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Novel Helicoma and Neohelicosporium (Tubeufiaceae, Tubeufiales) species and two new host records of Helicoma on tropical palms (Arecaceae) from China
expand article infoYinru Xiong§, Kevin D. Hyde§|, Li Lu§, Dulanjalee L. Harishchandra#, Ausana Mapook§, Biao Xu, Fatimah Alotibi¤, Ishara S. Manawasinghe
‡ Zhongkai University of Agriculture and Engineering, Guangzhou, China
§ Mae Fah Luang University, Chiang Rai, Thailand
| Kunming Institute of Botany, Chinese Academy of Science, Kunming, China
¶ Qujing Normal University, Qujing, China
# Chiang Mai University, Chiang Mai, Thailand
¤ King Saud University, Riyadh, Saudi Arabia
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Abstract

Asexual species of Tubeufiaceae are characterised as helicosporous hyphomycetes and are abundantly discovered in tropical and subtropical regions. The present study collected helicosporous fungal samples from rotting tissues of Caryota mitis, Elaeis guineensis and E. oleifera in Xishuangbanna, Yunan Province, China. Fungal isolates were identified, based on the morphological characteristics and multi-gene phylogeny with DNA sequence data of the internal transcribed spacer (ITS), part of the large subunit nuclear rRNA gene (LSU), translation elongation factor 1-alpha gene (tef 1-α) and RNA polymerase II second largest subunit gene (rpb2). Herein, we introduce three new species viz. Helicoma oleifera, Neohelicosporium guineensis and N. xishuangbannaensis. In addition, we introduce two new host records of Helicoma guttulatum and H. rufum on Caryota mitis. The illustrations of all identified species, detailed descriptions and in-depth phylogenetic analyses are provided. Our results add new knowledge of fungal species associated with palm hosts in southern China. Moreover, our data will contribute to the biodiversity of fungi in tropical China.

Key words

Caryota mitis, Elaeis guineensis, Elaeis oleifera, helicosporous fungi, phylogeny, saprobic fungi, taxonomy, three new species

Introduction

Regions in southern China exhibit characteristics of a monsoon climate and its weather patterns are additionally influenced by the geographical distribution and differentiation of land and sea (Wang et al. 1999; Peng et al. 2021). Therefore, the boundaries of China’s tropics are long and incoherent (Gongfu et al. 1990). In this fragmented tropical region spanning from south-eastern to south-western China, the flora shows certain differences depending on the geographical composition in different regions (Zhu 2017). There are 23 large plant families containing 100–200 species of tropical flora in different regions, amongst which 101 species and 18 genera are from Arecaceae (Zhu 2016, 2017).

Arecaceae species are commonly known as palms and they are common in tropical evergreen forests. These species are available in every ecological habitat in the Tropics and Sub-tropics and regulate the composition and climate in those ecosystems (Reichgelt et al. 2018; Fehr et al. 2020). They are rich in fungal diversity covering most major groups of fungi and have been widely reported and studied (Fröhlich and Hyde 1999, 2000; Taylor et al. 1999; Konta et al. 2023; Pereira and Phillips 2023). Amongst these, many Tubeufiaceae species are recorded in the history of studies of palm fungi (Pereira and Phillips 2023). A few examples are: Pirozynski (1972) reporting Helicoma ambiens on oil palm from Tanzania, Aquaphila ramdayalea reported by Bhat (2008) on Caryota urens from India and Berkleasmium corticola reported by Capdeet and Romero (2010) on Butia yatay and Syagrus romanzoffiana from Argentina.

Tubeufiaceae was introduced by Barr (1979), based on the type genus Tubeufia to accommodate bitunicate ascomycetes occurring as saprobes on decaying wood. Tubeufiaceae has fascinating and peculiar morphs of both sexual and asexual morphs (Zhao et al. 2007; Li et al. 2022). These species are prevalently distributed in temperate and tropical regions (Rossman 1987; Kirk et al. 2001; Lumbsch and Huhndorf 2010; Boonmee et al. 2014; Luo et al. 2017; Lu et al. 2018a, b). Although members of this family can be found in both terrestrial woody substrates and in aquatic habitats, an interesting phenomenon is that most asexual morphs of Tubeufiaceae are collected from freshwater habitats (Hyde et al. 2016, 2017; Brahamanage et al. 2017; Chaiwan et al. 2017; Lu et al. 2017a, b, c, 2018a, b; Liu et al. 2018; Hongsanan et al. 2020). The distinguishing characteristic of Tubeufiaceae is the asexual morph mostly found as helicosporous hyphomycetes, while some contain phragmosporous and chlamydosporous conidia (Lu et al. 2018b; Dong et al. 2020). Their sexual morphs are characterised by superficial ascomata, bitunicate asci and ascospores which are hyaline to pale brown, elongate, obovoid or oblong and septate (Barr 1980; Kodsueb et al. 2006; Boonmee et al. 2011, 2014; Brahamanage et al. 2017; Lu et al. 2018b). Following Wijayawardene et al. (2022) and Ma et al. (2023), 47 genera have been accepted in Tubeufiaceae including Helicoma and Neohelicosporium.

Helicoma was proposed by Corda (1837), with H. muelleri as the type species. It is one of the earliest genera of helicosporous hyphomycetes (Linder 1929; Moore 1955; Goos 1986; Boonmee et al. 2014; Lu et al. 2018b). Helicoma species are frequently reported as saprobes inhabiting terrestrial and aquatic environments (Zhao et al. 2007; Boonmee et al. 2011, 2014; Hyde et al. 2016; Lu et al. 2018b, 2023; Liu et al. 2019; Tian et al. 2022). Based on phylogenetic analysis of combined ITS, LSU, tef 1-α and rpb2, Lu et al. (2018b) accepted species that are different from typical-Helicoma morphs described by Goos (1986) as Helicoma. These species are characterised by conidiogenous cells that are intercalary, cylindrical, with denticles, arising laterally from the lower portion of conidiophores as tooth-like protrusions. Conidia are pleurogenous, helicoid, hygroscopic, tapering towards the apex and rounded at the tip, coiled 1½–5 times, becoming loosely coiled in water. Subsequently, H. hydei (Liu et al. 2019), H. wuzhishanense (Lu et al. 2022), H. acropleurogenum and H. liyui (Lu et al. 2023) were reported in this genus.

Neohelicosporium was introduced by Lu et al. (2018a) to accommodate taxa with special helicosporous spores based on morphology and phylogenetic analysis of combined ITS, LSU, tef 1-α, and rpb2. Their unique characteristics include branched or unbranched conidiophores arising from creeping hyphae, mono- to polyblastic, integrated, sympodial conidiogenous cells with denticles and acrogenous and/or acropleurogenous conidia, which are used to distinct Neohelicosporium from Helicosporium (Lu et al. 2018b). After synonymising several species from Helicoma, Helicomyces, Helicosporium, and Tubeufia, 25 species are accepted in Neohelicosporium (Lu et al. 2018b; Index Fungorum 2024).

To explore the relationship between Tubeufiaceae associated with various palms, the present study collected terrestrial decaying samples of Caryota mitis, Elaeis guineensis and E. oleifera. A total of 12 isolates were obtained, from which we introduce three new species: Helicoma oleifera, Neohelicosporium guineensis and N. xishuangbannaensis and two new host records: Helicoma guttulatum and H. rufum. Species descriptions, illustrations of macroscopic and microscopic morphology and phylogenetic analyses are provided to delineate new and known species.

Materials and methods

Samples collection and isolation

Samples were collected in 2023 from an unidentified forest area beside National Highway 219 in Xishuangbanna in Yunnan Province, China (21°93'20"N, 101°24'57"E, 549.6 m elev.). These samples were rotting materials from different palm species namely, Caryota mitis, Elaeis oleifera and E. guineensis. Samples were brought into the laboratory using plastic ziplock bags and relevant macro and micro-characteristics were photographed by a ZEISS SteREO Discovery V20 stereomicroscopy (Germany) and Nikon Eclipse 80i and the industrial DigitaL Sight DS-Fi1 (Panasonic, Japan) microscope. Following the methods of Senanayake et al. (2018, 2020), single-spore isolation was performed. Germinated spores were aseptically transferred into fresh potato dextrose agar (PDA) plates and incubated at 25 °C to obtain pure cultures (Senanayake et al. 2020). The cultures obtained during the study were deposited in the culture collection of Zhongkai University of Agriculture and Engineering (ZHKUCC). Herbarium materials were deposited at the Mycological Herbarium of Zhongkai University of Agriculture and Engineering (MHZU). Facesoffungi (FoF) numbers and Index Fungorum (IF) numbers were obtained as explained in Jayasiri et al. (2015) and Index Fungorum (2024).

