN645799	N/A	N/A	N/A
D. guttulata	CGMCC3.20100^T	MT385950	MT424685	MT424705	MW022470	MW022491
D. guttulata	GZCC 19-0371	MT797178	MT793021	MT793032	MW022471	MW022492
D. helianthi	CBS 592.81^T	KC343115	KC343841	KC344083	KC343357	KC343599
D. helianthi	CBS 344.94	KC343114	KC343840	KC344082	KC343356	KC343598
D. hordei	CBS 481.92^T	KC343120	KC343846	KC344088	KC343362	KC343604
D. infecunda	CBS 133812^T	KC343126	KC343852	KC344094	KC343368	KC343610
D. infertilis	CBS 230.52^T	KC343052	KC343778	KC344020	KC343294	KC343536
D. infertilis	CPC 20322	KC343053	KC343779	KC344021	KC343295	KC343537
D. juglandigena	GUCC 422.16^T	OP581229	OP688534	OP688559	N/A	N/A
D. juglandigena	GUCC 422.161	OP581230	OP688535	OP688560	N/A	N/A
D. kyushuensis	STE-U2675^T	AF230749	N/A	N/A	N/A	N/A
D. kyushuensis	ch-D-1	AB302250	N/A	N/A	N/A	N/A
D. leucospermi	CBS 111980^T	N/A	KY435632	KY435673	KY435663	KY435653
D. leucospermi	CAA763	MK792291	MK828064	MK837915	MK883823	MK871433
D. longicolla	FAU599^T	KJ590728	KJ590767	KJ610883	KJ612124	KJ659188
D. longicolla	CBS 100.87	KC343196	KC343922	KC344164	KC343438	KC343680
D. longispora	CBS 194.36^T	KC343135	KC343861	KC344103	KC343377	KC343619
D. lusitanicae	CBS 123213^T	KC343137	KC343863	KC344105	KC343379	KC343621
D. lusitanicae	CBS 123212	KC343136	KC343862	KC344104	KC343378	KC343620
D. machilii	SAUCC194.111^T	MT822639	MT855951	MT855836	MT855718	MT855606
D. mayteni	CBS 133185^T	KC343139	KC343865	KC344107	KC343381	KC343623
D. megalospora	CBS 143.27^T	KC343140	KC343866	KC344108	KC343382	KC343624
D. melonis	CBS 507.78^T	KC343142	KC343868	KC344110	KC343384	KC343626
D. melonis	FAU640	KJ590702	KJ590741	KJ610858	KJ612099	KJ659184
D. melonis	ZHKUCC 20-0014	MT355684	MT409338	MT409292	MT409314	N/A
*D. melonis*	MFLUCC 24-0522	OR936658	PQ774278	PQ774285	N/A	PQ774293
*D. melonis*	MFLUCC 23–0300	PQ777476	PQ774277	PQ774284	PQ774290	N/A
D. middletonii	BRIP 54884e^T	KJ197286	KJ197248	KJ197266	N/A	N/A
D. middletonii	BRIP 57329	KJ197285	KJ197247	KJ197265	N/A	N/A
D. minusculata	CGMCC3.20098^T	MT385957	MT424692	MT424712	MW022475	MW022499
D. minusculata	GZCC 19-0345	MT797184	MT793027	MT793038	MW022476	MW022500
D. monetii	MF Ha18-048^T	MW008493	MW008515	MW008504	MZ671938	MZ671964
D. monetii	MF Ha18-049	MW008494	MW008516	MW008505	MZ671939	MZ671965
D. morindendophytica	ZHKUCC 22-0069^T	ON322897	ON315053	ON315087	N/A	ON315027
D. morindendophytica	ZHKUCC 22-0070	ON322898	ON315054	ON315088	N/A	ON315028
D. myracrodruonis	URM 7972^T	MK205289	MK213408	MK205291	MK205290	N/A
D. neoarctii	CBS 109490^T	KC343145	KC343871	KC344113	KC343387	KC343629
D. novem	CBS 127270^T	KC343156	KC343882	KC344124	KC343398	KC343640
D. novem	CBS 127271	KC343157	KC343883	KC344125	KC343399	KC343641
D. novem	PL42	JQ697843	JQ697856	N/A	N/A	N/A
D. orixae	KUNCC 21-10714^T	OK283041	N/A	N/A	OK484485	OK484486
D. orixae	GZCC 21-1085	OL889852	OL944724	OL944726	N/A	N/A
D. ovalispora	CGMCC3.17256^T	KJ490628	KJ490507	KJ490449	N/A	KJ490570
D. oxe	CBS 133186^T	KC343164	KC343890	KC344132	KC343406	KC343648
D. oxe	CBS 133187	KC343165	KC343891	KC344133	KC343407	KC343649
D. pachirae	CDA 728^T	MG559537	MG559539	MG559541	MG559535	N/A
D. pachirae	CDA 730	MG559538	MG559540	MG559542	MG559536	N/A
D. paranensis	CBS 133184^T	KC343171	KC343897	KC344139	KC343413	KC343655
D. paranensis	LMICRO417	KY461115	KY461116	N/A	N/A	N/A
D. passiflorae	CBS 132527^T	JX069860	N/A	N/A	N/A	KY435654
D. passiflorae	CAA953	MN190308	MT309430	MT309456	MT309447	MT309439
D. pedratalhadensis	URM 8304^T	PP192073	PP430438	PP402232	N/A	PP421129
D. pedratalhadensis	FCCUFG 49	PP192075	N/A	N/A	PP408217	PP421131
D. phaseolorum	AR4203^T	KJ590738	KJ590739	KJ610893	KJ612135	KJ659220
D. pseudobauhiniae	MFLUCC 17-1669^T	MF190119	MF377598	N/A	N/A	N/A
