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Research Article
Melomastia (Dothideomycetes, Ascomycota) species associated with Chinese Aquilaria spp.
expand article infoTian-Ye Du‡§, Samantha C. Karunarathna§, Saowaluck Tibpromma§, Kevin D. Hyde‡, Somrudee Nilthong‡, Ausana Mapook‡, Xiang-Fu Liu§‡, Dong-Qin Dai§, Chen Niu|, Abdallah M. Elgorban, Ekachai Chukeatirote‡, Hao-Han Wang§
‡ Mae Fah Luang University, Chiang Rai, Thailand
§ Qujing Normal University, Qujing, China
| Chinese Academy of Tropical Agriculture Sciences, Wanning, China
¶ King Saud University, Riyadh, Saudi Arabia

Corresponding author: Tian-Ye Du ( fungitianyed@163.com )

Corresponding author: Ekachai Chukeatirote ( ekachai@mfu.ac.th )

Corresponding author: Hao-Han Wang ( wanghh@eastern-himalaya.cn )

Academic editor: Danushka Sandaruwan Tennakoon
© 2024 Tian-Ye Du, Samantha C. Karunarathna, Saowaluck Tibpromma, Kevin D. Hyde, Somrudee Nilthong, Ausana Mapook, Xiang-Fu Liu, Dong-Qin Dai, Chen Niu, Abdallah M. Elgorban, Ekachai Chukeatirote, Hao-Han Wang.
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: Du T-Y, Karunarathna SC, Tibpromma S, Hyde KD, Nilthong S, Mapook A, Liu X-F, Dai D-Q, Niu C, Elgorban AM, Chukeatirote E, Wang H-H (2024) Melomastia (Dothideomycetes, Ascomycota) species associated with Chinese Aquilaria spp. MycoKeys 111: 65-86. https://doi.org/10.3897/mycokeys.111.137898
Open Access

Abstract

This study is based on three terrestrial saprobic fungi associated with Aquilaria in Guangdong and Yunnan provinces in China. All isolated species matched with generic concepts of Melomastia. Detailed morphological characteristics and combined multigene phylogeny of LSU, SSU, and TEF revealed that the new isolates represent two new species (Melomastia guangdongensis and M. yunnanensis), and one new host and geographical record (M. sinensis). Melomastia guangdongensis is distinct from the phylogenetically closest species in having semi-immersed to immersed, globose to subglobose ascomata, and two strata of the peridium. Melomastia yunnanensis differs from the phylogenetically closest species in having immersed ascomata, conical ostiolar canals, and branched pseudoparaphyses. The discovery of these two new species and one new record collected expands the number of saprobic species associated with Aquilaria from 28 to 31. Descriptions, photo plates, and phylogenetic analyses of taxa are provided.

Key words

2 new species, Dyfrolomycetales, new records, Pleurotremataceae, saprobes, Thymelaeaceae

Introduction

Pleurotremataceae Walt. Watson was introduced by Watson (1929) to accommodate Pleurotrema Müll. Arg. with P. polysemum (Nyl.) Müll. Arg. as the type species. The familial placement of Pleurotrema has been controversial, as the mature asci are neither typically unitunicate nor bitunicate (Mathiassen 1989; Hyde 1992). The placement of Pleurotrema has been confirmed based on the re-examined feature of the type of species P. polysemum, and Maharachchikumbura et al. (2016) transferred Pleurotremataceae from Sordariomycetes O.E. Erikss. & Winka to Dothideomycetes O.E. Erikss. & Winka and synonymized Dyfrolomycetaceae K.D. Hyde, K.L. Pang, Alias, Suetrong & E.B.G. Jones under Pleurotremataceae based on morphological comparison. Currently, Pleurotremataceae is accepted as the type and only family in Dyfrolomycetales K.L. Pang, K.D. Hyde & E.B.G. Jones, with three genera, Dyfrolomyces K. D. Hyde, Melomastia Nitschke ex Sacc, and Pleurotrema in this family (Maharachchikumbura et al. 2016; Hongsanan et al. 2020; Wijayawardene et al. 2022; Hyde et al. 2024).

Melomastia was established by Saccardo (1875) to accommodate M. mastoidea (Fr.) J. Schröt. (=Melomastia friesii Nitschke) as the type species. Previously, relying solely on the morphological features of Melomastia type species, the genus was considered unresolved and classified under Ascomycota genera incertae sedis (Maharachchikumbura et al. 2016). Subsequently, Norphanphoun et al. (2017) assigned Melomastia to Pleurotremataceae based on the newly introduced taxon M. italica Norph., Camporesi, T.C. Wen & K.D. Hyde, supported by sequence data. Based on morphology and phylogenetic analyses, Li et al. (2022) synonymized Dyfrolomyces under Melomastia and simultaneously transferred 11 species from Dyfrolomyces to Melomastia. De Silva et al. (2022) reported two new records of Melomastia from Thailand. However, Kularathnage et al. (2023) maintained Dyfrolomyces to accommodate D. tiomanensis K.L. Pang, Alias, K.D. Hyde, Suetrong & E.B.G. Jones and D. chromolaenae Mapook & K.D. Hyde, based on morphology differences of ascospores and the phylogenetic analyses. Recently, some new taxa from Brazil, China and Thailand have been introduced, viz. M. beihaiensis T.Y. Du, K.D. Hyde & Tibpromma (Senanayake et al. 2023), M. loropetalicola Kular., W. Dong & K.D. Hyde (Dong et al. 2023), M. puerensis R.F. Xu & Tibpromma (Xu et al. 2024), M. pyriformis Kular. & Senan. (Kularathnage et al. 2023), M. septata J.Y. Zhang, K.D. Hyde & Y.Z. Lu (Hyde et al. 2023), and M. septemseptata Muxfeldt & Aptroot (Muxfeldt Naziazeno and Aptroot 2023). Currently, 66 epithets of Melomastia are listed in Index Fungorum (2024), while only 20 species have sequences available in GenBank.