Morphological characterisation

After the fungal samples were brought into the laboratory, the Cnoptec SZ650 series (Cnoptec, China) stereomicroscope was used to observe the macromorphological characteristics and photographs were taken using SteReo Discovery V20. Nikon Eclipse 80i and the industrial DigitaL Sight DS-Fi1 (Panasonic, Japan) microscope and imaging system were used to take pictures of micromorphological characters. Digital images of micromorphological structures, including shape, size and colour were recorded. The measurement of structures, including spore dimensions for each species was conducted using NIS-Elements BR 5.30.03. Adobe Photoshop CC 2019 and Adobe Illustrator CC 2019 software (Adobe Systems Inc., San Jose, America) were used to develop images and make photo plates. All pure cultures obtained in this study were grown on potato dextrose agar (PDA) at 25 °C in 12 hours of daylight for a week and the diameter of the culture was measured after six weeks. AxioVersion Rel. 4.8 was used to take photos of the cultures.

DNA extraction, PCR amplification and sequencing

The pure cultures were cultured on PDA plates for 1–2 weeks and about 500 mg of fresh fungal mycelia were scraped. Total genomic DNA was extracted from the mycelia using MagPure Plant AS Kit (Magen Biotech, China) following the manufacturer’s instructions. Four nuclear gene regions: internal transcribed spacer (ITS), large subunit nuclear rRNA gene (LSU), translation elongation factor 1-α (tef 1-α) and RNA polymerase II second largest subunit gene (rpb2) were amplified using the primers shown in Table 1. The PCR reaction mixture contained 25 μl of total volume, which consisted of 12.5 μl 2 × FastTaq Premix (mixture of FastTaq TM DNA Polymerase, buffer, dNTP Mixture and stabiliser) (Beijing Qingke Biological Technology Co., Ltd., Beijing, PR China), 1 μl of forward and reverse primers each, 9.5 μl ddH2O and 1 μl DNA. The polymerase chain reaction (PCR) was performed in a C1000 TouchTM thermal cycler. The PCR procedure is as follows: for ITS/LSU, the initial denaturation step is performed at 95 °C for 2 minutes, then 35 amplification cycles at 95 °C for 1 minute, 50 °C for 1 minute and 72 °C for 1 minute. Finally, extension for 10 minutes at 72 °C. For tef 1-α, an initial step of 2 minutes at 95 °C followed by 35 cycles of 1 minute at 95 °C, 1 minute at 52 °C, 1 minute at 72 °C and 7 minutes at 72 °C. For rpb2 PCR conditions, an initial denaturation step was performed at 95 °C for 5 minutes and then 30 cycles at 94 °C for 1 minute, 53 °C for 30 seconds, 72 °C for 90 seconds and finally 72 °C for 10 minutes. After PCR amplification, the product was observed on a 1% agarose gel under ultraviolet light. DNA sequencing was completed in Tianyi (Guangzhou, China) Co., Ltd. New sequences are deposited in GenBank. The sequences used for analyses with accession numbers are given in Table 2.

Table 1.

Genes and corresponding primers used in this study.

Gene Primer Sequence (5’-3’) Reference
ITS ITS5 GGAAGTAAAAGTCGTAACAAGG White et al. (1990)
ITS4 TCCTCCGCTTATTGATATGC
LSU LR0R ACCCGCTGAACTTAAGC Vilgalys and Hester (1990)
LR5 TCCTGAGGGAAACTTCG
tef 1-α EF1-983F GCYCCYGGHCAYCGTGAYTTYAT Carbone and Kohn (1999)
EF1-2218R TACTTGAAGGAACCCTTACC
rpb2 fRPB2-5F GAYGAYMGWGATCAYTTYGG O’Donnell et al. (2007)
RPB2-7cr CCCATRGCTTGYTTRCCCAT
Table 2.

Taxon names, strain numbers and corresponding GenBank accession numbers of the taxa used in the Tubeufiaceae phylogenetic analyses.

