Two new species of Diaporthe (Diaporthaceae, Diaporthales) associated with tree cankers in the Netherlands.

Diaporthe (Diaporthaceae, Diaporthales) is a common fungal genus inhabiting plant tissues as endophytes, pathogens and saprobes. Some species are reported from tree branches associated with canker diseases. In the present study, Diaporthe samples were collected from Alnusglutinosa, Fraxinusexcelsior and Quercusrobur in Utrecht, the Netherlands. They were identified to species based on a polyphasic approach including morphology, pure culture characters, and phylogenetic analyses of a combined matrix of partial ITS, cal, his3, tef1 and tub2 gene regions. As a result, four species (viz.  Diaporthepseudoalnea sp. nov. from Alnusglutinosa, Diaporthesilvicola sp. nov. from Fraxinusexcelsior, D.foeniculacea and D.rudis from Quercusrobur) were revealed from tree branches in the Netherlands. Diaporthepseudoalnea differs from D.eres (syn. D.alnea) by its longer conidiophores. Diaporthesilvicola is distinguished from D.fraxinicola and D.fraxini-angustifoliae by larger alpha conidia. Ning Jiang, Hermann Voglmayr, Chun-Gen Piao, Yong Li.

Introduction

(syn. ) is the type genus of in , commonly occurring as plant endophytes, pathogens and saprobes (Udayanga et al. 2014, 2015; Guarnaccia et al. 2017, 2018a, 2018b; Tibpromma et al. 2018; Yang et al. 2020; Dissanayake et al. 2020; Jiang et al. 2021). The sexual morph is characterized by immersed perithecial ascomata and an erumpent pseudostroma with more or less elongated perithecial necks, unitunicate clavate to cylindrical asci, and fusoid, ellipsoid to cylindrical, hyaline uni- to bicellular ascospores (Udayanga et al. 2011; Senanayake et al. 2017). The asexual morph is characterized by ostiolate conidiomata, with cylindrical phialides producing up to three types of hyaline, aseptate conidia (Udayanga et al. 2011; Gomes et al. 2013; Yang et al. 2018), and was previously classified as . Following the “one fungus one name” nomenclature, Rossman et al. (2015) recommended to use based on priority, necessitating the transfer of numerous species to .

Species of are known to cause plant diseases including dieback, canker, leaf spot, fruit rot, pod blights and seed decay. For example, , and caused melanose and stem end rot diseases of spp. (Udayanga et al. 2014), while caused leaf spot disease of in China (Jiang et al. 2021). Up to 19 species were confirmed to be associated with pear cankers in China (Guo et al. 2020), and eight species of were found to be the casual agents of Chinese grapevine dieback (Manawasinghe et al. 2019). Seven species were reported from blueberry twig blight and dieback diseases in Portugal (Hilário et al. 2020). and an additional six species were identified as endophytes from healthy tissues in China (Huang et al. 2015). and an additional 11 species were isolated as saprobes from dead wood in karst formations in China (Dissanayake et al. 2020).

species were previously classified mainly based on host association and morphology (Rehner and Uecker 1994; Santos and Phillips 2009; Udayanga et al. 2011, 2014). However, several taxonomic studies of proved that phylogeny based on multiple genes is suitable to separate species (Voglmayr et al. 2012, 2017; Fan et al. 2018; Jiang et al. 2019, 2020; Jaklitsch and Voglmayr 2019, 2020). Species of are now characterised and circumscribed both by morphology and phylogeny of multi-locus DNA data, which revealed many cryptic species in recent years (Diogo et al. 2010; Lombard et al. 2014; Gao et al. 2016, 2017; Long et al. 2019; Yang et al. 2020, 2021; Zapata et al. 2020; Huang et al. 2021). To clarify the species boundaries of the complex, the Genealogical Phylogenetic Species Recognition principle (GCPSR) and the coalescent-based model Poisson Tree Processes (PTPs) were employed, which suggested that the species complex actually represents only a single species, (Hilário et al. 2021).

In the present study, samples from cankered branches of several tree species were collected in the Netherlands, and identified based on modern taxonomic approaches. As a result, two new species and two known species were identified, and the new species are described and illustrated herein.

Materials and Methods

Collection, examination and isolation

The fresh specimens of cankered branches were sampled from , and in Utrecht, the Netherlands. Morphological characteristics of the conidiomata were determined under a Nikon AZ100 dissecting stereomicroscope. More than 20 conidiomata were sectioned, and 50 conidia were randomly selected for measurement using a Leica compound microscope (LM, DM 2500). Isolates were obtained by removing a mucoid conidial mass from conidiomata, spreading the suspension onto the surface of 1.8 % potato dextrose agar (PDA), and incubated at 25 °C for up to 24 h. Single germinating conidia were removed and plated onto fresh PDA plates. Cultural characteristics of isolates incubated on PDA in the dark at 25 °C were recorded, including the colony color and conidiomata structures. The cultures were deposited in the China Forestry Culture Collection Center (CFCC; http://www.cfcc-caf.org.cn/), and the specimens in the herbarium of the Chinese Academy of Forestry (CAF; http://museum.caf.ac.cn/).

DNA extraction, PCR amplification and phylogenetic analyses

Genomic DNA was extracted from colonies grown on cellophane-covered PDA using a cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). DNA was checked by electrophoresis in 1 % agarose gel, and the quality and quantity were measured using a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). Five partial loci, including the 5.8S nuclear ribosomal DNA gene with the two flanking internally transcribed spacer (ITS) regions, the calmodulin (), the histone H3 (), the translation elongation factor 1-alpha () and the beta-tubulin () genes were amplified by the primer pairs and polymerase chain reaction (PCR) process listed in Table 1. The PCR products were assayed via electrophoresis in 2 % agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminator Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Table 1.

Genes used in this study with PCR primers and process.

Locus	PCR primers	PCR: thermal cycles: (Annealing temp. in bold)	Reference
ITS	ITS1/ITS4	(95 °C: 30 s, 48 °C: 30 s, 72 °C: 1 min) × 35 cycles	White et al. 1990
cal	CAL228F/CAL737R	(95 °C: 15 s, 54 °C: 20 s, 72 °C: 1 min) × 35 cycles	Carbone and Kohn 1999
his3	CYLH3F/H3-1b	(95 °C: 30 s, 57 °C: 30 s, 72 °C: 1 min) × 35 cycles	Crous et al. 2004 Glass and Donaldson 1995
tef1	EF1-728F/EF1-986R	(95 °C: 15 s, 54 °C: 20 s, 72 °C: 1 min) × 35 cycles	Carbone and Kohn 1999
tub2	T1(Bt2a)/Bt2b	(95 °C: 30 s, 55 °C: 30 s, 72 °C: 1 min) × 35 cycles	Glass & Donaldson 1995;O’Donnell and Cigelnik 1997

The quality of the amplified nucleotide sequences was checked and the sequences assembled using SeqMan v.7.1.0. Reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), based on recent publications on the genus (Dissanayake et al. 2021; Gao et al. 2021; Huang et al. 2021; Sun et al. 2021, Wang et al. 2021; Yang et al. 2021). Sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and corrected manually using MEGA 7.0.21. The best-fit nucleotide substitution models for each gene were selected using jModelTest v. 2.1.7 (Darriba et al. 2012) under the Akaike Information Criterion.

