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. 2026 Aug 7;138:209–236. doi: 10.3897/mycokeys.138.203334

Additions to Diaporthe (Sordariomycetes, Diaporthaceae) and Neptunomyces (Dothideomycetes, Didymosphaeriaceae) species associated with Itea in Thailand

Jutamart Monkai 1,2, Rungtiwa Phookamsak 3,4,5, Surapong Khuna 6, Darbhe Jayarama Bhat 7,8, Toe Swe Zin Ei 2, Jianchu Xu 3,4,5,9, Sinang Hongsanan 6,✉, Saisamorn Lumyong 2,10,11,✉
PMCID: PMC13476751  PMID: 42604090

Abstract

Itea is an economically and medicinally important plant, widely distributed across China, Japan and Southeast Asia, noted for producing rare sugars. Despite this, endophytic fungi associated with this plant remain poorly studied. This study focuses on the taxonomy and phylogeny of fungi isolated from healthy tissues of Itea japonica and I. riparia in Northern Thailand. By incorporating morphology, multiloci phylogenetic analyses and nucleotide polymorphism comparison, we establish two novel species, Diaporthe iteae (Diaporthaceae, Sordariomycetes) and Neptunomyces iteae (Didymosphaeriaceae, Dothideomycetes) as well as report a new host and a new geographical record for Diaporthe coracoralinae. Diaporthe iteaesp. nov. was collected from I. japonica, and is recognized within the Diaporthe siamensis species complex (section Sojae). In addition, D. coracoralinae is reported on I. japonica in Thailand for the first time, occupying a position within section Sojae with an undetermined species complex. Neptunomyces iteaesp. nov. was collected from I. riparia and is classified within the family Didymosphaeriaceae. Updated phylogenetic trees and comprehensive morphological comparisons with closely related taxa are provided and discussed for better resolution of species delimitation. This study enhances our understanding of fungal diversity on Itea, and provides a taxonomic foundation for future biodiversity, ecological and applied studies.

Key words: Coelomycetous asexual morph, Diaporthe siamensis species complex, endophytic fungi, Itea-inhabiting fungi, novel species, polyphasic taxonomy, species identification

Introduction

Diaporthe (Diaporthaceae, Sordariomycetes) is an important genus of coelomycetous Ascomycota established by Nitschke (1870) with the type species, D. eres. Diaporthe commonly exists as endophytes, pathogens, and saprobes and is associated with diverse hosts and has a broad geographic distribution and their relationships within the Diaporthales still warrant further investigations (Rossman et al. 2007; Udayanga et al. 2011; Gomes et al. 2013; Senanayake et al. 2017, 2018; Perera et al. 2018; Huang et al. 2021; Ferro et al. 2024; Gomzhina and Gasich 2026). Previously, asexual morph in Diaporthe was treated as Phomopsis. According to the “one fungus one name” nomenclature (Rossman et al. 2015), Diaporthe has priority over the synonym Phomopsis. The asexual morph of this genus is characterized by ostiolate conidiomata, cylindrical phialides, aseptate, hyaline and three types of conidia (i.e. alpha, gamma, and beta) (Udayanga et al. 2011; Gomes et al. 2013), while the sexual morph is characterized by pseudostromatic ascomata with perithecial necks, unitunicate asci, septate or aseptate, hyaline, ellipsoid to fusiform ascospores, occasionally with appendages (Wehmeyer 1933; Senanayake et al. 2018). The classification of Diaporthe species was primarily based on host specificity and morphology (Uecker 1988; Udayanga et al. 2011). However, the main morphological characteristics such as conidiogenous cells and conidia were shared by species that are not closely related phylogenetically (Dissanayake et al. 2017; Gao et al. 2017; Norphanphoun et al. 2022). Therefore, polyphasic taxonomy incorporating morphology, multiloci phylogeny and ecological data has been extensively accepted for species boundaries (Udayanga et al. 2012; Perera et al. 2018; Dissanayake et al. 2020; Hongsanan et al. 2023; Monkai et al. 2023; Ferro et al. 2024; Aumentado et al. 2026). At present, more than 1300 species epithets of Diaporthe are listed in Index Fungorum (2026) with a significant increase of species number in recent years (Bhunjun et al. 2022; Aumentado et al. 2026).

Neptunomyces (Didymosphaeriaceae, Dothideomycetes) was proposed by Gonçalves et al. (2019) with N. aureus designated as the type species. Neptunomyces is a member of Didymosphaeriaceae, which is a large family in Pleosporales (Gonçalves et al. 2019; Hongsanan et al. 2020). Didymosphaeriaceae exhibit a remarkably wide range of hosts and habitats with worldwide distribution, predominantly existing as saprobes or as endophytes and pathogens (Ariyawansa et al. 2014a, 2014b; Yuan et al. 2020; Tennakoon et al. 2022; Ren et al. 2022; Htet et al. 2024; Wanasinghe et al. 2025). Morphologically, the sexual morph of Didymosphaeriaceae is characterized by ostiolate ascomata, trabeculate pseudoparaphyses, uni-septate, brown ascospores, where its asexual morphs may be coelomycetes and hyphomycetes (Ariyawansa et al. 2014a, 2014b; Wanasinghe et al. 2016; Tennakoon et al. 2022, 2025). Recently, molecular phylogeny coupled with morphology has been applied to resolve the boundaries of taxa in Didymosphaeriaceae (Ren et al. 2022; Tennakoon et al. 2022, 2025; Htet et al. 2024; Wanasinghe et al. 2024, 2025). Neptunomyces can be distinguished from other genera in Didymosphaeriaceae by producing coelomycetous, aseptate, hyaline to golden yellow conidia with round apices and also forms a monophyletic clade within this family (Gonçalves et al. 2019; Tennakoon et al. 2025). Currently, Neptunomyces comprises seven species according to Index Fungorum (2026).

Endophytic fungi have recently been redefined and they are known to constitute symbiotic interaction with host plants and are recognized as prolific sources of bioactive substances, which modulate the accumulation of secondary metabolites (Palwe et al. 2021; Reyes et al. 2021; Yu et al. 2024; Liao et al. 2025). These fungi foster plant growth and increase host resistance to diseases and environmental pressures (Yu et al. 2024; Xu et al. 2026). Itea species are particularly valued for their nutritional and therapeutic qualities, which are largely attributed to their ability to accumulate rare sugars and important bioactive compounds (Luo et al. 2014; Zeng et al. 2015; Lang et al. 2022; Feng et al. 2024). Studying the fungal communities within Itea is crucial for searching for novel sources of these compounds. Despite the widespread distribution of Itea throughout East Asia (Song and Zhang 2024; POWO 2026), research on endophytic fungi associated with this plant remains limited. To address this gap, we carried out taxonomic studies to characterize endophytic isolates from Itea riparia and I. japonica in Thailand (Monkai et al. 2025, 2026). During our observations, we found four coelomycetous strains that produced distinct conidial exudates in culture. The objectives of this study are to identify these taxa based on multiloci phylogeny, nucleotide sequence comparisons and morphology, and clarify taxonomic positions of these and related taxa within the genus and family.

Materials and methods

Fungal Collection and Isolation

Fresh plant samples of Itea japonica and I. riparia were collected from Hang Dong District and Mae Rim District, Chiang Mai Province, Thailand. Endophytic fungi were isolated from healthy tissues using the surface sterilization method described in a previous study (Monkai et al. 2025). The plant segments (approximately 5 mm2) were excised and surface-sterilized by rinsing in sterile distilled water for 1 min, immersing in 70% ethanol for 30 s, followed by 2% NaOCl for 30 s, then air-dried and placed onto potato dextrose agar (PDA) plates. The isolated plates were incubated at 25 °C for one to two days, and then the single-hyphal tips were transferred to fresh PDA for purification and further morphological observation. Pure cultures were deposited and permanently preserved in a metabolically inactive state at the Mae Fah Luang University Culture Collection (MFLUCC), Mae Fah Luang University, Chiang Rai Province, Thailand and the Culture Collection of Microbial Shenzhen University (MBSZU), Shenzhen University, China. Dried cultures were prepared for fungarium specimens (Monkai et al. 2025) and deposited in the herbarium of Mae Fah Luang University (MFLU), Mae Fah Luang University, Chiang Rai Province, Thailand.