Species Strain numbers ITS LSU tef 1-α rpb2
Acanthohelicospora aurea GZCC 16-0060 KY321323 KY321326 KY792600 MF589911
Acanthohelicospora pinicola MFLUCC 10-0116 KF301526 KF301534 KF301555 NA
Aquaphila albicans BCC 3543 DQ341096 DQ341101 NA NA
Aquaphila albicans MFLUCC 16-0010 KX454165 KX454166 KY117034 MF535255
Berkleasmium fusiforme MFLUCC 17-1979 MH558694 MH558821 MH550885 MH551008
Berkleasmium longisporum MFLUCC 17-1990 MH558697 MH558824 MH550888 MH551011
Botryosphaeria agaves T MFLUCC 10-0051 JX646790 JX646807 JX646855 NA
Botryosphaeria dothidea CBS 115476 NA NG_027577 NA NA
Chlamydotubeufia cylindrica T MFLUCC 16-1130 MH558702 MH558830 MH550893 MH551018
Chlamydotubeufia huaikangplaensis T MFLUCC 10-0926 JN865210 JN865198 NA NA
Chlamydotubeufia krabiensis T MFLUCC 16-1134 KY678767 KY678759 KY792598 MF535261
Dematiohelicomyces helicosporus T MFLUCC 16-0213 KX454169 KX454170 KY117035 MF535258
Dematiohelicomyces helicosporus MFLUCC 16-0003 MH558703 MH558831 MH550894 MH551019
Helicoarctatus aquaticus T MFLUCC 17-1996 MH558707 MH558835 MH550898 MH551024
Helicodochium aquaticum MFLUCC 16-0008 MH558708 MH558836 MH550899 MH551025
Helicodochium aquaticum T MFLUCC 17-2016 MH558709 MH558837 MH550900 MH551026
Helicohyalinum aquaticum T MFLUCC 16-1131 KY873625 KY873620 KY873284 MF535257
Helicohyalinum infundibulum T MFLUCC 16-1133 MH558712 MH558840 MH550903 MH551029
Helicoma acropleurogenum T GZCC 22-2035 OP806857 OP806854 OP821894 OP821897
Helicoma ambiens UAMH 10533 AY916451 AY856916 NA NA
Helicoma ambiens UAMH 10534 AY916450 AY856869 NA NA
Helicoma aquaticum T MFLUCC 17-2025 MH558713 MH558841 MH550904 MH551030
Helicoma brunneisporum T MFLUCC 17-1983 MH558714 MH558842 MH550905 MH551031
Helicoma dennisii NBRC 30667 AY916455 AY856897 NA NA
Helicoma freycinetiae T MFLUCC 16-0363 MH275062 MH260295 MH412770 NA
Helicoma fusiforme T MFLUCC 17-1981 MH558715 NA MH550906 NA
Helicoma guttulatum T MFLUCC 16-0022 KX454171 KX454172 MF535254 MH551032
Helicoma guttulatum GZCC 22-2004 OP508739 OP508779 OP698090 OP698079
Helicoma guttulatum GZCC 22-2024 OP508733 OP508773 OP698084 OP698073
Helicoma guttulatum GZCC 22-2025 OP508737 OP508777 OP698088 OP698077
Helicoma guttulatum MFLUCC 21-0152 OL545456 OL606150 OL964521 OL964527
Helicoma guttulatum ZHKUCC 24-0139 PP860094 PP860106 PP858054 PP858066
Helicoma guttulatum ZHKUCC 24-0140 PP860095 PP860107 PP858055 PP858067
Helicoma hongkongense MFLUCC 17-2005 MH558716 MH558843 MH550907 MH551033
Helicoma hydei MFLUCC 18-1270 MH747101 MH747116 MH747100 NA
Helicoma inthanonense T MFLUCC 11-0003 JN865211 JN865199 NA NA
Helicoma khunkornensis T MFLUCC 10-0119 JN865203 JN865191 KF301559 NA
Helicoma linderi NBRC 9207 AY916454 AY856895 NA NA
Helicoma liyui GZCC 22-2033 OP806858 OP806855 OP821895 NA
Helicoma longisporum GZCC 22-2005 OP508740 OP508780 OP698091 OP698080
Helicoma longisporum MFLUCC 16-0211 MH558719 MH558845 MH550910 MH551036
Helicoma longisporum T MFLUCC 17-1997 MH558720 MH558846 MH550911 MH551037
Helicoma miscanthi T MFLUCC 11-0375 KF301525 KF301533 KF301554 NA
Helicoma muelleri CBS 964.69 AY916453 AY856877 NA NA
Helicoma muelleri UBC F13877 AY916452 AY856917 NA NA
Helicoma multiseptatum T GZCC 16-0080 MH558721 MH558847 MH550912 MH551038
Helicoma nematosporum T MFLUCC 16-0011 MH558722 MH558848 MH550913 MH551039
Helicoma oleifera T ZHKUCC 24-0121 PP860086 PP860098 PP858056 PP858068
Helicoma oleifera ZHKUCC 24-0122 PP860087 PP860099 PP858057 PP858069
Helicoma oleifera ZHKUCC 24-0766 PP860088 PP860100 PP858058 PP858070
Helicoma oleifera ZHKUCC 24-0767 PP860089 PP860101 PP858059 PP858071
Helicoma rubriappendiculatum T MFLUCC 18-0491 MH558723 MH558849 MH550914 MH551040
Helicoma rufum T MFLUCC 17-1806 MH558724 MH558850 MH550915 NA
Helicoma rufum ZHKUCC 24-0143 PP860096 PP860108 PP858060 PP858072
Helicoma rufum ZHKUCC 24-0144 PP860097 PP860109 PP858061 PP858073
Helicoma rugosum GZCC 22-2034 OP806859 OP806856 OP821896 NA
Helicoma rugosum ANM 196 GQ856138 GQ850482 NA NA
Helicoma rugosum ANM 953 GQ856139 GQ850483 NA NA
Helicoma rugosum ANM 1169 NA GQ850484 NA NA
Helicoma rugosum JCM 2739 NA AY856888 NA NA
Helicoma septoconstrictum MFLUCC 17-1991 MH558725 MH558851 MH550916 MH551041
Helicoma septoconstrictum T MFLUCC 17-2001 MH558726 MH558852 MH550917 MH551042
Helicoma siamense T MFLUCC 10-0120 JN865204 JN865192 KF301558 NA
Helicoma sp. HKUCC 9118 NA AY849966 NA NA
Helicoma tectonae T MFLUCC 12-0563 KU144928 KU764713 KU872751 NA
Helicoma vaccinii CBS 216.90 AY916486 AY856879 NA NA
Helicoma wuzhishanense GZCC 22-2003 OP508732 OP508772 OP698083 OP698072
Helicomyces chiayiensis T BCRC FU30842 LC316604 NA NA NA
Helicomyces hyalosporus MFLUCC 17-0051 MH558731 MH558857 MH550922 MH551047
Helicomyces torquatus MFLUCC 16-0217 MH558732 MH558858 MH550923 MH551048
Helicosporium flavum T MFLUCC 16-1230 KY873626 KY873621 KY873285 NA
Helicosporium luteosporum T MFLUCC 16-0226 KY321324 KY321327 KY792601 MH551056
Helicosporium vesicarium T MFLUCC 17-1795 MH558739 MH558864 MH550930 MH551055
Helicotruncatum palmigenum NBRC 32663 AY916480 AY856898 NA NA
Helicotruncatum palmigenum KUMCC 21-0474 OM102542 OL985959 OM355488 OM355492
Helicotubeufia guangxiensis T MFLUCC 17-0040 MH290018 MH290023 MH290028 MH290033
Helicotubeufia hydei T MFLUCC 17-1980 MH290021 MH290026 MH290031 MH290036
Helicotubeufia jonesii T MFLUCC 17-0043 MH290020 MH290025 MH290030 MH290035
Kamalomyces mangrovei MFLUCC 17-0407 MH878781 MH878779 MH886508 NA
Kamalomyces thailandicus MFLUCC 13-0233 MF506884 MF506882 MF506886 NA
Muripulchra aquatica KUMCC 15-0245 KY320533 KY320550 KY320563 MH551057
Muripulchra aquatica KUMCC 15-0276 KY320534 KY320551 KY320564 MH551058
Neoacanthostigma fusiforme T MFLUCC 11-0510 KF301529 KF301537 NA NA
Neochlamydotubeufia fusiformis T MFLUCC 16-0016 MH558740 MH558865 MH550931 MH551059
Neochlamydotubeufia khunkornensis MFLUCC 16-0025 MH558742 MH558867 MH550933 MH551061
Neohelicomyces aquaticus KUMCC 15-0463 KY320529 KY320546 KY320562 MH551065
Neohelicomyces grandisporus T KUMCC 15-0470 KX454173 KX454174 NA MH551067
Neohelicomyces submersus T MFLUCC 16-1106 KY320530 KY320547 NA MH551068
Neohelicosporium abuense CBS 101688 AY916470 NA NA NA
Neohelicosporium acrogenisporum T MFLUCC 17-2019 MH558746 MH558871 MH550937 MH551069
Neohelicosporium aquaticum T MFLUCC 17-1519 MF467916 MF467929 MF535242 MF535272
Neohelicosporium astrictum T MFLUCC 17-2004 MH558747 MH558872 MH550938 MH551070
Neohelicosporium aurantiellum ANM 718 GQ856140 GQ850485 NA NA
Neohelicosporium bambusicola T MFLUCC 21-0156 OL606157 OL606146 OL964517 OL964523
Neohelicosporium ellipsoideum T MFLUCC 16-0229 MH558748 MH558873 MH550939 MH551071
Neohelicosporium fluviatile MFLUCC 15-0606 NA OP377957 OP473050 OP473111
Neohelicosporium fusisporum T MFUCC 16-0642 MG017612 MG017613 MG017614 NA
Neohelicosporium griseum CBS 961.69 AY916474 AY856884 NA NA
Neohelicosporium griseum CBS 113542 AY916475 AY916088 NA NA
Neohelicosporium guangxiense GZCC 16-0042 MF467920 MF467933 MF535246 MF535276
Neohelicosporium guangxiense MFLUCC 17-0054 MH558750 MH558875 MH550941 MH551073
Neohelicosporium guineensis T ZHKUCC 24-0113 PP860090 PP860102 PP858062 PP858074
Neohelicosporium guineensis ZHKUCC 24-0114 PP860091 PP860103 PP858063 PP858075
Neohelicosporium hyalosporum T GZCC 16-0076 MF467923 MF467936 MF535249 MF535279
Neohelicosporium hyalosporum GZCC 16-0063 MH558751 MH558876 MH550942 MH551074
Neohelicosporium irregulare T MFLUCC 17-1796 MH558752 MH558877 MH550943 MH551075
Neohelicosporium irregulare MFLUCC 17-1808 MH558753 MH558878 MH550944 MH551076
Neohelicosporium krabiense T MFLUCC 16-0224 MH558754 MH558879 MH550945 MH551077
Neohelicosporium laxisporum T MFLUCC 17-2027 MH558755 MH558880 MH550946 MH551078
Neohelicosporium morganii CBS 281.54 AY916468 AY856876 NA NA
Neohelicosporium morganii CBS 222.58 AY916469 AY856880 NA NA
Neohelicosporium ovoideum T GZCC 16-0064 MH558756 MH558881 MH550947 MH551079
Neohelicosporium ovoideum GZCC 16-0066 MH558757 MH558882 MH550948 MH551080
Neohelicosporium panacheum CBS 257.59 AY916471 AY916087 NA NA
Neohelicosporium parvisporum GZCC 16-0078 MF467924 MF467937 MF535250 MF535280
Neohelicosporium parvisporum MFLUCC 17-2010 MH558763 MH558888 MH550954 MH551086
Neohelicosporium sp. CBS 189.95 AY916472 AY856882 NA NA
Neohelicosporium sp. HKUCC 10235 NA AY849942 NA NA
Neohelicosporium suae CGMCC 3.23541 OP184079 OP184068 OP186052 OP265702
Neohelicosporium submersum MFLUCC 17-2376 MT627738 MN913738 NA NA
Neohelicosporium taiwanense T BCRC FU30841 LC316603 NA NA NA
Neohelicosporium thailandicum T MFLUCC 16-0221 MF467928 MF467941 MF535253 MF535283
Neohelicosporium xishuangbannaensis T ZHKUCC 24-0119 PP860092 PP860104 PP858064 PP858076
Neohelicosporium xishuangbannaensis ZHKUCC 24-0120 PP860093 PP860105 PP858065 PP858077
Neotubeufia krabiensis T MFLUCC 16-1125 MG012031 MG012024 MG012010 MG012017
Parahelicomyces aquaticus T MFLUCC 16-0234 MH558766 MH558891 MH550958 MH551092
Parahelicomyces chiangmaiensis T MFLUCC 21-0159 OL697884 OL606145 OL964516 OL964522
Parahelicomyces talbotii MFLUCC 17-2021 MH558765 MH558890 MH550957 MH551091
Pleurohelicosporium hyalinum T GZCC 20-0489 OP377816 OP377915 OP472996 OP473089
Pleurohelicosporium parvisporum T MFLUCC 17-1982 MH558764 MH558889 MH550956 MH551088
Pseudohelicoon gigantisporum BCC 3550 AY916467 AY856904 NA NA
Pseudohelicoon subglobosum T BCRC FU30843 LC316607 LC316610 NA NA
Thaxteriellopsis lignicola MFLUCC 10-0123 JN865207 JN865195 KF301562 NA
Thaxteriellopsis lignicola MFLUCC 10-0124 JN865208 JN865196 KF301561 NA
Tubeufia abundata T MFLUCC 17-2024 MH558769 MH558894 MH550961 MH551095
Tubeufia aquatica T MFLUCC 16-1249 KY320522 KY320539 KY320556 MH551142
Tubeufia bambusicola T MFLUCC 17-1803 MH558771 MH558896 MH550963 MH551097
Tubeufia chlamydospora T MFLUCC 16-0223 MH558775 MH558900 MH550967 MH551101
Tubeufia cocois T MFLUCC 22-0001 OM102541 OL985957 OM355486 OM355491
Tubeufia sympodilaxispora T MFLUCC 17-0048 MH558808 MH558932 MH551001 MH551135