Melomastia is characterized by immersed to semi-immersed, globose to subglobose, coriaceous to carbonaceous, ostiolate ascomata, dark brown peridium, filamentous pseudoparaphyses, bitunicate, cylindrical, 8-spored asci, and ascospores are fusiform to oblong, ovoid, or cylindrical, hyaline, 1–10-septate, with rounded or acute ends, with or without gelatinous sheath; while, the asexual morph of Melomastia is undetermined (Dayarathne et al. 2020; de Silva et al. 2022; Li et al. 2022; Kularathnage et al. 2023; Xu et al. 2024). Most Melomastia species have been recorded as saprobes from various habitats, such as terrestrial, freshwater, marine, and mangrove ecosystems (Hyde 1992; Hyde et al. 2017; Norphanphoun et al. 2017; Dayarathne et al. 2020; Phukhamsakda et al. 2020; Li et al. 2022; Hyde et al. 2023; Tian et al. 2024; Xu et al. 2024). Melomastia is a geographically widely distributed genus with a broad host range, which has been systematically documented in Li et al. (2022) and Kularathnage et al. (2023), viz. members of Melomastia have wide geographical distribution in Africa, Asia, Australia, Europe, and South America, while the reported hosts of Melomastia belong to Acanthaceae Juss., Asteraceae Bercht. & J. Presl, Euphorbiaceae Juss., Hamamelidaceae R. Br., Oleaceae Hoffmanns. & Link, Ranunculaceae Juss., Rhizophoraceae Pers., Theaceae Mirb., and Vitaceae Juss.

Aquilaria Lam. is an important agarwood resin-producing tree genus in Thymelaeaceae Juss. Agarwood resin is high-valued and very rare, and its formation is primarily due to injury, followed by microbial infection (Rasool and Mohamed 2016; Azren et al. 2018; Wang et al. 2018). So far, many reports have been published on the pathogenic and endophytic fungi associated with Aquilaria, while saprobic fungi have been neglected (Liu et al. 2020; Du et al. 2022a). Prior to 2022, there were only eight records of saprobic fungi associated with Aquilaria (Punithalingam and Gibson 1978; Subansenee et al. 1985), and molecular data and comprehensive morphological descriptions were lacking. Recently, 20 saprobic fungal species have been reported from Aquilaria spp. by Du et al. (2022b, 2023, 2024), Chethana et al. (2023), and Hyde et al. (2023, 2024) based on both morphological and molecular evidence. Therefore, so far only 28 records of Aquilaria-associated fungi have been found. This study focuses on filling the gap in research on the saprobic fungi associated with Aquilaria, and enriching the diversity of fungi associated with Aquilaria.

In this study, Aquilaria plant specimens with black ascomycetous fungal fruiting bodies were collected from Yunnan and Guangdong provinces in China. Based on phylogenetic and morphological analyses, these fungal collections were identified as two new species and one new record of the Melomastia. Full descriptions, illustrations, photo plates, and phylogenetic trees to indicate the placement of new taxa are provided.

Materials and methods

Sampling, examination, and isolation

Dead fallen branches of Aquilaria spp. with ascomycetous fungal fruiting bodies were collected from subtropical parts of Guangdong and Yunnan provinces in China. After recording important information (Rathnayaka et al. 2024), samples were transported to the laboratory in plastic bags. Morphological structures were examined by using an OPTEC SZ650 dissecting stereomicroscope (Chongqing, China), and an OLYMPUS DP74 (Tokyo, Japan) digital camera on an OLYMPUS optical microscope (Tokyo, Japan) was used to observe and photograph the microstructure of fungi. Micro-morphological structures were measured in Tarosoft ® Image Framework program v. 1.3, and photo plates were edited in Adobe Photoshop CS3 Extended version 22.0.0 software (Adobe Systems, California, the USA).

Fungi were isolated using single-spore isolation, as described by Senanayake et al. (2020). The fruiting bodies were cut by sterilized blades, and the ascospores were picked up by sterilized needles and cultured in potato dextrose agar (PDA) at 23–28 °C for 24–48 hours. The single germinated ascospores were picked up and transferred to PDA at 23–28 °C with recording culture characters.

Specimens were deposited at the Guizhou Medical University (GMB-W) and Mycological Herbarium of Zhongkai University of Agriculture and Engineering (MHZU), China. Living cultures are deposited in the Guizhou Medical University Culture Collection (GMBCC), Guizhou Culture Collection (GZCC), and Zhongkai University of Agriculture and Engineering Culture Collection (ZHKUCC), China. Facesoffungi (FoF) numbers were registered as described in Jayasiri et al. (2015), and MycoBank numbers (MB) were registered as outlined in MycoBank (2024).

