Fungal Species from Rhododendron sp.: Discosia rhododendricola sp.nov, Neopestalotiopsis rhododendricola sp.nov and Diaporthe nobilis as a New Host Record.

Abstract

In the present study, we report two new asexual fungal species (i.e., Discosia rhododendricola, Neopestalotiopsis rhododendricola (Sporocadaceae) and a new host for a previously described species (i.e., Diaporthe nobilis; Diaporthaceae). All species were isolated from Rhododendron spp. in Kunming, Yunnan Province, China. All taxa are described based on morphology, and phylogenetic relationships were inferred using a multigenic approach (LSU, ITS, RPB2, TEF1 and TUB2). The phylogenetic analyses indicated that D. rhododendronicola sp. nov. is phylogenetically related to D. muscicola, and N. rhododendricola sp. nov is related to N. sonnaratae. Diaporthe nobilis is reported herein as a new host record from Rhododendron sp. for China, and its phylogeny is depicted based on ITS, TEF1 and TUB2 sequence data.

1. Introduction

Rhododendron, a genus of shrub and small to large trees belonging to Ericaceae, is an indicator of health for forest areas [1], commonly found in low-quality acidic soil and sterile conditions. This plant is mainly distributed in India and southeastern Asia, extending from the northwest Himalayas (Arunachal Pradesh) to Bhutan, eastern Tibet, Nepal, north Myanmar, Sikkim, and west central China [2]. Rhododendron flowers are used as food, to produce fermented wine, and to make herbal tea due to their distinctive flavor and color [3,4]. Fungi colonizing Rhododendron include Alternaria alternata, Aspergillus brasiliensis, Chrysomyxa dietelii, C. succinea [5], Diaporthe nobilis [6], Epicoccum nigrum, Mucor hiemalis, Pestalotiopsis sydowiana and Trichoderma koningii [7]. However, given the economic importance of this plant, it is imperative to assess the fungal species associated with it.

Discosia was introduced in Discosiaceae by Maharachchikumbura et al. [8] to accommodate the type genus Discosia and the type species D. artocreas. Senanayake et al. [9,10] introduced Adisciso, Discosia, Discostroma, Immersidiscosia, Sarcostroma and Seimatosporium in the Discosiaceae family. Jaklitsch et al. [11] considered Discosiaceae a synonym of Sporocadaceae based on DNA sequence analyses with strong phylogenetic support. Wijayawardene et al. [12] accepted Discosia species belonging to the family Sporocadaceae. Libert introduced Discosia in 1837, with Discosia strobilina being the lectotype [9,12]. Liu et al. [13] reviewed the generic description of Discosia, an updated morphology, and the phylogenetic relationships based on ITS sequence data [13]. There are 118 epithets of Discosia in Index Fungorum 2022 [14]. Discosia has been identified as an asexual fungus and is characterized by uni- to multilocular conidiomata with muti-layered walls. Conidiogenous cells are monoblastic and phialidic to annellidic. Conidial types are bipolar, polar and subpolar appendages, and usually hyaline to pale brown [9].

The genus Neopestalotiopsis, introduced by Maharachchikumbura et al., (2014) [15], belongs to the family Sporocadaceae (Amphisphaeriales, Sordariomycetes) [8,15,16], with N. protearum being the type species. Neopestalotiopsis species have been reported on saprobes, trees or plant pathogens causing postharvest diseases (fruit rots and leaf blights) [17,18]. The sexual morph of Neopestalotiopsis species remains unknown [15,18,19,20]. Neopestalotiopsis species have a worldwide distribution. This genus has also been reported in caves in China [15,17,18,19,20,21,22]. Studies related to the taxonomy of Neopestalotiopsis included DNA sequence analyses and phylogeny of the ITS, TEF1 and TUB2 [22].

The genus Diaporthe introduced by Nitschke [23] belongs to the families Diaporthaceae, Diaporthales, and Sordariomycetes [8,9]. Diaporthe species have a worldwide distribution [6,24,25,26,27,28]. This genus has been associated with several grapevine diseases in Europe [29] and was detected in Uruguayan deciduous fruit tree (Malus domestica ‘Gala’) wood disease [30]. Studies related to the taxonomy of Diaporthe included DNA sequence analyses and phylogeny of the ITS, TEF1, TUB2 and CAL loci [6,31]. Dissanayake et al. [32] provided phylogenetic relationships of 171 Diaporthe species currently known from culture or direct sequencing, and are linked to their holotype, epitype, isotype or neotype and that can now be recognized with DNA sequence data, essential to species identification [33].

In this study, we introduce the new species D. rhododendricola, N. rhododendricola and a new host record of Diaporthe nobilis, collected from dead leaves of Rhododendron species in China. We further provide descriptions, illustrations, and DNA sequence-based phylogeny to verify identification and placement.

2. Materials and Methods

2.1. Sample Collection, Morphological Observation, and Fungal Isolation

Isolation was performed as described by Senanayake et al. [34]. Dead leaves of Rhododendron spp. were collected from Kunming, Yunnan Province, China and brought to the laboratory in labelled paper envelopes. A light microscope (Nikon ECLIPSE 80i compound microscope, Melville, NY, USA) was used to observe the specimens. Spore mass fruiting bodies were isolated on potato dextrose agar (PDA) plates and incubated at 25 °C.

The isolates were transferred to new PDA plates, incubated at 25 °C, and photographed using a Canon EOS 600D digital camera fitted to the microscope. The Tarosoft (R) Image Frame Work program measured the morphological characteristics. The figures were processed using Adobe Photoshop CS6 Extended version 10.0 (Adobe Systems, San Jose, CA, USA).

The specimens were deposited at the Herbarium of Mae Fah Luang University (Herb. MFLU) and Herbarium of Kungming Institute of Botany (KUN), Chinese Academy of Science, Kunming, China. Living cultures were deposited at the Culture Collection of Mae Fah Luang University (MFLUCC), Chiang Rai, Thailand and the Culture Collection of Kungming Institute of Botany (KUN), Chinese Academy of Science, Kunming, China. Faces of Fungi and Index Fungorum data are also provided [14,35]. New species were established based on guidelines provided by Jeewon and Hyde [36].

2.2. DNA Extraction, PCR Amplification, and Sequencing

Fungal cultures were grown on PDA at 25 °C for 2–4 weeks. The Biospin Fungus Genomic DNA Extraction Kit-BioFlux (BioFlux^®, Hangzhou, China) was used to extract DNA from the mycelium. PCR amplification was performed using primer pairs, ITS4/ITS5 for the internal transcribed spacer region of ribosomal DNA [37], LR0R/LR5 for large subunit nuclear ribosomal DNA [38], EF-728F/EF-986R for translation elongation factor 1-alpha gene [39], fRPB2-5f/fRPB2-7cR for the second largest subunit of RNA polymerase [40] and Bt2a/Bt2b for beta-tubulin [41]. The PCR conditions were based on the methodology as described by Chaiwan et al. [42].

2.3. Phylogenetic Analyses

The sequence alignment and phylogenetic analyses were performed as outlined by Dissanayake et al. [43] and Chaiwan et al. [42,44,45]. Phylogenetic analyses were performed using a combined Discosia dataset of ITS, LSU, RPB2, TEF1 and TUB2 sequence data and a combined Neopestalotiopsis and Diaporthe dataset of ITS, TEF1 and TUB2 sequence data. Taxa used in the analyses were obtained through recent publications [16,28,46]. The phylogenetic analyses were carried out using maximum parsimony (MP), maximum likelihood (ML) and Bayesian posterior probabilities (BYPP). PAUP v4.0b10 was used to conduct the parsimony analysis to obtain the phylogenetic trees [47]. Trees were inferred using the heuristic search option with 1000 random sequence additions. Maxtrees were set to 1000, branches of zero length were collapsed and all multiple parsimonious trees were saved. Descriptive tree statistics for parsimony—tree length (TL), consistency index (CI), retention index (RI), relative consistency index (RC) and homoplasy index (HI)—were calculated for trees generated following the Kishino-Hasegawa test (KHT) criteria [48], which was performed in order to determine whether trees were significantly different. Maximum-parsimony bootstrap values equal or greater than 60% are given as the second set of numbers above the nodes.