Morphological Characterization

After incubation at 25 °C for one to two months, fungal PDA cultures were examined for sporulation, colony features, pigment production and growth rate. Macro-morphology was examined using a stereo microscope (Nikon SMZ800N, Tokyo, Japan). Micro-morphological characteristics including conidiomata, conidiomatal wall, ostioles, conidiophores, conidiogenous cells and conidia were observed using a compound microscope equipped with Nikon DS-Ri2 camera (Nikon Eclipse Ni U, Tokyo, Japan). Measurements were conducted by the Tarosoft (R) Image Frame Work program with 10 measurements taken for conidiomata, conidiomatal wall, ostioles, conidiophores, conidiogenous cells and 30 measurements for conidia. The morphological description was prepared coupled with photo plates, which were organized by Adobe Photoshop version 21.2.4 (Adobe Systems, San Jose, CA, USA). The newly introduced taxa were registered to obtain Index Fungorum numbers (Index Fungorum 2026).

DNA extraction, amplification, and sequencing

The fungal mycelium (grown on PDA at 25 °C for one week) was processed for DNA extraction using an E.Z.N.A® Tissue DNA Kit (Omega, USA). Polymerase chain reaction (PCR) was used to amplify the following genomic regions including the internal transcribed spacer region of ribosomal DNA (ITS), the partial 28S large subunit nuclear ribosomal DNA (LSU), 18S small subunit rDNA (SSU), the translation elongation factor 1-α (tef1-α), beta-tubulin (tub2), calmodulin (cal), and histone H3 (his3). The specific primers and PCR thermal cycling programs for each gene region are listed in Table 1. PCR amplifications were conducted in a total volume of 20 μl, which contained 7 μl of deionized water, 10 μl of 2× Phanta Max Master Mix (Dye Plus) (Vazyme, China), 1 μl of genomic DNA, and 1 μl of each forward and reverse primer. The PCR products were purified using the E.Z.N.A® Gel Extraction Kit (Omega, USA), and assessed using 1% agarose gel electrophoresis and a Nanodrop 2000 Spectrophotometer (Thermo Scientific, USA). The DNA sequencing of purified PCR samples was performed by BGI-Shenzhen Company (Shenzhen, Guangdong, China).

Table 1.

The specific primer and PCR thermal cycling program of each gene region used in this study.

Gene region Primer References PCR thermal cycling program
ITS ITS4/ITS5 White et al. (1990) An initial denaturation step of 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 50 s at 52 °C and 1 min at 72 °C, and a final elongation step of 7 min at 72 °C
LSU LR0R/LR5 Vilgalys and Hester (1990)
SSU NS1/NS4 White et al. (1990)
tef1-α EF1-983F/EF1-2218R Rehner (2001) An initial denaturation step of 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 50 s at 54 °C and 1 min at 72 °C, and a final elongation step of 7 min at 72 °C
EF1-728F/EF1-986R Carbone and Kohn (1999)
tub2 Bt2a/Bt2b Glass and Donaldson (1995)
cal CAL228F/CAL737R Carbone and Kohn 1999
his3 CYLH3F/H3-1b Glass and Donaldson (1995); Crous et al. (2004)
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Phylogenetic analyses

The consensus sequence was processed using SeqMan v. 7.0.0 (DNASTAR, Madison, WI). The obtained nucleotide sequence was initially checked by the NCBI nucleotide BLAST search (www.ncbi.nlm.nih.gov/blast/; accessed on 1 March 2026) to identify the closest matches. The reference nucleotide sequences were obtained based on the results of the BLAST search and recently published data of Diaporthe section Sojae (Dissanayake et al. 2024; Ferro et al. 2024; Aumentado et al. 2026) and Neptunomyces (Yasanthika et al. 2024; Chen et al. 2025; Tennakoon et al. 2025) (Tables 2, 3). The single gene sequences were aligned using the MAFFT v7.307 online version (Katoh et al. 2017; https://mafft.cbrc.jp/alignment/server/, accessed on 1 October 2025) and corrected manually using BioEdit version 7.0.5.3 (Hall 1999). The aligned sequence data of ITS, tef1-α, tub2, cal and his3 were concatenated for phylogenetic analysis of Diaporthe section Sojae. While the alignment of ITS, LSU, SSU and tef1-α was concatenated for phylogenetic analysis of Didymosphaeriaceae. Phylogenetic trees were constructed using Maximum likelihood (ML) and Bayesian inference (BI) analyses via the CIPRES Science Gateway platform V3.3 (Miller et al. 2010). For ML analysis, ML node support was estimated with 1,000 bootstrap (BS) replicates and the GTRGAMMA model using RAxML-HPC2 on XSEDE (v.8.2.12) (Stamatakis 2014). Model selection of BI analysis was implemented by MrModelTest v.2.3 based on the Akaike Information Criterion (AIC) (Nylander 2004). The results of the best-fit model for each gene region are shown in Table 4. For BI analysis, Bayesian posterior probability (BYPP) was evaluated with six simultaneous Markov chains and sampling tree every 1000th generation using MrBayes v.3.2.6 (Ronquist et al. 2012). The burn in phase of initial 25% generated trees were discarded, while the remaining trees were applied to calculate posterior probabilities (PPs), when the average standard deviation of split frequencies dropped below 0.01 (Rambaut et al. 2018). The phylograms were visualized and refined in FigTree v1.4.0 (Rambaut and Drummond 2012) and Adobe Illustrator version 24.3 (Adobe Systems, San Jose, CA, the USA). The information on newly described strains was submitted for acquiring GenBank accession numbers. The concatenated alignments and phylogenetic trees are available in Figshare (https://doi.org/10.6084/m9.figshare.33091040).

Table 2.

GenBank accession numbers of Diaporthe section Sojae used in multiloci phylogenetic analyses.