DNA extraction, PCR amplification, and sequencing

Molecular studies were carried out according to Dissanayake et al. (2020). Total genomic DNA was extracted from one-month-old fresh fungal mycelium (grew on PDA) using a DNA Extraction Kit-BSC14S1 (BioFlux, Hangzhou, P.R. China) following the manufacturer’s instructions. Polymerase chain reactions (PCR) were carried out using the following primers: 28S nrRNA gene (LSU) was amplified by using the primers LR0R and LR5 (Vilgalys and Hester 1990), 18S ribosomal RNA (SSU) was amplified using the primers NS1 and NS4 (White et al. 1990), and translation elongation factor 1-alpha (TEF) was amplified using the primers EF1-983F and EF1-2218R (Rehner 2001). The DNA amplification procedure was performed by PCR in a 25 μL containing 12.5 μL 2×Master Mix (mixture of Easy Taq TM DNA Polymerase, dNTPs, and optimized buffer (Beijing Trans Gen Biotech Co., Chaoyang District, Beijing, China)), 8.5 μL ddH2O, 2 μL of DNA template, and 1 μL of each forward and reverse primer (10 pM). The PCR thermal cycle programs for LSU, SSU, and TEF were as follows: an initialization step of 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, an annealing step at 55 °C for 50 s, an elongation step at 72 °C for 1 min and a final extension step of 72 °C for 10 min. Purification and sequencing of PCR products were carried out by Sangon Biotech Co., Kunming, China.

Phylogenetic analyses

A combined gene dataset of LSU, SSU, and TEF was used for the phylogenetic analyses. Newly generated contigs were used to carry out the BLASTn search in NCBI to identify the most similarities taxa of our strains. The additional sequences included in the analysis were collected from previous publications (Li et al. 2022; Kularathnage et al. 2023; Xu et al. 2024) and downloaded from GenBank (Benson et al. 2014). Phylogenetic analyses were carried out with 50 sequences (Table 1). The FASTA file used for constructing the Randomized Accelerated Maximum Likelihood (RAxML) and Bayesian Inference analyses (BI) was performed using the OFPT (Zeng et al. 2023) with the protocol. Then, the FASTA file was converted to PHYLIP and NEXUS formats for RAxML and BI phylogenetic analyses in ALTER, respectively (Glez-Peña et al. 2010).

Table 1.
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Taxa names, strain numbers, and corresponding GenBank accession numbers of the taxa included in the present study.

Taxa Names Strain Numbers GenBank Accession Numbers
LSU SSU TEF
Acrospermum adeanum M133 EU940104 EU940031 —
Anisomeridium phaeospermum MPN539 JN887394 JN887374 JN887418
A. ubianum MPN94 — JN887379 JN887421
Dyfrolomyces chromolaenae MFLUCC 17-1434 T KY111905 MT214413 MT235800
D. tiomanensis MFLUCC 13-0440 T KC692156 KC692155 KC692157
Melomastia aquilariae ZHKUCC 23-0073 T OR807856 OR807854 OR832867
M. aquilariae ZHKUCC 23-0088 OR807857 OR807855 OR832868
M. beihaiensis KUMCC 21-0084 T MZ726990 MZ727002 OK043822
M. clematidis MFLUCC 17-2092 T MT214607 MT226718 MT394663
M. distoseptata MFLUCC 21-0102 MT860427 — —
M. fulvicomae MFLUCC 17-2083 T MT214608 MT226719 MT394664
M. fusispora CGMCC 3.20618 T OK623464 OK623494 OL335189
M. fusispora UESTCC 21.0001 OK623465 OK623495 OL335190
M. guangdongensis GMBCC1046 T PQ530970 PQ530975 PQ559185
M. guangdongensis ZHKUCC 23-0040 PQ530971 PQ530976 PQ559186
M. italica MFLUCC 15-0160 T MG029458 MG029459 —
M. loropetalicola ZHKUCC 22-0174 T OP791870 OP739334 —
M. maolanensis GZCC 16-0102 T — — KY814762
M. maomingensis ZHKUCC 23-0038 T PP809724 PP809704 PP812255
M. maomingensis GZCC 23-0619 PP809725 PP809705 PP812256
M. neothailandica MFLU 17-2589 T MN017857 — —
M. oleae CGMCC 3.20619 T OK623466 OK623496 OL335191
M. oleae UESTCC 21.0003 OK623467 OK623497 OL335192
M. oleae UESTCC 21.0005 OK623468 OK623498 OL335193
M. oleae UESTCC 21.0006 — OK623499 OL335194
M. phetchaburiensis MFLUCC 15-0951 T MF615402 MF615403 —
M. puerensis ZHKUCC 23-0802 T OR922309 OR922340 OR966284
M. puerensis ZHKUCC 23-0803 OR922310 OR922341 OR966285
M. pyriformis ZHKUCC 22-0175 T OP791870 OP739334 OQ718392
M. rhizophorae BCC15481 — KF160009 —
M. rhizophorae JK 5456A GU479799 — GU479860
M. septata MFLUCC 22-0112 T OP749870 — OP760198
M. sichuanensis CGMCC 3.20620 T OK623469 OK623500 OL335195
M. sichuanensis UESTCC 21.0008 OK623470 OK623501 OL335196
M. sinensis MFLUCC 17-1344 T MG836699 MG836700 —
M. sinensis MFLUCC 17-2606 OL782048 — OL875098
M. sinensis MFLU 17-0777 NG_064507 — —
M. sinensis GMBCC1008 PQ530972 PQ530977 PQ559187
M. thailandica MFLU 17-2610 MN017858 MN017923 MN077069
M. thamplaensis KUMCC 21-0671 OQ170875 OQ168226 OR613415
M. thamplaensis MFLUCC 15-0635 T KX925435 KX925436 KY814763
M. winteri CGMCC 3.20621 OK623471 OK623502 OL335197
M. yunnanensis GMBCC1009 T PQ530973 PQ530978 PQ559188
M. yunnanensis GZCC 23-0621 PQ530974 PQ530979 PQ559189
Muyocopron heveae MFLUCC 17-0066 T MH986832 MH986828 —
Mu. lithocarpi MFLUCC 14-1106 T KU726967 KU726970 MT136755
Palawania thailandense MFLU 16-1873 KY086494 — —
P. thailandense MFLUCC 14-1121 T KY086493 KY086495 —
Stigmatodiscus oculatus AP161116 — — MH756086
S. oculatus AP171116 — — MH756087