The phylogenetic analyses of the combined gene regions were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML was implemented on the CIPRES Science Gateway portal (https://www.phylo.org) using RAxML-HPC BlackBox 8.2.10 (Stamatakis 2014), employing a GTRGAMMA substitution model with 1000 bootstrap replicates. While BI was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0 (Ronquist et al. 2003). Two MCMC chains, started from random trees for 1000000 generations and trees, were sampled every 100th generation, resulting in a total of 10000 trees. The first 25 % of trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v.1.3.1 and processed by Adobe Illustrator CS5. The nucleotide sequence data of the new taxa were deposited in GenBank and are listed in Table 2.

Table 2.

Isolates and GenBank accession numbers used in the phylogenetic analyses of .

Species	Strain	Host	Origin	GenBank accession numbers
				ITS	cal	his3	tef1	tub2
Diaportheacaciigena	CBS 129521	Acaciaretinodes	Australia	KC343005	KC343247	KC343489	KC343731	KC343973
D.acericola	MFLUCC 17-0956	Acernegundo	Italy	KY964224	KY964137	NA	KY964180	KY964074
D.acerigena	CFCC 52554	Acertataricum	China	MH121489	MH121413	MH121449	MH121531	NA
D.acerigena	CFCC 52555	Acertataricum	China	MH121490	MH121414	MH121450	MH121532	NA
D.acuta	PSCG 047	Pyruspyrifolia	China	MK626957	MK691125	MK726161	MK654802	MK691225
D.acutispora	LC6161	Coffea	China	KX986764	KX999274	KX999235	KX999155	KX999195
D.alangii	CFCC 52556	Alangiumkurzii	China	MH121491	MH121415	MH121451	MH121533	MH121573
D.alangii	CFCC 52557	Alangiumkurzii	China	MH121492	MH121416	MH121452	MH121534	MH121574
D.albosinensis	CFCC 53066	Betulaalbosinensis	China	MK432659	MK442979	MK443004	MK578133	MK578059
D.albosinensis	CFCC 53067	Betulaalbosinensis	China	MK432660	MK442980	MK443005	MK578134	MK578060
D.alleghaniensis	CBS 495.72	Betulaalleghaniensis	Canada	MH121502	MH121426	MH121462	MH121544	MH121584
D.ambigua	CBS 114015	Pyruscommunis	South Africa	KC343010	KC343252	KC343494	KC343736	KC343978
D.ampelina	STE-U 2660	Vitisvinifera	France	NA	AY745026	NA	AY745056	NA
D.amygdali	CBS 126679	Prunusdulcis	Portugal	MH864208	KC343264	KC343506	KC343748	KC343990
D.anacardii	CBS 720.97	Anacardiumoccidentale	East Africa	KC343024	KC343266	KC343508	KC343750	KC343992
D.angelicae	CBS 111592	Heracleumsphondylium	Austria	KC343027	KC343269	KC343511	KC343753	KC343995
D.apiculatum	CFCC 53068	Rhuschinensis	China	MK432651	MK442973	MK442998	MK578127	MK578054
D.apiculatum	CFCC 53069	Rhuschinensis	China	MK432652	MK44297	MK442999	MK578128	MK578055
D.aquatica	IFRDCC 3051	Aquatic habitat	China	JQ797437	NA	NA	NA	NA
D.arctii	DP0482	Arctiumlappa	Austria	KJ590736	KJ612133	KJ659218	KJ590776	KJ610891
D.arecae	CBS 161.64	Arecacatechu	India	KC343032	KC343274	KC343516	KC343758	KC344000
D.arengae	CBS 114979	Arengaengleri	Hong Kong	MF773664	KC343276	KC343518	KC343760	KC344002
D.aseana	MFLUCC 12-0299a	Unknown	Thailand	KT459414	KT459464	NA	KT459448	KT459432
D.asheicola	CBS 136967	Vacciniumashei	Chile	KJ160562	KJ160542	NA	KJ160594	KJ160518
D.aspalathi	CBS 117169	Aspalathuslinearis	South Africa	KC343036	KC343278	KC343520	KC343762	KC344004
D.australafricana	CBS 111886	Vitisvinifera	Australia	KC343038	KC343280	KC343522	KC343764	KC344006
D.australiana	CBS 146457	Macadamia	Australia	MN708222	NA	NA	MN696522	MN696530
D.baccae	CBS 136972	Vacciniumcorymbosum	Italy	MK370623	MG281695	MF418264	KJ160597	MF418509
D.batatas	CBS 122.21	Ipomoeabatatas	USA	KC343040	KC343282	KC343524	KC343766	KC344008
D.bauhiniae	CFCC 53071	Bauhiniapurpurea	China	MK432648	MK442970	MK442995	MK578124	MK578051
D.bauhiniae	CFCC 53072	Bauhiniapurpurea	China	MK432649	MK442971	MK442996	MK578125	MK578052
D.bauhiniae	CFCC 53073	Bauhiniapurpurea	China	MK432650	MK442972	MK442997	MK578126	MK578053
D.beilharziae	BRIP 54792	Indigoferaaustralis	Australia	JX862529	NA	NA	JX862535	KF170921
D.benedicti	SBen914	Diaporthebenedicti	USA	KM669929	KM669862	NA	KM669785	NA
D.betulae	CFCC 50469	Betulaplatyphylla	China	KT732950	KT732997	KT732999	KT733016	KT733020
D.betulae	CFCC 50470	Betulaplatyphylla	China	KT732951	KT732998	KT733000	KT733017	KT733021
D.betulicola	CFCC 51128	Betulaalbo-sinensis	China	KX024653	KX024659	KX024661	KX024655	KX024657
D.betulicola	CFCC 51129	Betulaalbo-sinensis	China	KX0246554	KX024660	KX024662	KX0246556	KX024658
D.betulina	CFCC 52560	Betulaalbo-sinensis	China	MH121495	MH121419	MH121455	MH121537	MH121577