Species name Strain number ITS tef1-α tub2 cal his3
Diaporthe ambigua CBS 114015 MH862953 KC343736 KC343978 KC343252 KC343494
D. ambigua CBS 117167 KC343011 KC343737 KC343979 KC343253 KC343495
D. biguttulata CFCC 52584 MH121519 MH121561 MH121598 MH121437 MH121477
D. biguttulata CFCC 52585 MH121520 MH121562 MH121599 MH121438 MH121478
D. brasiliensis CBS 133183 KC343042 KC343768 KC344010 KC343284 KC343526
D. brasiliensis LGMF926 KC343043 KC343769 KC344011 KC343285 KC343527
D. caatingaensis URM7485 KY085927 KY115604 KY115601 KY115598 KY115605
D. caatingaensis URM7484 KY085928 N/A KY115602 KY115599 KY115606
D. caryae CFCC 52563 MH121498 MH121540 MH121580 MH121422 MH121458
D. caryae CFCC 52564 MH121499 MH121541 MH121581 MH121423 MH121459
D. cerradensis CMRP4331 MN173198 MT311685 MW751671 MW751655 MW751663
D. cerradensis CMRP4324 MN173195 MT311681 MW751667 MW751651 MW751659
D. chiangraiensis MFLUCC 17-1669 MF190119 MF377598 N/A N/A N/A
D. chiangraiensis MFLUCC 17-1670 MF190118 MF377599 N/A N/A N/A
D. cinnamomi CFCC 52569 MH121504 MH121546 MH121586 N/A MH121464
D. cinnamomi CFCC 52570 MH121505 MH121547 MH121587 N/A MH121465
D. citriasiana ZJUD30 JQ954645 JQ954663 KC357459 KC357491 N/A
D. citriasiana ZJUD81 KJ490616 KJ490495 KJ490437 N/A KJ490558
D. coracoralinae URM 8912 PP192078 PP430449 PP402241 PP408214 PP421133
D. coracoralinae IR7 PP192079 N/A PP402242 PP408215 PP421134
D. coracoralinae IR6A PP192077 N/A PP402239 PP408213 N/A
D. coracoralinae AR11B N/A N/A PP402236 PP408212 PP421132
D. coracoralinae MFLUCC 26-0215 PZ458814 PZ501085 PZ501081 PZ501083 PZ501089
D. cyatheae YMJ-1364 JX570889 KC465406 KC465403 KC465410 N/A
D. discoidispora ZJUD89 KJ490624 KJ490503 KJ490445 N/A KJ490566
D. eleutherrhenae 01 OK017069 OK017070 OK017071 N/A N/A
D. eleutherrhenae 02 OK648457 OK648458 OK648459 N/A N/A
D. eres (D. alnea) CBS 146.46 KC343008 KC343734 KC343976 KC343250 KC343492
D. eres (D. nobilis) CBS 587.79 KC343153 KC343879 KC344121 KC343395 KC343637
D. fici-septicae NCYUCC 19-0108 MW114349 MW192212 MW148269 N/A N/A
D. fici-septicae MFLUCC 20-0178 MW114348 MW192211 MW148268 N/A N/A
D. foliorum CMRP 1321 MT576688 MT584310 MT584327 MT584341 MT584338
D. foliorum CMRP 1320 MT576671 MT584309 MT584328 MT584342 MT584340
D. ganjae CBS 180.91 KC343112 KC343838 KC344080 KC343354 KC343596
D. ganjae PSCG489 MK626955 MK654897 MK691287 MK691202 MK726204
D. goulteri BRIP 55657a KJ197290 KJ197252 KJ197270 N/A N/A
D. infecunda CBS 133812 KC343126 KC343852 KC344094 KC343368 KC343610
D. infecunda LGMF908 KC343127 KC343853 KC344095 KC343369 KC343611
D. iteae MFLUCC 26-0214 PZ458813 PZ501084 PZ501080 PZ501082 PZ501088
D. juglandigena GUCC 422.16 OP581229 OP688534 OP688559 N/A N/A
D. juglandigena GUCC 422.161 OP581230 OP688535 OP688560 N/A N/A
D. kyushuensis STE-U2675 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 N/A KY435632 KY435673 KY435663 KY435653
D. leucospermi CAA483 KY435635 KY435625 KY435666 KY435656 KY435645
D. longispora CBS 194.36 KC343135 KC343861 KC344103 KC343377 KC343619
D. mangroviorum MFLUCC 24-0445 PQ699353 PV769004 PV753771 PV753687 PV752668
D. mangroviorum MFLUCC 24-0446 PQ699362 PV769015 PV753782 PV753689 PV752682
D. mayteni CBS 133185 KC343139 KC343865 KC344107 KC343381 KC343623
D. middletonii BRIP 54884e KJ197286 KJ197248 KJ197266 N/A N/A
D. middletonii BRIP 57329 KJ197285 KJ197247 KJ197265 N/A N/A
D. minusculata CGMCC3.20098 MT385957 MT424692 MT424712 MW022475 MW022499
D. minusculata GZCC 19-0345 MT797184 MT793027 MT793038 MW022476 MW022500
D. orixae KUNCC 21-10714 OK283041 N/A N/A OK484485 OK484486
D. orixae GZCC 21-1085 OL889852 OL944724 OL944726 N/A N/A
D. oxe CBS 133186 KC343164 KC343890 KC344132 KC343406 KC343648
D. oxe CBS 133187 KC343165 KC343891 KC344133 KC343407 KC343649
D. pachirae CDA 728 MG559537 MG559539 MG559541 MG559535 N/A
D. pachirae CDA 730 MG559538 MG559540 MG559542 MG559536 N/A
D. paranensis CBS 133184 KC343171 KC343897 KC344139 KC343413 KC343655
D. paranensis LMICRO417 KY461115 KY461116 N/A N/A N/A
D. passiflorae CBS 132527 JX069860 N/A N/A N/A KY435654
D. passiflorae DJY16A1-5 MH595929 MH621353 MH621349 N/A N/A
D. quercicola CSUFTCC104 ON076567 ON081659 N/A ON081670 ON081667
D. quercicola CSUFTCC105 ON076568 ON081660 N/A ON081671 ON081668
D. raonikayaporum CBS 133182 KC343188 KC343914 KC344156 KC343430 KC343672
D. raonikayaporum (D. neoraonikayaporum) MFLUCC 14-1136 KU712449 KU749369 KU743988 KU749356 N/A
D. rhizophoracearum MFLUCC 24-0449 PQ699356 PV769008 PV753774 PV753681 PV752671
D. rhizophoracearum MFLUCC 24-0450 PQ699357 PV769009 PV753775 PV753682 PV752672
D. sclerotioides CBS 296.67 MH858974 KC343919 KC344161 KC343435 KC343677
D. sclerotioides CBS 710.76 KC343194 KC343920 KC344162 KC343436 KC343678
D. sclerotioides (D. columnaris) AR3612 AF439625 N/A N/A N/A N/A
D. sclerotioides (D. columnaris) ICMP 16836 KC145883 KC145999 N/A N/A N/A
D. serafiniae BRIP 55665a KJ197274 KJ197236 KJ197254 N/A N/A
D. serafiniae BRIP 54136 KJ197273 KJ197235 KJ197253 N/A N/A
D. siamensis MFLUCC 12-0300 KT459417 KT459451 KT459435 KT459467 N/A
D. siamensis MFLUCC 10-0573a JQ619879 JX275393 JX275429 JX197423 N/A
D. submersa GZCC 19-0129 OP056717 OP150556 OP150633 OP150710 OP150786
D. submersa GZCC 22-0007 OP056718 OP150557 OP150634 OP150711 OP150787
D. tulliensis BRIP 62248a KR936130 KR936133 KR936132 N/A N/A
D. tulliensis RST-1 MN911382 MT121969 N/A N/A N/A
D. yunnanensis CGMCC3.18289 KX986796 KX999188 KX999228 KX999290 KX999267
D. yunnanensis LC8107 KY491542 KY491552 KY491562 KY491572 N/A
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Notes: Sequences shaded in blue were isolated in this study. Sequences in bold are the ex-type strains. Unavailable sequences are indicated as “N/A”.

Table 3.

GenBank accession numbers of taxa in Didymosphaeriaceae used in multiloci phylogenetic analyses.