Phylogenetic analyses

The quality of the DNA sequences was checked from their chromatograms and the sequences generated by forward and reverse primers were combined using Geneious Prime v. 2021.0.3 (Biomatters Ltd., San Diego, CA, USA). The BLASTn tool (Basic Local Alignment Search Tool) in the search engine of the National Center for Biotechnology Information (NCBI) to analyse the sequences is used in this study (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Based on the BLASTn results, we identified that our isolates belong to Helicoma and Neohelicosporium. Phylogenetic analyses for Tubeufiaceae were performed following Yang et al. (2023). The sequences for the phylogenetic analysis were downloaded from GenBank and listed in Table 2. MAFFT v. 7 (https://mafft.cbrc.jp/alignment/server/) was used to align and adjust the sequence datasets of the four gene regions. BioEdit 7.0.9.0 was used to improve the alignment manually when necessary. Using Alignment Transformation Environment online (https://sing.ei.uvigo.es/ALTER/), files were converted to run phylogenetic trees. Phylogenetic analysis was conducted using Maximum Likelihood (ML) inferred in RAxML v. 8.2.12 (Stamatakis 2014), Maximum Parsimony (MP) implied on PAUP v. 4.0b10 (Swofford 2003) and Bayesian Inference (BI) on MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001).

Maximum parsimony analysis was performed in PAUP (phylogenetic analysis using parsimony) v.4.0b10 (Swofford 2003) using the heuristic search option with tree bisection-reconnection (TBR) branch swapping and 1,000 random sequence additions. Ambiguous regions in the alignment were excluded and gaps were treated as missing data. The stability of the trees was evaluated by 1,000 bootstrap replications. Branches of zero length were collapsed and all multiple parsimonious trees were saved. Descriptive statistics, including tree length (TL), consistency index (CI), retention index (RI), relative consistency index (RC) and homoplasy index (HI) were calculated.

Maximum Likelihood analyses were accomplished using RAxML-HPC2 on XSEDE v. 8.2.8 (Stamatakis et al. 2008; Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) using the GTR+I+G model of evolution with 1,000 non-parametric bootstrapping iterations. MrBayes v.3.0b4 (Huelsenbeck and Ronquist 2001) used for the Bayesian analyses, implemented in MrMTgui (Nuin 2007), was used to determine the best-fit evolution model for Bayesian Inference analyses using the Akaike Information Criterion (AIC). The Markov Chain Monte Carlo sampling (BMCMC) analysis was conducted with four simultaneous Markov chains. The best model of evolution determined for LSU, ITS, rpb2 and tef 1-α by MrModelTest v. 2.2 was GTR+I+G. They were run for 1,000,000 generations, sampling the trees at every 100th generation. From the 10,000 trees obtained, the first 2,000 representing the burn-in phase were discarded. The remaining 8,000 trees were used to calculate posterior probabilities in the majority rule consensus tree. The phylogenetic tree was visualised in FigTree v. 1.4.2. Taxonomic novelties were submitted to the Facesoffungi database (Jayasiri et al. 2015), Index Fungorum (http://www.indexfungorum.org) and Palm Fungi (Xiong et al. 2024) databases. Species delineation was based on criteria set by Chethana et al. (2021) and Pem et al. (2021).

Results

Phylogenetic analyses

Phylogenetic trees were generated by ML, MP and BI of combined ITS (971 bp), LSU (1,172 bp), rpb2 (1,045 bp) and tef 1-α (912 bp) sequence data. The tree topologies generated by these three methods were similar and close to the topology of Yang et al. (2023); the best-scoring ML tree is shown in Fig. 1. The sequence alignment comprised 139 taxa of representative strains of Tubeufiaceae, including 12 isolates obtained in this study. Botryosphaeria agaves (MFLUCC 10-0051) and B. dothidea (CBS 115476) were used as the outgroup taxa. Maximum parsimony analysis consisted of 2,223 constant characters and 1,628 informative characters resulting in 368 equally parsimonious trees (Fig. 1) (CI = 0.293, RI = 0.752, RC = 0.220, HI = 0.707). The best-scoring ML tree had an optimisation likelihood value of -51369.027254. The matrix had 2,120 distinct alignment patterns with a 32.53% proportion of gaps and completely undetermined characters. Estimated base frequencies were as follows: A = 0.245833, C = 0.250339, G = 0.261605, T = 0.242223; substitution rates: AC = 1.153811, AG = 5.616698, AT = 2.094588, CG = 0.769981, CT = 8.981963, GT = 1.0; gamma distribution shape parameter α = 0.237020. Incomplete portions at the ends of the sequences were excluded from the analysis. Our 12 new isolates are distributed in five clades, of which eight were distributed in Helicoma and four were distributed in Neohelicosporium. Based on the phylogenetic evidence and morphology, here we introduce three novel species and two new host records.

Figure 1. 

Maximum Likelihood majority rule consensus tree for Tubeufiaceae using ITS, LSU, rpb2 and tef 1-α sequence dataset with Botryosphaeria agaves (MFLUCC 10-0051) and B. dothidea (CBS 115476) as the outgroup taxa. Bootstrap support for Maximum Likelihood (ML) and Maximum Parsimony (MP) equal to or greater than 75% and Bayesian Inference posterior probability (BIPP) equal to or greater than 0.90 are indicated above branches as MP/ML/BIPP. The scale bar indicates 0.2 nucleotide changes per site. Isolates from this study are marked in blue and ex-type strains are marked in bold.

Taxonomy

Helicoma oleifera Y.R. Xiong, Manawas. & K.D. Hyde, sp. nov.

Fig. 2

Etymology

Species epithet refers to the host species name “oleifera” from which the fungus was isolated.

Holotype

MHZU 23-0157.