D. pseudobauhiniae	MFLUCC 17-1670	MF190118	MF377599	N/A	N/A	N/A
D. quercicola	CSUFTCC104^T	ON076567	ON081659	N/A	ON081670	ON081667
D. quercicola	CSUFTCC105	ON076568	ON081660	N/A	ON081671	ON081668
D. racemosae	CBS 143770^T	MG600223	MG600225	MG600227	MG600219	MG600221
D. raonikayaporum	CBS 133182^T	KC343188	KC343914	KC344156	KC343430	KC343672
D. raonikayaporum	MFLUCC 14-1133	KU712448	KU749368	KU743987	KU749355	N/A
D. raonikayaporum	MFLUCC 14-1136	KU712449	KU749369	KU743988	KU749356	N/A
D. rosae	MFLUCC 17-2658^T	MG828894	N/A	MG843878	MG829273	N/A
D. rosae	MFLUCC 17-2574	MG906793	MG968954	MG968952	N/A	N/A
D. rosiphthora	COAD 2914^T	N/A	QOI91674	N/A	QOI91672	N/A
D. sackstonii	BRIP 54669b^T	KJ197287	KJ197249	KJ197267	N/A	N/A
D. schini	CBS 133181^T	KC343191	KC343917	KC344159	KC343433	KC343675
D. schini	LGMF 910	KC343192	KC343918	KC344160	KC343434	KC343676
D. schoeni	MFLU 15-1279^T	KY964226	KY964182	KY964109	KY964139	N/A
D. schoeni	MFLU 15-2609	KY964229	KY964185	KY964112	KY964141	N/A
D. sclerotioides	CBS 296.67^T	MH858974	KC343919	KC344161	KC343435	KC343677
D. sclerotioides	CBS 710.76	KC343194	KC343920	KC344162	KC343436	KC343678
D. serafiniae	BRIP 55665a^T	KJ197274	KJ197236	KJ197254	N/A	N/A
D. serafiniae	BRIP 54136	KJ197273	KJ197235	KJ197253	N/A	N/A
D. siamensis	MFLUCC 10-0573a^T	JQ619879	JX275393	JX275429	JX197423	N/A
D. siamensis	MFLUCC 12-0300	KT459417	KT459451	KT459435	KT459467	N/A
D. sojae	FAU635^T	KJ590719	KJ590762	KJ610875	KJ612116	KJ659208
D. sojae	CBS 116019	KC343175	KC343901	KC344143	KC343417	KC343659
D. stewartii	CBS 193.36	MH867279	GQ250324	JX275421	JX197415	N/A
D. stewartii	MN1	KX668416	KX852355	N/A	N/A	N/A
D. submersa	CGMCC3.24297^T	OP056717	OP150556	OP150633	OP150710	OP150786
D. submersa	GZCC 22-0007	OP056718	OP150557	OP150634	OP150711	OP150787
D. subordinaria	CBS 464.90^T	KC343214	KC343940	KC344182	KC343456	KC343698
D. subordinaria	CBS 101711	KC343213	KC343939	KC344181	KC343455	KC343697
D. tarchonanthi	CBS 146073^T	MT223794	N/A	MT223733	N/A	MT223759
D. tecomae	CBS 100547^T	KC343215	KC343941	KC344183	KC343457	KC343699
D. tectoendophytica	MFLUCC 13-0471^T	KU712439	KU749367	KU743986	KU749354	N/A
D. tectonendophytica	LC8115	KY491550	KY491560	KY491570	N/A	N/A
D. terebinthifolii	CBS 133180^T	KC343216	KC343942	KC344184	KC343458	KC343700
D. terebinthifolii	LGMF907	KC343217	KC343943	KC344185	KC343459	KC343701
*D. thailandica*	MFLUCC 24-0523 ^T	OR946374	PQ774276	PQ774283	N/A	PQ774292
*D. thailandica*	MFLUCC 23–0299	PQ777475	PQ774275	PQ774282	PQ774289	N/A
D. thunbergiicola	MFLUCC 12-0033^T	KP715097	KP715098	N/A	N/A	N/A
D. tulliensis	BRIP 62248a^T	KR936130	KR936133	KR936132	N/A	N/A
D. tulliensis	JZB320128	MK335814	MK523573	MK500152	MK500240	N/A
D. tulliensis	MFLUCC 14-1139	KU712438	KU749366	KU743985	KU749353	N/A
*D. tulliensis*	MFLUCC 24-0524	PQ777478	PQ774280	PQ774287	N/A	PQ774294
*D. tulliensis*	MFLUCC 23–0301	PQ777477	PQ774279	PQ774286	PQ774291	N/A
D. ueckerae	FAU656^T	KJ590726	KJ590747	KJ610881	KJ612122	KJ659215
D. ueckerae	BRIP 54736j	KJ197282	KJ197244	KJ197262	N/A	N/A
D. unshiuensis	ZJUD50^T	KJ490585	KJ490464	KJ490406	N/A	KJ490527
D. unshiuensis	PSCG339	MK626928	MK654879	MK691300	MK691181	MK726188
D. vangoghii	MF Ha18-045^T	MW008491	MW008513	MW008502	MZ671936	MZ671962
D. vangoghii	MF Ha18-046	MW008492	MW008514	MW008503	MZ671937	MZ671963
D. vargemgrandensis	URM 8784^T	PP192069	PP430456	PP421092	PP421068	PP421135
D. vexans	CBS 127.14	KC343229	KC343955	KC344197	KC343471	KC343713
D. vexans	FAU597	KJ590734	KJ590774	KJ610889	KJ612131	KJ659216
D. vochysiae	LGMF1583^T	MG976391	MK007526	MK007527	MK007528	MK033323
D. yunnanensis	CGMCC3.18289^T	KX986796	KX999188	KX999228	KX999290	KX999267
D. yunnanensis	LC8107	KY491542	KY491552	KY491562	KY491572	N/A