D.betulina	CFCC 52561	Betulaalbo-sinensis	China	MH121496	MH121420	MH121456	MH121538	MH121578
D.biconispora	ZJUD62	Citrusmaxima	China	KJ490597	NA	KJ490539	KJ490476	KJ490418
D.biguttulata	ZJUD47	Citruslimon	China	KJ490582	NA	KJ490524	KJ490461	KJ490403
D.bohemiae	CBS 143347	Vitisvinifera	Czech Republic	MK300012	MG281710	MG281361	MG281536	MG281188
D.brasiliensis	CBS 133183	Aspidospermatomentosum	Brazil	KC343042	KC343284	KC343526	KC343768	KC344010
D.caatingaensis	URM7485	Tacingainamoena	Brazil	KY085927	KY115598	NA	KY115604	KY115601
D.camelliae-sinensis	SAUCC194.92	Camelliasinensis	China	MT822620	MT855699	MT855588	MT855932	MT855817
D.canthii	CPC 19740	Canthiuminerme	South Africa	JX069864	NA	NA	NA	NA
D.caryae	CFCC 52563	Caryaillinoinensis	China	MH121498	MH121422	MH121458	MH121540	MH121580
D.caryae	CFCC 52564	Caryaillinoinensis	China	MH121499	MH121423	MH121459	MH121541	MH121581
D.cassines	CPC 21916	Cassineperagua	South Africa	KF777155	NA	NA	KF777244	NA
D.caulivora	CBS 127268	Glycinemax	Croatia	MH864501	KC343287	KC343529	KC343771	KC344013
D.cercidis	CFCC 52565	Cercischinensis	China	MH121500	MH121424	MH121460	NA	MH121582
D.cercidis	CFCC 52566	Cercischinensis	China	MH121501	MH121425	MH121461	NA	MH121583
D.chamaeropis	CBS 454.81	Chamaeropshumilis	Greece	KC343048	KC343290	KC343532	KC343774	KC344016
D.charlesworthii	BRIP 54884m	Rapistrumrugostrum	Australia	KJ197288	NA	NA	KJ197250	KJ197268
D.chensiensis	CFCC 52567	Abieschensiensis	China	MH121502	MH121426	MH121462	MH121544	MH121584
D.chensiensis	CFCC 52568	Abieschensiensis	China	MH121503	MH121427	MH121463	MH121545	MH121585
D.chongqingensis	PSCG 435	Pyruspyrifolia	China	MK626916	MK691209	MK726257	MK654866	MK691321
D.chrysalidocarpi	SAUCC194.35	Chrysalidocarpuslutescens	China	MT822563	MT855646	MT855532	MT855760	MT855876
D.cichorii	MFLUCC 17-1023	Cichoriumintybus	Italy	KY964220	KY964133	NA	KY964176	KY964104
D.cinnamomi	CFCC 52569	Cinnamomum	China	MH121504	NA	MH121464	MH121546	MH121586
D.cinnamomi	CFCC 52570	Cinnamomum	China	MH121505	NA	MH121465	MH121547	MH121587
D.cissampeli	CPC 27302	Cissampeloscapensis	South Africa	KX228273	NA	KX228366	NA	KX228384
D.citri	AR3405	Citrus	USA	KC843311	KC843157	KJ420881	KC843071	KC843187
D.citri	CFCC 53079	Citrussinensis	China	MK573940	MK574579	MK574595	MK574615	MK574635
D.citriasiana	CGMCC 3.15224	Citrusunshiu	China	JQ954645	KC357491	KC490515	JQ954663	KC357459
D.citrichinensis	CGMCC 3.15225	Citrus	China	JQ954648	KC357494	NA	JQ954666	NA
D.collariana	MFLU 17-2770	Magnoliachampaca	Thailand	MG806115	MG783042	NA	MG783040	MG783041
D.compactum	LC3083	Camelliasinensis	China	KP267854	NA	KP293508	KP267928	NA
D.conica	CFCC 52571	Alangiumchinense	China	MH121506	MH121428	MH121466	MH121548	MH121588
D.conica	CFCC 52572	Alangiumchinense	China	MH121507	MH121429	MH121467	MH121549	MH121589
D.constrictospora	CGMCC 3.20096	Unknown	China	MT385947	MT424718	MW022487	MT424682	MT424702
D.convolvuli	CBS 124654	Convolvulusarvensis	Turkey	KC343054	KC343296	KC343538	KC343780	KC344022
D.coryli	CFCC 53083	Corylusmandshurica	China	MK432661	MK442981	MK443006	MK578135	MK578061
D.coryli	CFCC 53084	Corylusmandshurica	China	MK432662	MK442982	MK443007	MK538176	MK578062
D.corylicola	CFCC 53986	Corylusheterophylla	China	MW839880	MW836684	MW836717	MW815894	MW883977
D.corylicola	CFCC 53987	Corylusheterophylla	China	MW839867	MW836685	MW836718	MW815895	MW883978
D.crotalariae	CBS 162.33	Crotalariaspectabilis	USA	MH855395	JX197439	KC343540	GQ250307	KC344024
D.crousii	CAA 823	Vacciniumcorymbosum	Portugal	MK792311	MK883835	MK871450	MK828081	MK837932
D.cucurbitae	DAOM 42078	Cucumis	Canada	KM453210	NA	KM453212	KM453211	KP118848
D.cuppatea	CBS 117499	Aspalathuslinearis	South Africa	MH863021	KC343299	KC343541	KC343783	KC344025
D.cynaroidis	CBS 122676	Proteacynaroides	South Africa	KC343058	KC343300	KC343542	KC343784	KC344026
D.cytosporella	FAU461	Citruslimon	Italy	KC843307	KC843141	NA	KC843116	KC843221
D.diospyricola	CPC 21169	Diospyroswhyteana	South Africa	KF777209	NA	NA	NA	NA
D.discoidispora	ZJUD89	Citrusunshiu	China	KJ490624	NA	KJ490566	KJ490503	KJ490445
D.dorycnii	MFLUCC 17-1015	Dorycniumhirsutum	Italy	KY964215	NA	NA	KY964171	KY964099
D.drenthii	CBS 146453	Macadamia	Australia	MN708229	NA	NA	MN696526	MN696537
D.elaeagni-glabrae	LC4802	Elaeagnusglabra	China	KX986779	KX999281	KX999251	KX999171	KX999212
D.ellipicola	CGMCC 3.17084	Lithocarpusglaber	China	KF576270	NA	NA	KF576245	KF576294