Species name Strain number ITS LSU SSU tef1-α
Alloconiothyrium aptrootii CBS 980.95 JX496121 JX496234 N/A N/A
A. aptrootii CBS 981.95 JX496122 JX496235 N/A N/A
A. encephalarti CPC: 35980 MN562102 MN567610 N/A N/A
Austropleospora archidendri MFLUCC 17-2429 MK347757 MK347974 MK347863 MK360044
A. archidendri MFLU 22-0042 OP058964 OP059055 OP059006 OP135941
Bambusistroma didymosporum MFLU 15-0057 KP761733 KP761730 KP761737 KP761727
B. didymosporum MFLU 15-0058 KP761734 KP761731 KP761738 KP761728
Bimuria novae-zelandiae CBS 107.79 MH861181 AY016356 AY016338 DQ471087
Chromolaenicola nanensis MFLUCC 17-1473 MN325015 MN325003 MN325009 MN335648
C. nanensis MFLUCC 17-1477 MN325014 MN325002 MN325008 MN335647
C. sapindi KUMCC 21-0564 OP058967 OP059058 OP059009 OP135943
Cylindroaseptospora leucaenae MFLUCC 17-2424 NR_163333 NG_066310 MK347856 MK360047
C. siamensis MFLUCC 17-2527 MK347760 MK347976 MK347866 MK360048
Deniquelata barringtoniae MFLUCC 11-0422 NR_111779 NG_042696 JX254656 N/A
Dictyoarthrinium vittalii NFCCI4249 MF406218 MF182395 MF622059 MF182398
D. hydei SQUCC 13296 MW077145 N/A MW077161 MW075771
Didymocrea sadasivanii CBS 438.65 MH858658 DQ384103 N/A N/A
Didymosphaeria rubi-ulmifolii MFLUCC 14-0023T N/A KJ436586 NG_063557 N/A
D. rubi-ulmifolii MFLUCC 14-0024 N/A KJ436585 KJ436587 N/A
Kalmusia italica MFLUCC 14-0560 KP325440 KP325441 KP325442 N/A
K. erioi MFLU 18-0832 NR_169984 MN473052 MN473046 MN481599
Kalmusibambusa triseptata MFLUCC 13-0232 KY682697 KY682695 KY682696 N/A
Karstenula lancangensis KUMCC 21-0670 OP058969 OP059060 OP059011 N/A
K. lancangensis KUMCC 21-0677 OP058970 OP059061 OP059012 N/A
Laburnicola hawksworthii MFLUCC 13-0602 KU743194 KU743195 KU743196 N/A
L. muriformis MFLUCC 14-0921 KU743200 KU743201 KU743202 N/A
Letendraea cordylinicola MFLUCC 11-0150 KM213996 KM213999 KM214002 N/A
L. cordylinicola MFLUCC 11-0148 NR_154118 NG_059530 KM214001 N/A
Montagnula donacina KUMCC 21-0653 OP058961 OP059052 OP059003 OP135938
M. thailandica MFLUCC 17-1508 MT214352 NG070949 NG070158 MT235774
Neokalmusia brevispora KT 1466 LC014573 AB524600 AB524459 AB539112
N. scabrispora KT 1023 LC014575 AB524593 AB524452 AB539106
Neptunomyces aureus CMG 10A = MUM 19.38 MK912119 N/A N/A MK948000
Neptunomyces sp. YNE01044 NMDCN0007RII NMDCN0007RIV N/A NMDCN0007RJ6
N. chinensis SZU25-020 PV730047 PV730026 PV730037 PV749903
N. chinensis SZU25-021 PV730048 PV730027 PV730038 PV749904
N. iteae MFLUCC 26-0216 PZ458815 PZ458817 PZ458811 PZ501086
N. iteae MFLUCC 26-0217 PZ458816 PZ458818 PZ458812 PZ501087
N. jeanbriggsiae BRIP 75897a PQ431208 PQ431200 N/A N/A
N. juncicola CPC 45436 PP791436 PP791464 N/A PP780627
N. litoralis BRIP 75555a OR271911 OR259052 N/A N/A
N. soli MFLUCC 24-0272 PP993910 PQ306558 N/A N/A
N. soli MFLUCC 24-0279 PQ306559 PQ306560 N/A N/A
N. yunnanensis YNE00523 NMDCN0007RIH NMDCN0007RIP N/A NMDCN0007RJC
Paracamarosporium fagi CPC 24890 KR611886 KR611904 N/A N/A
P. fagi CPC 24892 KR611887 KR611905 N/A N/A
Paramassariaosphaeria anthostomoides MFLUCC 16-0172 KU743206 KU743207 KU743208 N/A
Paraphaeosphaeria rosae MFLUCC 17-2547 MG828935 MG829044 MG829150 MG829222
P. rosae MFLUCC 17-2549 MG828937 MG829046 MG829152 MG829223
Phaeodothis winteri CBS 182.58 N/A GU301857 GU296183 N/A
Pseudocamarosporium propinquum MFLUCC 13-0544 KJ747049 KJ813280 KJ819949 N/A
P. pteleae MFLUCC 17-0724 NR_157536 MG829061 MG829166 MG829233
Pseudopithomyces entadae MFLUCC 17-0917 N/A NG_066305 MK347835 MK360083
P. rosae MFLUCC 15-0035 MG828953 MG829064 MG829168 N/A
Septofusispora thailandica KUMCC 21-0647 OP058971 OP059062 OP059013 OP135945
S. thailandica KUMCC 21-0652 OP058972 OP059063 OP059014 N/A
Spegazzinia bromeliacearum URM 8084 MK804501 MK809513 N/A N/A
S. camelliae WNA03 MZ538526 MZ538560 N/A MZ567102
S. camelliae CMU328 MH734522 MH734521 MH734523 MH734524
Tremateia arundicola MFLU 16-1275 KX274241 KX274248 KX274254 KX284706
T. guiyangensis GZAAS01 KX274240 KX274247 KX274253 KX284705
T. murispora GZCC 18-2787 NR_165916 MK972751 MK972750 MK986482
Verrucoconiothyrium nitidae CBS 119209 EU552112 EU552112 N/A N/A
Xenocamarosporium acaciae CBS 139895 NR_137982 NG_058163 N/A N/A
X. acaciae MFLUCC 17-2432 MK347766 MK347983 MK347873 MK360093
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Notes: Sequences shaded in blue were isolated in this study. Sequences in bold are the ex-type strains. Unavailable sequences are indicated as “N/A”. The National Microbiology Data Center (NMDC) receives accession numbers for genomic loci that start with “NMDCXXXXXXXX,” while the National Center for Biotechnology Information (NCBI) GenBank receives all other accession numbers.

Table 4.

The result of best-fit model for each gene region.

Gene region/loci Diaporthe Didymosphaeriaceae
ITS GTR+I+G GTR+G
LSU – GTR+I+G
SSU – GTR+I+G
tef1-α HKY+I+G GTR+I+G
tub2 HKY+I+G –
cal GTR+I+G –
his3 GTR+I+G –
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Results

Phylogenetic Analyses

Phylogenetic analysis of 84 taxa from Diaporthe, section Sojae was performed using a concatenated ITS, tef1-α, tub2, cal and his3 sequence dataset including the outgroup taxa, D. eres (= D. alnea) CBS 146.46 and D. eres (= D. nobilis) CBS 587.79 (Fig. 1). The combined alignment dataset contained 2609 total characters (ITS: 1–561 bp, tef1-α: 562–904 bp, tub2: 905–1672 bp, cal: 1673–2151 bp, his3: 2152–2609 bp), including gaps. The RAxML analysis generated a best scoring tree with a final ML optimization likelihood value of −23431.396860. The matrix is composed of 1361 distinct alignment patterns, with 29.37% undetermined characters or gaps. Estimated base frequencies were recorded as follows: A = 0.213190, C = 0.326225, G = 0.238836, T = 0.221749; substitution rates AC = 1.227081, AG = 3.737206, AT = 1.377917, CG = 1.142710, CT = 5.072988, and GT = 1.000000. The gamma distribution shape parameter (α value) was equal to 0.315160. For Bayesian analysis, the average standard deviation of split frequencies was 0.009337.

Figure 1.

Figure 1.

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Maximum likelihood phylogram based on combined ITS, tef1-α, tub2, cal and his3 sequence data. Isolates from present study are in red and ex-type strains are indicated in bold. Bootstrap support values ≥ 40% (left) and Bayesian posterior probabilities ≥ 0.80 (right) are shown at the nodes (ML/BYPP).

