Literature DB >> 30271260

High diversity of Diaporthe species associated with dieback diseases in China, with twelve new species described.

Qin Yang¹, Xin-Lei Fan¹, Vladimiro Guarnaccia^2,3, Cheng-Ming Tian¹.

Abstract

Diaporthe species have often been reported as important plant pathogens, saprobes and endophytes on a wide range of plant hosts. Although several Diaporthe species have been recorded in China, little is known about species able to infect forest trees. Therefore, extensive surveys were recently conducted in Beijing, Heilongjiang, Jiangsu, Jiangxi, Shaanxi and Zhejiang Provinces. The current results emphasised on 15 species from 42 representative isolates involving 16 host genera using comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions, as well as their morphological features. Three known species, D.biguttulata, D.eres and D.unshiuensis, were identified. In addition, twelve novel taxa were collected and are described as D.acerigena, D.alangii, D.betulina, D.caryae, D.cercidis, D.chensiensis, D.cinnamomi, D.conica, D.fraxinicola, D.kadsurae, D.padina and D.ukurunduensis. The current study improves the understanding of species causing diebacks on ecological and economic forest trees and provides useful information for the effective disease management of these hosts in China.

Entities: CellLine Chemical Disease Species

Keywords: DNA phylogeny; Dieback; Systematics; Taxonomy

Year: 2018 PMID： 30271260 PMCID： PMC6160862 DOI： 10.3897/mycokeys.39.26914

Source DB: PubMed Journal: MycoKeys ISSN： 1314-4049 Impact factor: 2.984

Introduction

The genus Nitschke represents a cosmopolitan group of fungi occupying diverse ecological behaviour as plant pathogens, endophytes and saprobes (Muralli et al. 2006, Rossman et al. 2007, Garcia-Reyne et al. 2011, Udayanga et al. 2011, 2012a, b, 2014a, b, 2015, Gomes et al. 2013, Fan et al. 2015, Du et al. 2016, Dissanayake et al. 2017b, Guarnaccia and Crous 2017, Yang et al. 2017a, b, 2018, Guarnaccia et al. 2018, Marin-Felix et al. 2018). species are responsible for diseases on a wide range of plant hosts, including agricultural crops, forest trees and ornamentals, some of which are economically important. Several symptoms such as root and fruit rots, dieback, stem cankers, leaf spots, leaf and pod blights and seed decay are caused by spp. (Uecker 1988, Rehner and Uecker 1994, Mostert et al. 2001, Santos et al. 2011, Thompson et al. 2011, Udayanga et al. 2011). For example, , the causal agent of cane and leaf spot, is known as a severe pathogen of grapevines (Hewitt and Pearson 1988), infecting all green tissues and causing yield reductions of up to 30% in temperate regions (Erincik et al. 2001). is another well-known pathogen exclusively found on spp. causing melanose, stem-end rot and gummosis in all the citrus production areas except Europe (Mondal et al. 2007, Udayanga et al. 2014a, Guarnaccia and Crous 2017, 2018). Similarly, stem canker, attributed to several spp., is one of the most important diseases of sunflower () worldwide (Muntañola-Cvetković et al. 1981, Thompson et al. 2011). Several species of include a broad number of endophytes associated with hosts present in temperate and tropical regions (Udayanga et al. 2011). Gomes et al. (2013) considered that is a sterile endophyte on and based on molecular phylogeny. Huang et al. (2015) distinguished seven undescribed species associated with citrus in China. Moreover, some endophytes have been shown to act as opportunistic plant pathogens. For instance, has been found as both endophyte and opportunistic pathogen on various herbaceous weeds, ornamentals and fruit trees (Udayanga et al. 2014a, Guarnaccia et al. 2016). The genus (syn. ) was established by Nitschke (1870). Species identification criteria in were originally based on host association, morphology and culture characteristics (Mostert et al. 2001, Santos and Phillips 2009, Udayanga et al. 2012). As a consequence, a broad increase in the number of proposed species occurred. More than 1000 epithets for and 950 for were listed in Index Fungorum (2018) (http://www.indexfungorum.org/) (accessed 1 March 2018). The abolishment of the dual nomenclature system for pleomorphic fungi raised the question about which generic name to use. Given that both names are well known amongst plant pathologists and