D.endophytica	CBS 133811	Schinusterebinthifolius	Brazil	KC343065	KC343307	KC343549	KC343791	KC344033
D.eres	CBS 146.46	Alnus	Netherlands	KC343008	KC343250	KC343492	KC343734	KC343976
D.eres	CBS 121004	Juglans	USA	KC343134	KC343376	KC343618	KC343860	KC344102
D.eres	CGMCC 3.17081	Lithocarpusglabra	China	KF576282	NA	NA	KF576257	KF576306
D.eres	CFCC 51632	Camptothecaacuminata	China	KY203726	KY228877	KY228881	KY228887	KY228893
D.eres	CBS 139.27	Celastrus	USA	KC343047	KC343289	KC343531	KC343773	KC344015
D.eres	CBS 143349	Vitisvinifera	United Kingdom	MG281017	MG281712	MG281363	MG281538	MG281190
D.eres	AR5193	Ulmus	Germany	KJ210529	KJ434999	KJ420850	KJ210550	KJ420799
D.eres	CFCC 52575	Castaneamollissima	China	MH121510	NA	MH121470	MH121552	MH121592
D.eres	CFCC 52576	Castaneamollissima	China	MH121511	MH121432	MH121471	MH121553	MH121593
D.eres	CFCC 52577	Acanthopanaxsenticosus	China	MH121512	MH121433	MH121472	MH121554	MH121594
D.eres	CFCC 52578	Sorbus	China	MH121513	MH121433	MH121473	MH121555	MH121595
D.eres	CFCC 52579	Juglansregia	China	MH121514	NA	MH121474	MH121556	NA
D.eres	CFCC 52580	Meliaazedarace	China	MH121515	NA	MH121475	MH121557	MH121596
D.eres	CFCC 52581	Rhododendronsimsii	China	MH121516	NA	MH121476	MH121558	MH121597
D.eres	MAFF 625034	Pyruspyrifolia	Japan	NA	KJ435023	KJ420868	NA	KJ420819
D.eres	AR5211	Hederahelix	France	KJ210538	KJ435043	KJ420875	KJ210559	KJ420828
D.eres	CGMCC 3.17089	Lithocarpusglabra	China	KF576267	NA	NA	KF576242	KF576291
D.eres	MFLUCC 17-0963	Lonicera	Italy	KY964190	KY964116	NA	KY964146	KY964073
D.eres	DAOM 695742	Picearuben	Canada	KU552025	NA	NA	KU552023	KU574615
D.eres	MFLUCC 16-0113	Prunuspersica	China	KU557563	NA	KU557611	KU557631	KU55758
D.eres	CBS 144.27	Spiraea	USA	KC343144	KC343386	KC343628	KC343870	KC344112
D.eres	CBS 587.79	Pinusparvifloravar	Japan	KC343153	KC343395	KC343637	KC343879	KC344121
D.eres	CBS 338.89	Hederahelix	Yugoslavia	KC343152	KC343394	KC343636	KC343878	KC344120
D.eres	MFLU 17-0646	Rosa	United Kingdom	MG828895	MG829274	NA	MG829270	MG843877
D.eucalyptorum	CBS 132525	Eucalyptus	China	MH305525	NA	NA	NA	NA
D.foeniculacea	CBS 111553	Foeniculumvulgare	Spain	MH854926	KC343343	KC343585	KC343827	KC344069
D.foeniculacea	CFCC 54192	Quercusrobur	Netherlands	MZ727033	NA	MZ753474	MZ816339	MZ753483
D.foeniculacea	M35	Quercusrobur	Netherlands	MZ727034	NA	MZ753475	MZ816340	MZ753484
D.foeniculacea	M40-1	Quercusrobur	Netherlands	MZ727035	NA	MZ753476	MZ816341	MZ753485
D.foeniculacea	M84	Quercusrobur	Netherlands	MZ727036	NA	MZ753477	MZ816342	MZ753486
D.fraxini-angustifoliae	BRIP 54781	Fraxinusangustifolia	Australia	JX862528	KT459462	NA	JX862534	NA
D.fraxinicola	CFCC 52582	Fraxinuschinensis	China	MH121517	MH121435	NA	MH121560	NA
D.fraxinicola	CFCC 52583	Fraxinuschinensis	China	MH121518	MH121436	NA	MH121559	NA
D.fulvicolor	PSCG 051	Pyruspyrifolia	China	MK626859	MK691132	MK726163	MK654806	MK691236
D.fusicola	CGMCC 3.17087	Lithocarpusglabra	China	KF576281	KF576233	NA	KF576256	KF576305
D.ganjae	CBS 180.91	Cannabissativa	USA	KC343112	KC343354	KC343596	KC343838	KC344080
D.ganzhouensis	CFCC 53087	Unknown	China	MK432665	MK442985	MK443010	MK578139	MK578065
D.ganzhouensis	CFCC 53088	Unknown	China	MK432666	MK442986	MK443011	MK578140	MK578066
D.garethjonesii	MFLUCC 12-0542a	Unknown	Thailand	KT459423	KT459470	NA	KT459457	KT459441
D.goulteri	BRIP 55657a	Helianthusannuus	Australia	KJ197290	NA	NA	KJ197252	KJ197270
D.grandiflori	SAUCC194.84	Heterostemmagrandiflorum	China	MT822612	MT855691	MT855580	MT855809	MT855924
D.guangxiensis	JZB320087	Vitisvinifera	China	MK335765	MK736720	NA	MK500161	MK523560
D.gulyae	BRIP 54025	Helianthusannuus	Australia	NA	NA	NA	JN645803	KJ197271
D.guttulata	CGMCC 3.20100	Unknown	China	MT385950	MW022470	MW022491	MT424685	MT424705
D.helianthi	CBS 592.81	Helianthusannuus	Serbia	KC343115	KC343357	KC343599	KC343841	KC344083
D.heliconiae	SAUCC194.77	Heliconiametallica	China	MT822605	MT855684	MT855573	MT855802	MT855917
D.heterophyllae	CPC 26215	Acaciaheterophylla	France	MG600222	MG600218	MG600220	MG600224	MG600226
D.heterostemmatis	SAUCC194.85	Heterostemmagrandiflorum	China	MT822613	MT855692	MT855581	MT855810	MT855925
D.hickoriae	CBS 145.26	Caryaglabra	USA	KC343118	KC343360	NA	KC343844	KC344086
D.hispaniae	CBS 143351	Vitisvinifera	Spain	MG281123	MG281820	MG281471	MG281644	MG281296
D.hongkongensis	CBS 115448	Dichroafebrifuga	China	MK304388	KC343361	KC343603	KC343845	KC344087