Remarks: The newly generated sequences are indicated in bold, the superscript T indicates ex-type, and “—” indicates information unavailable.

CIPRES Science Gateway platform was used to carry out the Randomized Accelerated Maximum Likelihood (RAxML) and Bayesian Inference analyses (BI) (Miller et al. 2010). The RAxML tree analyzed with 1,000 bootstrap replicates was generated using RAxML-HPC2 on XSEDE (8.2.12) (Stamatakis et al. 2008; Stamatakis 2014) with GTR+I+G model of evolution and bootstrap supports. The BI tree was performed with MrBayes on XSEDE (3.2.7a) (Ronquist et al. 2012) by the Markov Chain Monte Carlo (MCMC) method to evaluate posterior probabilities (BYPP) (Richard and Lippmann 1991; Rannala and Yang 1996; Zhaxybayeva and Gogarten 2002). The best-fit nucleotide substitution models for each dataset were then selected based on the Bayesian information criterion (BIC) from twenty-two common DNA substitution models with rate heterogeneity by ModelFinder (Kalyaanamoorthy et al. 2017). The best model for LSU was TN+F+G4, TIM2e+I for SSU, and TN+F+I+G4 for TEF. Six simultaneous Markov chains were run for 2,000,000 generations, and a tree was sampled every 100th generation. The phylogenetic tree was visualized in FigTree v.1.4.2 (Rambaut 2012), and edited by Microsoft Office PowerPoint 2021 and Adobe Photoshop CS3 Extended version 22.0.0 software (Adobe Systems, California, the USA). All newly generated sequences in this study were deposited to the GenBank (https://www.ncbi.nlm.nih.gov/WebSub/?form=history&tool=genbank).

Results

Phylogenetic analyses

The phylogenetic trees obtained from RAxML and BI analyses provided essentially similar topologies. The RAxML analyses of the combined dataset yielded the best scoring tree (Fig. 1), which comprised 2912 base pairs of LSU = 899, SSU = 1069, and TEF = 944. The final ML optimization likelihood value was -11933.909808. The matrix had 871 distinct alignment patterns, with 23.14% being undetermined characters or gaps. Parameters for the GTR+I+G model of the combined LSU, SSU, and TEF were as follows: estimated base frequencies A = 0.239382, C = 0.262893, G = 0.291472, T = 0.206253; substitution rates AC = 0.831502, AG = 1.991603, AT = 1.062650, CG = 0.930785, CT = 8.413262, GT = 1.000000; proportion of invariable sites I = 0.495458; and gamma distribution shape parameter α = 0.612808. The final RAxML tree is shown in Fig. 1.

Figure 1. 

Phylogram generated from maximum likelihood analysis based on combined LSU, SSU, and TEF sequence data of 50 taxa. Bootstrap support values for maximum likelihood (ML) equal to or greater than 60% and clade credibility values greater than 0.90 from Bayesian inference analysis are labelled at each node. The tree is rooted with Anisomeridium phaeospermum (MPN539) and A. ubianum (MPN94). The new isolates are indicated in red, and the ex-type strains are in bold.

In this phylogenetic tree, Melomastia was resolved as two clades, Melomastia sensu lato (15 species) and Melomastia sensu stricto (nine species), and the results are similar to those reported by Li et al. (2022), Kularathnage et al. (2023), and Xu et al. (2024). Kularathnage et al. (2023) have separated Melomastia into two clades, Melomastia sensu lato, and Melomastia sensu stricto; this was done due to Melomastia sensu stricto’s close resemblance to the type species M. mastoidea, while Melomastia sensu lato still needs more collections, sequences, and phenotypic data to support Kularathnage et al. (2023). Our two new species, M. guangdongensis (GMBCC1046 and ZHKUCC 23-0040) and M. yunnanensis (GMBCC1009 and GZCC 23-0621), and a new record M. sinensis (GMBCC1008) clustered within Melomastia sensu lato.

New species M. guangdongensis (GMBCC1046 and ZHKUCC 23-0040) was well separated from M. thamplaensis (KUMCC 21-0671 and MFLUCC 15-0635) in an independent lineage with 75% ML/0.91 PP statistical support; M. yunnanensis (GMBCC1009 and GZCC 23-0621) was well separated from M. sinensis (GMBCC1008, MFLU 17-0777, MFLUCC 17-1344 and MFLUCC 17-2606) in a distinct lineage with 100% ML/1.00 PP statistical support. The new record M. sinensis (GMBCC1008) was grouped within three strains of M. sinensis with 99% ML/0.93 PP statistical support.