Phylogenetic analysis of 65 taxa in Didymosphaeriaceae was performed using a combined ITS, LSU, SSU and tef1-α sequence dataset including the outgroup taxa, Bambusistroma didymosporumMFLU 15-0057 and B. didymosporumMFLU 15-0058 (Fig. 2). The combined alignment dataset contained 3459 total characters (ITS: 1–604 bp, LSU: 605–1506 bp, SSU: 1507–2531 bp, tef1-α: 2532–3459 bp), including gaps. The RAxML analysis generated a best scoring tree with a final ML optimization likelihood value of −20608.400551. The matrix is composed of 1131 distinct alignment patterns, with 27.46% undetermined characters or gaps. Estimated base frequencies were recorded as follows: A = 0.238889, C = 0.251162, G = 0.272199, T = 0.237751; substitution rates AC = 1.520570, AG = 2.266778, AT = 1.554952, CG = 1.154130, CT = 6.226753, and GT = 1.000000. The gamma distribution shape parameter (α value) was equal to 0.191358. For Bayesian analysis, the average standard deviation of split frequencies was 0.009683.

Figure 2.

Figure 2.

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Maximum likelihood phylogram based on combined ITS, LSU, SSU and tef1-α sequence data. Isolates from present study are in red and ex-type strains are indicated in bold. Bootstrap support values ≥ 60% (left) and Bayesian posterior probabilities ≥ 0.90 (right) are shown at the nodes (ML/BYPP).

The phylograms derived from ML and BI analyses were topologically comparable. Thus, the RAxML trees were displayed as backbone trees. The phylogram of Diaporthe section Sojae revealed that Diaporthe iteae sp. nov. (MFLUCC 26-0214) constitutes a distinct sister clade with D. kyushuensis (STE-U2675), D. fici-septicae (MFLUCC 20-0178) and D. citriasiana (ZJUD30) (45% ML, 0.86 BYPP) within Diaporthe siamensis species complex (Fig. 1). The new strain MFLUCC 26-0215 clusters with the D. coracoralinae (URM8912, ex-type, AR11B, IR7 and IR6A) with high support (100% ML, 1.00 BYPP; Fig. 1). Overall phylogenetic placements of Diaporthe section Sojae are well-resolved, except D. cyatheae, D. infecunda, D. iteae and D. mayteni (Fig. 1).

While the phylogram of Didymosphaeriaceae showed that two strains of Neptunomyces iteae sp. nov. (MFLUCC 26-0217, ex-type and MFLUCC 26-0216) clusters together with high support (100%ML, 1.00 BYPP; Fig. 2) and closely related with N. jeanbriggsiae (BRIP 75897a) (Fig. 2). In addition, many taxa in Didymosphaeriaceae (e.g. Alloconiothyrium aptrootii, Cylindroaseptospora leucaenae, Didymocrea sadasivanii, Kalmusibambusa triseptata, Paramassariaosphaeria anthostomoides, Phaeodothis winteri, and Verrucoconiothyrium nitidae) constitute poor support lineages (Fig. 2). Particularly, several Neptunomyces species (e.g. Neptunomyces jeanbriggsiae, N. litoralis, N. soli and N. yunnanensis) occupy unresolved placements (Fig. 2).

Taxonomy

Diaporthe iteae

Monkai, Bhat, Hongsanan & S. Lumyong sp. nov.

3A52DDC3-6A65-5559-A1F4-41F10F9BBE3A

Fig. 3

Figure 3.

Figure 3.

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Diaporthe iteae (MFLUCC 26-0214, ex-type). A, B. Colony after one month on PDA at 28 °C (surface and reverse); C, D. Conidiomata and conidial masses; E. Ostioles; F. Pycnidia; G, H. Conidiomata wall; I. Conidiogenous cells and conidia; J. Conidia. Scale bars: 1000 μm (C, D); 200 μm (E, F); 10 μm (G–J).

Etymology.

Referring to the host genus Itea from which the holotype was collected.

Holotype.

MFLU 26-0232.

Description.

Endophytic on healthy leaves of Itea japonica. Sexual morph: undetermined. Asexual morph: Coelomycetous. Conidiomata on PDA pycnidial, 500–1800 μm diam., stromatic, semi-immersed, scattered on PDA, subglobose to ovoid, dark brown to black, multilocular with several prominent black necks extending above the stroma, with conidial drops exuding in white to pale-yellow. Ostiolar neck 800–1400 μm high × 250–400 μm diam., elongated, conical, dark brown to black, papilate. Conidiomatal wall 80–180 μm diam., comprising multiple layers of brown to reddish brown, arranged in textura prismatica to textura globosa. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 17–38 × 1.5–2.3 μm (x– = 26 × 2 μm, n = 10), phialidic, cylindrical, densely aggregated, terminal, slightly tapering towards the apex, hyaline. Alpha conidia not observed. Beta conidia 21.8–35.5 × 1–1.8 μm (x– = 28.5 × 1.5 μm, n = 30), aseptate, filiform, tapering towards the apex, truncate at the base, sinuous at one end, hyaline, eguttulate, smooth-walled.

Culture characteristics.

Colonies on PDA reached at 8 cm diam. in 5 d at 28 °C, raised elevation, effuse, dense aerial mycelium, cottony to floccose, undulate margin, initial white, becoming pale brown due to pigment formation, in reverse, pale brown to reddish brown, sporulated after one month, with white to pale yellowish conidial drops exuding from the ostioles.

Material examined.

Thailand • Chiang Mai Province, Hang Dong District, on living leaves of Itea japonica (Iteaceae), 7 February 2023, J. Monkai, IT26 (MFLU 26-0232, holotype), ex-type living culture, MFLUCC 26-0214 = MBSZU 26-101, dried culture permanently preserved in a metabolically inactive state.

Notes.

In the phylogenetic analysis, Diaporthe iteae forms a distinct clade and has a close relationship with D. kyushuensis, D. fici-septicae and D. citriasiana (45% ML, 0.86 BYPP; Fig. 1). Morphologically, D. iteae resembles D. fici-septicae and D. citriasiana in having stromatic conidiomata with ostioles (Tennakoon et al. 2021). Diaporthe iteae can be distinguished from D. citriasiana and D. kyushuensis by having shorter beta conidia (21.8–35.5 × 1–1.8 vs. 24–42.5 × 1–2 vs. 25–55 × 1–2 μm) (Kajitani and Kanematsu 2000; Huang et al. 2013). However, D. fici-septicae produced only alpha conidia (Tennakoon et al. 2021). Thus, it was unable to compare their conidial morphology. A pairwise nucleotide comparison between D. iteae and D. citriasiana indicates 3.2%, 5.3%, 3.7%, 6.3%, and 5% bp difference in ITS, tef1-α, tub2, cal, his3, respectively (Table 5). Diaporthe iteae can be distinguisted from D. fici-septicae in 2%, 2.4%, and 2.5% bp difference in ITS, tef1-α, tub2 respectively, and D. kyushuensis in 3.4% bp difference in ITS. These substantial nucleotide differences across the selected gene regions (greater than 1.5% bp divergence) further support the distinction of the species, in accordance with the guidelines of Jeewon and Hyde (2016), Chethana et al. (2021) and Maharachchikumbura et al. (2021), which have been widely applied in previous studies (Monkai et al. 2023; Li et al. 2025; Aumentado et al. 2026; Vasighzadeh et al. 2026). Based on its morphological characteristics, phylogenetic analysis and pairwise nucleotide difference, D. iteae is described here as a novel species.

Table 5.

A pairwise nucleotide comparison between Diaporthe iteae (MFLUCC 26-0214T) and closely related taxa.

Taxa Gene region/locus
ITS tef1-α tub2 cal his3*
D. citriasiana (ZJUD30T, ZJUD81*) 3.2% (19/592) 5.3% (18/338) 3.7% (20/543) 6.3% (25/399) 5.0% (23/463)
D. fici-septicae (MFLUCC 20-0178) 2.0% (10/507) 2.4% (8/336) 2.5% (11/446) NA NA
D. kyushuensis (STE-U2675T) 3.4% (16/465) NA NA NA NA
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Note: The data represented by % base-pair difference (the number of different nucleotides/number of all nucleotides). Tex-type strain, *For his3 gene region, D. citriasiana strain ZJUD81 was used for comparison.