D.hubeiensis	JZB320123	Vitisvinifera	China	MK335809	MK500235	NA	MK523570	MK500148
D.incompleta	LC6754	Camelliasinensis	China	KX986794	KX999289	KX999265	KX999186	KX999226
D.inconspicua	CBS 133813	Maytenusilicifolia	Brazil	NA	KC343365	KC343607	KC343849	KC344091
D.infecunda	CBS 133812	Schinusterebinthifolius	Brazil	KC343126	KC343368	KC343610	KC343852	KC344094
D.irregularis	CGMCC 3.20092	Unknown	China	MT385951	MT424721	NA	MT424686	MT424706
D.isoberliniae	CPC 22549	Isoberliniaangolensis	Zambia	KJ869190	NA	NA	NA	KJ869245
D.juglandicola	CFCC 51134	Juglansmandshurica	China	KU985101	KX024616	KX024622	KX024628	KX024634
D.kadsurae	CFCC 52586	Kadsuralongipedunculata	China	MH121521	MH121439	MH121479	MH121563	MH121600
D.kadsurae	CFCC 52587	Kadsuralongipedunculata	China	MH121522	MH121440	MH121480	MH121564	MH121601
D.kochmanii	BRIP 54033	Helianthusannuus	Australia	NA	NA	NA	JN645809	NA
D.kongii	BRIP 54031	Helianthusannuus	Australia	NA	NA	NA	NA	KJ197272
D.lenispora	CGMCC 3.20101	Unknown	China	MT385952	MW022472	MW022493	MT424687	MT424707
D.litchicola	BRIP 54900	Litchichinensis	Australia	LC041036	NA	NA	JX862539	NA
D.litchii	SAUCC194.22	Litchichinensis	China	MT822550	MT855635	MT855519	MT855747	MT855863
D.lithocarpus	CGMCC 3.15175	Lithocarpusglabra	China	KC135104	KF576235	NA	KC153095	KF576311
D.longicolla	FAU599	Glycinemax	USA	KJ590728	KJ612124	KJ659188	KJ590767	KJ610883
D.longispora	CBS 194.36	Ribes	Canada	MH855769	KC343377	KC343619	KC343861	KC344103
D.lusitanicae	CBS 123212	Foeniculumvulgare	Portugal	MH863279	KC343378	KC343620	KC343862	KC344104
D.lutescens	SAUCC194.36	Chrysalidocarpuslutescens	China	MT822564	MT855647	MT855533	MT855761	MT855877
D.macadamiae	CBS 146455	Macadamia	Australia	MN708230	NA	NA	MN696528	MN696539
D.macintoshii	BRIP 55064a	Rapistrumrugosum	Australia	KJ197289	NA	NA	KJ197251	KJ197269
D.mahothocarpus	CGMCC 3.15181	Lithocarpusglabra	China	KC153096	NA	NA	KC153087	KF576312
D.malorum	CAA 734	Malusdomestica	Portugal	KY435638	KY435658	KY435648	KY435627	KY435668
D.masirevicii	BRIP 54256	Glycinemax	Australia	KJ197277	NA	NA	KJ197238	KJ197256
D.mayteni	CBS 133185	Maytenusilicifolia	Brazil	KC343139	KC343381	KC343623	KC343865	KC344107
D.maytenicola	CPC 21896	Maytenusacuminata	South Africa	KF777157	NA	NA	NA	KF777250
D.mediterranea	SAUCC194.111	Machiluspingii	China	MT822639	MT855718	MT855606	MT855836	MT855951
D.melastomatis	SAUCC194.55	Melastomamalabathricum	China	MT822583	MT855664	MT855551	MT855780	MT855896
D.melonis	CBS 435.87	Glycinesoja	Indonesia	KC343141	KC343383	KC343625	KC343867	KC344109
D.middletonii	BRIP 54884e	Rapistrumrugosum	Australia	KJ197286	NA	NA	KJ197248	KJ197266
D.minima	CGMCC 3.20097	Unknown	China	MT385953	MT424722	MW022496	MT424688	MT424708
D.minusculata	CGMCC 3.20098	Unknown	China	MT385957	MW022475	MW022499	MT424692	MT424712
D.miriciae	BRIP 54736j	Helianthusannuus	Australia	KJ197282	NA	NA	KJ197244	KJ197262
D.multigutullata	CFCC 53095	Citrusmaxima	China	MK432645	MK442967	MK442992	MK578121	MK578048
D.multigutullata	CFCC 53096	Citrusmaxima	China	MK432646	MK442968	MK442993	MK578122	MK578049
D.musigena	CBS 129519	Musa	Australia	KC343143	KC343385	KC343267	KC343869	KC344111
D.neoarctii	CBS 109490	Ambrosiatrifida	USA	KC343145	KC343387	KC343629	KC343871	KC344113
D.neoraonikayaporum	MFLUCC 14-1136	Tectonagrandis	Thailand	KU712449	KU749356	NA	KU749369	KU743988
D.nothofagi	BRIP 54801	Nothofaguscunninghamii	Australia	JX862530	NA	NA	JX862536	KF170922
D.novem	CBS 127269	Glycinemax	Croatia	KC343155	KC343397	KC343639	KC343881	KC344123
D.ocoteae	CPC 26217	Ocoteabullata	France	KX228293	NA	NA	NA	KX228388
D.oraccinii	LC3166	Camelliasinensis	China	KP267863	NA	KP293517	KP267937	KP293443
D.ovalispora	ZJUD93	Citruslimon	China	KJ490628	NA	KJ490570	KJ490507	KJ490449
D.ovoicicola	CGMCC 3.17093	Lithocarpusglabra	China	KF576265	KF576223	NA	KF576240	KF576289
D.oxe	CBS 133186	Maytenusilicifolia	Brazil	KC343164	KC343406	KC343648	KC343890	KC344132
D.padina	CFCC 52590	Padusracemosa	China	MH121525	MH121443	MH121483	MH121567	MH121604
D.padina	CFCC 52591	Padusracemosa	China	MH121526	MH121444	MH121484	MH121568	MH121605
D.pandanicola	MFLUCC 17-0607	Pandanaceae	Thailand	MG646974	NA	NA	NA	MG646930
D.paranensis	CBS 133184	Maytenusilicifolia	Brazil	KC343171	KC343413	KC343655	KC343897	KC344139
D.parapterocarpi	CPC 22729	Pterocarpusbrenanii	Zambia	KJ869138	NA	NA	NA	KJ869248
D.parvae	PSCG 035	Pyrusbretschneideri	China	MK626920	MK691169	MK726211	MK654859	MK691249