Taxonomy

Melomastia guangdongensis T.Y. Du, K.D. Hyde, Tibpromma & Karun., sp. nov.

MycoBank No: 856407
Facesoffungi Number: FoF16958
Fig. 2

Etymology

Named after the type locality “Guangdong, China”.

Holotype

MHZU 23-0021

Description

Saprobic on a dead branch of Aquilaria sinensis. Sexual morph: Ascomata (excluding neck) 180–360 µm high × 200–300 µm diam. (x– = 267 × 245 µm, n = 10), visible as black dots on the host surface, black, solitary, scattered to gregarious, semi-immersed to immersed, uniloculate, globose to subglobose, coriaceous to carbonaceous, ostiolate. Ostiolar canal 190–240 µm high × 120–160 µm wide (x– = 214 × 140 µm, n = 10), central, black, cylindrical, coriaceous to carbonaceous, filled with hyaline cells. Peridium 30–60 µm wide (x– = 40 µm, n = 20), comprising dense, several layers, outer layers brown to dark brown, thick-walled cells of textura angularis to textura globulosa, inner layers hyaline, thin-walled cells of textura angularis to textura prismatica, not fusion well with host tissue. Hamathecium comprising 1.5–3 µm wide, numerous filamentous, filiform, septate, sometimes branched, hyaline, pseudoparaphyses, attached to the base and between the asci, embedded in a gelatinous matrix. Asci 120–168 × 5.5–7.5 µm (x– = 144 × 6.5 µm, n = 30), bitunicate, 8-spored, cylindrical, short pedicel, rounded in apex, with an obvious ocular chamber. Ascospores (18.7–)20–26 × 5–7 µm (x– = 23 × 6 µm, n = 30), overlapping-uniseriate, hyaline, 3-septate at maturity, fusiform with acute ends, slightly constricted at the middle septum, smooth-walled, not surrounded by a mucilaginous sheath. Asexual morph: Undetermined.

Culture characteristics

Ascospores germinated on PDA after 24 hours, germ tubes were produced from both ends. Colonies on PDA reaching 3 cm diam., after two weeks at 23–28 °C. Colonies obverse: dense, circular, white, velvety, slightly raised at the center, entire edge. Colonies reverse: yellow, cream at the margin.

Material examined

China • Guangdong Province, Maoming City, Dianbai District, Poxin, 21°34'28"N, 111°7'39"E, on a dead branch of Aquilaria sinensis (Thymelaeaceae), 3 June 2022, T.Y. Du, MMA14, (MHZU 23-0021, holotype), ex-type, GMBCC1046, other living culture, ZHKUCC 23-0040.

Figure 2. 

Melomastia guangdongensis (MHZU 23-0021, holotype) A–C appearance of ascomata on the host (the arrows indicate ascomata) D, E vertical sections through the ascomata F ostiole G–J asci (I, J asci stained with cotton blue, and arrows indicate ocular chambers) K pseudoparaphyses stained with cotton blue L–O ascospores (O ascospore stained with cotton blue) P germinated ascospore Q colony on PDA obverse and reverse view. Scale bars: 200 µm (D–F); 50 µm (G–J); 10 µm (K–P).

Notes

In the phylogenetic analyses, our new collection, M. guangdongensis formed a sister branch with M. thamplaensis strains (HKAS122773, KUMCC 21-0671, and MFLUCC 15-0635) in Melomastia sensu lato clade (Fig. 1) with a 75% ML/0.91 PP bootstrap support. NCBI BLASTn searches of our collection, M. guangdongensis showed 99.88% similarity to M. thamplaensis (HKAS122773) in the LSU sequence, 100% similarity to M. thamplaensis (AND9) in the SSU sequence, and 98.17% similarity to M. thamplaensis (KUMCC 21-0671) in the TEF sequence. Our new collection, M. guangdongensis shares similar morphology with M. thamplaensis in the shape of asci and ascospores. However, M. thamplaensis differs from M. guangdongensis in having clypeate, raised spots, immersed, subglobose to obpyriform, some with broad, flattened base ascomata, and three strata of peridium (Zhang et al. 2017), while M. guangdongensis has semi-immersed to immersed, globose to subglobose ascomata, and two strata of peridium. Base pair differences of the LSU and SSU genes between our new collection M. guangdongensis (GMBCC1046, ex-type) and M. thamplaensis (MFLUCC 15-0635, ex-type) showed that there are no nucleotide differences, while the TEF has 1.6% nucleotide differences (14/865 bp, without gaps), and a comparison of the TEF nucleotides between new collections and another strain of M. thamplaensis (KUMCC 21-0671) resulted in 1.7% differences (15/865 bp, without gaps) (Zhang et al. 2017; Ren et al. 2024). Therefore, we introduce our collection, M. guangdongensis, as a new species on a dead branch of Aquilaria sinensis from terrestrial habitats in China, based on both morphology and phylogenetic analyses following the guidelines of Maharachchikumbura et al. (2021).