Diaporthe coracoralinae

L.O. Ferro, Souza-Motta & J.D.P., Bezerra, Fungal Syst. Evol. 14: 259 (2024)

7BB366CC-0B58-5585-804A-EAD4B76670A1

Fig. 4

Figure 4.

Figure 4.

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Diaporthe coracoralinae (MFLUCC 26-0215). A, B. Colony after one month on PDA at 28 °C (surface and reverse); C. Conidiomata and conidial masses; D, E. Section of conidiomata; F. Conidiomata wall; G. Ostioles; H, I. Conidiogenous cells and conidia; J. Conidia. Scale bars: 1000 μm (C); 500 μm (D, E); 100 μm (F, G); 10 μm (H, I).

Description.

Endophytic on healthy leaves of Itea japonica. Sexual morph: undetermined. Asexual morph: Coelomycetous. Conidiomata on PDA pycnidial, 600–3500 μm diam., stromatic, semi-immersed, solitary or aggregated, subglobose to discoid, dark brown to black, multilocular with several locules embedded in the stroma, ostiolate, with conidial drops exuding in white to pale-yellow. Ostioles up to 200 μm diam., conical, dark brown to black, papilate. Conidiomatal wall 50–120 μm diam., comprising several layers of brown to dark brown, arranged in textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 11.5–16 × 1.6–2.4 μm (x– = 14 × 2 μm, n = 10), phialidic, cylindrical, densely aggregated, terminal, slightly tapering towards the apex, hyaline. Alpha conidia not observed. Beta conidia 22.3–31.5 × 1.2–2 μm (x– = 27 × 1.5 μm, n = 30), aseptate, filiform, tapering towards the apex, truncate at the base, slightly curved or sinuous at one end, hyaline, eguttulate, smooth-walled.

Culture characteristics.

Colonies on PDA reached at 8 cm diam. in 5 d at 28 °C, flat elevation, effuse, sparse aerial mycelium, floccose to strongly rugose, filiform margin, initial white, becoming pale brown due to pigment formation, in reverse, pale brown to dark brown, sporulated after one month, with white to pale yellowish conidial drops exuding from the ostioles.

Material examined.

Thailand • Chiang Mai Province, Hang Dong District, on living leaves of Itea japonica (Iteaceae), 7 February 2023, J. Monkai, IT27 (MFLU 26-0233), living culture, MFLUCC 26-0215 = MBSZU 26-102, dried culture permanently preserved in a metabolically inactive state.

Notes.

Diaporthe coracoralinae was described as an endophyte on healthy branches of Brosimum gaudichaudii from Brazil (Ferro et al. 2024). In our phylogenetic analysis, strain MFLUCC 26-0215, isolated from leaves of Itea japonica from Thailand, clusters with the type strain (URM8912) and other representative strains of D. coracoralinae with strong statistical support (100% ML, 1.00 BYPP; Fig. 1). A pairwise nucleotide comparison of ITS, tef1-α, tub2, cal and his3 between our collection and the type strain of D. coracoralinae (URM8912) indicates that they are identical, except for a 2 bp difference (0.5%) in his3 (Ferro et al. 2024). Ferro et al. (2024) reported that this species produce hyaline, aseptate, fusiform alpha conidia (5–7 × 2–3 μm). Whereas, beta and gamma conidia were not observed. Our study first time reports the production of beta conidia on D. coracoralinae which are hyaline, aseptate, filiform and eguttulate. Hence, we identify the new collection as D. coracoralinae, marking a new host record on Itea and a new geographical record from Thailand.

Neptunomyces iteae

Monkai, Bhat, Hongsanan & S. Lumyong sp. nov.

C00C669C-E6AF-552F-A40F-2121AAFF4AE0

Fig. 5

Figure 5.

Figure 5.

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Neptunomyces iteae [MFLUCC 26-0217, ex-type (A, B), MFLUCC 26-0216 (C, D)]. A–D. Colony after one month on PDA at 28 °C (surface and reverse); E, F. Conidiomata and conidial masses on toothpick and PDA; G, H. Pycnidia and conidiomata wall; I–K. Conidiophores, conidiogenous cells and conidia; L, M. Conidia. Scale bars: 500 μm (E, F); 200 μm (G–I); 10 μm (J–P).

Etymology.

Referring to the host genus Itea from which the holotype was collected.

Holotype.

MFLU 26-0235.

Description.

Endophytic on healthy stems and leaves of Itea riparia. Sexual morph: undetermined. Asexual morph: Coelomycetous. Conidiomata on PDA pycnidial, 200–800 μm diam., erumpent, immersed to semi-immersed, covered by dense mycelium, globose to subglobose, dark brown to black, solitary to gregarious, releasing black conidial masses. Conidiomatal wall 22–58 μm wide (x– = 41 μm), comprising a few layers of brown to dark brown pseudoparenchymatous cells, arranged in textura angularis. Conidiophores 1.7–4 μm diam., aggregated in clusters on hyphae, smooth, septate, branched, straight or flexuous, hyaline to pale brown. Conidiogenous cells 5–15.5 × 1.5–3 μm (x– = 8 × 2.3 μm, n = 10), holoblastic, discrete, subcylindrical to clavate, hyaline, smooth. Conidia 4.8–7 × 3–4.3 μm (x– = 6 × 3.7 μm, n = 30), aseptate, ellipsoidal to limoniform, apex acute to apiculate, widest in the middle, tapering towards a narrowly truncate base, brown to golden brown, guttulate, smooth, thick-walled.

Culture characteristics.

Colonies on PDA reached at 5 cm diam. in 7 d at 28 °C, raised elevation, cottony, entire margin, initial white, becoming yellowish-white with age, slightly immersed into agar, in reverse, whitish to yellowish in the middle, sporulated after one to two months, with numerous black conidial masses.

Material examined.

Thailand • Chiang Mai Province, Mae Rim District, on living stems of Itea riparia (Iteaceae), 10 July 2023, J. Monkai, IT82 (MFLU 26-0235, holotype), ex-type living culture, MFLUCC 26-0217 = MBSZU 26-103, dried culture permanently preserved in a metabolically inactive state; • ibid., on living leaves of Itea riparia (Iteaceae), 10 July 2023, J. Monkai, IT60 (MFLU 26-0234), living culture, MFLUCC 26-0216 = MBSZU 26-104, dried culture permanently preserved in a metabolically inactive state.

Notes.

Based on multiloci phylogeny, Neptunomyces iteae constitutes an independent lineage sister to N. jeanbriggsiae (Fig. 2). Besides, two new isolates cluster together with robust statistically support (100% ML, 1.00 BYPP; Fig. 2). A pairwise nucleotide comparison between N. iteae (MFLUCC 26-0217) and N. jeanbriggsiae (BRIP 75897a) indicates 9.9% (58/587) bp difference in ITS and 1% (9/870) bp difference in LSU, which further distinguishes the two species. The morphological description of N. jeanbriggsiae was not provided (Tan et al. 2024), thus it is not possible to compare its morphological characteristics with our new species. Neptunomyces iteae has morphological affinities with N. chinensis, N. soli and N. yunnanensis in having immersed to semi-immersed, globose to subglobose conidiomata and golden yellow to golden brown, ellipsoidal to limoniform conidia with an acute to apiculate apex (Yasanthika et al. 2024; Chen et al. 2025; Tennakoon et al. 2025) (Table 6). However, N. iteae is distinct from those species by the presence of conidiophores and longer conidiogenous cells (5–15.5 × 1.5–3 μm in N. iteae vs. 4–7 × 3–4 μm in N. chinensis vs. 3–7 × 2.5–7 μm in N. soli vs. 4.5–6 × 3.9–4.2 μm in N. yunnanensis) (Yasanthika et al. 2024; Chen et al. 2025; Tennakoon et al. 2025) (Table 6). Therefore, N. iteae is proposed as a new species based on its morphology, phylogenetic placement and pairwise nucleotide difference.