have been equally used, Rossman et al. (2015) proposed that the name (Nitschke 1870) has priority over (Saccardo and Roumeguère 1884) and has been adopted as the generic name in recent major studies (Gomes et al. 2013, Udayanga et al. 2014a, b, 2015, Fan et al. 2015, Huang et al. 2015, Du et al. 2016, Gao et al. 2017, Yang et al. 2017a, b, c, 2018). The sexual morph of is characterised by immersed ascomata and an erumpent pseudostroma with elongated perithecial necks. Asci are unitunicate, clavate to cylindrical. Ascospores are fusoid, ellipsoid to cylindrical, hyaline, biseriate to uniseriate in the ascus and sometimes with appendages (Udayanga et al. 2011). The asexual morph is characterised by ostiolate conidiomata, with cylindrical phialides producing three types of hyaline, aseptate conidia (Udayanga et al. 2011). Previously, species identification of was largely referred to the assumption of host-specificity, leading to the proliferation of names (Gomes et al. 2013). More than one species of can colonise a single host, while one species can be associated with different hosts (Santos and Phillips 2009, Diogo et al. 2010, Santos et al. 2011, Gomes et al. 2013). In addition, considerable variability of the phenotype characters is present within a species (Rehner and Uecker 1994, Mostert et al. 2001, Santos et al. 2010, Udayanga et al. 2011, 2012a). Species identification is essential for understanding the epidemiology and plant diseases management and to guide the implementation of phytosanitary measures (Santos and Phillips 2009, Udayanga et al. 2011, Santos et al. 2017). Thus, molecular data are necessary to resolve taxonomy and, during the recent years, many species have been described through a polyphasic approach together with morphology (Gomes et al. 2013, Udayanga et al. 2014a, b, 2015, Huang et al. 2015, Gao et al. 2017, Guarnaccia and Crous 2017, Yang et al. 2018). Santos et al. (2017) revealed that the use of a five-loci dataset (ITS-cal-his3-tef1-tub2) is the optimal combination for species delimitation, showing the ribosomal ITS locus as the least informative, which is contrary to the result of Santos et al. (2010). Although the classification of has been on-going, species are currently being identified based on a combination of morphological, cultural, phytopathological and phylogenetical analyses (Gomes et al. 2013, Huang et al. 2013, 2015, Udayanga et al. 2014a, b, 2015, Fan et al. 2015, Du et al. 2016, Gao et al. 2016, 2017, Guarnaccia and Crous 2017, Hyde et al. 2017, 2018, Guarnaccia et al. 2018, Jayawardena et al. 2018, Perera et al. 2018a, b, Tibpromma et al. 2018, Wanasinghe et al. 2018). However, fungi isolated from forest trees in China were recorded in old fungal literature without any living culture and molecular data (Teng 1963, Tai 1979, Wei 1979). The current study aimed to investigate the major ecological or economic trees in China by large-scale sampling and to identify isolates via morphology and multi-locus phylogeny based on modern taxonomic concepts. From 2015 to 2017, several surveys were conducted in six Provinces representing 16 host genera. The objectives of the present study were (i) to provide a multi-gene phylogeny for the genus based on a large set of freshly collected specimens in China; (ii) to identify taxa associated with disease symptoms or non-symptomatic tissues of various host genera distributed over six Provinces in China; (iii) to define the species limits of and closely related species based on multi-gene genealogies.

Materials and methods

Isolates

From 2015 to 2017, fresh specimens of were collected from symptomatic or non-symptomatic twigs or branches from Beijing, Heilongjiang, Jiangsu, Jiangxi, Shaanxi and Zhejiang Provinces in China (Table 1). A total of 105 isolates were obtained by removing a mucoid spore mass from conidiomata and spreading the suspension on the surface of 1.8% potato dextrose agar (PDA) in a Petri dish and incubating at 25 °C for up to 24 h. Single germinating conidia were transferred on to fresh PDA plates. Forty-two representative strains were selected based on cultural characteristics on PDA, conidia morphology and ITS sequence data. Specimens were deposited in the Museum of the Beijing Forestry University (BJFC). Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Table 1.