Table 2.

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XLSX

GenBank accession numbers of the taxa used in the phylogenetic analyses of Xylariales.

Species	Strain	ITS	LSU	rpb2	tub2
Albicollum vincensii	CBS 147286^T	ON869297	ON869297	ON808475	ON808519
Amphirosellinia nigrospora	HAST 91092308^T	GU322457	N/A	GQ848340	GQ495951
Anthostomella helicofissa	MFLUCC 14-0173^T	MW240653	MW240583	KP340534	KP406617
Anthostomella lamiacearum	MFLU18-0101^T	MW240669	MW240599	MW658648	N/A
Anthostomelloides brabeji	CBS 110128	EU552098	EU552098	N/A	N/A
Anthostomelloides krabiensis	MFLUCC 15-0678^T	KX305927	KX305928	KX305929	N/A
Anthostomelloides leucospermi	CBS 110126^T	EU552100	EU552100	N/A	N/A
Barrmaelia macrospora	CBS 142768^T	KC774566	KC774566	MF488995	MF489014
Biscogniauxia nummularia	MUCL 51395^T	KY610382	KY610427	KY624236	KX271241
Chaetomium elatum	CBS 374.66	KC109758	KC109758	KF001820	KC109776
Circinotrichum circinatum	CBS 148326	ON400743	ON400796	ON399328	N/A
Circinotrichum maculiforme	CBS 140016^T	KR611874	KR611895	ON399338	N/A
Clypeosphaeria mamillana	CBS 140735^T	KT949897	KT949897	MF489001	MH704637
Clypeosphaeria mamillana	WU 33599	KT949898	KT949898	N/A	N/A
Clypeosphaeria oleae	CPC 36779	MN562130	MN567637	N/A	N/A
Coniocessia maxima	CBS 593.74^T	GU553332	MH878275	N/A	N/A
Coniocessia nodulisporioides	CBS 281.77^T	MH861061	MH872831	N/A	N/A
Dematophora bunodes	CBS 124028	MN984619	MN984625	N/A	MN987245
Didymobotryum rigidum	JCM 8837^T	LC228650	LC228707	N/A	N/A
Digitodochium amoenum	CBS 147285^T	ON869303	ON869303	ON808481	ON808525
Digitodochium rhodoleucum	NBRC 32296	LC146732	LC146732	N/A	N/A
Emarcea castanopsidicola	CBS 117105	AY603496	MK762717	MK791285	MK776962
Emarcea eucalyptigena	CBS 139908	KR476733	MK762718	MK791286	MK776963
Entalbostroma erumpens	ICMP 21152^T	KX258206	N/A	KX258204	KX258205
Entoleuca mammata	JDR 100	GU300072	N/A	GQ844782	GQ470230
Entosordaria perfidiosa	CBS 142773^T	MF488993	MF488993	MF489003	MF489021
Fasciatispora arengae	MFLUCC 15-0326a^T	MK120275	MK120300	MK890794	MK890793
Fasciatispora cocoes	MFLUCC 18-1445^T	MN482680	MN482675	MN481517	MN505154
Graphostroma platystomum	CBS 270.87^T	JX658535	DQ836906	KY624296	HG934108
Gyrothrix verticillata	CBS 148806	ON400759	ON400813	ON399318	N/A
Halorosellinia krabiensis	MFLU 17-2596^T	MN047119	MN017883	N/A	MN431493
Hansfordia pruni	CBS 194.56^T	MK442585	MH869122	KU684307	N/A
Hansfordia pulvinata	CBS 144422	MK442587	MK442527	N/A	N/A
Helicogermslita clypeata	MFLU 18-0852^T	MW240666	MW240596	MW658647	MW775614
Hypocopra rostrata	NRRL 66178	KM067909	KM067909	N/A	N/A
Hypocopra zeae	MFLU 18-0809^T	MW240671	MW240601	MW658650	MW775616
Hypocreodendron sanguineum	J.D.R.169^T	GU322433	N/A	GQ844819	GQ487710
Hypomontagnella monticulosa	MFLUCC 18-0362	MN337231	MN336235.2	MN366246	MN509783
Muscodor thailandicus	MFLUCC 17-2669	MK762707	MK762714	MK791283	MK776960
Muscodor ziziphi	MFLUCC 17-2662	MK762705	MK762712	MK791281	MK776958
Jackrogersella multiformis	CBS 119016^T	KC477234	KY610473	KY624290	KX271262
Kretzschmaria deusta	CBS 163.93	KC477237	KY610458	KY624227	KX271251
Kretzschmariella culmorum	JDR 88	KX430043	N/A	KX430045	KX430046
Linosporopsis ischnotheca	CBS 145761^T	MN818952	MN818952	MN820708	MN820715
Magnostiolata mucida	MFLU 19-2133^T	MW240673	MW240603	MW658652	MW775618
Melanographium phoenicis	MFLUCC 18-1481^T	MN482677	MN482678	N/A	N/A
Melanographium smilacis	MFLU 21-0075^T	MZ538514	MZ538548	N/A	N/A
Nemania serpens	HAST 235	GU292820	N/A	GQ844773	GQ470223
Neoanthostomella fici	MFLU 19-2765^T	MW114390	MW114445	MW177711	N/A
Neoxylaria juruensis	HAST 92042501	GU322439	N/A	GQ844825	GQ495932
Nigropunctata bambusicola	MFLU 19-2145^T	MW240664	MW240594	MW658646	N/A
Nigropunctata nigrocircularis	MFLU 19-2130^T	MW240661	MW240591	N/A	MW775612
Occultitheca ananasi	MFLU 23-0251^T	OR438426	OR438886	N/A	N/A
Occultitheca ananasi	MFLUCC 23-0120	OR438427	OR438887	OR634962	OR538094
*Occultitheca chiangraiensis*	MFLU 24-0414 ^T	PQ777479	PQ778042	PQ774295	PQ774288
Occultitheca rosae	HKAS 102393^T	MW240672	MW240602	MW658651	MW775617
Podosordaria mexicana	WSP 176	GU324762	N/A	GQ853039	GQ844840
Poronia punctata	CBS 656.78^T	KT281904	KY610496	KY624278	KX271281
Pseudoanthostomella pini-nigrae	MFLUCC 16-0478^T	KX533453	KX533454	KX789492	N/A
Pseudoceratocladium polysetosum	FMR 10750^T	KY853430	KY853490	ON399348	N/A
Rosellinia chiangmaiensis	MFLUCC 15-0015^T	KU246226	KU246227	N/A	N/A
Rosellinia lamprostoma	HAST 89112602	EF026118	N/A	GQ844778	EF025604
Sarcoxylon compunctum	CBS 359.61	KT281903	KT281898	KY624230	KX271255
Sordaria fimicola	CBS 723.96	MH862606	MH874231	DQ368647	N/A
Spiririma gaudefroyi	CBS 147284^T	ON869320	ON869320	ON808497	ON808541
Spirodecospora melnikii	MAFF 247746^T	LC731937	LC731946	LC731955	N/A
Spirodecospora paulospiralis	MAFF 247749^T	LC731940	LC731949	LC731957	N/A
Stromatoneurospora phoenix	BCC 82040	MT703666	MT735133	MT742605	MT700438
Vamsapriya indica	MFLUCC 12-0544	KM462839	KM462840	KM462841	KM462838
Xenoanthostomella chromolaenae	MFLUCC 17-1484^T	MN638863	MN638848	MN648729	N/A
Xenoanthostomella cycadis	CBS 137969^T	KJ869121	KJ869178	ON399350	N/A
Xylaria acuminatilongissima	HAST 95060506^T	EU178738	N/A	GQ853028	GQ502711
Xylaria arbuscula	CBS 126415	KY610394	KY610463	KY624287	KX271257
Xylaria botuliformis	HAST 89091627	MN089652	N/A	MN095399	MN095400
Xylaria brunneovinosa	HAST 720^T	EU179862	N/A	GQ853023	GQ502706
Xylaria ellisii	DAOM 628556^T	MN218820	MN218817	MN216186	N/A
Xylaria eucalypti	CPC 36723	MN562127	MN567634	N/A	N/A
Xylaria hypoxylon	CBS 122620^T	AM993141	KM186301	KM186302	KM186300

Alignments and phylogenetic analyses

Consensus sequences were assembled using Geneious Prime 2025 (Biomatters Ltd., Auckland, New Zealand) and were used for BLASTn search against the NCBI nucleotide non-redundant database (Sayers et al. 2022). For Diaporthe, sequences were downloaded from GenBank (Table 1) following the classification in Dissanayake et al. (2024). For Xylariales, related sequences were downloaded from GenBank (Table 2) based on recent publications (Samarakoon et al. 2022; Sugita et al. 2022; Voglmayr et al. 2022). The sequences were aligned using MAFFT version 7 (Katoh et al. 2019) with minimal adjustment of any ambiguous nucleotides using AliView version 1.26 (Larsson 2014). The alignments were concatenated using SequenceMatrix version 1.8 (Vaidya et al. 2011).

Maximum likelihood analyses (ML), including 1000 bootstrap pseudoreplicates, were performed at the CIPRES web portal (Miller et al. 2017) using RAxML version 8.2.12 (Stamatakis 2014). The general time reversible (GTR) model with a discrete gamma distribution plus invariant site (GTR + I + G) was used as the nucleotide substitution model. The best model for each gene was determined in JModelTest version 2.1.10 (Darriba et al. 2012) for the Bayesian analysis. The Bayesian inference posterior probabilities (BPP) distribution (Zhaxybayeva and Gogarten 2002) was estimated by Markov Chain Monte Carlo sampling (MCMC) in MrBayes version 3.2.2 on XSEDE (Ronquist et al. 2012) with four runs of MCMC for 1,000,000 generations, sampling trees every 100^th generation. The first 25% of trees were excluded as burn-in, and the remaining trees were used to calculate posterior probabilities (BPP). The trees were visualised using FigTree version 1.4.4 (Rambaut 2012) and edited using Adobe Illustrator® CS6 (Adobe Systems, USA).