Description

Saprobic on the rotting petiole of Elaeis oleifera. Sexual morph: Not observed. Asexual morph: Hyphomycetous, helicosporous. Colonies on the substratum superficial, effuse, gregarious, brown. Mycelium composed of partly immersed, partly superficial, hyaline, septate, branched hyphae. Conidiophores 145–360 µm long, 6.5–7.5 µm wide (x̄ = 210 × 6.5 μm, n = 20), macronematous, mononematous, cylindrical, unbranched or branched at base, straight to slightly bent, septate, deep brown at root part, brown at apex, pale brown at middle part mixing with some brown areas, smooth-walled with irregular inclusion. Conidiogenous cells 13–22 µm long, 5–7.5 µm wide (x̄ = 17 × 6.4 μm, n = 20), monoblastic, integrated, sympodial, terminal, cylindrical or fertile at the apex of conidiophores, brown, smooth-walled with irregular inclusion; with denticles, 1.3–2.3 µm long, 1.4–2.5 µm wide (x̄ = 1.6 × 1.8 μm, n = 20), arising from the apex portion of conidiophores as tooth-like and papillate protrusions, exposed or imbedded in the apex of conidiophore, mono- to polyblastic, brown, smooth-wall. Conidia 18–22.5 μm diam. (x̄ = 20.4 μm, n = 40) and conidial filament 6.8–9 μm wide (x̄ = 8.2 μm, n = 40), 45–55 μm long (x̄ = 50.6 μm, n = 40), solitary, acrogenous, helicoid, rounded at tip, tapering towards flat end, conic truncate at base, tightly coiled 1½ times, 8-septate, not becoming loose in water, guttulate, hyaline to pale brown, smooth-walled, the third cell shrinking and producing the root canal.

Culture characteristics

Conidia germinating on water agar and germ tubes produced from conidia within 12 h. Colonies growing on PDA attaining 2.5 cm diam. after six weeks at 25 °C, irregular, undulate, rough, superficial and partially immersed, brown aerial mycelium mixed with pale brown, deep brown at up and down junction area; reverse brown with pale brown.

Material examined

China, Yunnan Province, Xishuangbanna City, an unidentified forest beside National Highway 219 (21°93'N, 101°24'E, 549.6 m elev.), rotting petiole of the Elaeis oleifera, 5 February 2023, Y.R. Xiong and Li Lu, XG198 (MHZU 23-0157, holotype); ex-type culture, ZHKUCC 24-0121, other living cultures ZHKUCC 24-0122, ZHKUCC 24-0766, ZHKUCC 24-0767.

Notes

Four isolates obtained in this study from the rotting petiole of the Elaeis oleifera clustered in an independent clade in the phylogenetic tree with 78% ML, 76% MP bootstrap support and 1.00 BIPP bootstrap support. The nucleotide differences between Helicoma oleifera and its phylogenetically related species were checked, excluding gaps: H. acropleurogenum (GZCC 22-2035) - ITS: 3.53% (18/510 base pairs), LSU: 0.71% (6/844 base pairs), tef 1-α: 2.85% (26/912 base pairs), rpb2: 3.92% (41/1045 base pairs); H. dennisii (NBRC 30667) - ITS: 4.36% (25/573 base pairs), LSU: 0.35% (2/564 base pairs), tef 1-α and rpb2 sequence unavailable; H. hydei (MFLUCC 18-1270) - ITS: 3.50% (26/744 base pairs), LSU: 0.71% (6/847 base pairs), tef 1-α: 2.74% (25/912 base pairs), rpb2 sequence is unavailable; H. inthanonense (MFLUCC 11-0003) - ITS: 4.56% (26/570 base pairs), LSU: 1.63% (14/860 base pairs), tef 1-α and rpb2 sequence is unavailable. Helicoma oleifera is different from related species not only in the size of conidia and conidiophores (Table 3), but also in conidia, which shrink and produce the tubular structure at the third cell (Fig. 2q, r, s), while other species do not produce any deformation. In addition, H. acropleurogenum (Lu et al. 2023) has intercalary and mostly monoblastic, rarely polyblastic conidiogenous cells; however, H. oleifera has terminal and monoblastic or polyblastic conidiogenous cells. Helicoma dennisii (Tsui et al. 2006) has intercalary and polyblastic conidiogenous cells and fertile denticle structure at several cells on the upper end of the conidiophore. However, H. oleifera only has fertile denticle structures at the apex cell of the conidiophore. Furthermore, H. oleifera differs from H. hydei (Liu et al. 2019) by having an embedded denticle structure, while H. hydei (Liu et al. 2019) has an exposed denticle structure. Furthermore, H. inthanonense (Boonmee et al. 2011) has acropleurogenous and brown conidia with 7-septate and produces an asexual morph from MEA culture, while H. oleifera has acrogenous and hyaline to pale brown conidia with 8-septate. Therefore, we introduce H. oleifera as a new species.

Table 3.

Comparison of asexual morph characteristics of Helicoma species in this study; the names of strains in this study are indicated in bold.

Species names and culture accession numbers Conidiophores Conidiogenous cells Conidia Septate number Colour Coiled times References
Helicoma acropleurogenum GZCC 22-2035 118–389 μm long, 5.5–8.5 μm wide (x̄ = 219 × 6.5 μm, n = 20) 20–32 μm long, 5–8 μm wide (x̄ = 25 × 6 μm, n = 20) 21–24 μm diam. and conidial filament 8.5–10.5 μm wide (x̄ = 22.0 × 9.5 μm, n = 20), 48–58 μm long 6–7 pale brown tightly coiled 1½–1¾ times Lu et al. (2023)
Helicoma inthanonense MFLUCC 11-0003 (14.5–)26.5–34(−42) μm in diam., 3 μm wide NA (10–)13–20 μm in diam., 4–7 μm wide (x̄ = 14 × 6 μm) 7 hyaline to brown NA Boonmee et al. (2011)
Helicoma hydei MFLUCC 18-1270 135–310 μm long, 4.5–7.0 μm wide 13–37 μm long, 4.5–7.0 μm wide 19–30 μm diam. (x̄ = 25.0 μm, n = 20), conidial filament 6–12 μm wide (x̄ = 8.1 μm, n = 20) NA pale brown to brown tightly coiled 1–1½ times Liu et al. (2019)
Helicoma dennisii NBRC 30667 3.5–5 µm wide at the basal part and tapering to 3–3.5 µm wide at the apical part, up to 190 µm long 1–1.5 × 0.5–1 µm 10–15 (13.5) µm in diam.; conidial filament hyaline to dilute fuscous, 4–5.5 (4.5) µm thick 6–9 (8) Hyaline tightly coiled 1¼–1¾ (1½) times Zhao et al. (2007)
Helicoma oleifera ZHKUCC 24-0121 145–360 μm long, 6.5–7.5 μm wide (x̄ = 235 × 6.8 μm, n = 20) 13–22 μm long, 5–7.5 μm wide, tiny tooth–like protrusions (1.3–2.3 μm long, 1.4–2.5 μm wide) 18–22.5 μm diam. and conidial filament 6.8–9 μm wide (x̄ = 20.4 μm diam., 8.2 μm wide, n = 50), 45–55 μm long 8 pale brown to brown tightly coiled 1½ times This study
Helicoma guttulatum MFLUCC 16-0022 74–182 (197) μm long, 4–6 μm wide (x̄ = 120 × 5 μm, n = 20) NA 18–23 μm diam. and conidial filament 6–8 μm wide (x̄ = 20 × 7 μm, n = 20) 8–9 hyaline to pale brown tightly coiled 1–1½ times Hyde et al. (2016)
Helicoma guttulatum ZHKUCC 24-0139 75–225 μm long, 5.5–6 μm wide (x̄ = 152 × 5.7 μm, n = 20) 10–29 μm long, 5–8.8 μm wide, tiny tooth–like protrusions (1.6–3.5 μm long, 1.6–2.5 μm wide) 21–30 μm diam. and conidial filament 7.2–10 μm wide (x̄ = 25 μm diam., 8.4 μm wide, n = 50), 48–69 μm long 8–9 pale brown tightly coiled 1½ times This study
Helicoma rufum MFLUCC 17-1806 110–210 μm long, 7–8.5 μm wide 9–14 μm long, 5.5–8.5 μm wide, tiny tooth–like protrusions (2.5–3.6 μm long, 1.5–2 μm wide) 35–45 μm diam. and conidial filament 4–5.5 μm wide (x = 41 × 4.5 μm, n = 20), 240–410 μm long 27–37 hyaline to pale brown coiled 2–3 times, becoming loosely coiled in water Lu et al. (2018b)
Helicoma rufum ZHKUCC 24-0143 150–270 µm long, 4–7.5 µm thick (x̄ = 225 × 5.9 μm, n = 20) 7–15 μm long, 4–7 μm wide, tiny tooth–like protrusions (3–6 μm long, 1.5–3 μm wide) 21–47 μm diam. and conidial filament 2–5 μm wide (x̄ = 36 × 3.8 μm, n = 40), 145–345 μm long 25–35 hyaline tightly coiled 3–4.5 coils This study
Neohelicosporium hyalosporum GZCC 16-0076 up to 540 μm long, 4–5.5 μm wide 9–13 μm long, 4–5.5 μm wide 25–33 μm diam. and conidial filament 3–4 μm wide (x̄ = 28 μm diam., 3.5 μm wide, n = 50), 125–225 μm long NA hyaline tightly coiled 2.5–3.5 times, becoming loosely coiled in water Lu et al. (2018a)
Neohelicosporium ovoideum GZCC 16-0064 up to 420 μm long, 4–6 μm wide 10–15 μm long, 4–6 μm wide 25–35 μm diam. and conidial filament 3–4 μm wide (x̄ = 28 × 3.5 μm, n = 50), 180–230 μm long multi–septate hyaline tightly coiled 2–3 times, becoming loosely coiled in water Lu et al. (2018b)
Neohelicosporium guineensis ZHKUCC 24-0113 50–160 μm long, 4–6 μm wide (x̄ = 120 × 5.2 μm, n = 10) 11.5–20 μm long, 3.5–5.5 μm wide, tiny tooth–like protrusions (1.4–2.7 μm long, 1.2–2 μm wide) 16–20 μm diam. and conidial filament 1.8–3 μm wide (x̄ = 18 μm diam., 2.4 μm wide, n = 50), 90–130 μm long 11–12 hyaline tightly coiled 2½–3½ times, loosely coiled in water This study
Neohelicosporium fusisporum MFUCC 16-0642 NA 12–20 μm long, 1.5–2.5 μm wide 18–22 μm diam. and conidial filament 1.5–2.5 μm wide (x̄ = 18 μm × 2 μm, n = 50), 100–150 μm long multi–septate hyaline tightly coiled 2½–3¼ times, loosely coiled in water Jayasiri et al. (2017)
Neohelicosporium xishuangbannaensis ZHKUCC 24-0119 40–125 μm long, 3–6 μm wide (x̄ = 68.4 × 4.4 μm, n = 20) 7–14 μm long, 2.5–5.5 μm wide, tiny tooth–like protrusions (1.8–3.3 μm long, 1.1–2.3 μm wide) 16.5–20.5 μm diam. and conidial filament 1.8–3.2 μm wide (x̄ = 18.5 μm diam., 2.4 μm wide, n = 50), 90–125 μm long 9–13 hyaline tightly coiled 2–3¼ times, loosely coiled in water This study
Figure 2. 