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

Species	Isolate	Host	Location	GenBank accession numbers
Species	Isolate	Host	Location	ITS	cal	his3	tef1	tub2
D. acaciarum	CBS 138862	Acacia tortilis	Tanzania	KP004460	N/A^a	N/A^a	N/A^a	KP004509
D. acaciigena	CBS 129521	Acacia retinodes	Australia	KC343005	KC343247	KC343489	KC343731	KC343973
D. acericola	MFLUCC 17-0956	Acer negundo	Italy	KY964224	KY964137	N/A^a	KY964180	KY964074
D. acerigena	CFCC 52554	Acer tataricum	China	MH121489	MH121413	MH121449	MH121531	N/A^a
D. acerigena	CFCC 52555	Acer tataricum	China	MH121490	MH121414	MH121450	MH121532	N/A^a
D. acutispora	CGMCC 3.18285	Coffea sp.	China	KX986764	KX999274	N/A^a	KX999155	KX999195
D. alangii	CFCC 52556	Alangium kurzii	China	MH121491	MH121415	MH121451	MH121533	MH121573
	CFCC 52557	Alangium kurzii	China	MH121492	MH121416	MH121452	MH121534	MH121574
	CFCC 52558	Alangium kurzii	China	MH121493	MH121417	MH121453	MH121535	MH121575
	CFCC 52559	Alangium kurzii	China	MH121494	MH121418	MH121454	MH121536	MH121576
D. alleghaniensis	CBS 495.72	Betula alleghaniensis	Canada	KC343007	KC343249	KC343491	KC343733	KC343975
D. alnea	CBS 146.46	Alnus sp.	Netherlands	KC343008	KC343250	KC343492	KC343734	KC343976
D. ambigua	CBS 114015	Pyrus communis	South Africa	KC343010	KC343252	KC343494	KC343736	KC343978
D. ampelina	STEU2660	Vitis vinifera	France	AF230751	AY745026	N/A^a	AY745056	JX275452
D. amygdali	CBS 126679	Prunus dulcis	Portugal	KC343022	KC343264	KC343506	AY343748	KC343990
D. anacardii	CBS 720.97	Anacardium occidentale	East Africa	KC343024	KC343266	KC343508	KC343750	KC343992
D. angelicae	CBS 111592	Heracleum sphondylium	Austria	KC343027	KC343269	KC343511	KC343753	KC343995
D. apiculatum	CGMCC 3.17533	Camellia sinensis	China	KP267896	N/A^a	N/A^a	KP267970	KP293476
D. aquatica	IFRDCC 3051	Aquatic habitat	China	JQ797437	N/A^a	N/A^a	N/A^a	N/A^a
D. arctii	CBS 139280	Arctium lappa	Austria	KJ590736	KJ612133	KJ659218	KJ590776	KJ610891
D. arecae	CBS 161.64	Areca catechu	India	KC343032	KC343274	KC343516	KC343758	KC344000
D. arengae	CBS 114979	Arenga enngleri	Hong Kong	KC343034	KC343276	KC343518	KC343760	KC344002
D. aseana	MFLUCC 12-0299a	Unknown dead leaf	Thailand	KT459414	KT459464	N/A^a	KT459448	KT459432
D. asheicola	CBS 136967	Vaccinium ashei	Chile	KJ160562	KJ160542	N/Aa	KJ160594	KJ160518
D. aspalathi	CBS 117169	Aspalathus linearis	South Africa	KC343036	KC343278	KC343520	KC343762	KC344004
D. australafricana	CBS 111886	Vitis vinifera	Australia	KC343038	KC343280	KC343522	KC343764	KC344006
D. baccae	CBS 136972	Vaccinium corymbosum	Italy	KJ160565	N/A^a	MF418264	KJ160597	N/A^a
D. batatas	CBS 122.21	Ipomoea batatas	USA	KC343040	KC343282	N/A^a	KC343766	KC344008