Maximum likelihood analysis was performed by using RAxML-HPC2, New Orleans, LA on XSEDE (8.2.8) [45,48,49,50]. The search strategy was set to rapid bootstrapping and the analysis was carried out using the GTRGAMMAI model of nucleotide substitution. Maximum likelihood bootstrap values equal to or greater than 60% are given as the first set of numbers above the nodes.

Bayesian inference (BI) analysis was conducted with MrBayes v. 3.1.2 to evaluate the posterior probabilities (BYPP) using Markov chain Monte Carlo sampling [51]. Two parallel runs were conducted using the default settings, but with the following adjustments: six simultaneous Markov chains were run for 2,000,000 generations and trees were sampled every 200 generations. The distribution of log-likelihood scores were examined to determine stationary phase for each search and to decide if extra runs were required to achieve convergence, using the program Tracer 1.4 [52]. The first 10% of generated trees were discarded and the remaining 90% of trees were used to calculate posterior probabilities (PP) of the majority rule consensus tree. The phylogenetic trees were viewed in FigTree v. 1.4 [53] and edited using Microsoft Office Power Point 2007 and Adobe Photoshop CS6 Extended [42].

2.4. Genealogical Concordance Phylogenetic Species Recognition (GCPSR) Analysis

The related species were analyzed using the Genealogical Concordance Phylogenetic Species Recognition model. The pairwise homoplasy index (PHI) [54] is a model test based on the fact that multiple gene phylogenies will be concordant between species and discordant due to recombination and mutations within a species. The data were analyzed by the pairwise homoplasy index (PHI) test [54]. The test was performed in SplitsTree4 [55,56] as described by Quaedvlieg [57] to determine the recombination level within phylogenetically closely related species using a five-locus concatenated dataset to determine the recombination level within phylogenetically closely related species. If the PHI is below the 0.05 threshold (Φw < 0.05), it indicates that there is significant recombination in the dataset. This means that related species in a group and recombination level are not different. If the PHI is above the 0.05 threshold (Φw > 0.05), it indicates that it is not significant, which means the related species in a group level are different. The new species and its closely related species were analyzed using this model. The relationships between closely related species were visualized by constructing a split graph, using both the LogDet transformation and splits decomposition options.

2.5. Discosia, Habitat and Known Distribution Checklist Associated with Rhododendron sp.

An updated checklist of Discosia based on the SMML database (https://nt.ars-grin.gov/fungaldatabases/) (accessed on 10 June 2022) is provided [58]. Those species for which molecular data are available are indicated. The distribution information regarding the type or original descriptions available and the locality from which Discosia have been recorded on Rhododendron spp. is provided, including all the specimens encountered during this study.

3. Results

3.1. Phylogenetic Analyses

The combined sequence alignments of Discosia comprised 54 taxa (Table 1), with Immersidiscosia eucalypti MFLU16-1372 and NBRC 104195 as the outgroup taxa. The dataset comprised 4364 characters including alignment gaps (LSU, ITS, RPB2, TEF1 and TUB2 sequence data). The MP analysis for the combined dataset had 430 parsimony-informative, 3522 constant, and 412 parsimony-uninformative characters, and yielded a single most parsimonious tree (TL = 1353, CI = 0.777, RI = 0.764, RC = 0.594; HI = 0.223). The RAxML analysis of the combined dataset yielded a best scoring tree with a final ML optimization likelihood value of −22,013.917605. The matrix had 840 distinct alignment patterns, with 66% undetermined characters or gaps. Bayesian posterior probabilities from Bayesian inference analysis were assessed with a final average standard deviation of split frequencies = 0.009983. The phylogenetic tree in this study showed that our strain (Discosia rhododendricola KUN-HKAS 123205 and MFLU20-0486) is related to D. muscicola with high support value in the phylogenbetic tree (Figure 1). Sequence alignments are deposited in TreeBASE.

Table 1.

Culture collection numbers and GenBank accession numbers for Discosia used in this study. The type species are indicated in bold. The newly generated sequences are indicated in red. Instances where the GenBank Accession No. did not show the molecular data are marked with a dash.

Species Name	Culture Collection No.	Substrate/Host	Country	GenBank Accession No					References
				LSU	ITS	TUB2	TEF1	RPB2
Discosia pleurochaeta	KT 2179	-	-	KT281912	KT284775	-	-	-	[9]
Discosia pleurochaeta	KT 2188	-	-	AB593713	AB594781	AB594179	-	-	[9]
Discosia pleurochaeta	KT 2192	-	-	AB593714	AB594782	AB594180	-	-	[9]
Discosia artocreas	CBS 124848	Fagus sylvatica	Germany	MH554213	MH553994	MH554662	MH554420	MH554903	[13]
Discosia brasiliensis	MFLUCC 12-0429	Dead leaf	Thailand	KF827436	KF827432	KF827469	KF827465	KF827473	[59]
Discosia brasiliensis	MFLUCC 12-0431	Dead leaf	Thailand	KF827437	KF827433	KF827470	KF827466	KF827474	[59]
Discosia brasiliensis	MFLUCC 12-0435	Dead leaf	Thailand	KF827438	KF827434	KF827471	KF827467	KF827475	[59]
Discosia fagi	MFLU 14-0299A	Fagus sylvatica	Italy	KM678048	KM678040	-	-	-	[60]
Discosia fagi	MFLU 14-0299B	Fagus sylvatica	Italy	KM678047	KM678039	-	-	-	[60]
Discosia fagi	MFLU 14-0299C	Fagus sylvatica	Italy	KM678048	KM678040	-	-	-	[60]
Discosia italica	MFLU 14-0298B	Fagus sylvatica	Italy	KM678045	KM678042	-	-	-	[60]
Discosia italica	MFLU 14-0298C	Fagus sylvatica	Italy	KM678044	KM678041	-	-	-	[60]
Discosia macrozamiae	CPC 32109	-	-	MH327856	MH327820	MH327895	MH327884	-	[61]
Discosia muscicola	CBS 109.48	-	-	MH867828	-	-	-	-	[62]
Discosia neofraxinea	NTIT 469	Fagus sylvatica	Italy	KF827439	KF827435	KF827472	KF827468	KF827476	[59]
Discosia neofraxinea	MFLUCC 13-0204	Fagus sylvatica	Italy	KR072672	KR072673	-	-	-	[10]
Discosia pseudoartocreas	CBS 136438	Tilia sp.	Austria	KF777214	KF777161	MH554672	MH554430	MH554913	[63]
Discosia pini	MAFF 410149	Pinus densiflora	Japan	AB593708	AB594776	AB594174	-	-	[9]
Discosia querci	MFLUCC 16-0642	-	-	MG815830	MG815829	-	-	-	[64]
Discosia ravennica	MFLU 18-0131	Pyrus sp.	Italy	MT376617	MT376615	MT393594	-	-	[46]
Discosia rhododendricola	KUN-HKAS 123205	Rhododendron sp.	China	MT741963	MT741959	-	-	MW143037	This study
	MFLU20-0486	Rhododendron sp.	China	OP162409	OP162414	-	-	OP169687	This study
Discosia rubi	CBS 143893	Rubus phoenicolasius	USA	MH554334	MH554131	MH554804	MH554566	MH555038	[13]
Discosia sp.	F 233	-	-	-	KU751876	-	-	-	[13]
Discosia sp.	3T30CF	-	-	-	FJ861385	-	-	-	[65]
Discosia sp.	3T9A	-	-	-	FJ861386	-	-	-	[65]
Discosia sp.	3T9C	-	-	-	FJ861387	-	-	-	[65]
Discosia sp.	FIHB 571	-	-	-	DQ536523	-	-	-	[66]
Discosia sp.	HKUCC 6626	-	-	AF382381	AF405303	-	-	-	[67]
Discosia sp.	JSP0111c42	-	-	-	KR093849	-	-	-	[68]
Discosia sp.	KT 2193	-	-	AB593706	AB594774	-	-	-	[9]
Discosia sp.	OT1 143c	-	-	-	KT804147	-	-	-	[13]
Discosia sp.	OT2 143a	-	-	-	KT804075	-	-	-	[13]
Discosia sp.	OT3 176b	-	-	-	KT804146	-	-	-	[13]
Discosia sp.	P4 A7 53	-	-	-	KU325138	-	-	-	[13]
Discosia sp.	P8 A7-852	-	-	-	KU325418	-	-	-	[13]
Discosia sp.	R 158	-	-	-	JN689956	-	-	-	[13]
Discosia sp.	UNH ID260	-	-	-	KX459431	-	-	-	[13]
Discosia sp.	UWR 012	-	-	-	KX426948	-	-	-	[13]
Discosia sp.	UWR 040	-	-	-	KX426977	-	-	-	[13]
Discosia sp.	KT 2109	-	-	-	MT236494	-	-	-	[69]
Discosia sp.	SH 125	-	-	-	JF449727	-	-	-	[13]
Discosia sp.	SH 288	-	-	-	AB594783	-	-	-	[9]
Discosia sp.	MAFF 236709	-	-	-	KU751876	-	-	-	[13]
Discosia sp.	CBS 241.66	Acacia karroo	South Africa	MH554244	MH554022	MH554698	-	-	[13]
Discosia sp.	CBS 684.70	Aesculus hippocastanum	Netherlands	MH554277	MH554064	MH554740	-	-	[13]
Discosia tricellularis	MAFF 237478	-	-	AB593730	AB594798	AB594189	-	-	[9]
Discosia tricellularis	NBRC 32705	Rhododendron indicum	Japan	AB593728	AB594796	AB594188	-	-	[9]
Discosia yakushimensis	MAFF 242774	Symplocos prunifolia	Japan	AB593721	AB594789	AB594187	-	-	[9]
Sporocadus cornicola	MFLUCC 14-0448	Cornus sanguinea	Italy	-	KU974967	-	-	-	[70]
Sporocadus rosarum	MFLUCC 14-0466	Rosa canina	Italy	KT281912	KT284775	-	-	-	[70]