Helicoma oleifera (MHZU 23-0157, holotype) a specimen observed b, c colony on decaying Elaeis oleifera d, e conidiophores f, g conidiogenous cell with attached conidia h–l conidiogenous cells m–p conidia q–s conidia produce the tubular structure at the third cell t germinated conidium u, v culture on PDA from above and reverse. Scale bars: 100 μm (d, e); 20 μm (f–t).

Helicoma guttulatum Y.Z. Lu, Boonmee & K.D. Hyde, Fungal Diversity 80: 1–270 (2016)

Fig. 3

Description

Saprobic on the rotting petiole of Caryota mitis. Sexual morph: Not observed. Asexual morph: Hyphomycetous, helicosporous. Colonies on the substratum superficial, effuse, gregarious, brown. Mycelium composed of partly immersed, partly superficial, brown, septate hyphae. Conidiophores 75–225 μm long, 5.5–6 μm wide (x̄ = 152 × 5.7 μm, n = 20), macronematous, crowded, erect, straight to slightly bent, brown, deep brown towards the base, septate, branched, smooth-walled with irregular inclusion. Conidiogenous cells 10–29 μm long, 5–8.8 μm wide (x̄ = 18 × 6.3 μm, n = 20), mono- to polyblastic, integrated, cylindrical, terminal, pale brown to brown, smooth-walled with irregular inclusion; with denticles, 1.6–3.5 μm long, 1.6–2.5 μm wide (x̄ = 2.4 × 2.1 μm, n = 20), arising from the apex portion of conidiophores as tooth-like protrusions, mono- to polyblastic, brown, smooth-wall. Conidia 21–30 μm diam. (x̄ = 25.1 μm, n = 40) and conidial filament 7.2–10 μm wide (x̄ = 8.4 μm, n = 40), 48–69 μm long (x̄ = 57.4 μm, n = 40), solitary, acropleurogenous, tightly coiled 1½ times, guttulate, not becoming loose in water, hyaline to pale brown, tapering towards flat end, 8–9-septate, rounded at the apex, conic truncate at the base, smooth-walled.

Figure 3. 

Helicoma guttulatum (MHZU 23-0166, new host record) a specimen observed b, c colony on decaying Caryota mitis d conidiophores e–j conidiogenous cells, thereinto j with attached conidia k–o conidia p germinated conidium q, r culture on PDA from above and reverse. Scale bars: 50 μm (d); 20 μm (e–j); 10 μm (k–o); 20 μm (p).

Culture characteristics

Conidia germinating on water agar and germ tubes produced from conidia within 12 h. Colonies growing on PDA attaining 3 cm diam. after six weeks at 25 °C, irregular, undulate, rough, superficial and partially immersed, brown aerial mycelium mixed with pale brown; reverse brown with pale brown.

Material examined

China, Yunnan Province, Xishuangbanna City, an unknown forest beside National Highway 219 (21°93'N, 101°24'E, 549.6 m elev.), rotting petiole of the Caryota mitis, 5 February 2023, Y.R. Xiong and Li Lu, XG215 (MHZU 23-0166, new host record; living culture, ZHKUCC 24-0139, ZHKUCC 24-0140).

Notes

Two isolates on rotting petiole of the Caryota mitis obtained in this study clustered with the H. guttulatum clade, based on the phylogenetic tree with 100% ML, 100% MP bootstrap support and 0.91 BIPP bootstrap support. The nucleotide differences excluding gaps between H. guttulatum (ZHKUCC 24-0139) and H. guttulatum (MFLUCC 16-0022) in ITS is 3.60% (17/472 base pairs), while there is no difference in LSU and one base pair difference in tef 1-α and rpb2. Our two isolates are similar to H. guttulatum (Hyde et al. 2016) in shape, colour and size of conidia (Table 3). Although the conidiophores are longer than in previous collections, this might be due to the branching of the conidiophore of the isolates in this study, whereas the previous collections are unbranched. In addition, the location of the denticles is the same. Therefore, based on morphology and phylogenetic analysis, we identified our isolates as H. guttulatum and this is a new record of H. guttulatum on Caryota mitis. Helicoma guttulatum was first introduced on decaying wood from Thailand by Hyde et al. (2016), based on morphology and phylogeny. Tian et al. (2022) reported a new collection of H. guttulatum on decaying wood of an unidentified host from Thailand.