Table 6.

Synopsis of Neptunomyces species. The new species is indicated in bold.

Neptunomyces species Hosts and habitats Countries Sexual morphological characters Asexual morphological characters References
Conidiomata Conidiophores Conidiogenous cells Conidia
N. aureus Gracilaria gracilis Portugal UD Immersed or rarely superficial, dark brown, globose to subglobose, aggregated or solitary Reduced to conidiogenous cells 5.2 ± 0.3 × 2.0 ± 0.6 μm, hyaline, ampulliform to subcylindrical 7.0 ± 0.6 × 2.7 ± 0.2 μm, aseptate, golden yellow, subcylindrical with rounded apices Gonçalves et al. (2019)
N. chinensis Dead leaves of Phragmites australis China (Guangdong Province) UD 90–110 × 80–150 μm, pycnidial, immersed to semi-immersed, dark brown to black, globose to sub-globose, solitary to aggregated Reduced to conidiogenous cells 4–7 × 3–4 μm, holoblastic, discrete, hyaline, globose to doliiform 6.5–8 × 4–5 μm, aseptate, hyaline to pale brown, ellipsoidal to limoniform, apex acute to apiculate Tennakoon et al. (2025)
N. iteae Healthy stems and leaves of Itea riparia Thailand UD 200–800 μm diam., pycnidial, immersed to semi-immersed, dark brown to black, globose to subglobose, solitary to gregarious 1.7–4 μm diam., septate, branched, hyaline to pale brown 5–15.5 × 1.5–3 μm, holoblastic, discrete, hyaline, subcylindrical to clavate 4.8–7 × 3–4.3 μm, aseptate, brown to golden brown, ellipsoidal to limoniform, apex acute to apiculate This study
N. jeanbriggsiae Unidentified pink basidiocarp on the trunk of an unidentified tree Australia UD UD UD UD UD Tan et al. (2024)
N. juncicola Culms of Juncus maritimus Netherlands Pseudothecial, subglobose, papillate, asci fusoid-ellipsoid, ascospores tri-to multiseriate, fusoid-ellipsoid, 5–7 transverse septa, golden brown 180–220 μm diam., pycnidial, ostiolate, brown, globose 3–5 × 4–5 μm, phialidic, hyaline, ampulliform UD 7–8 × 2–2.5 μm, aseptate, golden brown, subcylindrical to fusoid-ellipsoid, apex subobtuse, hilum truncate Crous et al. (2024)
N. litoralis Sand Australia UD UD UD UD UD Tan et al. (2023)
N. soli Soil Thailand UD Brown to dark brown, globose to subglobose, superficial to semi-immersed UD 3–7 × 2.5–7 μm, hyaline to subhyaline, doliiform to obovoid 5–6.5 × 3–5 μm, aseptate, hyaline to golden yellow, ellipsoidal to fusiform Yasanthika et al. (2024)
N. yunnanensis Healthy leaves of Oryza meyeriana subsp. granulata China (Yunnan Province) UD Up to 400 μm diam., pycnidial, immersed, dark brown to black, subglobose, solitary or aggregated UD 4.5–6 × 3.9–4.2 μm, phialidic, hyaline, ampulliform 3.9–6 × 2.6–3.9 μm, aseptate, golden brown, ellipsoid to fusoid Chen et al. (2025)
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Discussion

The present study adopts a dual approach including morphological comparison, multiloci phylogenetic construction coupled with nucleotide polymorphism analyses to characterize endophytic coelomycetous fungi associated with Itea from Thailand. As a result, two new species are described: Diaporthe iteae from Itea japonica and Neptunomyces iteae from I. riparia. Additionally, we report the first record of D. coracoralinae from Itea in Thailand, which was previously isolated from Brosimum in Brazil.

Taxonomic circumscription of Diaporthe has earlier been extremely ambiguous in view of intraspecific variability and phenotypic plasticity among species (Dissanayake et al. 2017). The concept of species complex was proposed by Norphanphoun et al. (2022) which delineated Diaporthe into 13 species complexes based on phylogenetic analyses of individual and combined five loci (ITS, tef1-α, tub2, cal and his3) sequence datasets. Aligning with this phylogenetic reevaluation, Hongsanan et al. (2023) offered a comprehensive review of recognized or suspected Diaporthe species and proposed taxonomic modifications for select species based on updated and robust information. Subsequently, Dissanayake et al. (2024) applied a sectional framework to reassess Diaporthe species into seven phylogenetically distinct sections including 15 species-complexes and 13 species using integrative phylogenetic approaches. Our study follows the framework of Dissanayake et al. (2024) by applying this concatenated five loci phylogeny, the placements of two species (D. iteae and D. coracoralinae) belong to section Sojae (Fig. 1). Within the section, a new species, D. iteae is placed under the Diaporthe siamensis species complex and together they form distinct lineage sister to D. kyushuensis, D. fici-septicae and D. citriasiana (Fig. 1). Whereas strain MFLUCC 26-0215 exhibits a close affinity with D. coracoralinae and the lineage occupies outside the refined species-complexes (Fig. 1). Notably, the clade comprises D. cerradensis, D. coracoralinae, D. mayteni, D. foliorum, D. raonikayaporum and D. rhizophoracearum is closely related with the D. ganjae species complex and the D. sclerotioides species complex (Fig. 1); thus, it could be introduced as a novel species-complex by further accurate systematic framework. Moreover, we indicate rather poor support for the branch of D. iteae (45% ML, 0.86 BYPP; Fig. 1). This is probably due to the lack of cal and his3 sequence data in D. fici-septicae and the lack of tef1-α, tub2, cal and his3 sequence data in D. kyushuensis (Table 5). Incomplete sequence data, particularly tub2, cal and his3, have been found in many Diaporthe species (Norphanphoun et al. 2022; Dissanayake et al. 2024). These protein-coding loci are now considered to be effective phylogenetic markers for species delineation (Norphanphoun et al. 2022; Monkai et al. 2023; Dissanayake et al. 2024; Li et al. 2025; Vasighzadeh et al. 2026). Consequently, additional sequence data from both existing and new collections are necessitated to resolve uncertain placements of Diaporthe species.

Neptunomyces is an ecologically diverse genus that occupies both marine and terrestrial niches (Gonçalves et al. 2019; Tennakoon et al. 2025). The type species, N. aureus was originally discovered as an algicolous fungus (epiphytic or endophytic) in Portugal (Gonçalves et al. 2019). The ecological distribution of this genus has been expanded to include soil, sand, and a variety of terrestrial plants in which it serves as endophytes and saprobes (Gonçalves et al. 2019; Tan et al. 2023, 2024; Crous et al. 2024; Yasanthika et al. 2024; Chen et al. 2025; Tennakoon et al. 2025). In this study, we introduce Neptunomyces iteae as an endophyte of Itea riparia in Thailand. Our discovery, together with existing records from Australia, China, Thailand and the Netherlands, points to a cosmopolitan distribution for the genus, covering temperate and tropical climates across three continents (Gonçalves et al. 2019; Tan et al. 2023, 2024; Crous et al. 2024; Yasanthika et al. 2024; Chen et al. 2025; Tennakoon et al. 2025).