Melomastia sinensis (Samarak., Tennakoon & K.D. Hyde) W.L. Li, Maharachch. & Jian K. Liu (2022)

MycoBank No: 842093
Facesoffungi Number: FoF03935
Fig. 3

Description

Saprobic on a dead branch of Aquilaria sp. Sexual morph: Ascomata (excluding neck) 400–600 µm high × 430–580 µm diam. (x– = 515 × 520 µm, n = 10), solitary, scattered to gregarious, semi-immersed to immersed, erumpent through host tissue, globose to subglobose, black, coriaceous to carbonaceous, ostiolate. Ostiolar canal 230–365 µm high × 200–260 µm wide (x– = 303 × 230 µm, n = 10), central, black, conical, coriaceous to carbonaceous, filled with hyaline sparse periphyses. Peridium 30–120 µm wide (x– = 75 µm, n = 20), comprising dense, several layers of thick-walled cells of textura angularis to textura prismatica, outer layers brown to dark brown, becoming lighter inwardly. Hamathecium comprising 2.5–6.5 µm wide, numerous filamentous, filiform, septate, unbranched, hyaline pseudoparaphyses, attached to the base and between the asci, embedded in a gelatinous matrix. Asci 175–220 × 8.5–11.5 µm (x– = 195 × 10.5 µm, n = 30), bitunicate, 8-spored, cylindrical, long pedicel, thickened and rounded apex, with an obvious ocular chamber. Ascospores (17.5–)20–26.5 × 7–9 µm (x– = 24 × 8 µm, n = 30), overlapping-uniseriate, hyaline, when ascospores gather together, they appear light yellow, mostly 6–7-septate at maturity, cylindrical, with rounded ends, slightly constricted at the septum, often similar width of cells with several small guttules, not surrounded by a mucilaginous sheath. Asexual morph: Undetermined.

Culture characteristics

Ascospores germinated on PDA after 24 hours, germ tubes were produced from most cells, germinated ascospores appear light yellow. Colonies on PDA reaching 3 cm diam., after two weeks at 23–28 °C. Colonies obverse: dense, circular or irregular, umbonate, cream, light yellow at the center, entire or undulate edge. Colonies reverse: dark gray, yellow at the margin.

Figure 3. 

Melomastia sinensis (GMB-W 1006, new host and geographical record) A–C appearance of ascomata on the host (A the arrows indicate ascomata) D vertical sections through the ascoma E ostiole F peridium G pseudoparaphyses H asci I ascus with an ocular chamber J, K ascospores L germinated ascospore M, N colony on PDA obverse and reverse view. Scale bars: 200 µm (D, E); 100 µm (H); 50 µm (F); 20 µm (I–L); 10 µm (G).

Material examined

China • Yunnan Province, Xishuangbanna, Jinghong City, Naban River Nature Reserve, 22°7'48"N, 100°40'24"E, on a dead branch of Aquilaria sp. (Thymelaeaceae), 14 September 2021, Tianye Du, YNA41 (GMB-W 1006, new host and geographical record), living culture, GMBCC1008.

Host and distribution

Aquilaria sp. (China; this study), Camellia sinensis (Thailand; Hyde et al. 2018), and Hevea brasiliensis (Thailand; Senwanna et al. 2021).

Notes

In the phylogenetic analyses, our new collection (GMBCC1008) isolated from a dead branch of Aquilaria sp. grouped with Melomastia sinensis strains (MFLUCC 17-1344, MFLUCC 17-2606 and MFLU 17-0777) in Melomastia sensu lato, with a 99% ML/0.93 PP bootstrap support (Fig. 1). NCBI BLASTn searches of our collection showed 99.78% similarity to M. sinensis (MFLUCC 17-2606) in the LSU sequence, 99.21% similarity to M. oleae (UESTCC 21.0006) in the SSU sequence, and 99.67% similarity to M. sinensis (MFLUCC 17-2606) in the TEF sequence.

Melomastia sinensis (=Dyfrolomyces sinensis Samarak., Tennakoon & K.D. Hyde) was introduced by Hyde et al. (2018) as a saprobic on Camellia sinensis (L.) Kuntze stems. Our new collection shares a similar morphology with M. sinensis (MFLU 17-0777, holotype) in cylindrical ascospores with 6–7-septate ascospores. Our new collection has semi-immersed to immersed ascomata, differs from M. sinensis (MFLU 17-0777, holotype) in having superficial ascomata (Hyde et al. 2018) and differs from immersed ascomata in M. sinensis (MFLU 19-0232) (Senwanna et al. 2021). However, the nucleotide base pair differences between our new collection (GMBCC1008) and M. sinensis (MFLUCC 17-1344, ex-type) showed that the LSU and SSU gene has no nucleotide differences, while the TEF gene of M. sinensis (MFLUCC 17-1344, ex-type) is unavailable in NCBI (Hyde et al. 2018). The comparison of the TEF nucleotides between the new collection and another strain of M. sinensis (MFLUCC 17-2606) resulted in 0.3% differences (3/873 bp, without gaps) (Senwanna et al. 2021). This study first discovered M. sinensis on Aquilaria sp. in China. Therefore, we introduce our new collection as a new host and geographical record of M. sinensis based on both morphological study and phylogenetic analyses.

Melomastia yunnanensis T.Y. Du, K.D. Hyde, Tibpromma & Karun., sp. nov.

MycoBank No: 856408
Facesoffungi Number: FoF16959
Fig. 4

Etymology

Named after the type location “Yunnan, China”.