Figure 1.

Phylogram generated from maximum parsimony analysis of LSU, ITS, RPB2, TEF1 and TUB2 gene regions. Bootstrap support values for MP and ML equal to or greater than 60% and Bayesian posterior probabilities (PP) equal to or greater than 0.90 are defined as MP/ML/PP above or below the nodes. Taxonomic novelty is indicated in red. The tree is rooted with Immersidiscosia eucalypti (MFLU 16-1372) and (NBRC 104195).

The combined sequence alignments of Neopestalotiopsis comprised 89 taxa (Table 2), with Monochaetia monochaeta CBS115004 and M. ilexae CBS101009 as the outgroup taxa. The dataset comprised 2634 characters including alignment gaps (ITS, TUB2 and TEF1 sequence data). The MP analysis for the combined dataset had 631 parsimony-informative, 1524 constant, and 479 parsimony-uninformative characters, and yielded a single most parsimonious tree (TL = 2304, CI = 0.679, RI = 0.813, RC = 0.552; HI = 0.321). The RAxML analysis of the combined dataset yielded a best scoring tree with a final ML optimization likelihood value of −24,500.881631. The matrix had 1268 distinct alignment patterns, with 35.77% undetermined characters or gaps. Bayesian posterior probabilities from Bayesian inference analysis were assessed with a standard deviation of split frequencies = 0.024223. The phylogenetic tree in this study showed that N. rhododendricola KUN-HKAS 123204 and MFLU20-0046 belonged to a separate clade, phylogenetically related to N. sonneratae, N. coffeae-arabicae and N. thailandica with 88% MP support (Figure 2). Sequence alignments are deposited in TreeBASE.

Table 2.

Culture collection numbers and GenBank accession numbers for Neopestalotiopsis used in this study. The type species are indicated in bold. The newly generated sequences are indicated in red. Instances where the GenBank Accession No. did not show the molecular data are marked with the dash.