Genealogical concordance phylogenetic species recognition analysis

The closely related strains were further analysed using the genetic distances by performing a pairwise homoplasy index test (Φw) (Taylor et al. 2000; Bruen et al. 2006). A pairwise homoplasy index (PHI) test was performed in SplitsTree (CE 6.0.0) using Kimura’s two-parameter (K2P) models for low genetic distance datasets (Huson and Bryant 2024). LogDet transformation was applied for the average of nucleotide frequencies and splits decomposition graph options (Gu and Li 1996a, b; Taylor et al. 2000; Bruen et al. 2006; Huson and Bryant 2006; Gioan and Paul 2012; Nishimaki and Sato 2019). The standard deviation of split frequencies PHI test result (Φw) < 0.05 indicates significant recombination within the dataset.

Results

Phylogenetic analyses of section sojae

The phylogeny represents taxa from section sojae based on the concatenated dataset of ITS, tef1, tub2, cal, and his3 sequences. The combined sequence alignment comprised 159 taxa with 3279 characters, including gaps (ITS: 1–587, tef1: 588–1131, tub2: 1132–2067, cal: 2068–2682, his3: 2683–3279). The ML and BI analyses showed similar topologies (Fig. 1). The best scoring ML tree had a final likelihood value of -47785.673. The matrix had 1463 constant sites, 1408 parsimony informative sites, and 2202 distinct site patterns. Estimated base frequencies were as follows: A = 0.213, C = 0.325, G = 0.239, T = 0.224, substitution rates: AC = 1.16629, AG = 3.42597, AT = 1.16629, CG = 1.00000, CT = 4.44139, GT = 1.000, gamma distribution shape parameter = 0.954, and tree length = 7.506.

Figure 1.

Phylogram of the Diaporthe sojae species complex generated from a maximum likelihood analysis based on the combined ITS, tef1, tub2, cal, and his3 sequence data. Diaporthella corylina (CBS 121124) was used as the outgroup. Bootstrap support values ≥ 60% ML/≥0.90 BPP are given at the nodes. The newly generated taxa are indicated in red. The holotype/ex-type strains are denoted with ^T.

Sordariomycetes O.E. Erikss. & Winka

Diaporthales Nannf.

Diaporthaceae Höhn. ex Wehm.

Diaporthe melonis Beraha & M.J. O’Brien, Phytopath. Z. 94(3): 205 (1979)

Index Fungorum: IF312933

Facesoffungi Number: FoF17285

Fig. 2

Description.

Saprobic on dead unidentified branch. Sexual morph: not observed. Asexual morph: Conidiomata 148–374 × 128–338 µm high (x̄ = 250 × 225 µm, n = 15), pycnidial, mostly scattered, immersed, slightly erumpent through the host surface, discoid or subglobose, with a solitary undivided locule. Conidiophores reduced to conidiogenous cells. Alpha conidiogenous cells 5.7–25 × 1.1–2.5 µm (x̄ = 15.6 × 1.7 µm, n = 50), hyaline, rarely branched, mostly aseptate, densely aggregated, cylindrical, straight to slightly curved and smooth. Alpha conidia 5–7.3 × 1.9–2.7 µm (x̄ = 6.3 × 2.3 µm, n = 40), unicellular, fusiform to ellipsoidal, apex and base rounded, hyaline, smooth, bi-guttulate. Beta conidiogenous cells 6.2–16 × 1.6–2.6 µm (x̄ = 9.4 × 2.1 µm, n = 40), phialidic, subcylindrical, tapering towards the apex, hyaline. Beta conidia 19–27 × 1–2 µm (x̄ = 23 × 1.5 µm, n = 40), filiform, aseptate, hyaline, smooth-walled, straight from base, and curve at apex. Gamma conidia not observed.

Culture characteristics.

Colonies on PDA, reaching 20 mm diam., after 3 weeks at 25 °C, initially white, turning beige after 7–10 days, flat, felty with a thick texture at the centre and marginal area, lacking aerial mycelium; reverse, glossy grey, radiating outwardly.

Material examined.

Thailand, Chiang Rai Province, Muang District, on a dead unidentified dicot branch, 16 January 2023, J. Louangphan, CR1-02 (MFLU 23–0474); living culture MFLUCC 24–0522 = MFLUCC 23–0300.

Hosts.

Annona squamosa (Annonaceae), Berberis aristata (Berberidaceae), Carapa guianensis (Meliaceae), Citrus grandis cv. Tomentosa (Rutaceae), Cucumis melo (Cucurbitaceae), Glottidium sp. (Fabaceae), Glycine max, G. soja (Fabaceae), unidentified branch (Dong et al. 2021a, b; Hongsanan et al. 2023; This study).

Figure 2.

Diaporthe melonis (MFLU 23–0474) a host substrate b conidiomata on substrate c transverse section of conidioma d, e vertical section through conidiomata f, g conidiophores and conidiogenous cells h, i beta conidia j alpha conidia k a germinated conidium l front and reverse view of the colony on PDA. Scale bars: 200 μm (b–d); 100 μm (e); 20 μm (f, k); 10 μm (g, h–j).

Distribution.

China, Myanmar, India, Indonesia, Japan, Thailand, the United States (Dong et al. 2021a, b; Hongsanan et al. 2023; this study).

Notes.

Our isolates (MFLUCC 23–0300 and MFLUCC 24–0522) clustered with D. melonis isolates (CBS 507.78, FAU640, and ZHKUCC 20-0014) with 100% ML/1.00 BPP support (Fig. 1). Our isolate has a similar morphology to D. melonis but differs in having smaller conidiomata (148–374 µm vs. 100–500 µm diam.) and smaller alpha conidia (6.3 × 2.3 µm vs. 8.3 × 2.6 µm) (Beraha and O’Brien 1979). Our isolate has a beige culture compared to the brown culture of D. melonis (Beraha and O’Brien 1979). Our isolate also differs from D. melonis (D. guangdongensis ZHKUCC 20-0014) in the size of conidiomata (128–338 × 148–374 µm vs. 130–515 × 100–390 µm), alpha conidia (5–7.3 × 1.9–2.7 µm vs. 6–8 × 2–4 µm), and beta conidia (19–27 × 1–2 µm vs. 14–35 × 1–2 µm) (Dong et al. 2021a, b). Therefore, we report our isolate as a new geographical record of D. melonis from Thailand.

Diaporthe thailandica Louangphan, Phukhams., K.D. Hyde & Bhunjun, sp. nov.

Index Fungorum: IF903043

Facesoffungi Number: FoF17286

Figs 3, 4

Etymology.

The name refers to the country where the holotype was collected.

Holotype.

MFLU 23–0473.