Holotype

GMB-W 1007

Description

Saprobic on a dead branch of Aquilaria sp. Sexual morph: Ascomata (excluding neck) 400–500 µm high × 300–480 µm diam. (x– = 458 × 395 µm, n = 10), solitary, scattered to gregarious, immersed to erumpent through host tissue, globose, black, carbonaceous, ostiolate. Ostiolar canal 100–160 µm high × 120–230 µm wide (x– = 130 × 184 µm, n = 10), central, black, conical, carbonaceous, filled with hyaline sparse periphyses. Peridium 25–75 µm wide (x– = 55 µm, n = 10), comprising of dense, several layers of brown to dark brown, thick-walled cells of textura angularis to textura prismatica. Hamathecium comprising 2.5–7.5 µm wide, numerous filamentous, filiform, septate, sometimes branched, hyaline pseudoparaphyses, attached to the base and between the asci, embedded in a gelatinous matrix. Asci 180–220 × 7.5–10.5 µm (x– = 195.5 × 9 µm, n = 30), bitunicate, 8-spored, cylindrical, short pedicel, thickened and rounded apex, with an obvious ocular chamber. Ascospores 20–24.5 × 6–8 µm (x– = 22.5 × 7 µm, n = 30), overlapping-uniseriate, hyaline, when ascospores gather together, they appear light yellow, mostly 6–8-septate at maturity, mostly 7-septate, cylindrical, with rounded ends, slightly constricted at the septum, often similar width of cells with several small guttules, not surrounded by a mucilaginous sheath. Asexual morph: Undetermined.

Figure 4. 

Melomastia yunnanensis (GMB-W 1007, holotype) A–C appearance of ascomata on the host (the arrows indicate ascomata) D vertical sections through the ascoma E ostiole F peridium G–I asci J asci ocular chamber K germinated ascospore L pseudoparaphyses M–Q ascospores R, S colonies on PDA obverse and reverse view. Scale bars: 200 µm (D); 100 µm (G–I); 50 µm (E, F); 20 µm (J, K, M–Q); 10 µm (L).

Culture characteristics

Ascospores germinated on PDA after 24 hours, germ tubes were produced from both ends, germinated ascospores appear light brown. Colonies on PDA reaching 2 cm diam., after two weeks at 23–28 °C. Colonies obverse: dense, circular, umbonate, gray at the center, cream, and entire edge. Colonies reverse: gray brown, light brown at the margin.

Material examined

China • Yunnan Province, Xishuangbanna, Jinghong City, Naban River Nature Reserve, 22°7'51"N, 100°40'21"E, on a dead branch of Aquilaria sp. (Thymelaeaceae), 14 September 2021, Tianye Du, YNA51 (GMB-W 1007, holotype), ex-type, GMBCC1009, other living culture, GZCC 23-0621.

Notes

In the phylogenetic analyses, our new collection, M. yunnanensis formed a sister branch with M. sinensis (MFLUCC 17-1344, MFLUCC 17-2606, MFLU 17-0777, and GMBCC1008) in Melomastia sensu lato with a 100% ML/1.00 PP bootstrap support (Fig. 1). NCBI BLASTn searches of our collection M. yunnanensis showed 99.23% similarity to M. sinensis (MFLUCC 17-2606) in the LSU sequence, 98.92% similarity to M. thamplaensis (AND9) in the SSU sequence, and 96.34% similarity to M. sinensis (MFLUCC 17-2606) in the TEF sequence. Our new collection, M. yunnanensis shares similar morphology with M. sinensis in cylindrical and septate ascospores. However, M. sinensis differs from M. yunnanensis in having superficial, semi-immersed to immersed ascomata, cylindrical or conical ostiolar canal, and unbranched pseudoparaphyses (Hyde et al. 2018), while our M. yunnanensis has immersed ascomata, conical ostiolar canal, and pseudoparaphyses sometimes branched. In addition, the nucleotide base pair differences between our new collection M. yunnanensis (GMBCC1009, ex-type) and M. sinensis (MFLUCC 17-1344, ex-type) showed the LSU gene has 0.5% nucleotide differences (4/760 bp, without gaps), the SSU gene has 0.5% nucleotide differences (4/813 bp, without gaps), while the TEF gene of M. sinensis (MFLUCC 17-1344, ex-type) is unavailable (Hyde et al. 2018). We compared the TEF nucleotides between the new collection and another collection of M. sinensis (MFLUCC 17-2606), which resulted in 3.8% differences (33/873 bp, without gaps) (Senwanna et al. 2021). Therefore, we introduce our new collection, M. yunnanensis, as a new species on a dead branch of Aquilaria sp. from terrestrial habitats in China, based on both morphological study and phylogenetic analyses following the guidelines of Maharachchikumbura et al. (2021).

Discussion

Based on the morphological study and phylogenetic analyses, this study identifies, describes, and introduces two new species, Melomastia guangdongensis and M. yunnanensis, and a new host and geographical record of M. sinensis from Aquilaria spp. These findings significantly contribute to the understanding of the diversity and distribution of agarwood resin-producing tree-associated fungi.