D.pascoei	BRIP 54847	Perseaamericana	Australia	MK111097	NA	NA	JX862538	KF170924
D.passiflorae	CPC 19183	Passifloraedulis	Netherlands	JX069860	NA	NA	NA	NA
D.passifloricola	CPC 27480	Passiflorafoetida	Malaysia	KX228292	NA	KX228367	NA	KX228387
D.penetriteum	LC3215	Camelliasinensis	China	KP267879	NA	NA	KP293532	KP267953
D.perjuncta	CBS 109745	Ulmusglabra	Austria	KC343172	KC343414	KC343656	KC343898	KC344140
D.perseae	CBS 151.73	Perseagratissima	Netherlands	KC343173	KC343415	NA	NA	NA
D.pescicola	MFLUCC 16-0105	Prunuspersica	China	KU557555	KU557603	NA	KY400831	KU557579
D.phaseolorum	AR4203	Phaseolusvulgaris	USA	KJ590738	KJ612135	KJ659220	KJ590739	KJ610893
D.phillipsii	CAA 817	Vacciniumcorymbosum	Portugal	MK792305	MK883831	MK871445	MK828076	MN000351
D.podocarpi-macrophylli	LC6155	Podocarpusmacrophyllus	Japan	KX986774	KX999278	KX999246	KX999167	KX999207
D.pometiae	SAUCC194.72	Pometiapinnata	China	MT822600	MT855679	MT855568	MT855797	MT855912
D.pseudoalnea	CFCC 54190	Alnusglutinosa	Netherlands	MZ727037	MZ753468	MZ781302	MZ816343	MZ753487
D.pseudoalnea	M2A	Alnusglutinosa	Netherlands	MZ727038	MZ753469	MZ753478	MZ816344	MZ753488
D.pseudomangiferae	CBS 101339	Mangiferaindica	Dominican Republic	KC343181	KC343423	KC343665	KC343907	KC344149
D.pseudophoenicicola	CBS 176.77	Mangiferaindica	Iraq	KC343183	KC343425	KC343667	KC343909	KC344151
D.pseudotsugae	MFLU 15-3228	Pseudotsugamenziesii	Italy	KY964225	KY964138	NA	KY964181	KY964108
D.psoraleae	CPC 21634	Psoraleapinnata	South Africa	KF777158	NA	NA	KF777245	KF777251
D.psoraleae-pinnatae	CPC 21638	Psoraleapinnata	South Africa	KF777159	NA	NA	NA	KF777252
D.pterocarpicola	MFLUCC 10-0580a	Pterocarpusindicus	Thailand	JQ619887	JX197433	NA	JX275403	JX275441
D.pungensis	SAUCC194.112	Elaeagnuspungens	China	MT822640	MT855719	MT855607	MT855837	MT855952
D.pyracanthae	CAA483	Pyracanthacoccinea	Portugal	KY435635	KY435645	KY435656	KY435625	KY435666
D.racemosae	CPC 26646	Euclearacemosa	South Africa	MG600223	MG600219	MG600221	MG600225	MG600227
D.raonikayaporum	CBS 133182	Spondiasmombin	Brazil	KC343188	KC343430	KC343672	KC343914	KC344156
D.ravennica	MFLUCC 16-0997	Clematisvitalba	Italy	NA	NA	NA	MT394670	NA
D.rhusicola	CPC 18191	Rhuspendulina	South Africa	JF951146	NA	NA	NA	NA
D.rosae	MFLUCC 17-2658	Rosa	United Kingdom	MG828894	MG829273	NA	NA	MG843878
D.rosiphthora	COAD 2914	Rosa	Brazil	MT311197	MT313691	NA	MT313693	NA
D.rossmaniae	CAA 762	Vacciniumcorymbosum	Portugal	MK792290	MK883822	MK871432	MK828063	MK837914
D.rostrata	CFCC 50062	Juglansmandshurica	China	KP208847	KP208849	KP208851	KP208853	KP208855
D.rostrata	CFCC 50063	Juglansmandshurica	China	KP208848	KP208850	KP208852	KP208854	KP208856
D.rudis	AR3422	Laburnumanagyroides	Austria	KC843331	KC843146	NA	KC843090	KC843177
D.rudis	CFCC 54193	Quercusrobur	Netherlands	MZ727039	MZ753470	MZ753479	MZ816345	MZ753489
D.rudis	M86	Quercusrobur	Netherlands	MZ727040	MZ753471	MZ753480	MZ816346	MZ753490
D.saccarata	CBS 116311	Protearepens	South Africa	KC343190	KC343432	KC343674	KC343916	KC344158
D.sackstonii	BRIP 54669b	Helianthusannuus	Australia	KJ197287	NA	NA	KJ197249	KJ197267
D.salicicola	BRIP 54825	Salixpurpurea	Australia	JX862531	NA	NA	JX862537	KF170923
D.sambucusii	CFCC 51986	Sambucuswilliamsii	China	KY852495	KY852499	KY852503	KY852507	KY852511
D.sambucusii	CFCC 51987	Sambucuswilliamsii	China	KY852496	KY852500	KY852504	KY852508	KY852512
D.schimae	CFCC 53103	Schimasuperba	China	MK442640	MK442962	MK442987	MK578116	MK578043
D.schimae	CFCC 53104	Schimasuperba	China	MK442641	MK442963	MK442988	MK578117	MK578044
D.schimae	CFCC 53105	Schimasuperba	China	MK442642	MK442964	MK442989	MK578118	MK578045
D.schini	CBS 133181	Schinusterebinthifolius	Brazil	KC343191	KC343433	KC343675	KC343917	KC344159
D.schisandrae	CFCC 51988	Schisandrachinensis	China	KY852497	KY852501	KY852505	KY852509	KY852513
D.schisandrae	CFCC 51989	Schisandrachinensis	China	KY852498	KY852502	KY852506	KY852510	KY852514
D.schoeni	MFLU 15-1279	Schoenusnigricans	Italy	KY964226	KY964139	NA	KY964182	KY964109
D.sclerotioides	CBS 296.67	Cucumissativus	Netherlands	MH858974	KC343435	KC343677	KC343919	KC344161
D.searlei	CBS 146456	Macadamia	Australia	MN708231	NA	NA	NA	MN696540
D.sennae	CFCC 51636	Sennabicapsularis	China	KY203724	KY228875	NA	KY228885	KY228891
D.sennae	CFCC 51637	Sennabicapsularis	China	KY203725	KY228876	NA	KY228886	KY228892
D.sennicola	CFCC 51634	Sennabicapsularis	China	KY203722	KY228873	KY228879	KY228883	KY228889