Description.

Saprobic on decaying dicot, visible as black necks immerging through the host surface. Sexual morph: Ascomata 328–495 × 303–371 µm (x̄ = 400 × 343 µm, n = 10), immersed in the host epidermis, globose to sub-globose, solitary or occur in clusters, black, ostiolate, papillate. Ostiole neck 220 × 86 µm, long, filled with periphysate. Peridium 20–50 µm wide, composed of several layers of cells of textura angularis, outer layers dark brown and inner layers hyaline to brown, thin-walled. Paraphyses 3.2–6.6 µm (n = 20), thin-walled, 2–4-septate, hyaline, wide at base, tapering towards the apex. Asci 45–58.9 × 8.6–12.7 µm (x̄ = 51.5 × 10.5 µm, n = 40), unitunicate, 8-spored, clavate to subclavate, straight to slightly curved, sessile, with a J-, apical ring. Ascospores 11–15.5 × 3.9–5.6 µm (x̄ = 13.5 × 4.7 µm, n = 40) L/W = 2.8, overlapping uniseriate to biseriate, 1-septate, constricted at the septum, ellipsoidal, smooth-walled, 2–4-guttulate, straight, hyaline, without appendages or a mucilaginous sheath. Asexual morph on PDA: Conidiomata 500–700 × 300–600 µm (x̄ = 580 × 480 µm, n = 10), pycnidial, scattered or aggregated, globose or variable in shape, ostiolate with prominent neck, dark brown to black, pycnidal wall brown, consisting of thick-walled cells of textura angularis, conidial mass globose, initially hyaline to yellowish, becoming white to cream, conidial droplets exuding from central ostioles. Conidiophores 10–29.5 × 1.3–2.5 μm (x̄ = 16.9 × 1.9 µm, n = 40), ampulliform to subcylindrical, filiform, branched to unbranched, 1–3-septate, hyaline, smooth, straight or slightly curved, wider at base, tapering towards the apex. Conidiogenous cells 2.1–8.1 × 1–2.3 μm (x̄ = 4.2 × 1.5 µm, n = 40), subcylindrical, filiform, straight to curved, tapering towards the apex, collarette not flared, hyaline. Alpha conidia 5.3–8.8 × 2.3–3.5 μm (x̄ = 7.3 × 2.9 μm, n = 40), ellipsoid, apex bluntly rounded, base obtuse to subtruncate, smooth, hyaline, bi- to multi-guttulate. Beta conidia 8.5–18.5 × 1.2–2 μm (x̄ = 13.3 × 1.7 μm, n = 40), filiform, flexible to slightly curved, hyaline, base subtruncate, and aseptate. Gamma conidia not observed.

Figure 3.

Diaporthe thailandica (MFLU 23–0473, holotype) a host substrate b, c ascomata on host substrate d vertical section through ascoma e peridium f ostiole g hamathecium h–j asci k–n ascospores o a germinated ascospore p front and reverse view of the colony on PDA. Scale bars: 200 μm (b); 100 μm (c, d); 20 μm (e–j); 10 μm (k–o).

Figure 4.

Diaporthe thailandica (Asexual morph, MFLUCC 24–0523) a culture on pda b, c conidiomata sporulating on pda d, e conidiogenous cells giving rise to conidia f alpha conidia g beta conidia h alpha and beta conidia. Scale bars: 500 µm (b, c); 10 µm (d–h).

Culture characteristics.

Colonies on PDA, reaching 40 mm diam., after 2 weeks at 25 °C, initially white, turning pale brown after 7–10 days, radiating to the edge, margin undulate, medium dense, flat or umbonate; reverse, cream, radiating white outwardly with grey patches.

Material examined.

Thailand, Chiang Rai Province, Muang District, on a dead unidentified dicot, 16 January 2023, J. Louangphan, CR1-09 (MFLU 23–0473, holotype); ex-type MFLUCC 24–0523 = MFLUCC 23–0299.

Host.

Unidentified branch (this study).

Distribution.

Thailand (this study).

Notes.

Diaporthe thailandica (MFLUCC 23-0299 and MFLUCC 24–0523) formed a sister clade with isolates of D. raonikayaporum (CBS 133182, MFLUCC 14–1133, and MFLUCC 14–1136) with 100% ML/1.00 BPP support (Fig. 1). Diaporthe thailandica differs from D. raonikayaporum in its conidiomata (500–700 × 300–600 µm vs. 110–200 × 50–130 μm), conidiophores (10–29.5 × 1.3–2.5 μm vs. 16–26 × 2–3 μm), conidiogenous cells (2.1–8.1 × 1–2.3 μm vs. 5–10 × 2–3 μm), and beta conidia (8.5–18.5 vs. 7–13 μm) (Gomes et al. 2013). Diaporthe thailandica differs from D. raonikayaporum (=D. neoraonikayaporum MFLUCC 14–1133) in its conidiomata (500–700 × 300–600 µm vs. 690–1190 × 805–1285 μm), conidiophores (10–29.5 μm vs. 15–23 μm), alpha conidia (5.3–8.8 × 2.3–3.5 μm vs. 4–6 × 2–3 μm), and beta conidia (8.5–18.5 μm vs. 13–21 μm) (Doilom et al. 2017). Gamma conidia was observed in D. raonikayaporum (= D. neoraonikayaporum) but not in D. thailandica (Doilom et al. 2017). Diaporthe thailandica further differs from D. raonikayaporum, which has only been reported as an asexual morph (Gomes et al. 2013; Doilom et al. 2017). Our strain differs significantly (> 2.5%) compared to the sequence data of D. raonikayaporum (Table 3). However, our isolate does not have cal sequence data, while his3 sequence data is not available for D. raonikayaporum (MFLUCC 14–1133 and MFLUCC 14–1136). A pairwise homoplasy index showed Φw = 1.0 when a genealogical correlation model was applied between Diaporthe thailandica and D. raonikayaporum (Fig. 5). Thus, Diaporthe thailandica is reported as a new species based on morphology and molecular evidence.

Figure 5.

The splits graph from the pairwise homoplasy index (PHI) test generated from the combined ITS, tef1, tub2, cal, and his3 sequence data of Diaporthe thailandica (indicated in red) and closely related taxa using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicate significant recombination within the dataset.

Table 3.

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Pairwise comparison of the sequences of Diaporthe thailandica and D. raonikayaporum isolates (excluding gaps).

Sequences	D. raonikayaporum (CBS 133182)	D. raonikayaporum (MFLUCC 14-1133)	D. raonikayaporum (MFLUCC 14-1136)
ITS	3.4% (18/529)	2.6% (14/529)	3.0% (16/529)
tef1	6.5% (21/323)	7.4% (24/323)	8.4% (27/323)
tub2	4.3% (18/415)	6.5% (27/415)	7.7% (32/415)
cal	−	−	−
his3	3.7% (17/450)	−	−

Diaporthe tulliensis R.G. Shivas, Vawdrey & Y.P. Tan, Persoonia 35: 301 (2015)

Index Fungorum: IF812896

Facesoffungi Number: FoF16300

Fig. 6

Description.