Species Name	Culture Collection No.	Substrate/Host	Country	GenBank Accession No			References
				ITS	TUB2	TEF1
Monochaetia ilexae	CBS 101009	Air	Japan	MH55395	MH554612	MH554371	[13]
M. monochaeta	CBS 115004	Quercus robur	Netherlands	AY853243	MH554639	MH554398	[13]
Neopestalotiopsis acrostichi	MFLUCC 17-1754	Acrostichum aureum	Thailand	MK764272	MK764338	MK764316	[21]
N. acrostichi	MFLUCC 17-1755	Acrostichum aureum	Thailand	MK764273	MK764339	MK764317	[21]
N. alpapicalis	MFLUCC 17-2544	Rhizophora mucronata	Thailand	MK357772	MK463545	MK463547	[71]
N. alpapicalis	MFLUCC 17-2545	Symptomatic Rhizophora apiculata leaves	Thailand	MK357773	MK463546	MK463548	[71]
N. aotearoa	CBS 367.54	Canvas	New Zealand	KM199369	KM199454	KM199526	[15]
N. asiatica	MFLUCC 12-0286	Prunus dulcis	China	JX398983	JX399018	JX399049	[15]
N. australis	CBS 114159	Telopea sp.	Australia	KM199348	KM199432	KM199537	[15]
N. brachiata	MFLUCC 17-555	Rhizophora apiculata	Thailand	MK764274	MK764340	MK764318	[21]
N. brasiliensis	COAD 2166	Psidium guajava	Brazil	MG686469	MG692400	MG692402	[72]
N. cavernicola	KUMCC 20-0269	Cave	China	MW545802	MW557596	MW550735	[22]
N. chiangmaiensis	MFLUCC 18-0113	Pandanus sp.	Thailand	-	MH412725	MH388404	[73]
N. chrysea	MFLUCC 12-0261	Dead leaves	China	JX398985	JX399020	JX399051	[74]
N. chrysea	MFLUCC 12-0262	Dead plant	China	JX398986	JX399021	JX399052	[74]
N. clavispora	MFLUCC 12-0280	Magnolia sp.	China	JX398978	JX399013	JX399044	[74]
N. clavispora	MFLUCC 12-0281	Magnolia sp.	China	JX398979	JX399014	JX399045	[74]
N. cocoës	MFLUCC 15-0152	Cocos nucifera	Thailand	KX789687	-	KX789689	[17]
N. coffeae-arabicae	HGUP4015	Coffea arabica	China	KF412647	KF412641	KF412644	[75]
N. coffeae-arabicae	HGUP4019	Coffea arabica	China	KF412649	KF412643	KF412646	[75]
N. cubana	CBS 600.96	Leaf Litter	Cuba	KM199347	KM199438	KM199521	[15]
N. dendrobii	MFLUCC 14-0099	Dendrobium cariniferum	Thailand	MK993570	MK975834	MK975828	[76]
N. dendrobii	MFLUCC 14-0106	Dendrobium cariniferum	Thailand	MK993571	MK975835	MK975829	[76]
N. egyptiaca	CBS H 22294	Mangifera indica	Egypt	KP943747	KP943746	KP943748	[77]
N. ellipsospora	MFLUCC 12-0283	Dead plant materials	China	JX398980	JX399016	JX399047	[74]
N. eucalypticola	CBS 264.37	Eucalyptus globulus	-	KM199376	KM199431	KM199551	[15]
N. foedans	CGMCC 3.9123	Mangrove plant	China	JX398987	JX399022	JX399053	[74]
N. foedans	CGMCC 3.9178	Neodypsis decaryi	China	JX398989	JX399024	JX399055	[74]
N. formicidarum	CBS 115.83	Plant debris	Cuba	KM199344	KM199444	KM199519	[78]
N. formicidarum	CBS 362.72	Dead Formicidae (ant)	Cuba	KM199358	KM199455	KM199517	[78]
N. honoluluana	CBS 111535	Telopea sp.	USA	KM199363	KM199461	KM199546	[15]
N. honoluluana	CBS 114495	Telopea sp.	USA	KM199364	KM199457	KM199548	[15]
N. hydeana	MFLUCC 20-0132	Artocarpus heterophyllus	Thailand	MW266069	MW251119	MW251129	[79]
N. iranensis	CBS 137767	Fragaria ananassa	Iran	KM074045	KM074056	KM074053	[80]
N. iranensis	CBS 137768	Fragaria ananassa	Iran	KM074048	KM074057	KM074051	[81]
N. javaensis	CBS 257.31	Cocos nucifera	Java	KM199357	KM199437	KM199543	[15]
N. keteleeria	MFLUCC 13-0915	Keteleeria pubescens	China	KJ023087	KJ023088	KJ023089	[75]
N. macadamiae	BRIP 63737c	Macadamia integrifolia	Australia	KX186604	KX186654	KX186627	[81]
N. macadamiae	BRIP 63742a	Macadamia integrifolia	Australia	KX186599	KX186657	KX186629	[82]
N. magna	MFLUCC 12-652	Pteridium sp.	France	KF582795	KF582793	KF582791	[15]
N. mesopotamica	CBS 299.74	Eucalyptus sp.	Turkey	KM199361	KM199435	KM199541	[15]
N. mesopotamica	CBS 336.86	Pinus brutia	Iraq	KM199362	KM199441	KM199555	[15]
N. musae	MFLUCC 15-0776	Musa sp.	Thailand	KX789683	KX789686	KX789685	[17]
N. natalensis	CBS 138.41	Acacia mollissima	South Africa	KM199377	KM199466	KM199552	[15]
N. nebuloides	BRIP 66617	Sporobolus elongatus	Australia	MK966338	MK977632	MK977633	[82]
N. pandanicola	KUMCC 17-0175	Pandanus sp.	China	-	MH412720	MH388389	[73]
N. pernambucana	URM7148	Vismia guianensis	Brazil	KJ792466	-	KU306739	[83]
N. pernambucana	RV02	Vismia guianensis	Brazil	KJ792467	-	KU306740	[83]
N. petila	MFLUCC 17-1737	Rhizophora mucronata	Thailand	MK764275	MK764341	MK764319	[21]
N. petila	MFLUCC 17-1738	Rhizophora mucronata	Thailand	MK764276	MK764342	MK764320	[21]
N. phangngaensis	MFLUCC 18-0119	Pandanus sp.	Thailand	MH388354	MH412721	MH388390	[73]
N. piceana	CBS 254.32	Cocos nucifera	Indonesia	KM199372	KM199452	KM199529	[15]
N. piceana	CBS 394.48	Picea sp.	UK	KM199368	KM199453	KM199527	[15]
N. protearum	CBS 114178	Leucospermum cuneiforme cv. “Sunbird”	Zimbabwe	JN712498	KM199463	LT853201	[15]
N. rhizophorae	MFLUCC 17-1550	Rhizophora mucronata	Thailand	MK764277	MK764343	MK764321	[21]
N. rhizophorae	MFLUCC 17-1551	Rhizophora mucronata	Thailand	MK764278	MK764344	MK764322	[21]
N. rhododendricola	KUN-HKAS 123204	Rhododendron sp.	China	OK283069	OK274147	OK274148	This study
	MFLU20-0046	Rhododendron sp.	China	OP11897554	OP169689	OP169688	This study
N. rosae	CBS 101057	Rosa sp.	New Zealand	KM199359	KM199429	KM199523	[15]
N. rosae	CBS 124745	Paeonia suffruticosa	USA	KM199360	KM199430	KM199524	[15]
N. rosicola	CFCC 51992	Rosa chinensis	China	KY885239	KY885245	KY885243	[84]
N. rosicola	CFCC 51993	Rosa chinensis	China	KY885240	KY885246	KY885244	[84]
N. samarangensis	CBS 115451	Unidentified tree	China	KM199365	KM199447	KM199556	[85]
N. saprophytica	MFLUCC 12-0282	Magnolia sp.	China	JX398982	JX399017	JX399048	[74]
N. sichuanensis	CFCC 54338	Castanea mollissima	China	MW166231	MW218524	MW199750	[86]
N. sichuanensis	SM15-1C	Castanea mollissima	China	MW166232	MW218525	MW199751	[86]
N. sonneratae	MFLUCC 17-1744	Sonneronata alba	Thailand	MK764279	MK764345	MK764323	[21]
N. sonneratae	MFLUCC 17-1745	Sonneronata alba	Thailand	MK764280	MK764346	MK764324	[21]
N. steyaertii	IMI 192475	Eucalyptus viminalis	Australia	KF582796	KF582794	KF582792	[15]
N. surinamensis	CBS 450.74	Soil under Elaeis guineensis	Suriname	KM199351	KM199465	KM199518	[15]
N. thailandica	MFLUCC 17-1730	Rhizophora mucronata	Thailand	MK764281	MK764347	MK764325	[21]
N. thailandica	MFLUCC 17-1731	Rhizophora mucronata	Thailand	MK764282	MK764348	MK764326	[21]
N. umbrinospora	MFLUCC 12-0285	Unidentified plant	China	JX398984	JX399019	JX399050	[74]
N. vitis	MFLUCC 15-1265	Vitis vinifera cv. “Summer black”	China	KU140694	KU140685	KU140676	[87]
N. vitis	MFLUCC 15-1270	Vitis vinifera cv. “Kyoho”	China	KU140699	KU140690	KU140681	[87]
N. zimbabwana	CBS 111495	Leucospermum cunciforme	Zimbabwe	JX556231	KM199456	KM199545	[15]
Pestalotiopsis adusta	ICMP6088	On refrigerator door PVC gasket	Fiji	JX399006	JX399037	JX399070	[74]
P. adusta	MFLUCC10-0146	Syzygium sp.	Thailand	JX399007	JX399038	JX399071	[74]
P. anacardiacearum	IFRDCC 2397	Mangifera indica	China	KC247154	KC247155	KC247156	[88]
P. humus	CBS 115450	Ilex cinerea	China	KM199319	KM199418	KM199487	[15]
P. humus	CBS 336.97	Soil	Papua New Guinea	KM199317	KM199420	KM199484	[15]
P. hydei	MFLUCC 20135	Litsea petiolata	Thailand	MW266063	MW251112	MW251113	[79]
N. thailandica	MFLUCC 17-1730	Rhizophora mucronata	Thailand	MK764281	MK764347	MK764325	[21]
N. thailandica	MFLUCC 17-1731	Rhizophora mucronata	Thailand	MK764282	MK764348	MK764326	[21]
N. umbrinospora	MFLUCC 12-0285	Unidentified plant	China	JX398984	JX399019	JX399050	[74]
P. hydei	E-72-02	Eucalyptus grandis	Brazil	KU926708	KU926716	KU926712	[79]
P. inflexa	MFLUCC12-0270	Unidentifified tree	China	JX399008	JX399039	JX399072	[74]
P. linearis	MFLUCC12-0271	Trachelospermum sp.	China	JX398992	JX399027	JX399058	[74]
Pseudopestalotiopsis cocos	CBS 272.29	Cocos nucifera	Indonesia	KM199378	KM199467	KM199553	[15]
Ps. indica	CBS 459.78	Hibiscus rosa-sinensis	India	KM199381	KM199470	KM199560	[15]
Ps. theae	MFLUCC12-0055 T	Camellia sinensis	Thailand	JQ683727	JQ683711	JQ683743	[15]
Ps. theae	SC011	Camellia sinensis	Thailand	JQ683726	JQ683710	JQ683742	[15]

Figure 2.

RAxML tree based on a combined dataset of ITS, TUB2 and TEF1 gene regions. Bootstrap support values for ML and MP equal to or greater than 60% and Bayesian posterior probabilities (PP) equal to or greater than 0.90 are defined as ML/MP/PP above or below the nodes. Our new taxon is indicated in red. The tree was rooted with Monochaetia monochaeta strains (CBS115004) and Monochaetia ilexae strains (CBS101009).