D. beilharziae	BRIP 54792	Indigofera australis	Australia	JX862529	N/A^a	N/A^a	JX862535	KF170921
D. benedicti	BPI 893190	Salix sp.	USA	KM669929	KM669862	N/A^a	KM669785	N/A^a
D. betulae	CFCC 50469	Betula platyphylla	China	KT732950	KT732997	KT732999	KT733016	KT733020
D. betulae	CFCC 50470	Betula platyphylla	China	KT732951	KT732998	KT733000	KT733017	KT733021
D. betulicola	CFCC 51128	Betula albo-sinensis	China	KX024653	KX024659	KX024661	KX024655	KX024657
D. betulicola	CFCC 51129	Betula albo-sinensis	China	KX024654	KX024660	KX024662	KX024656	KX024658
D. betulina	CFCC 52560	Betula albo-sinensis	China	MH121495	MH121419	MH121455	MH121537	MH121577
	CFCC 52561	Betula costata	China	MH121496	MH121420	MH121456	MH121538	MH121578
	CFCC 52562	Betula platyphylla	China	MH121497	MH121421	MH121457	MH121539	MH121579
D. bicincta	CBS 121004	Juglans sp.	USA	KC343134	KC343376	KC343618	KC343860	KC344102
D. biconispora	CGMCC 3.17252	Citrus grandis	China	KJ490597	KJ490539	KJ490539	KJ490476	KJ490418
D. biguttulata	CGMCC 3.17248	Citrus limon	China	KJ490582	N/A^a	KJ490524	KJ490461	KJ490403
	CFCC 52584	Juglans regia	China	MH121519	MH121437	MH121477	MH121561	MH121598
	CFCC 52585	Juglans regia	China	MH121520	MH121438	MH121478	MH121562	MH121599
D. biguttusis	CGMCC 3.17081	Lithocarpus glabra	China	KF576282	N/A^a	N/A^a	KF576257	KF576306
D. bohemiae	CPC 28222	Vitis vinifera	Czech Republic	MG281015	MG281710	MG281361	MG281536	MG281188
D. brasiliensis	CBS 133183	Aspidosperma tomentosum	Brazil	KC343042	KC343284	KC343526	KC343768	KC344010
D. caatingaensis	CBS 141542	Tacinga inamoena	Brazil	KY085927	N/A^a	N/A^a	KY115603	KY115600
D. camptothecicola	CFCC 51632	Camptotheca acuminata	China	KY203726	KY228877	KY228881	KY228887	KY228893
D. canthii	CBS 132533	Canthium inerme	South Africa	JX069864	KC843174	N/A^a	KC843120	KC843230
D. caryae	CFCC 52563	Carya illinoensis	China	MH121498	MH121422	MH121458	MH121540	MH121580
D. caryae	CFCC 52564	Carya illinoensis	China	MH121499	MH121423	MH121459	MH121541	MH121581
D. cassines	CPC 21916	Cassine peragua	South Africa	KF777155	N/A^a	N/A^a	KF777244	N/A^a
D. caulivora	CBS 127268	Glycine max	Croatia	KC343045	KC343287	N/A^a	KC343771	KC344013
D. celeris	CPC 28262	Vitis vinifera	Czech Republic	MG281017	MG281712	MG281363	MG281538	MG281190
D. celastrina	CBS 139.27	Celastrus sp.	USA	KC343047	KC343289	KC343531	KC343773	KC344015
D. cercidis	CFCC 52565	Cercis chinensis	China	MH121500	MH121424	MH121460	MH121542	MH121582
D. cercidis	CFCC 52566	Cercis chinensis	China	MH121501	MH121425	MH121461	MH121543	MH121583
D. chamaeropis	CBS 454.81	Chamaerops humilis	Greece	KC343048	KC343290	KC343532	KC343774	KC344016