Saprobic on decaying Bambusa. Sexual morph: Undetermined. Asexual morph: Conidiomata 91–148 × 311–974 μm (x̄ = 120 × 583 μm, n = 20), pycnidial, scattered or aggregated, embedded in host surface, slightly erumpent through host surface, 1–3-locular conidioma, nearly flat, elongated, discoid, or variable in shape, black, consisting of hyaline, thin-walled cells of textura angularis, outer layer thick walled. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 4–16 × 1.2–2.6 μm (x̄ = 7.5 × 1.8 μm, n = 40), cylindrical, unbranched, aseptate, smooth, straight or slightly curved, tapering towards the apex, wider at base, hyaline. Alpha conidia 4.1–7.8 × 1.7–3.1 μm (x̄ = 5.8 × 2.5 μm, n = 40), apex bluntly rounded, 1–2-guttulate, mostly bi-guttulate, oval or oblong to ellipsoid, hyaline, smooth, base obtuse to subtruncate. Beta and Gamma conidia not observed.

Figure 6.

Diaporthe tulliensis (MFLU 23–0475) a, b conidiomata on host c longitudinal section through conidioma d, e transverse section of conidioma f, g conidiogenous cells giving rise to conidia h, i alpha conidia j, k germinated conidia l front and reverse view of the colony on PDA. Scale bars: 200 µm (b–d); 20 µm (e); 10 µm (f–k).

Culture characteristics.

Colonies on PDA, reaching 40 mm diam., after 2 weeks at 25 °C, initially white, turning olivaceous grey after 7–10 days, darker at the centre and marginal area, lacking aerial mycelium; reverse, olivaceous grey bordered by dark margins.

Material examined.

Thailand, Chiang Mai Province, Mae Taeng District, on dead terrestrial stem of Bambusa (Poaceae), 19 November 2022, J. Louangphan, MJ11 (MFLU 23–0475); living culture, MFLUCC 24–0524 = MFLUCC 23–0301.

Hosts.

Actinidia spp. (Actinidiaceae), Alangium kurzii (Cornaceae), Bambusa sp. (Poaceae), Bougainvillea glabra (Nyctaginaceae), Celtis formosana (Ulmaceae), Morinda officinalis (Rutaceae), Tectona grandis (Lamiaceae), Theobroma cacao (Malvaceae), Soil, Vitis vinifera (Vitaceae) (Chang et al. 2005; Crous et al. 2015; Bai et al. 2017; Doilom et al. 2017; Yang et al. 2018; Manawasinghe et al. 2019; Tennakoon et al. 2021; Luo et al. 2022; this study).

Distribution.

Australia, China, Korea, Thailand (Chang et al. 2005; Crous et al. 2015; Bai et al. 2017; Doilom et al. 2017; Yang et al. 2018; Manawasinghe et al. 2019; Tennakoon et al. 2021; Luo et al. 2022; this study).

Notes.

In the phylogenetic analysis, our isolates (MFLUCC 23–0301 and MFLUCC 24–0524) clustered with D. tulliensis isolates (MFLUCC 14–1139, JZB320128, and BRIP 62248a) with 100% ML/1.00 BPP support (Fig. 1). Our isolate has a similar morphology to D. tulliensis isolates but differs from D. tulliensis in the size of conidiomata (up to 500 µm (= D. celtidis and D. tulliensis) vs. up to 510 µm (= D. hubeiensis) vs. 135–330 μm (= D. alangii) vs. 50–380 μm (= D. morindae) vs. 725–820 μm diam. (= D. tectonae)) (Crous et al. 2015; Doilom et al. 2017; Yang et al. 2018; Manawasinghe et al. 2019; Luo et al. 2022). Our isolate also differs due to the absence of beta conidia, which has been reported in some D. tulliensis isolates (Chang et al. 2005; Crous et al. 2015; Doilom et al. 2017; Manawasinghe et al. 2019). Therefore, we report our isolate as a new host record of D. tulliensis.

Phylogenetic analyses of Xylariales

The phylogeny represents selected taxa in Xylariales based on the concatenated dataset of ITS, LSU, rpb2, and tub2 sequences. The combined sequence alignment comprised 78 strains with 3624 characters, including gaps (ITS: 1–578, LSU: 579–1432, rpb2: 1433–2573, tub2: 2574–3624). The ML and BI analyses of single and multi-gene showed similar topologies. The best scoring ML tree with a final likelihood value of -54607.950 (Fig. 7). The matrix had 1992 constant sites, 1349 parsimony informative sites, and 2026 distinct site patterns. Estimated base frequencies were as follows: A = 0.2405, C = 0.2618, G = 0.2637, T = 0.2337, substitution rates: AC = 1.4137, AG = 3.9195, AT = 1.4543, CG = 1.1208, CT = 7.4615, GT = 1.000, gamma distribution shape parameter = 0.759631, and tree length = 7.506.

Figure 7.

Phylogram generated from maximum likelihood analyses based on combined ITS-LSU-rpb2-tub2 datasets. The tree is rooted with Chaetomium elatum (CBS 374.66) and Sordaria fimicola (CBS 723.96) as the outgroup taxa. Bootstrap support values ≥ 60% ML/≥0.90 BPP are given at the nodes. The newly generated taxa are indicated in red. The holotype/ex-type strains are denoted with ^T.

Xylariales Nannf.

Xylariaceae Tul. & C. Tul.

Occultitheca J.D. Rogers & Y.M. Ju

Occultitheca chiangraiensis Louangphan, Phukhams., K.D. Hyde & Bhunjun, sp. nov.

Index Fungorum: IF903235

Facesoffungi Number: FoF17287

Fig. 8

Etymology.

The name refers to the province where the holotype was collected.

Holotype.

MFLU 24–0414.

Description.

Saprobic on early decaying branch. Sexual morph: Ascomata 220–342 × 228–395 μm (x̄ = 290 × 324 μm, n = 15), immersed, solitary, scattered, globose to subglobose, erumpent through host surface, visible as black dot of ostiole, surrounded by a whitish halo. Clypeus carbonaceous, rudimentary, thick-walled, the ostiolar opening surrounded with black cells. Ostioles centric, ostiolar canal periphysate. Peridium 17–30 μm (x̄ = 23 μm, n = 20) wide, tightly attached to the host tissue, with two cell layers, outer layer thick-walled, comprising yellowish brown cells of textura angularis, inner layer thin, composed of hyaline cells of textura angularis. Paraphyses 3.7–7.6 μm (x̄ = 5.4 μm, n = 25) wide, wider at the base, longer than the asci, filamentous, septate, constricted at the septa, embedded in gelatinous matrix. Asci 112–158 × 8.5–13.7 μm (x̄ = 131 × 11 μm, n = 25), 8-spored, unitunicate, cylindrical, short pedicellate, apically rounded, with 3.9–5.6 × 2.5–3.7 μm (x̄ = 5 × 3 μm, n = 18), rectangular to slightly obconic, apical ring, J+ in Melzer’s reagent. Ascospores 14.5–17.6 × 6–7.4 μm (x̄ = 16.3 × 6.7 μm, n = 30), L/W 2.4, oblong to ellipsoidal, uniseriate, brown, inequilaterally unicellular, apical cell 13–15.5 μm (x̄ = 14.5 μm, n = 30) long, usually with large guttules, brown cell with a mucilaginous sheath covering most of the spore length when mature, with a small, hyaline, rounded, basal cell, 1.3–2.3 μm (x̄ = 1.8 μm, n = 30), lack of germ slit. Asexual morph: Undetermined.