The combined sequence alignments of Diaporthe comprised 56 taxa (Table 3), with Diaporthella corylina CBS 121124 used as the outgroup taxon. The dataset comprised 2350 characters, including alignment gaps (ITS, TEF1 and TUB2 sequence data). After alignment, 641 characters were derived from ITS, 916 from TEF1, and 793 from TUB2. The MP analysis for the combined dataset had 730 parsimony-informative, 1216 constant, and 404 parsimony-uninformative characters, and yielded a single most parsimonious tree (TL = 3968, CI = 0.480, RI = 0.622, RC = 0.298; HI = 0.520). The RAxML analysis of the combined dataset yielded a best scoring tree with a final ML optimization likelihood value of −21,299.667012. The matrix had 1319 distinct alignment patterns, with 37.51% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.226983, C = 0.316389, G = 0.231894, T = 0.224734; substitution rates AC = 1.153998, AG = 3.111864, AT = 1.039115, CG = 0.869376, CT = 4.271324, GT = 1.000000; gamma distribution shape parameter α = 0.376625. Bayesian posterior probabilities from Bayesian inference analysis were assessed with a standard deviation of split frequencies = 0.009867. The phylogenetic tree in this study showed that D. nobilis KUN-HKAS 123203 grouped with the ex-type strain of D. nobilis, and formed a supported clade with 0.99 PP (Figure 3). Sequence alignments are deposited in TreeBASE.

Table 3.

Culture collection numbers and GenBank accession numbers for Diaporthe used in this study. The type species are indicated in bold. The newly generated sequences are indicated in red. Instances where the GenBank Accession No. did not show the molecular data are marked with the dash.

Species Name	Culture Collection No.	Substrate/Host	Country	GenBank Accession No			References
				ITS	TUB2	TEF1
Diaporthe acaciigena	CBS 129521	Acacia retinodes	-	KC343005	KC343973	KC343731	[6]
Diaporthe alleghaniensis	CBS 495.72	Betula alleghaniensis	-	KC343007	KC343975	KC343733	[6]
Diaporthe alnea	CBS 146.46	Alnus sp.	-	KC343008	KC343976	KC343734	[6]
Diaporthe ambigua	CBS 187.87	Helianthus annuus	Italy	KC343015	KC343983	KC343741	[6]
Diaporthe ampelina	CBS 111888	Vaccinium vinifera	USA	KC343016	KC343984	KC343742	[6]
Diaporthe amygdali	CBS 126679	Prunus dulcis	-	KC343022	KC343990	AY343748	[6]
Diaporthe anacardii	CBS 720.97	Anacardium ocidentale	-	KC343024	KC343992	KC343750	[6]
Diaporthe arecae	CBS 161.64	Areca catechu	-	KC343032	KC344000	KC343758	[6]
Diaporthe arengae	CBS 114979	Arenga engleri	-	KC343034	KC344002	KC343760	[6]
Diaporthe australafricana	CBS 111886	Vaccinium vinifera	Australia	KC343038	KC344006	KC343764	[6]
Diaporthe baccae	CBS 136972	Vaccinium corymbosum	-	KJ160565	-	KJ160597	[45]
Diaporthe bicincta	CBS 121004	Juglans sp.	-	KC343134	KC344102	KC343860	[6]
Diaporthe bohemiae	CBS 143347	Vitis spp.	Czech Republic	MG281015	MG281188	MG281536	[29]
Diaporthe carpini	CBS 114437	Carpinus betulus	Sweden	KC343044	KC344012	KC343770	[6]
Diaporthe celastrina	CBS 139.27	Celastrus scandens	-	KC343047	KC344015	KC343773	[6]
Diaporthe celeris	CBS 143349	Vaccinium vinifera	UK	MG281017	MG281190	MG281538	[29]
Diaporthella corylina	CBS 121124	Corylus sp.	-	KC343004	KC343972	KC343730	[6]
Diaporthe citri	AR 3405	-	-	KC843311	KC843187	KC843071	[89]
Diaporthe cucurbitae	DAOM 42078	Cucumis sativus	-	KM453210	KP118848	KM453211	[89]
Diaporthe decedens	CBS 109772	Corylus avellana	Austria	KC343059	KC344027	KC343785	[6]
Diaporthe detrusa	CBS 109770	Berberis vulgaris	Austria	KC343061	KC344029	KC343787	[6]
Diaporthe elaeagni	CBS 504.72	Eleagnus sp.	Netherlands	KC343064	KC344032	KC343790	[6]
Diaporthe nobilis	KUN-HKAS 123203	Rhododendron sp.	China	MT741962	MW150988	MW248138	This study
Diaporthe nobilis	CBS 338.89	Hedera helix	-	KC343152	KC344120	KC343878	[6]
Diaporthe nobilis	CBS 200.39	Laurus nobilis	Germany	KC343151	KC344119	KC343877	[6]
Diaporthe nobilis	CBS 113470	-	-	KC343146	-	-	[6]
Diaporthe nobilis	CBS 116953	-	-	KC343147	-	-	[6]
Diaporthe nobilis	CBS 124030	-	-	KC343149	-	-	[6]
Diaporthe nobilis	CBS 129167	-	-	KC343150	-	-	[6]
Diaporthe nobilis	CBS 587.79	Pinus pantepella	-	KC343153	KC344121	KC343879	[6]
Diaporthe fibrosa	CBS 109751	-	-	KC343099	KC344067	KC343825	[6]
Diaporthe foeniculacea	CBS 187.27	-	-	KC343107	KC344075	KC343833	[6]
Diaporthe helianthi	CBS 592.81	Helianthus annuus	-	KC343115	KC344083	KC343841	[6]
Diaporthe nitschkei	AR 5211	Hedera helix	-	KJ210538	KJ420828	KJ210559	[89]
Diaporthe hispaniae	CBS 143351	-	-	MG281124	MG281296	MG281645	[29]
Diaporthe hongkongensis	CBS 115448	Dichroa febrífuga	-	KC343119	KC344087	KC343845	[6]
Diaporthe hungariae	CPC 30129	-	-	-	-	MG281646	[29]
Diaporthe impulse	CBS 114434	-	-	KC343122	KC344089	KC343847	[6]
Diaporthe inconspicua	CBS 133813	Maytenus ilicifolia	-	KC343123	KC344091	KC343849	[6]
Diaporthe infecunda	CBS 133812	Schinus terebinthifolius	-	KC343126	KC344094	KC343852	[6]
Diaporthe neilliae	CBS 144. 27	Spiraea sp.	-	KC343144	KC344112	KC343870	[90]
Diaporthe nothofagi	BRIP 54801	Nothofagus cunninghamii	-	JX862530	KF170922	JX862536	[91]
Diaporthe novem	CBS 127271	-	-	HM347710	-	HM347698	[6]
Diaporthe oncostoma	CBS 589.78	-	-	KC343162	KC344130	KC343888	[6]
Diaporthe perjuncta	CBS 109745	Ulmus glabra	-	KC343172	KC344140	KC343898	[6]
Diaporthe perseae	CBS 151.73	Persea gratissima	-	KC343173	KC344141	KC343899	[6]
Diaporthe pseudomangiferae	CBS 101339	Mangifera indica	-	KC343181	KC344149	KC343907	[6]
Diaporthe pseudophoenicicola	CBS 462.69	Phoenix dactylifera	-	KC343183	KC344151	KC343909	[6]
Diaporthe rudis	CBS 2665	-	-	-	KM396309	KM396311	[6]
Diaporthe saccarata	CBS 116311	Protea repens	-	KC343190	KC344158	KC343916	[6]
Diaporthe schini	CBS 133181	Schinus terebinthifolius	-	KC343191	KC344159	KC343917	[6]
Diaporthe sterilis	CBS 136969	Vaccinium corymbosum	-	KJ160579	KJ160528	KJ160611	[92]
Diaporthe subclavata	ZJUD 95	-	-	KJ490630	KJ490451	KJ490509	[93]
Diaporthe toxica	CBS 534.93	Lupinus angustifolius	-	KC343220	KC344188	KC343946	[6]
Diaporthe vaccinii	CBS 160.32	Vaccinium macrocarpon	-	AF317578	JX270436	GQ250326	[92]
Phomopsis sp.	FH 2012b	-	-	JQ954649	-	JQ954667	[93]

Figure 3.

RAxML tree based on a combined dataset of ITS, TEF1 and TUB2 gene regions. Bootstrap support values for ML and MP equal to or greater than 60% and Bayesian posterior probabilities (PP) equal to or greater than 0.90 are defined as ML/MP/PP above or below the nodes. Our new taxon is indicated in red. The tree was rooted with Diaporthella corylina (CBS 121124).

3.2. Taxonomy

3.2.1. Discosia rhododendricola Chaiwan & K.D. Hyde, sp. Nov. (Figure 4)

MycoBank number: 845145; Facesoffungi number: FoF 09452

Etymology: name reflects the host from which the fungus was isolated.