Our phylogenetic analysis based on LSU, SSU, and TEF also showed that the results are similar to those of Kularathnage et al. (2023) and Xu et al. (2024), who have divided Melomastia into two clades, Melomastia sensu lato and Melomastia sensu stricto. However, the majority of species are clustered in Melomastia sensu lato, and only 20 out of 66 listed records in Index Fungorum (2024) have available sequences, posing a challenge for the study of phylogenetic analysis in this genus. To address this, we believe it is necessary to explore and collect more samples of new and known species of Melomastia and supplement our research with molecular studies. In addition, relevant information about Melomastia, such as life mode, habitat, host, geographical location, and ecological niche, must be collected and analyzed to enhance our knowledge of this genus.

Morphologically, most species in Melomastia have fusiform or ellipsoidal ascospores, while two species (M. marinospora and M. sinensis) show cylindrical ascospores (Li et al. 2022). Previously, the ascospores of this genus are usually reported 3-septate (e.g. M. aquatica, M. clematidis, M. distoseptata, M. fusispora, M. maolanensis, M. marinospora, M. oleae, M. sichuanensis, M. thamplaensis, and M. winteri) (Li et al. 2022). Current studies as more new taxa were introduced into this genus reveal multi-septate ascospores, while these taxa with similar characteristics do not cluster together on the phylogenetic tree (Fig. 1), such as M. mangrovei (7–9-septate, no molecular data available in NCBI), M. phetchaburiensis (1–10-septate, in Melomastia sensu lato), M. rhizophorae (4–6-septate, in Melomastia sensu stricto), M. sinensis (6–7-septate, in Melomastia sensu lato), and M. thailandica (3–5-septate, in Melomastia sensu stricto) (Li et al. 2022). In this study, M. guangdongensis shows the fusiform with 3-septate ascospores, while M. yunnanensis shows the cylindrical with 6–8-septate ascospores, both of these new taxa belong to Melomastia sensu lato. Therefore, more studies are needed to discuss the morphological and phylogenetic connections of this genus. In addition, in this study, we also found Melomastia from the same host genus Aquilaria, but when we compare ascomata, semi-immersed to immersed ascomata in M. guangdongensis and M. sinensis. In contrast, ascomata of M. yunnanensis are immersed to erumpent through host tissue. Further research is needed to explore whether the attachment mode of ascomata on the substrate is influenced by the host, environment, or other factors.

In recent years, many studies on saprobic fungi in economic crops, such as rice, sugarcane, rubber, coffee, mango, and macadamia nuts, have been published (Yang et al. 2022, Lu et al. 2024, Tian et al. 2024, Xu et al. 2024, Zhang et al. 2024). However, there is a noticeable lack of research on saprobic fungi in Aquilaria spp. This study introduces three saprobic fungal taxa, expanding the previous record of 28 saprobic fungi associated with Aquilaria to 31. It also highlights the urgent need for further, more in-depth investigations. We believe that future studies with a broader geographical range will be crucial in enhancing our understanding of the distribution and diversity of fungi in Aquilaria.

Acknowledgments

The authors are grateful to the High-Level Talent Recruitment Plan of Yunnan Province (“High-End Foreign Experts” Program and “Young Talents” Program). Tianye Du extends her heartfelt gratitude to Mae Fah Luang University for granting a tuition-fee scholarship for her Ph.D. study. Shaun Pennycook is thanked for his assistance in selecting species epithets for the new species.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

The authors are grateful to the Special Basic Cooperative Research Innovation Programs of Qujing Science and Technology Bureau & Qujing Normal University (Grant No. KJLH2022YB03), the Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities (Grant No. 202101BA070001-209, 202101BA070001-279), the Yunnan Fundamental Research projects [202201AU070017], Mee-mann Chang Academician Workstation in Yunnan Province (Grant No. 202205AF150002), and Yunnan Province Young and Middle-aged Academic and Technical Leaders Reserve Talents Program (Grant No.202305AC350252), and General Programs of the Provincial Department of Science and Technology (Grant No. 202101BA070001-076) for support. The authors also extend their appreciation to the Researchers Supporting Project number (RSP2025R56), King Saud University, Riyadh, Saudi Arabia.

Author contributions

Conceptualization: SCK, ST. Data curation: TYD. Formal analysis: SCK. Funding acquisition: DQD, ST, HHW. Investigation: SCK, ST, TYD. Methodology: SCK, ST, XFL, TYD. Project administration: ST, DQD, HHW. Resources: TYD. Software: TYD. Validation: SCK, ST, AM, EC, KDH. Visualization: TYD. Writing – original draft: TYD. Writing – review and editing: SN, KDH, AM, XFL, CN, AME, EC, SCK, ST, TYD, DQD, HHW.

Author ORCIDs

Tian-Ye Du https://orcid.org/0000-0003-2105-1803

Samantha C. Karunarathna https://orcid.org/0000-0001-7080-0781

Saowaluck Tibpromma https://orcid.org/0000-0002-4706-6547

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

Somrudee Nilthong https://orcid.org/0000-0002-7454-5826

Ausana Mapook https://orcid.org/0000-0001-7929-2429

Xiang-Fu Liu https://orcid.org/0000-0003-0100-2094

Dong-Qin Dai https://orcid.org/0000-0001-8935-8807

Chen Niu https://orcid.org/0009-0008-4633-9719

Abdallah M. Elgorban https://orcid.org/0000-0003-3664-7853

Ekachai Chukeatirote https://orcid.org/0000-0002-9968-5841

Hao-Han Wang https://orcid.org/0000-0002-2128-7894

Data availability

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

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