Morphologically, Neptunomyces is typically classified by its asexual morph with globose to subglobose, immersed to semi-immersed, pycnidial conidiomata, holoblastic or phialidic conidiogenous cells and ellipsoid or fusiform to limoniform, initially hyaline, later golden yellow to golden brown conidia with round apices (Gonçalves et al. 2019; Crous et al. 2024; Yasanthika et al. 2024; Chen et al. 2025; Tennakoon et al. 2025) (Table 6). Notably, N. iteae produces conidiophores, a character not rarely observed in other species. This finding contributes an important dimension to the generic description. Sexual morphs are still uncommon as they have only been recorded in N. juncicola (Crous et al. 2024). The absence of morphological data for N. jeanbriggsiae and N. litoralis (Tan et al. 2023, 2024), emphasizes the necessity of employing molecular data to delineate species.

Previous studies have applied various combinations of ribosomal DNA regions (e.g. ITS, LSU and/or SSU) and protein-coding loci (e.g. tef1-α, tub2 and/or rpb2) for the phylogenetic reconstruction of taxa within Didymosphaeriaceae (Gonçalves et al. 2019; Ren et al. 2022; Wanasinghe et al. 2024; Chen et al. 2025; Tennakoon et al. 2025). In accordance with Tennakoon et al. (2025) and based on available reference sequence data, we utilized a four-loci dataset of ITS, LSU, SSU and tef1-α to perform the phylogenetic analyses of Neptunomyces. However, our phylogenetic tree demonstrates several unresolved positions (Fig. 2). Neptunomyces iteae clusters with N. jeanbriggsiae with low support, forming a branch significantly distant from the well-supported core clade (97% ML, 1.00 BYPP, Fig. 2). In addition, we observe that N. yunnanensis and N. soli share a close relationship with high support (80% ML, 1.00 BYPP, Fig. 2). A pairwise nucleotide comparison of ITS and LSU between the type strains of N. yunnanensis and N. soli indicates that they are identical and conspecific (Yasanthika et al. 2024; Chen et al. 2025). However, tef1-α sequence data for N. soli are lacking (Yasanthika et al. 2024). Morphologically, N. yunnanensis resembles N. soli in having phialidic, hyaline, doliiform to ampulliform conidiogenous cells with overlapping size ranges (4.5–6 × 3.9–4.2 vs. 3–7 × 2.5–7 μm) and golden yellow to golden brown, ellipsoidal to fusiform conidia with the same size (3.9–6 × 2.6–3.9 vs. 5–6.5 × 3–5 μm) (Yasanthika et al. 2024; Chen et al. 2025). We recommend treating N. yunnanensis as a synonym of N. soli until tef1-α sequence data are acquired and verified. Unstable placements and weak support for these taxa are likely caused by missing sequence data. The lack of SSU and tef1-α sequences across all type strains limits the resolution of the Didymosphaeriaceae backbone tree (Wanasinghe et al. 2016, 2024; Gonçalves et al. 2019; Tennakoon et al. 2025). Future investigations need to focus on protein-coding markers (e.g. tef1-α, tub2, rpb2) and extensive sampling to clarify species identification and establish a resolved and stable phylogenetic framework for taxa in Didymosphaeriaceae.

Taxonomic investigation into the endophytic fungal diversity associated with Itea in Northern Thailand has revealed a significant and previously underestimated level of species richness. Our ongoing studies have successfully isolated several noteworthy taxa from Itea japonica and I. riparia in Thailand including the discovery of three new species (i.e. Neopestalotiopsis iteae, Nigrospora iteae and Pseudopestalotiopsis iteae) alongside four new host records from Itea (i.e. Apiospora vietnamensis, Neopestalotiopsis chrysea, N. haikouensis, and Pestalotiopsis jinchanghensis (Monkai et al. 2025, 2026). This work considerably advances our knowledge of fungal communities associated with these plant species. Hence, future research is essential to expand sampling efforts throughout various geographical regions in Thailand and China to explore additional fungal lineages. Furthermore, examining the pathogenic potential of these isolates is crucial to characterize disease dynamics and developing sustainable management plans for the conservation of medicinal plant hosts and forest resources within this ecologically significant area.

Supplementary Material

XML Treatment for Diaporthe iteae
XML Treatment for Diaporthe coracoralinae
XML Treatment for Neptunomyces iteae

Acknowledgements

Jutamart Monkai gratefully acknowledges Chiang Mai University for providing financial support and laboratory facilities. Assist. Prof. Dr. Verasak Sahachaisaree is thanked for his valuable help with sample collection. Dr. Shaun Pennycook is thanked for the nomenclatural review. Rungtiwa Phookamsak sincerely acknowledges the Talent Introduction Plan of Kunming Institute of Botany, Chinese Academy of Sciences, Yunnan Revitalization Talent Support Program: High-end Foreign Expert Project (Grant No. XDYC-GDWZ-2024-0016), and Yunnan Intelligence Union Program for Young Scientists, Yunnan, China (Grant No. 202503AM140005). Jianchu Xu expresses sincerely thank to Yunnan Department of Sciences and Technology of China (Grant No: 202302AE090023, 202303AP140001). Darbhe Jayarama Bhat gratefully acknowledges the financial support provided under the Distinguished Scientist Fellowship Programme-2025, King Saud University, Riyadh, Saudi Arabia.

Citation

Monkai J, Phookamsak R, Khuna S, Bhat DJ, Swe Zin Ei T, Xu J, Hongsanan S, Lumyong S (2026) Additions to Diaporthe (Sordariomycetes, Diaporthaceae) and Neptunomyces (Dothideomycetes, Didymosphaeriaceae) species associated with Itea in Thailand. MycoKeys 138: 209–236. https://doi.org/10.3897/mycokeys.138.203334

Contributor Information

Sinang Hongsanan, Email: sinang333@gmail.com.

Saisamorn Lumyong, Email: scboi009@gmail.com.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Artificial Intelligence (AI) use

The authors accept full responsibility for the content of the manuscript, including the disclosure of any use of AI.

No AI tools were used in the preparation of this manuscript.

Funding

The research was supported by the CMU Proactive Researcher program, Chiang Mai University, Chiang Mai, Thailand (grant number 814/2567). This research was also funded by the National Research Center of Thailand (No. 42A650198).

Author contributions

Conceptualization: J.M., S.H. and S.L. Data curation: J.M. and R.P. Funding acquisition: J.X., S.H. and S.L. Investigation: J.M. and T.S.Z.E. Methodology: J.M. and S.K. Project administration, J.M., R.P. and S.K. Resources: J.X., S.H. and S.L. Supervision: J.X. and S.L. Validation: R.P., D.J.B., S.H. and S.L. Original draft preparation: J.M. Review and editing: J.M., R.P., S.K., D.J.B., T.S.Z.E., J.X., S.H. and S.L. All authors have read and agreed to the published version of the manuscript.

Author ORCIDs

J. Monkai https://orcid.org/0000-0001-6043-0625

R. Phookamsak https://orcid.org/0000-0002-6321-8416

S. Khuna https://orcid.org/0000-0003-1999-4001

D.J. Bhat https://orcid.org/0000-0002-3800-5910

T. Swe Zin Ei https://orcid.org/0009-0005-9585-8863

J. Xu https://orcid.org/0000-0002-2485-2254

S. Hongsanan https://orcid.org/0000-0003-0550-3152

S. Lumyong https://orcid.org/0000-0002-6485-414X

Data availability

All of the data that support the findings of this study are available in the main text. The concatenated alignments and phylogenetic trees are available in Figshare (https://doi.org/10.6084/m9.figshare.33091040).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

XML Treatment for Diaporthe iteae
mycokeys.138.203334-treatment1.xml (30.3KB, xml)
XML Treatment for Diaporthe coracoralinae
mycokeys.138.203334-treatment2.xml (15.4KB, xml)
XML Treatment for Neptunomyces iteae
mycokeys.138.203334-treatment3.xml (32.9KB, xml)

Data Availability Statement

All of the data that support the findings of this study are available in the main text. The concatenated alignments and phylogenetic trees are available in Figshare (https://doi.org/10.6084/m9.figshare.33091040).

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