Holotype: KUN-HKAS 123205

Figure 4.

Discosia rhododendricola (KUN-HKAS 123205, holotype). (a–c) Appearance on host surface; (d) vertical section of conidioma; (e–i) conidiogenous cells and developing conidia; (j–o) conidia from holotype. Scale bars: (b) 500 μm; (c) 200 μm; (d) 100 μm; (g–i) 50 μm; (e,j–o) 20 μm; (f) 10 μm.

Saprobic on dead leaves of Rhododendron sp. Sexual morph: Undetermined. Asexual morph: Conidiomata 200–250 × 30–75 μm, pycnidial, cervular, applanate to disc-like, partly immersed or superficial, black, rounded to irregular in outline, glabrous, unilocular or divided into several locules by tissue conspicuous at the surface. Conidiophores were observed arising from the base, hyaline, filiform to cylindrical, smooth, and reduced to conidiogenous cells. Conidiogenous cells appeared subcylindrical, flask-shaped, hyaline, smooth, phialidic, each producing a single unbranched conidium. Conidia 20–30 × 4–5 μm ($\overline{\mathrm{x}}$ = 25 × 4.5 μm, n = 30), subcylindrical, slightly curved, 3-septate, with slight constrictions at the septa, brown, smooth-walled with unequal cells; bipolar appendages; with a long, tubular base, two median cells subcylindrical, second cell joined to the base, 10–15 μm ($\overline{\mathrm{x}}$ = 12.5 μm) long, the third cell joined to the apex, 11–15 μm ($\overline{\mathrm{x}}$ = 13 μm) long; apical cell subconical with a rounded apex; apical and basal cells each with a subapical, unbranched, filiform, straight appendage; apical appendage, 9–11 μm ($\overline{\mathrm{x}}$ = 10 μm), basal appendage, 20–25 μm ($\overline{\mathrm{x}}$ = 22.5 μm).

Culture characteristics: Colonies grown on PDA were filamentous, raised, filiform margin, reached 4–5 cm in 5 days at 25 °C, brown to black, mycelium superficial, branched, septate, white mycelium with aerial on the surface, and produced black spore mass.

Material examined: CHINA, Kunming Yunnan Province; on dead leaves of Rhododendron sp. (Ericaceae), 28 July 2018, Napalai Chaiwan, KIB009 (KUN-HKAS 123205, holotype; isolate MFLU20-0486; Ex-type living culture KUNCC22-10804, isolate MFLUCC21-0004.

Notes: Discosia rhododendricola is similar to D. macrozamiae CPC 32109 [94] with regards to conidiomata size (D. rhododendricola a: 200–250 μm diam., 30–75 μm high vs. D. macrozamiae CPC 32109: 250 μm diam, 50 μm height). Discosia rhododendricola and D. artocreas (type species) share similar conidiophores lining the inner cavity (0–2-septate, rarely branched at base). There are also similar in conidial characteristics (conidial dimensions between 30 and 32 μm; the second cell joining to the base was 10–15 μm in length ($\overline{\mathrm{x}}$ = 12.5 μm) in D. rhododendricola; 10–11 μm ($\overline{\mathrm{x}}$ = 10.5 μm) in D. macrozamiae CPC 32109; the third cell joining to the apex was 11–15 μm in length ($\overline{\mathrm{x}}$ = 13 μm) in D. rhododendria and 4–5 μm ($\overline{\mathrm{x}}$ = 4.5 μm) in D. macrozamiae CPC 32109. The apical appendage of D. rhododendricola was 9–11 μm in length ($\overline{\mathrm{x}}$ = 10 μm), while in D. macrozamiae (CPC 32109) it was 7–11 μm ($\overline{\mathrm{x}}$ = 9 μm). The basal appendage in D. rhododendricola was 20–25 μm ($\overline{\mathrm{x}}$ = 22.5 μm) in length, and in D. macrozamiae (CPC 32109) 10–16 μm ($\overline{\mathrm{x}}$ = 13 μm).

Discosia rhododendricola differs from the type species, D. artocreas, in ascomatal size (D. rhododendricola: 200–250 μm diam., 30–75 μm high; D. artocreas 150–500 μm diam, 60 μm high). The two species share similar conidiophores and conidiogenous cells characteristics. However, D. rhododendricola has hyaline to pale brown conidiogenous cells and conidia, whereas D. artocreas has hyaline conidiogenous cells and conidia. The second cell joining to the base measured 10–15 μm in length ($\overline{\mathrm{x}}$ = 12.5 μm) in D. rhododendricola but 5–9 μm ($\overline{\mathrm{x}}$ = 7.5 μm) in D. artocreas. The third cell joining to the apex was 11–15 μm in length ($\overline{\mathrm{x}}$ = 13 μm) in D. rhododendria and 3–6 μm ($\overline{\mathrm{x}}$ = 4.5 μm) in D. artocreas. The apical appendage of D. rhododendricola was 9–11 μm in length ($\overline{\mathrm{x}}$ = 10 μm), while in D. artocreas it was 6–12 μm ($\overline{\mathrm{x}}$ = 10 μm). The basal appendage in D. rhododendricola was 20–25 μm in length ($\overline{\mathrm{x}}$ = 22.5 μm), while in D. artocreas it was 7–12 μm ($\overline{\mathrm{x}}$ = 10 μm).

The NCBI BLAST search of ITS sequence D. rhododendricola presented 95.32% similarity with Immersdiscosia eucalypti. A comparison of the 542 ITS (+5.8S) nucleotides of D.rhododendricola sp. nov. and I. eucalypti reveals 21 (3.87%) nucleotides differencess. We compared 876 LSU nucleotides of D. rhododendricola with D. muscicola CBS 109.48, and a 0.34% bp difference was observed (a difference of 3 bp in a total 879 bp) (Table 4).

Table 4.

LSU and ITS nucleotides comparisons of Discosia species related to our new taxon.

	LSU								ITS
	Base Pair Positions								Base Pair Positions
	70	369	379	407	502	646	872	873	939	959	1003	1004	1019	1039	1303	1402	1404
D. rhododendricola (KUN-HKAS 123205)	G	A	T	T	G	A	C	T	C	T	A	C	A	T	A	C	T
D. macrozamiae CPC 32109	A	A	T	T	G	G	-	-	C	G	G	T	T	T	A	G	A
D. muscicola CBS 109.48	G	G	T	C	A	A	-	-	-	-	-	-	-	-	-	-	-
D. pleurochaeta KT2179	A	A	C	T	G	G	T	C	C	G	G	T	T	T	A	G	A
D. pleurochae KT 2188	A	A	T	T	G	G	T	C	C	G	G	T	T	T	A	G	A
D. pleurochae KT 2192	A	A	T	T	G	G	T	C	C	G	G	T	T	T	A	G	A
D. tricellularis MAFF237478	-	-	-	-	-	-	-	-	G	G	G	T	T	A	G	G	A
D. tricellularis NCBR32705	-	-	-	-	-	-	-	-	G	G	G	T	T	A	G	G	A
D. yakushimensis MAFF 242774	-	-	-	-	-	-	-	-	C	G	G	T	T	T	A	G	A

When analyzing the sequences, D. rhododendricola sp. nov. (KUN-HKAS123205 and MFLU20-0486) were found to be phylogenetically related to D. macrozamiae CPC 32109, D. muscicola CBS 109.48, D. pleurochaeta KT2179, D. pleurochae KT 2188 and KT 2192, while D. tricellularis MAFF237478 and NCBR32705 and D. yakushimensis MAFF 242774 were found to be in a clade. The two isolates of the new taxon (KUN-HKAS123205 and MFLU20-0486) have a high support value in the phylogenetic tree in a distinct clade (Figure 1). The ITS and LSU base pair differences between D. rhododendricola and other related species are shown in Table 4.

Discosia rhododendricola KUN-HKAS 123205 is closely related to the clade consisting of D. muscicola CBS 109.48, D. tricellularis MAFF 237478, NBRC 32705, and D. yakushimensis MAFF 242774 (Figure 5). The results of molecular analyses based on the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) also showed that D. rhododendricola KUN-HKAS 123205 can be distinguished as a separate species by genealogical concordance (PHI = 1.0).