D. charlesworthii	BRIP 54884m	Rapistrum rugostrum	Australia	KJ197288	N/A^a	N/A^a	KJ197250	KJ197268
D. chensiensis	CFCC 52567	Abies chensiensis	China	MH121502	MH121426	MH121462	MH121544	MH121584
D. chensiensis	CFCC 52568	Abies chensiensis	China	MH121503	MH121427	MH121463	MH121545	MH121585
D. cichorii	MFLUCC 17-1023	Cichorium intybus	Italy	KY964220	KY964133	N/A^a	KY964176	KY964104
D. cinnamomi	CFCC 52569	Cinnamomum sp.	China	MH121504	N/A^a	MH121464	MH121546	MH121586
D. cinnamomi	CFCC 52570	Cinnamomum sp.	China	MH121505	N/A^a	MH121465	MH121547	MH121587
D. cissampeli	CBS 141331	Cissampelos capensis	South Africa	KX228273	N/A^a	KX228366	N/A^a	KX228384
D. citri	AR 3405	Citrus sp.	USA	KC843311	KC843157	N/A^a	KC843071	KC843187
D. citriasiana	CGMCC 3.15224	Citrus unshiu	China	JQ954645	KC357491	KJ490515	JQ954663	KC357459
D.citrichinensis	CGMCC 3.15225	Citrus sp.	China	JQ954648	KC357494	N/A^a	JQ954666	N/A^a
D. collariana	MFLU 17-2770	Magnolia champaca	Thailand	MG806115	MG783042	N/A^a	MG783040	MG783041
D. compacta	CGMCC 3.17536	Camellia sinensis	China	KP267854	N/A^a	KP293508	KP267928	KP293434
D. conica	CFCC 52571	Alangium chinense	China	MH121506	MH121428	MH121466	MH121548	MH121588
	CFCC 52572	Alangium chinense	China	MH121507	MH121429	MH121467	MH121549	MH121589
	CFCC 52573	Alangium chinense	China	MH121508	MH121430	MH121468	MH121550	MH121590
	CFCC 52574	Alangium chinense	China	MH121509	MH121431	MH121469	MH121551	MH121591
D. convolvuli	CBS 124654	Convolvulus arvensis	Turkey	KC343054	KC343296	KC343538	KC343780	KC344022
D. crotalariae	CBS 162.33	Crotalaria spectabilis	USA	KC343056	KC343298	KC343540	KC343782	KC344024
D. cucurbitae	CBS 136.25	Arctium sp.	Unknown	KC343031	KC343273	KC343515	KC343757	KC343999
D. cuppatea	CBS 117499	Aspalathus linearis	South Africa	KC343057	KC343299	KC343541	KC343783	KC344025
D. cynaroidis	CBS 122676	Protea cynaroides	South Africa	KC343058	KC343300	KC343542	KC343784	KC344026
D. cytosporella	FAU461	Citrus limon	Italy	KC843307	KC843141	N/A^a	KC843116	KC843221
D. diospyricola	CPC 21169	Diospyros whyteana	South Africa	KF777156	N/A^a	N/A^a	N/A^a	N/A^a
D. discoidispora	ZJUD89	Citrus unshiu	China	KJ490624	N/A^a	KJ490566	KJ490503	KJ490445
D. dorycnii	MFLUCC 17-1015	Dorycnium hirsutum	Italy	KY964215	N/A^a	N/A^a	KY964171	KY964099
D. elaeagni-glabrae	CGMCC 3.18287	Elaeagnus glabra	China	KX986779	KX999281	KX999251	KX999171	KX999212
D. ellipicola	CGMCC 3.17084	Lithocarpus glabra	China	KF576270	N/A^a	N/A^a	KF576245	KF576291
D. endophytica	CBS 133811	Schinus terebinthifolius	Brazil	KC343065	KC343307	KC343549	KC343791	KC343065
D. eres	AR5193	Ulmus sp.	Germany	KJ210529	KJ434999	KJ420850	KJ210550	KJ420799