Figure 8.

Occultitheca chiangraiensis (MFLU 24–0414, holotype) a host substrate b ascomata in host surface c, d horizontal and vertical section of ascoma e section through ascoma f peridium g ostiole h apical ring stained with melzer’s reagent i paraphyses j–m immature and mature asci n–r ascospores (r ascospores show mucilaginous sheath in indian ink) s a germinated spore t front and reverse view of the colony on PDA. Scale bars: 200 μm (b); 100 μm (c–e); 20 μm (f, g, i); 10 μm (h, n–s); 50 μm (j–m).

Culture characteristics.

Colonies on PDA, reaching 40 mm diam., after 21 days at 25 °C, circular, entire edge, smooth surface, flat, slightly woolly, smooth margin, above ash white from the centre to white at the edge with concentric rings of woolly; from below: light brown at the centre, white at the margin, with ash white mycelium.

Material examined.

Thailand, Chiang Rai, Mae Fah Luang District, Mae Salong Nok, on a dead unidentified dicot branch, 16 January 2023, J. Louangphan, CR1–19 (MFLU 24–0414, holotype); ex-type MFLUCC 25–0158.

Host.

Unidentified branch (this study).

Distribution.

Thailand (this study).

Notes.

Based on multi-gene phylogenetic analyses of ITS, LSU, rpb2, and tub2 sequences, Occultitheca chiangraiensis (MFLU 24–0414) clustered with O. rosae (HKAS 102393) and Clypeosphaeria oleae (CPC 36779) with 100% ML and 1.00 BPP support (Fig. 7). Clypeosphaeria oleae was reported only from the asexual morph (Crous et al. 2019); thus, we could not compare the morphology between the species as we could not obtain the asexual morph of our strain, and therefore the link between them cannot be confirmed. Furthermore, C. oleae lacks rpb2 and tub2 data, which is important to confirm its phylogenetic placement. Morphologically, Occultitheca chiangraiensis fits the generic concept of Occultitheca in having immersed ascomata, short pedicellate asci with a J+, apical ring, a long distance between the ascus apex and the uppermost ascospore, and hyaline basal cells attached to brown ascospores (Rogers and Ju 2003; Samarakoon et al. 2022). Our isolate was compared to Occultitheca species as detailed in Table 4. Occultitheca chiangraiensis differs from O. rosae by having smaller ascomata (x̄ = 290 × 324 μm vs. 370 × 385 μm), a lack of a germ slit, and possesses a thicker mucilaginous sheath compared to O. rosae (Samarakoon et al. 2022). Our strain differs from O. ananasi, which has uniseriate, olive-greenish ascospores becoming 2-seriate in the middle and thin mucilaginous sheath (Tian et al. 2024). Occultitheca chiangraiensis was also compared to the type species O. costaricensis as it lacks molecular data. Occultitheca chiangraiensis differs by having 1–2 individual ascomata and ascospores with a mucilaginous sheath, while O. costaricensis has 2–12 ascomata in a cluster and ascospores without a sheath (Rogers and Ju 2003). Additionally, some Anthostomella species have similar characteristics in terms of ascoma, asci, and ascospores with draft cells and lack germ slits, such as A. clypeata and A. clypeoides, but differ in a short space of the top ascospore and the ascus apex and shape of the sheath compared to Occultitheca species (Lu and Hyde 2000; Rogers and Ju 2003). Our strain differs by 6% in the ITS region (30/482, 4 gaps), 2% in LSU (14/745, 4 gaps), 15% in rpb2 (117/798, no gap), 16% in tub2 (120/754, 22 gap), and 3% in tef1 (31/915, 1 gap) sequences compared to O. rosae (HKAS 102393). Thus, Occultitheca chiangraiensis is reported as a new species based on morphology and phylogenetic evidence.

Table 4.

Download as

CSV

XLSX

Synopsis of Occultitheca species.

Species	O. costaricensis	O. rosae	O. ananasi	O. chiangraiensis
Host	Unidentified decaying wood	Rosa sp.	Ananas comosus	Unidentified decaying wood
Country	Costa Rica	China	Thailand	Thailand
Ascomata (μm)	400–600	360–385 × 350–420	190–230 × 160–260	220–342 × 228–395
Peridium (μm)	−	18–25	15–20	17–30
Paraphyses (μm)	−	3–6.5	3–5	3.7–7.6
Asci (μm)	185–190 × 10–10.5	90–140 × 11–13	70–90 × 5–10	112–158 × 8.5–13.7
Apical ring (μm)	6 × 3	3.5–4.5 × 2.8–3.2	−	3.9–5.6 × 2.5–3.7
Ascospores (μm)	14.5–23.5 × 7–10.5	16.5–20 × 6.5–8 L/W 2.6	10–12.5 × 3.5–4.5 L/W 2.9	14.5–17.6 × 6–7.4 L/W 2.4
Basal cell (μm)	1.5–4.5	1.5–2.2	−	1.3–2.3
Spore sheath	No	Thin	Thin	One side thick
Germ slit	Straight	Straight	Straight	No
References	Rogers and Ju (2003)	Samarakoon et al. (2022)	Tian et al. (2024)	This study

Discussion

In this study, two novel species, Diaporthe thailandica and Occultitheca chiangraiensis, along with a new host record of D. tulliensis and a new geographical record of D. melonis, are introduced based on morphology and molecular data. This study expands the known diversity of these taxa and highlights the importance of saprobic microfungi in ecological systems.

Diaporthe is a species-rich genus with a diverse host range and global distribution (Dissanayake et al. 2017; Phukhamsakda et al. 2020; Hongsanan et al. 2023; Bhunjun et al. 2024b). The species have overlapping morphological traits. There are more than 1200 epithets under Diaporthe in Index Fungorum (2024); thus, the boundaries of the species/species complexes within the genus have been revised by several studies. Diaporthe was recently restructured into seven sections and 15 species complexes based on molecular analyses. Several Diaporthe species have been synonymised under D. tulliensis (D. alangii, D. celtidis, D. glabrae, D. hubeiensis, D. morindae, and D. tectonae), and these taxa formed a clade in our trees (including in the backbone tree with 226 taxa; data not shown), similar to Dissanayake et al. (2024). Diaporthe melonis (= D. guangdongensis) also formed a clade in our trees (including in the backbone tree; data not shown), similar to Dissanayake et al. (2024). The implementation of the markers (ITS, tef-1, and tub) proved to be phylogenetically informative in this study, resulting in a similar topology as previous studies based on five-marker combinations. The GCPSR analysis was also used to support the novelty of the Diaporthe species in this study. Therefore, molecular data and morphological evidence are needed for accurate species identification, thus reinforcing the importance of integrative approaches.