Figure 5.

Results of the pairwise homoplasy index (PHI) test of closely related species using both LogDet transformation and splits decomposition. PHI test results (Φw) <0.05 indicate significant recombination within the dataset. The new taxon is in red bold type.

3.2.2. Neopestalotiopsis Rhododendricola Chaiwan & K.D. Hyde, sp. Nov. (Figure 6)

MycoBank number: 845144; Facesoffungi number: FoF 10475

Etymology: Name reflects the host from which the fungus was isolated.

Holotype: KUN-HKAS 123204

Figure 6.

Neopestalotiopsis rhododendricola (KUN-HKAS 123204). (a) The habitat of the host plant (Rhododendron sp.); (b) Pycnidia with drops of conidial exudate on the leaf surface; (c,d) colonies growing on PDA; (e,f) culture; (g–j) conidiogenous cells and developing conidia; (k–p) conidia. Scale bars: (c) 500 µm; (d) 200 µm; (h) 20 µm; (i–p) 10 µm.

Saprobic on dead leaves of Rhododendron sp. Sexual morph: Undetermined. Asexual morph: Conidiomata (on PDA) 60–80 × 50–75 μm, pycnidial, cervular, applanate to disc-like, partly immersed or superficial, globose to clavate, solitary or confluent, embedded or semi-immersed to erumpent, dark brown, exuding globose, dark brown to black conidial masses, rounded to irregular in outline, glabrous, and unilocular or divided into several locules by tissue cells. Conidiophores are indistinct, arising from the base, hyaline, filiform to cylindrical, smooth, and are often reduced to conidiogenous cells. Conidiogenous cells appeared subcylindrical, flaskshaped, hyaline, smooth, and phialidic, with each producing a single conidium. Conidia 20–30 × 5–7 μm ($\overline{\mathrm{x}}$ = 25 × 6 μm, n = 30), subcylindrical fusoid, ellipsoid, straight to slightly curved, 4-septate, (19–28) × 5–7 μm ($\overline{\mathrm{x}}$ = 23.5 × 6 μm, n = 30), μm; basal cell conic with a truncate base, hyaline, rugose and thin-walled, with constrictions at the septa, hyaline, smooth-walled; with a long, tubular base, two median cells subcylindrical, second cell joined to the base, 10–15 μm ($\overline{\mathrm{x}}$ = 12.5 μm) long, the third cell joined to the apex, 11–15 μm ($\overline{\mathrm{x}}$ = 13 μm) long; apical cell subconical with a rounded apex; apical and basal cells each with a subapical, unbranched, filiform, straight appendage; apical appendage, 9–11 μm ($\overline{\mathrm{x}}$ = 10 μm), basal appendage, 20–25 μm ($\overline{\mathrm{x}}$ = 22.5 μm).

Culture characteristics: Colonies grown on PDA, with an undulating edge, reached 4–5 cm in 5 days at 25 °C, mycelium superficial, branched, septate, white mycelium with aerial on the surface, and produced black spore mass.

Material examined: CHINA, Kunming Yunnan Province; on dead leaves of Rhododendron sp. (Ericaceae), 28 July 2018, Napalai Chaiwan, KIB008 (KUN-HKAS 123204, holotype; isolate MFLU20-0046; Ex-type living culture KUNCC22-10802; isolate MFLUCC22-0004).

Notes: Neopestalotiopsis rhododendricola (KUN-HKAS 123204 and MFLU20-0046) were isolated from a Rhododendron sp. in China. In the phylogenetic analyses, N. rhododendricola forms a distinct highly supported lineage sister to N. sonneratae (MFLUCC17-1745T, MFLUCC17-1744), N. coffeae-arabicae (HGUP4019T, HGUP4015), N. thailandica (MFLUCC17-1730T, MFLUCC17-1731) and N. macadamiae (Figure 2). Neopestalotiopsis sonneratae was reported from leaf spots on Sonneronata alba in Thailand [21], Neopestalotiopsis thailandica was reported from leaf spots on Rhizophora mucronata Lam. in Thailand [21], and N. coffeae-arabicae was found on leaves of Coffea arabica in China [75].

Neopestalotiopsis rhododendricola sp. nov. resembles N. thailandica in having similar conidial size [21], but the difference is that N. rhododendricola has two to three tubular appendages on the apical cell, while N. thailandica showed only one to two tubular appendages on the apical cell. Comparison of ITS sequence differences revealed 2 base pairs, comparison of TEF sequence differences revealed 15 base pairs, and comparison of TUB differences revealed 6 base pairs of N. rhododendricola and N. thailandica. Therefore, based on morphology and phylogeny, we justify the description of N. rhododendricola as a new species in the Neopestalotiopsis genus.

3.2.3. Diaporthe nobilis Tanaka & S. Endô, in Endô, J. Pl. Prot. Japan 13: (1927) (Figure 7)

Faces of Fungi number: FoF 02717

Saprobic on dead leaves of Rhododendron sp. Sexual morph: Undetermined. Asexual morph: Conidiomata pycnidial 50–100 × 25–75 μm. ($\overline{\mathrm{x}}$ = 75 × 50 μm, n = 10), globose to stromatic, multilocular, dark brown to black, scattered. Conidiophores were observed arising from the base, hyaline, filiform to cylindrical, smooth, straight. Conidiogenous cells, 35–40 × 1–2 μm ($\overline{\mathrm{x}}$ = 37.5 × 1.5 μm, n = 10), phialidic, cylindrical, terminal and lateral, slightly tapered towards the apex, with visible periclinal, thickening, hyaline, and smooth-walled. Beta conidia 16–20 × 1–2 μm ($\overline{\mathrm{x}}$ = 18 × 1.5 μm, n = 30), hyaline smooth, guttulate, fusoid to ellipsoid, straight, tapered towards both ends, apex sub obtuse, base sub truncate, and aseptate. Alpha conidia not found.

Culture characteristics: Colonies grew on PDA, filamentous, flattened, dense and felty, reaching 5–6 cm in 14 days at 25 °C, white to brown on the surface, mycelium superficial, branched, and septate.

Figure 7.

Diaporthe nobilis (KUN-HKAS 123203). (a) Habitat of host; (b,c) appearance of fungi on host surface; (d,e) culture characters on PDA; (g,h) conidiophore with attached conidium; (i–p) conidia. Scale bars: (b,c) 200 μm; (d–f,i–p) 10 μm.

Material examined: CHINA, Kunming Yunnan Province, on dead leaves of Rhododendron sp. (Ericaceae), 28 July 2018, Napalai Chaiwan, KIB003 (KUN-HKAS 123203, new host record; isolate MFLU20-0485; living culture KUNCC22-10803; isolate MFLUCC 18–1482.

Notes: Diaporthe nobilis KUN-HKAS 123203 clustered with D. nobilis CBS 587.79 and CBS113470 with high 0.99 PP bootstrap support. Conidiomata from the MFLUCC 18–1482 strain was acervular, semi-immersed, globose to eustromatic, and multilocular, while D. nobilis CBS 587.79 has pycnidia subcuticular, scattered to confluent, and uniloculate. Our strain was observed to share similar morphological characteristics with other Diaporthe nobilis strains in having conidiogenous cells formed at the apex of the conidiophores, cylindric, straight or curved hyaline and smooth-walled. Comparison of ITS, TEF1 and TUB2 sequence data of isolate KUN-HKAS 123203 and D. nobilis CBS113470, revealed 9 bp (1.41%) in 637 ITS (+5.8S) nucleotides, 2 bp (0.40%) in 496 TEF1 nucleotides and 6 bp (0.71%) in 844 TUB2 nucleotides. Therefore, we consider our strain (KUN-HKAS 123203) as D. nobilis and as a new host record from Rhododendron sp. in China.