	CFCC 52575	Castanea mollissima	China	MH121510	N/A^a	MH121470	MH121552	MH121592
	CFCC 52576	Castanea mollissima	China	MH121511	MH121432	MH121471	MH121553	MH121593
	CFCC 52577	Acanthopanax senticosus	China	MH121512	MH121433	MH121472	MH121554	MH121594
	CFCC 52578	Sorbus sp.	China	MH121513	MH121434	MH121473	MH121555	MH121595
	CFCC 52579	Juglans regia	China	MH121514	N/A^a	MH121474	MH121556	N/A^a
	CFCC 52580	Melia azedarace	China	MH121515	N/A^a	MH121475	MH121557	MH121596
	CFCC 52581	Rhododendron simsii	China	MH121516	N/A^a	MH121476	MH121558	MH121597
D. eucalyptorum	CBS 132525	Eucalyptus sp.	Australia	NR120157	N/Aa	N/Aa	N/Aa	N/Aa
D. foeniculacea	CBS 123208	Foeniculum vulgare	Portugal	KC343104	KC343346	KC343588	KC343830	KC344072
D. fraxini-angustifoliae	BRIP 54781	Fraxinus angustifolia	Australia	JX862528	N/A^a	N/A^a	JX862534	KF170920
D. fraxinicola	CFCC 52582	Fraxinus chinensis	China	MH121517	MH121435	N/A^a	MH121559	N/A^a
D. fraxinicola	CFCC 52583	Fraxinus chinensis	China	MH121518	MH121436	N/A^a	MH121560	N/A^a
D. fukushii	MAFF 625034	Pyrus pyrifolia	Japan	JQ807469	N/A^a	N/A^a	JQ807418	N/A^a
D. fusicola	CGMCC 3.17087	Lithocarpus glabra	China	KF576281	KF576233	N/A^a	KF576256	KF576305
D. ganjae	CBS 180.91	Cannabis sativa	USA	KC343112	KC343354	KC343596	KC343838	KC344080
D. garethjonesii	MFLUCC 12-0542a	Unknown dead leaf	Thailand	KT459423	KT459470	N/A^a	KT459457	KT459441
D. goulteri	BRIP 55657a	Helianthus annuus	Australia	KJ197290	N/A^a	N/A^a	KJ197252	KJ197270
D. gulyae	BRIP 54025	Helianthus annuus	Australia	JF431299	N/A^a	N/A^a	KJ197271	JN645803
D. helianthi	CBS 592.81	Helianthus annuus	Serbia	KC343115	KC343357	KC343599	KC343841	KC344083
D. helicis	AR5211	Hedera helix	France	KJ210538	KJ435043	KJ420875	KJ210559	KJ420828
D. heterophyllae	CBS 143769	Acacia heterohpylla	France	MG600222	MG600218	MG600220	MG600224	MG600226
D. hickoriae	CBS 145.26	Carya glabra	USA	KC343118	KC343360	KC343602	KC343844	KC344086
D. hispaniae	CPC 30321	Vitis vinifera	Spain	MG281123	MG281820	MG281471	MG281644	MG281296
D. hongkongensis	CBS 115448	Dichroa febrífuga	China	KC343119	KC343361	KC343603	KC343845	KC344087
D. incompleta	CGMCC 3.18288	Camellia sinensis	China	KX986794	KX999289	KX999265	KX999186	KX999226
D. inconspicua	CBS 133813	Maytenus ilicifolia	Brazil	KC343123	KC343365	KC343607	KC343849	KC344091
D. infecunda	CBS 133812	Schinus terebinthifolius	Brazil	KC343126	KC343368	KC343610	KC343852	KC344094
D. isoberliniae	CPC 22549	Isoberlinia angolensis	Zambia	KJ869133	N/A^a	N/A^a	N/A^a	KJ869245
D. juglandicola	CFCC 51134	Juglans mandshurica	China	KU985101	KX024616	KX024622	KX024628	KX024634