D.sennicola	CFCC 51635	Sennabicapsularis	China	KY203723	KY228874	KY228880	KY228884	KY228890
D.serafiniae	BRIP 55665a	Helianthusannuus	Australia	KJ197274	NA	NA	KJ197236	KJ197254
D.shaanxiensis	CFCC 53106	on branches of liana	China	MK432654	MK442976	MK443001	MK578130	NA
D.shaanxiensis	CFCC 53107	on branches of liana	China	MK432655	MK432977	MK432002	MK578131	NA
D.siamensis	MFLUCC 10-0573a	Dasymaschalon	Thailand	NA	JQ619897	NA	JX275393	JX275429
D.silvicola	CFCC 54191	Fraxinusexcelsior	Netherlands	MZ727041	MZ753472	MZ753481	MZ816347	MZ753491
D.silvicola	M79	Fraxinusexcelsior	Netherlands	MZ727042	MZ753473	MZ753482	MZ816348	MZ753492
D.sojae	FAU635	Glycinemax	USA	KJ590719	KJ612116	KJ659208	KJ590762	KJ610875
D.spartinicola	CPC 24951	Spartium junceμm	Spain	KR611879	NA	KR857696	NA	KR857695
D.spinosa	PSCG 383	Pyruspyrifolia	China	MK626849	MK691129	MK726156	MK654811	MK691234
D.sterilis	CBS 136969	Vacciniumcorymbosum	Italy	KJ160579	KJ160548	MF418350	KJ160611	KJ160528
D.stictica	CBS 370.54	Buxussampervirens	Italy	KC343212	KC343454	KC343696	KC343938	KC344180
D.subclavata	ZJUD95	Citrusunshiu	China	KJ490630	NA	KJ490572	KJ490509	KJ490451
D.subcylindrospora	KUMCC 17-0151	Unknown	China	MG746629	NA	NA	MG746630	MG746631
D.subellipicola	KUMCC 17-0153	Unknown	China	MG746632	NA	NA	MG746633	MG746634
D.subordinaria	CBS 464.90	Plantagolanceolata	South Africa	KC343214	KC343456	KC343698	KC343940	KC344182
D.taoicola	MFLUCC 16-0117	Prunuspersica	China	KU557567	NA	NA	KU557636	KU557591
D.tectonae	MFLUCC 12-0777	Tectonagrandis	Thailand	KU712430	KU749345	NA	KU749359	KU743977
D.tectonendophytica	MFLUCC 13-0471	Tectonagrandis	Thailand	KU712439	KU749354	NA	KU749367	KU743986
D.tectonigena	MFLUCC 12-0767	Camelliasinensis	China	KX986782	KX999284	KX999254	KX999174	KX999214
D.terebinthifolii	CBS 133180	Schinusterebinthifolius	Brazil	KC343216	KC343458	KC343700	KC343942	KC344184
D.ternstroemia	CGMCC 3.15183	Ternstroemiagymnanthera	China	KC153098	NA	NA	KC153089	NA
D.thunbergii	MFLUCC 10-0576a	Thunbergialaurifolia	Thailand	JQ619893	JX197440	NA	JX275409	NA
D.thunbergiicola	MFLUCC 12-0033	Thunbergialaurifolia	Thailand	KP715097	NA	NA	KP715098	NA
D.tibetensis	CFCC 51999	Juglandisregia	China	MF279843	MF279888	MF279828	MF279858	MF279873
D.tibetensis	CFCC 52000	Juglandisregia	China	MF279844	MF279889	MF279829	MF279859	MF279874
D.torilicola	MFLUCC 17-1051	Torilisarvensis	Italy	KY964212	KY964127	NA	KY964168	KY964096
D.toxica	CBS 534.93	Lupinusangustifolius	Australia	KC343220	KC343462	KC343704	KC343946	KC344188
D.tulliensis	BRIP 62248a	Theobromacacao	Australia	KR936130	NA	NA	KR936133	KR936132
D.ueckerae	FAU656T	Cucumismelo	USA	KJ590726	KJ612122	KJ659215	KJ590747	KJ610881
D.ukurunduensis	CFCC 52592	Acerukurunduense	China	MH121527	MH121445	MH121485	MH121569	NA
D.ukurunduensis	CFCC 52593	Acerukurunduense	China	MH121528	MH121446	MH121486	MH121570	NA
D.undulata	LC6624	Unknown	China	KX986798	NA	KX999269	KX999190	KX999230
D.unshiuensis	ZJUD52	Citrusunshiu	China	KJ490587	NA	KJ490529	KJ490466	KJ490408
D.unshiuensis	CFCC 52594	Caryaillinoensis	China	MH121529	MH121447	MH121487	MH121571	MH121606
D.unshiuensis	CFCC 52595	Caryaillinoensis	China	MH121530	MH121448	MH121488	MH121572	MH121607
D.vaccinii	CBS 160.32	Oxycoccusmacrocarpos	USA	MH121502	MH121426	MH121462	MH121544	MH121584
D.vangueriae	CBS 137985	Vangueriainfausta	Zambia	KJ869137	NA	NA	NA	KJ869247
D.vawdreyi	BRIP 57887a	Psidiumguajava	Australia	KR936126	NA	NA	KR936129	KR936128
D.velutina	LC4421	Neolitsea	China	KX986790	NA	KX999261	KX999182	KX999223
D.verniciicola	CFCC 53109	Verniciamontana	China	MK573944	MK574583	MK574599	MK574619	MK574639
D.verniciicola	CFCC 53110	Verniciamontana	China	MK573945	MK574584	MK574600	MK574620	MK574640
D.viniferae	JZB320071	Vitisvinifera	China	MK341551	MK500119	NA	MK500107	MK500112
D.virgiliae	CMW 40748	Virgiliaoroboides	South Africa	KP247556	NA	NA	NA	KP247575
D.xishuangbanica	LC6707	Camelliasinensis	China	KX986783	NA	KX999255	KX999175	KX999216
D.xunwuensis	CFCC 53085	Unknown	China	MK432663	MK442983	MK443008	MK578137	MK578063
D.xunwuensis	CFCC 53086	Unknown	China	MK432664	MK442984	MK443009	MK578138	MK578064
D.yunnanensis	LC6168	Unknown	China	KX986796	KX999290	KX999267	KX999188	KX999228
D.zaobaisu	PSCG 031	Pyrusbretschneideri	China	MK626922	NA	MK726207	MK654855	MK691245
Diaporthellacorylina	CBS 121124	Corylus	NA	KC343004	KC343246	KC343488	KC343730	KC343972