4. Discussion

Discosia species are distributed on various vascular plants and a wide range of hosts, and occur primarily in their asexual state as endophytes, saprobes and pathogens [20,58]. Host-specificity of species in this genus has not yet been established. Discosia species can be found on Fagus sylvatica (Fagaceae), Gaultheria procumbens (Ericaceae), Platanus orientalis (Platanaceae), Quercus sp. (Fagaceae), Syzygium cumini (Myrtaceae), Smilax rotundifolia (Smilacaceae), and leaves of undetermined plants [60]. Discosia blumencronii Bubák was reported from Rhododendron poniicum [92], while other species can be found on leaves of Beilschmeidia tarairi (Lauraceae), Brachychiton populneus (Malvaceae), Ceanothus fiedleri (Rhamnaceae), Eucalyptus sp. (Myrtaceae), Laurus nobilis (Lauraceae) and Phillyrea latifolia (Oleaceae) [9,60]. Discosia species is distributed in temperate regions, being previously reported in Algeria, Austria, Brazil, France, Germany, India, Italy, New Zealand, Portugal, the USA, Sweden, Tunisia and Turkey [9].

The new taxon, D. rhododendricola, was phylogenetically related to D. muscicola, described by Nicot-Toulouse Morelet (1968), and isolated from Cephalozia bicuspidate (Cephaloziaceae) in France. However, no morphological data are available for comparison [94]. Discosia rhododendricola sp. nov. was isolated from Rhododendron sp. and its morphology was compared. The ascomata and conidia of D. rhododendricola were larger than those of D. artocreas, whereas the sizes of conidiophores, conidiogenous cells and apical appendage were similar.

Discosia rhododendricola is similar to D. macrozamiae (CPC 32109) [62], but the phylogenetic tree showed that our species was closely related to D. muscicola CBS 109.48. However, for D. muscicola CBS 109.48, only rDNA sequence data were available (Figure 1). It should be pointed out that when the ITS DNA sequences of Discosia muscicola were subjected to a blast search, the closest hits were Aspergillus species similar to A. avenaceus. Our novel species have DNA sequence data from three regions (LSU, ITS, and RPB2), but we can only compare the LSU region for D. muscicola CBS 109.48, as there are no sequence data of the protein coding gene available for comparison. Based on the previous study of Wijayawardene et al. [16], 34 genera are recognized in Sporocadaceae. In this study, we introduce Discosia rhododendricola as a new species based on phylogenetic analyses and the pairwise homoplasy index.

Discosia species share similar morphological characters, but most characters are not meaningful in species delineation. In this study, our new species constitutes a different branching pattern in our phylogeny and appears distinct from extant species. A relationship among species based on similar conidial characters does not necessarily correlate with our phylogenetic relationships, and this indicates that morphology has little significance for reliable species identification.

Herein, we introduce a new species, Neopestalotiopsis rhododendricola KUN-HKAS 123204, within the Neopestalotiopsis genus that was separated from the other Neopestalotiopsis clade based on morphological and molecular phylogenetic analyses (Figure 2). Neopestalotiopsis are characterized by their conidia with versicolor median cells, by indistinct conidiogenous cells [15] and the ITS, TUB2 and TEF1 sequences. The newly described species is phylogenetically related to the group of N. sonneratae, N. coffeae-arabicae and N. thailandica in the phylogenetic tree (Figure 2), and the relationship is not strongly supported. Our new species was found on a Rhododendron sp. plant host from China, while N. sonneratae was reported on leaf spots on Sonneronata alba L. [21] and Neopestalotiopsis thailandica was reported on leaf spots of Rhizophora mucronata Lam. Both strains have been reported in Thailand [21], and N. coffeae-arabicae was found on leaves of Coffea arabica in China [75].

Diaporthe species have been reported as plant pathogens, saprobes and endophytes on many plant hosts [23,28,58]. Species of Diaporthe are not host-specific [6,28,40]. Substrates colonized by members of Diaporthe recorded to date are mainly dicotyledons of Ericaceae, Fagaceae, Pinaceae, Rhizophoraceae, Rosaceae and Theaceae. Some species of Diaporthe can be found on more than one host. For example, Diaporthe nobilis was reported on Camellia sinensis (Theaceae), Castanea sativa (Fagaceae), Malus pumila (Rosaceae), Pinus pantepella (Pinaceae), Pyrus pyrifolia (Rosaceae) and Rhododendron sp. (Ericaceae) [6,28,40,58]. Diaporthe is mostly presented in the asexual morph as coelomycetes [23]. Diaporthe nobilis complex [6] has alpha and beta conidia [28]. However, our strain was only found to have beta conidia.

Diaporthe have been reported on Rhododendron spp. from Europe (Latvia) [6]. The strain (KUN-HKAS 123203) was isolated from Asia (China), indicating that the species is distributed in different geographical locations on the host; however, there is a need for more collections of microfungi associated with Rhododendron, targeting a wide variety of geographical locations. A checklist for Discosia species associated with Rhododendron is also provided herein.

5. Discosia Species Associated with Rhododendron sp.: Habitat, Known Distribution and Checklist

The above information is based on the USDA Systematic Mycology and Microbiology Laboratory (SMML) database [58], relevant literature, date from this study while current names and fungal classifications used are according to Index Fungorum (2022) [14], and an outline of Ascomycota [16]. Species confirmed with DNA sequence data are marked with an asterisk.

1. Discosia artocreas (Tode) Fr., Summa veg. Scand., Sectio Post. (Stockholm): 423 (1849)

   = Sphaeria artocreas Tode, F. Meckl. 2: 77, 1791; Fries, Syst. Myc. 2: 523, 1823.

   Habitat: Rhododendron arboretum, R. campylocarpum, R. nudiflorum [95,96], R. catawbiense, R. maximum [97], R. ponticum [98,99] and Rhododendron sp. [95,96,100]

   Known distribution: Italy [98], Maryland [95,96,97], New York [97], United Kingdom [100], Turkey* [99], Washington [95,96].

2. Discosia blumencronii Bubák, in Handel-Mazzetti, Annln K. K. naturh. Hofmus. Wien 23: 106 (1910)

   Habitat: Rhododendron ponticum (on dead leaves) [101]

   Known distribution: Turkey [101]

3. Discosia himalayensis Died., Annls mycol. 14(3/4): 218 (1916)

   = Discosia strobilina Lib. ex Sacc., Syll. Fung. (Abellini) 3: 656 (1884)

   Habitat: Rhododendron arboretum, R. campanulatum (on dead leaves) [101,102,103]

   Known distribution: India [101,102,103]

4. Discosia rhododendri (Speschnew, Monit. Jard. Bot. Tiflis 4: 10 (1906)

   Habitat: Rhododendron albrechtii (on dead leaves)* [104], R. ponticum* [99], Rhododendron sp. (on leaves) [101]

   Known distribution: Japan* [104], Turkey* [99]

5. Discosia rhododendricola (This study *)

   Habitat: Rhododendron sp. (on dead leaves) (This study *)

   Known distribution: China (This study *)

6. Discosia sp.

   Habitat: Rhododendron sp.* [104]

   Known distribution: Japan* [104]

7. Discosia tricellularis (Okane, Nakagiri & Tad. Ito) F. Liu, L. Cai & Crous, in Liu, Bonthond, Groenewald, Cai & Crous, Stud. Mycol. 92: 322 (2018) (2019)

   Habitat: Rhododendron indicum [105]

   Known distribution: Japan [105]

8. Discosia vagans De Not., Atti Acad. Tor.: 354 (1849)

   Habitat: Rhododendron arboretum, R. nilagiricum, R. veitchianum* [59,103], R. ponticum* [61]

   Known distribution: India* [99,105], Scotland* [59]

Author Contributions

N.C., D.P., R.J., R.S.J., N.N., A.M., I.P. and K.D.H. designed the study and were involved in the writing of the paper. N.C. performed the sample collections. N.C. and R.S.J. were involved in phylogenetic analyses. N.N. contributed to paper planning and editing. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All sequences generated in this study were submitted to GenBank.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

We are grateful to the Thailand Research Fund (TRF) grant no PHD60K0147 and Kunming Institute of Botany for partially supporting this work. Shaun Pennycook is thanked for nomenclatural advice. K.D. Hyde would like to thank the Thailand Research Fund projects entitled ‘The future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species (No. DBG6080013)’ and ‘Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (No. RDG6130001)’. K.D. Hyde thanks Chiang Mai University for the award of Visiting Professor. Adam Kaplan is thanked for the English editing of the manuscript.