Morphologically, Occultitheca is considered an inconspicuous xylarialean and treated as anthostomella-like taxa in terms of having immersed, clypeate ascomata, asci with a J+, apical ring, ascospores with a large brown cell, and a basal hyaline dwarf cell (Daranagama et al. 2018; Samarakoon et al. 2022). Anthostomella-like taxa have now been split into five genera as suspected by Daranagama et al. (2015). These include Anthocanalis, Astrocystis, Brunneiperidium, Lunatiannulus, and Pyriformiascoma, and they differ in terms of ascomata and asexual morph characters. Occultitheca has a distinctive ostiole surrounded by a whitish halo and apiosporous ascospores with a small hyaline dwarf cell at one end and a dark brown, larger cell. There are only three species in Occultitheca, and all of them were found as saprobes as sexual morphs from terrestrial ecosystems. Occultitheca has a limited distribution and a narrow range of hosts, as only one species was found in Costa Rica (Rogers and Ju 2003), one in China, and one in Thailand (Samarakoon et al. 2022; Tian et al. 2024). Here, we provide a new addition to Occultitheca from unidentified decaying wood in Chiang Rai, Thailand. Occultitheca chiangraiensis was reported only as a sexual morph due to failure to obtain the asexual morphs from cultures. Thus, the link between the sexual and asexual morphs of this genus remains unknown. Due to the uncertainty of asexual and sexual morphologies and the low number of collections, Occultitheca has been placed under Xylariales genera incertae sedis (Samarakoon et al. 2022; Tian et al. 2024) and has also been considered to be part of Xylariaceae based on phylogenetic analyses (Voglmayr et al. 2022; this study). Expanding the sample collection will improve the representativeness of the genus.

This study provides a vital contribution to our understanding of Sordariomycetes diversity. Introducing new taxa is significant as they contribute to the broader understanding of fungal evolution, taxonomy, and ecology. It also contributes to the growing knowledge about the diversity of fungi associated with woody litter. It emphasises the necessity for continued exploration of fungal biodiversity across various habitats. As global ecosystems undergo rapid changes due to climate shifts and habitat destruction, understanding these dynamics will be crucial for conservation efforts and ecosystem management. Therefore, this study encourages further exploration in understudied substrates and regions, which could unveil additional species and enrich our comprehension of fungal ecology and taxonomy.

Acknowledgements

Chayanard Phukhamsakda would like to thank the Basic Research Fund (Fundamental Fund) supported by the National Science, Research, and Innovation Fund under Grant No. 672A16008 for financial support. The authors thank Dr. Shaun Pennycook from Manaaki Whenua, Landcare Research, New Zealand. Chitrabhanu S. Bhunjun would like to thank Thailand Science Research and Innovation (TSRI) and the National Science Research and Innovation Fund (NSRF) (Fundamental Fund: Grant no. 662A16047) entitled “Biodiversity, ecology, and applications of plant litter-inhabiting fungi for waste degradation.” Kevin David Hyde would like to thank the National Research Council of Thailand (NRCT) grant “Total fungal diversity in a given forest area with implications towards species numbers, chemical diversity, and biotechnology” (grant no. N42A650547). The authors extend their appreciation to the Researchers Supporting Project number (RSP2025R114), King Saud University, Riyadh, Saudi Arabia.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was funded by the Basic Research Fund (Fundamental Fund) supported by the National Science, Research, and Innovation Fund under Grant No. 672A16008, Thailand Science Research and Innovation (TSRI) and the National Science Research and Innovation Fund (NSRF) (Fundamental Fund: Grant no. 662A16047) entitled “Biodiversity, ecology, and applications of plant litter-inhabiting fungi for waste degradation”.

Author contributions

The authors confirm contributions to the paper as follows: Fungal specimen collection and isolation, fungal specimen deposition, and manuscript writing: Chayanard Phukhamsakda, Johnny Louangphan, Kedsara Navasit, Chitrabhanu S. Bhunjun; fungal identification and contributed to the revision of the manuscript: Chayanard Phukhamsakda, Kevin D. Hyde, Milan C. Samarakoon, Fatimah O. Alotibi, Chitrabhanu S. Bhunjun. All authors have read and agreed to the published version of the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Author ORCIDs

Chayanard Phukhamsakda https://orcid.org/0000-0002-1033-937X

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

Milan C. Samarakoon https://orcid.org/0000-0002-4815-125X

Johnny Louangphan https://orcid.org/0000-0003-3845-6145

Kedsara Navasit https://orcid.org/0009-0004-8167-0600

Fatimah Al-Otibi https://orcid.org/0000-0003-3629-5755

Data availability

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

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﻿Abstract

Key words:

﻿Introduction

﻿Materials and methods

﻿Fungal collection, isolation, and observation

﻿DNA extraction, amplification, and sequencing

﻿Alignments and phylogenetic analyses

﻿Genealogical concordance phylogenetic species recognition analysis

﻿Results

﻿Phylogenetic analyses of section sojae

﻿Sordariomycetes O.E. Erikss. & Winka

﻿ Diaporthe melonis Beraha & M.J. O’Brien, Phytopath. Z. 94(3): 205 (1979)

Description.

Culture characteristics.

Material examined.

Hosts.

Distribution.

Notes.

﻿ Diaporthe thailandica Louangphan, Phukhams., K.D. Hyde & Bhunjun, sp. nov.

Etymology.

Holotype.

Description.

Culture characteristics.

Material examined.

Host.

Distribution.

Notes.

﻿ Diaporthe tulliensis R.G. Shivas, Vawdrey & Y.P. Tan, Persoonia 35: 301 (2015)

Description.

Culture characteristics.

Material examined.

Hosts.

Distribution.

Notes.

﻿Phylogenetic analyses of Xylariales

﻿Xylariales Nannf.

﻿ Occultitheca chiangraiensis Louangphan, Phukhams., K.D. Hyde & Bhunjun, sp. nov.

Etymology.

Holotype.

Description.

Culture characteristics.

Material examined.

Host.

Distribution.

Notes.

﻿Discussion

Acknowledgements

﻿Additional information

Conflict of interest

Ethical statement

Funding

Author contributions

Author ORCIDs

Data availability

﻿References

Abstract

Introduction

Materials and methods

Fungal collection, isolation, and observation

DNA extraction, amplification, and sequencing

Alignments and phylogenetic analyses

Genealogical concordance phylogenetic species recognition analysis

Results

Phylogenetic analyses of section sojae

Sordariomycetes O.E. Erikss. & Winka

Diaporthe melonis Beraha & M.J. O’Brien, Phytopath. Z. 94(3): 205 (1979)

Diaporthe thailandica Louangphan, Phukhams., K.D. Hyde & Bhunjun, sp. nov.

Diaporthe tulliensis R.G. Shivas, Vawdrey & Y.P. Tan, Persoonia 35: 301 (2015)

Phylogenetic analyses of Xylariales

Xylariales Nannf.

Occultitheca chiangraiensis Louangphan, Phukhams., K.D. Hyde & Bhunjun, sp. nov.

Discussion

Additional information

References