Helicoma rufum Y.Z. Lu, J.C. Kang & K.D. Hyde, Fungal Diversity 92: 131–344 (2018)

Fig. 4

Description

Saprobic on the rotting inflorescence of Caryota mitis. Sexual morph: Not observed. Asexual morph: Hyphomycetous, helicosporous. Colonies on the substratum superficial, effuse, gregarious, pale brown. Mycelium composed of partly immersed, partly superficial, hyaline to brown, septate, branched hyphae. Conidiophores 150–270 µm long, 4–7.5 µm wide (x̄ = 225 × 5.9 μm, n = 20), macronematous, mononematous, cylindrical, erect, straight to slightly bent, pale brown to deep brown from top towards the base, apex hyaline, septate, mostly unbranched, smooth-walled. Conidiogenous cells 7–15 μm long, 4–7 μm wide (x̄ = 12 × 5.9 μm, n = 20), mono- to polyblastic, cylindrical, integrated, intercalary, brown, smooth-walled; with denticles, 3–6 μm long, 1.5–3 μm wide (x̄ = 4.6 × 2.5 μm, n = 20), arising from the lower portion of conidiophores as tooth-like protrusions, mono- to polyblastic, pale brown to brown, smooth-walled. Conidia 21–47 μm diam. (x̄ = 36.2 μm, n = 40) and conidial filament 2–5 μm wide (x̄ = 3.8 μm, n = 40), 145–345 μm long (x̄ = 257.7 μm, n = 40), solitary, pleurogenous, tightly coiled 3–4½ times, guttulate, become loose in water, hyaline to pale brown, 25–35-septate, smooth-walled.

Figure 4. 

Helicoma rufum (MHZU 23-0168, new host record) a specimen observed b, c colony on decaying Caryota mitis d, e conidiophores f–i conidiogenous cells j–l conidia m germinated conidium. Scale bars: 50 μm (d, e, m); 10 μm (f–i); 20 μm (j–l).

Culture characteristics

Conidia germinating on water agar and germ tubes produced from conidia within 12 h. Colonies growing on PDA attaining 3 cm diam. after six weeks at 25 °C, irregular, undulate, rough, superficial and partially immersed, brown aerial mycelium mixed with pale brown; reverse brown with pale brown.

Material examined

China, Yunnan Province, Xishuangbanna City, an unidentified forest beside National Highway 219 (21°93'N, 101°24'E, 549.6 m), rotting inflorescence of the Caryota mitis, 5 February 2023, Y.R. Xiong and Li Lu, XG217 (MHZU 23-0168, new host record; living culture, ZHKUCC 24-0143, ZHKUCC 24-0144).

Notes

Two isolates on rotting inflorescence of Caryota mitis obtained in this study clustered with the H. rufum clade in the phylogenetic tree with 96% ML, 95% MP bootstrap values and 0.99 BIPP bootstrap support. The nucleotide differences between H. rufum (ZHKUCC 24-0143) and H. rufum (MFLUCC 17-1806) are LSU: 0.09% (1/1171 base pairs), tef 1-α: 0.22% (2/912 base pairs), rpb2 sequence unavailable and no difference in ITS, excluding gaps. Our collection is similar to H. rufum (Lu et al. 2018b) in the shape, colour and size of conidia (Table 3). Although the conidiophores and conidia are longer than in previous collections, this might be because the collections came from a different area, resulting in branching at the top of the conidiophores. Therefore, based on phylogenetic and morphological analysis, we identified our isolates as a new host record of H. rufum on Caryota mitis. Helicoma rufum was introduced from decaying wood in Thailand by Lu et al. (2018b), based on the distinguished phylogenetic clade, wider conidiophores and larger tooth-like conidiogenous protrusions and larger conidia.

Neohelicosporium guineensis Y.R. Xiong, Manawas. & K.D. Hyde, sp. nov.

Fig. 5

Etymology

Species epithet refers to the host species name “guineensis” from which the fungus was isolated.

Holotype

MHZU 23-0153.

Description

Saprobic on the rotting petiole of Elaeis guineensis. Sexual morph: Not observed. Asexual morph: Hyphomycetous, helicosporous. Colonies on the substratum superficial, effuse, gregarious, brown. Mycelium composed of partly immersed, partly superficial, pale brown, glistening, septate, branched hyphae. Conidiophores 50–160 µm long, 4–6 µm wide (x̄ = 120 × 5.2 μm, n = 20), macronematous, mononematous, cylindrical, unbranched or branched at apex, straight, septate, pale brown, brown at root part, smooth-walled. Conidiogenous cells 11.5–20 µm long, 3.5–5.5 µm wide (x̄ = 15.5 × 4.8 μm, n = 20), mono- to polyblastic, integrated, sympodial, terminal or intercalary, cylindrical, yellowish to pale brown, smooth-walled; with denticles, 1.4–2.7 µm long, 1.2–2 µm wide (x̄ = 1.9 × 1.6 μm, n = 20), arising from the juncture portion of two conidiogenous cells as tooth-like protrusions, mono- to polyblastic, hyaline, smooth-walled. Conidia 16–20 μm diam. (x̄ = 18 μm, n = 40) and conidial filament 1.8–3 μm wide (x̄ = 2.4 μm, n = 40), 90–130 μm long (x̄ = 112.9 μm, n = 40), solitary, mostly pleurogenous, rarely acrogenous, helicoid, rounded at tip, obvious hump and constricted at septa, coiled 2½–3½ times, 11–12-septate, becoming loose in water, guttulate, hyaline, smooth-walled.

Figure 5. 

Neohelicosporium guineensis (MHZU 23-0153, holotype) a specimen observed b, c colony on decaying Elaeis guineensis d, e apical branches forming long connected conidiophores f–j conidiogenous cells k–q conidia r germinated conidium s, t culture on PDA from above and reverse. Scale bars: 50 μm (d, e); 10 μm (f–q); 20 μm (r).

Culture characteristics

Conidia germinating on water agar and germ tubes produced from conidia within 12 h. Colonies growing on PDA attaining 3.5 cm diam. after six weeks at 25 °C, irregular, undulate, umbonate, rough, superficial and partially immersed, white aerial mycelium, deep brown at immersed area; reverse white to deep brown.

Material examined

China, Yunnan Province, Xishuangbanna City, an unidentified forest beside National Highway 219 (21°93'N, 101°24'E, 549.6 m elev.), rotting petiole of the Elaeis guineensis, 5 February 2023, Y.R. Xiong and Li Lu, XG186 (MHZU 23-0153, holotype); ex-type, ZHKUCC 24-0113, other living culture ZHKUCC 24-0114.

Notes

Two isolates from this study formed a separate lineage and clustered with Neohelicosporium hyalosporum and N. ovoideum in the phylogenetic tree with 88% ML, 78% MP bootstrap support and 1.00 BIPP bootstrap support. The nucleotide differences excluding gaps between N. guineensis and its phylogenetically related species were checked: N. hyalosporum (GZCC 16-0076) - ITS: 1.56% (8/513 base pairs), LSU: 0.83% (7/840 base pairs), tef 1-α: 1.32% (12/912 base pairs), rpb2: 3.63% (38/1045 base pairs); N. ovoideum (GZCC 16-0064) - ITS: 1.50% (8/534 base pairs), LSU: 0.48% (4/826 base pairs), tef 1-α: 1.21% (11/912 base pairs), rpb2: 3.16% (33/1045 base pairs). Neohelicosporium guineensis differs from its closely-related species in the size of conidia and conidiophores (Table 3). Neohelicosporium hyalosporum and N. ovoideum are multi-septate and are not constricted at the septa, while N. guineensis are 11–12-septate and constricted at the septa (Lu et al. 2018a, b). Neohelicosporium hyalosporum (Lu et al. 2018a) has multi-denticles in one conidiogenous cell, while N. guineensis, has no more than three denticles (Fig. 5e, f) in one conidiogenous cell. Furthermore, N. ovoideum (Lu et al. 2018b) has 1–2 short-connecting cells between conidiophores, while N. guineensis has one long connecting cell (Fig. 5d, e) which connects conidiophores at the apex. Based on the phylogenetic placement and morphological variations, we introduce N. guineensis as a new species.

Neohelicosporium xishuangbannaensis Y.R. Xiong, Manawas., & K.D. Hyde, sp. nov.

Fig. 6

Etymology

Species epithet refers to the location name “Xishuangbanna” from where the holotype was collected.

Holotype

MHZU 23-0156.