Note: NA, not applicable. Strains in this study are marked in bold.

Results

Phylogenetic analyses

The five-gene sequence dataset (ITS, , , and ) was analysed to infer the interspecific relationships within . The dataset consisted of 307 sequences including one outgroup taxon, (CBS 121124). A total of 2649 characters including gaps (516 for ITS, 576 for , 526 for , 507 for and 524 for ) were included in the phylogenetic analysis. Of these characters, 844 were constant, 318 were variable and parsimony-uninformative, and 1487 were parsimony-informative. The topologies resulting from ML and BI analyses of the concatenated dataset were congruent (Fig. 1). Isolates from the present study formed four individual clades representing four species of , of which isolates CFCC 54192, M35, M40-1 and M84 from represent , while CFCC 54193 and M86 from represent . CFCC 54191 and M79 from and CFCC 54190 and M2A from represent two new species which are here described as and , respectively.

Figure 1.

Phylogram of resulting from a maximum likelihood analysis based on a combined matrix of ITS, , , and . Numbers above the branches indicate ML bootstraps (left, ML BS ≥ 50 %) and Bayesian Posterior Probabilities (right, BPP ≥ 0.75). The tree is rooted with . Isolates from present study are marked in blue.

Taxonomy

N. Jiang sp. nov.

ABC17B34-3B5B-5E2F-A337-152C8D79C8D9

840714

Fig. 2

Figure 2.

from A–C habit of conidiomata on branches D transverse section of conidiomata E longitudinal section through conidiomata F, G conidiophores and conidia H, I conidia. Scale bars: 2 mm (A), 500 μm (B, C, E), 200 μm (D), 10 μm (F–I).

Etymology.

With reference to , which was described from the same host genus, .

Description.

Conidiomata pycnidial, discoid, immersed in bark, scattered, erumpent through the bark surface, with a solitary locule. Locule 800–1250 μm diam., undivided. Conidiophores 22–68.5 × 1.5–3 μm (av. = 39.8 × 2.2 μm, n = 50), cylindrical, attenuate towards the apex, hyaline, slightly brown at base, phialidic, unbranched, straight or slightly curved. Alpha conidia (5.8–)7.1–8.9(–11.2) × (1.5–)1.8–2.2(–2.7) μm (av. = 7.9 × 2.0 μm, n = 50), L/W = 3.2–4.7 (av. = 3.8, n = 50), hyaline, aseptate, subcylindrical with a nearly rounded apex, multi-guttulate, sometimes acute at both ends. Beta conidia not observed.

Culture characters.

Colonies are initially white with fluffy aerial mycelium, becoming dirty white after 2 weeks, and conidiomata are randomly distributed with orange conidial drops oozing out of the ostioles.

Specimens examined.

NETHERLANDS. Utrecht City, on branches of , , 8 Apr. 2019, N. Jiang (holotype CAF800005 = JNH0001; ex-type living culture: CFCC 54190; other living culture: M2A).

Notes.

and were recorded from the host genus . Udayanga et al. (2014) investigated the lectotype of and revealed it as a species based on a well-developed ectostromata and the ascospores characteristics, and Jaklitsch and Voglmayr (2020) treated it as a synonym of . has been reported from the Czech Republic, Germany, the Netherlands and the USA, and both sexual and asexual morphs have been described (Udayanga et al. 2014). However, applying the GCPSR principle, has recently been considered to be a synonym of (Hilário et al. 2021), which has also been confirmed in our analyses where the ex-epitype isolate CBS 146.46 of is placed within the clade (Fig. 1). morphologically differs from (now ) by its longer conidiophores (22–68.5 × 1.5–3 μm in vs. 9–16 × 1–2 μm in ; Udayanga et al. 2014). In our multi-gene analyses, forms a distinct phylogenetic lineage which is placed remotely from the isolate CBS 146.46 of (Fig. 1).

N. Jiang sp. nov.

A563ADB6-35CD-5A06-9D98-8A27FCC9D26A

840715

Fig. 3

Figure 3.

from A–C habit of conidiomata on branches D transverse section of conidiomata E longitudinal section through conidiomata F, I conidia G, H conidiophores and conidia. Scale bars: 2 mm (A), 1 mm (B), 500 μm (C), 200 μm (D, E), 10 μm (F–I).

Name from “silva” = forest and “-cola” = inhabiting; with reference to its woody host.

Conidiomata pycnidial, conical, immersed in bark, scattered, erumpent through the bark surface, with a solitary locule. Locule 450–700 μm diam., undivided. Conidiophores 6.5–25 × 1.5–4 μm (av. = 15.4 × 2.4 μm, n = 50), cylindrical, attenuate towards the apex, hyaline, slightly brown, phialidic, unbranched, slightly curved. Alpha conidia (9.2–)10.1–12.3(–13.5) × (3.8–)4.2–4.9(–5.2) μm (av. = 11.5 × 4.5 μm, n = 50), L/W = 2.0–3.2 (av. = 2.5, n = 50), hyaline, aseptate, fusiform to oval, multi-guttulate, acute at both ends. Beta conidia not observed.

Colonies are initially white, aerial mycelium turning grey at edges of plate, yellowish pigmentation developing in centre, conidiomata not produced until 2 weeks.

NETHERLANDS. Utrecht City, on branches of in the forest ecosystem, , 6 Jun. 2019, N. Jiang (holotype CAF800006 = JNH0002; ex-type living culture: CFCC 54191; other living culture: M79).

was reported from cv. Claret Ash in Australia (Tan et al. 2013). was described from in China (Yang et al. 2018). However, from in Netherlands differs from and by obviously larger alpha conidia (9.2–13.5 × 3.8–5.2 μm in vs. 4–10 × 2–3 μm in vs. 7–10 × 2.9–3.2 μm in ; Tan et al. 2013; Yang et al. 2018).

Discussion

In this study, branch-inhabiting species were sampled from , and in Utrecht, the Netherlands. Ten isolates were obtained and identified based on five combined loci (ITS, , , and ), as well as morphological characters from the natural substrates. The phylogenetic and morphological analyses revealed sp. nov. from , sp. nov. from , and and from .

Phylogenetic analyses were conducted based on a combined DNA sequence matrix of five loci (ITS, , , and ) reported as useful markers to distinguish species of (Udayanga et al. 2014, 2015; Guarnaccia et al. 2017, 2018a, 2018b; Tibpromma et al. 2018; Yang et al. 2020; Dissanayake et al. 2020; Huang et al. 2021; Sun et al. 2021, Wang et al. 2021). The two novel species in this study can be distinguished from the other known species by all genes studied, but most effectively by , , and . The multi-locus phylogenetic analysis grouped the isolates in two new clades, which support the introduction of the new species.

The utility of host association for species identification is limited because several species have wide host ranges (e.g., inhabits 282 different hosts; inhabits 44 different hosts), and multiple species can infect a single host (e.g., nineteen species are associated with pear cankers in China) (Guo et al. 2020; Farr and Rossman 2021). Thus, a polyphasic approach of morphological, cultural, ecological and molecular data to identify samples or to introduce new species is essential.