Description

Saprobic on the rotting petiole of Elaeis guineensis. Sexual morph: Not observed. Asexual morph: Hyphomycetous, helicosporous. Colonies on the substratum superficial, effuse, gregarious, brown. Mycelium composed of partly immersed, partly superficial, brown, septate, unbranched hyphae. Conidiophores 40–125 μm long, 3–6 μm wide (x = 68.4 × 4.4 μm, n = 20), macronematous, mononematous, flexuous, long, cylindrical, branched, septate, smooth-walled. Conidiogenous cells 7–14 μm long, 2.5–5.5 μm wide (x̄ = 11.2 × 3.9 μm, n = 20), mono- to polyblastic, integrated, sympodial, terminal or intercalary, cylindrical, pale brown, smooth-walled; with denticles, 1.8–3.3 μm long, 1.1–2.3 μm wide (x̄ = 2.4 × 1.4 μm, n = 20), arising from the juncture portion of two conidiogenous cells as tooth-like and papillate protrusions, mono- to polyblastic, pale brown or hyaline, smooth-walled. Conidia 16.5–20.5 μm diam. (x̄ = 18.5 μm, n = 40) and conidial filament 1.8–3.2 μm wide (x̄ = 2.4 μm, n = 40), 90–125 μm long (x̄ = 107 μm, n = 40), solitary, acropleurogenous, helicoid, rounded at tip, coiled 2–3¼ times, 9–13-septate, becoming loose in water, guttulate, slightly constricted at septa, hyaline to pale brown, smooth-walled.

Culture characteristics

Conidia germinating on water agar and germ tubes produced from conidia within 12 h. Colonies growing on PDA attaining 2.5 cm diam. after six weeks at 25 °C, irregular, undulate, umbonate, rough, superficial and partially immersed, brown aerial mycelium mixed with pale brown, deep brown at up and down junction area; reverse brown with deep brown.

Material examined

China, Yunnan Province, Xishuangbanna City, an unidentified forest beside National Highway 219 (21°93'N, 101°24'E, 549.6 m elev.), rotting petiole of the Elaeis guineensis, 5 February 2023, Y.R. Xiong and Li Lu, XG197 (MHZU 23-0156, holotype); ex-type, ZHKUCC 24-0119, other living culture ZHKUCC 24-0120.

Notes

Two isolates obtained in this study developed an independent clade in the phylogenetic tree with 77% ML, 79% MP bootstrap support and 0.99 BIPP bootstrap support. The nucleotide differences excluding gaps between Neohelicosporium xishuangbannaensis and N. fusisporum (MFUCC 16-0642) are ITS: 2.81% (15/533 base pairs), LSU: 1.06% (9/852 base pairs), tef 1-α: 2.41% (22/912 base pairs) and rpb2 sequence is unavailable. Neohelicosporium fusisporum was reported as a sexual and asexual morph by Jayasiri et al. (2017). Neohelicosporium xishuangbannaensis is different from the asexual morph of N. fusisporum (Jayasiri et al. 2017) in the size of conidia and conidiogenous cells (Table 3). In addition, the asexual morph of N. fusisporum (Jayasiri et al. 2017) has an intercalary conidiogenous cell, while N. xishuangbannaensis has an intercalary (Fig. 6e, f) or terminal (Fig. 6g, h) conidiogenous cell. Furthermore, the asexual morph of N. fusisporum (Jayasiri et al. 2017) has denticles with tooth-like or long neck cells, while N. xishuangbannaensis has a denticle with tooth-like and papillate protrusions. Based on these differences, herein we introduce N. xishuangbannaensis as a new species.

Figure 6. 

Neohelicosporium xishuangbannaensis (MHZU 23-0156, holotype) a specimen observed b, c colony on decaying Elaeis guineensis d conidiophores e, f intercalary conidiogenous cells g, h terminal conidiogenous cells i–o conidia p germinated conidium q, r culture on PDA from above and reverse. Scale bars: 50 μm (d); 10 μm (e–o); 20 μm (p).

Discussion

In the present study, we identified and introduced three new species viz. Helicoma oleifera, Neohelicosporium guineensis and N. xishuangbannaensis with two new host records of Helicoma viz. H. guttulatum and H. rufum, which are associated with palms in tropical China. Xishuangbanna forests comprise numerous palm species, including Caryota sp., Calamus sp. and Elaeis sp. This humid tropical area near streams is also an ideal environment for Tubeufiaceae species (Lu et al. 2018b). In addition, most previous reports of this family are on unknown decaying woods (Lu et al. 2023) and we believe that there may be more undiscovered records of Tubeufiaceae on palms in tropical regions. Furthermore, in our comparison with closely-related species, we observed that H. anastomosanse (David 1931), H. divaricatum (Holubová-Jechová 1987) and H. westonii (David 1931) were reported to inhabit palms, but no molecular data were available for conducting phylogenetic analysis. The spores of H. anastomosanse (David 1931) have 18–25 septa, H. westonii (David 1931) have 11–14 septa and H. divaricatum (Holubová-Jechová 1987) have branched conidiophores and pleurogenous spores, which can be clearly distinguished from H. oleifera. However, the lack of molecular data for the above-mentioned three species has posed a significant challenge, forcing us to spend more time on morphological comparisons to identify H. oleifera. Similarly, almost all Tubeufiaceae reported on palm hosts in the early 20th century lack molecular data, which further complicates our task of sorting out the information on Tubeufiaceae on palm hosts.

Helicoma is one of the most typical helicosporous genera (Lu et al. 2023), although Goos (1986) and Lu et al. (2018b) successively revised this genus. In addition, the species of this genus cluster on the same large branch in phylogenetic analysis; some morphologically similar species are in different subordinate clades. In addition, we observed that Helicoma guttulatum (ZHKUCC 24-0139), which was identified, based on phylogenetic analysis has a different morphology compared to the type (Hyde et al. 2016). Helicoma guttulatum (ZHKUCC 24-0139) was observed to have branched conidiophores and is different from H. guttulatum (MFLU 21-0183) unbranched conidiophores (Tian et al. 2022). Since the two collections were collected from different locations and climates, we hypothesise that isolations could be influenced by different locations and climates. However, further collections and detailed analysis are required to confirm this hypothesis.

Neohelicosporium was introduced to accommodate helicosporous taxa with distinct conidiophores and is supported by molecular phylogenies, based on ITS, LSU, tef 1-α and rpb2 sequence data (Lu et al. 2018a). However, the bootstrap values of ML and MP are below 0.75% for some species (e.g. N. abuense, N. astrictum and N. bambusicola) within this genus (Lu et al. 2018a; Tian et al. 2022). The three new species and two new host records introduced in this study are significant as they expand our understanding of the diversity and distribution of Tubeufiaceae in tropical regions and provide valuable insights into their ecological roles and interactions with palm hosts. In addition, Zhang et al. (2023) identified four useful chemical compounds from N. guangxiense and they can play a vital role in drug design and functional group modification. This underscores the urgent need for future studies to explore the potential chemical composition and corresponding applications of this genus, a call to action for professional researchers.

Acknowledgements

Yinru Xiong would like to thank Mae Fah Luang University for the award of Tuition fee waiver scholarship for the PhD. Ishara Manawasinghe would like to acknowledge Zhongkai University of Agriculture and Engineering, talent funding (grant number KA210319288) and the Guangzhou Science and Technology Plan Project (2023A04J1427). Biao Xu thanks to the National Natural Science Foundation of China (Nos. 32370021) and the Innovative team program of the Department of Education of Guangdong Province (2022KCXTD015 and 2022ZDJS020). We would like to acknowledge the Innovative team programme of the Department of Education of Guangdong Province (2022KCXTD015 and 2022ZDJS020). The authors also extend their appreciation to the Researchers Supporting Project number (RSP2024R114), King Saud University, Riyadh, Saudi Arabia for funding this work.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was funded by the High-level Talents at Zhongkai University of Agriculture and Engineering, grant no: J2201080102 Researchers Supporting Project number (RSP2024R114), King Saud University, Riyadh, Saudi Arabia.

Author contributions

Data curation: LL, YX. Formal analysis: ISM. Funding acquisition: KDH, FA, XB. Investigation: LL, YX. Methodology: YX, LL. Project administration: KDH. Resources: KDH. Supervision: ISM. Visualization: ISM. Writing - original draft: YX. Writing - review and editing: DLH, ISM, AM, KDH.

Author ORCIDs

Yinru Xiong https://orcid.org/0000-0002-4673-606X

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

Li Lu https://orcid.org/0000-0003-0977-6414

Dulanjalee L. Harishchandra https://orcid.org/0000-0003-1538-4951

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

Ishara S. Manawasinghe https://orcid.org/0000-0001-5730-3596

Data availability

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

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