D. juglandicola	CFCC 51135	Juglans mandshurica	China	KU985102	KX024617	KX024623	KX024629	KX024635
D. kadsurae	CFCC 52586	Kadsura longipedunculata	China	MH121521	MH121439	MH121479	MH121563	MH121600
	CFCC 52587	Kadsura longipedunculata	China	MH121522	MH121440	MH121480	MH121564	MH121601
	CFCC 52588	Acer sp.	China	MH121523	MH121441	MH121481	MH121565	MH121602
	CFCC 52589	Acer sp.	China	MH121524	MH121442	MH121482	MH121566	MH121603
D. kochmanii	BRIP 54033	Helianthus annuus	Australia	JF431295	N/A^a	N/A^a	JN645809	N/A^a
D. kongii	BRIP 54031	Portulaca grandiflora	Australia	JF431301	N/A^a	N/A^a	JN645797	KJ197272
D. litchicola	BRIP 54900	Litchi chinensis	Australia	JX862533	N/A^a	N/A^a	JX862539	KF170925
D. lithocarpus	CGMCC 3.15175	Lithocarpus glabra	China	KC153104	KF576235	N/A^a	KC153095	KF576311
D. longicicola	CGMCC 3.17089	Lithocarpus glabra	China	KF576267	N/A^a	N/A^a	KF576242	KF576291
D. longicolla	ATCC 60325	Glycine max	USA	KJ590728	N/A^a	KJ659188	KJ590767	KJ610883
D. longispora	CBS 194.36	Ribes sp.	Canada	KC343135	KC343377	KC343619	KC343861	KC344103
D. lonicerae	MFLUCC 17-0963	Lonicera sp.	Italy	KY964190	KY964116	N/A^a	KY964146	KY964073
D. lusitanicae	CBS 123212	Foeniculum vulgare	Portugal	KC343136	KC343378	KC343620	KC343862	KC344104
D. macinthoshii	BRIP 55064a	Rapistrum rugostrum	Australia	KJ197289	N/A^a	N/A^a	KJ197251	KJ197269
D. mahothocarpus	CGMCC 3.15181	Lithocarpus glabra	China	KC153096	N/A^a	N/A^a	KC153087	KF576312
D. malorum	CAA734	Malus domestica	Portugal	KY435638	KY435658	KY435648	KY435627	KY435668
D. maritima	DAOMC 250563	Picea rubens	Canada	N/A^a	N/A^a	N/A^a	N/A^a	KU574616
D. masirevicii	BRIP 57892a	Helianthus annuus	Australia	KJ197277	N/A^a	N/A^a	KJ197239	KJ197257
D. mayteni	CBS 133185	Maytenus ilicifolia	Brazil	KC343139	KC343381	KC343623	KC343865	KC344107
D. maytenicola	CPC 21896*	Maytenus acuminata	South Africa	KF777157	N/A^a	N/A^a	N/A^a	KF777250
D. melonis	CBS 507.78	Cucumis melo	USA	KC343142	KC343384	KC343626	KC343868	KC344110
D. middletonii	BRIP 54884e	Rapistrum rugostrum	Australia	KJ197286	N/A^a	N/A^a	KJ197248	KJ197266
D. miriciae	BRIP 54736j	Helianthus annuus	Australia	KJ197282	N/A^a	N/A^a	KJ197244	KJ197262
D. momicola	MFLUCC 16-0113	Prunus persica	China	KU557563	KU557611	N/A^a	KU557631	KU55758
D. multigutullata	ZJUD98	Citrus grandis	China	KJ490633	N/A^a	KJ490575	KJ490512	KJ490454
D. musigena	CBS 129519	Musa sp.	Australia	KC343143	KC343385	KC343627	KC343869	KC344111
D. neilliae	CBS 144.27	Spiraea sp.	USA	KC343144	KC343386	KC343628	KC343870	KC344112
D. neoarctii	CBS 109490	Ambrosia trifida	USA	KC343145	KC343387	KC343629	KC343871	KC344113
D. neoraonikayaporum	MFLUCC 14-1136	Tectona grandis	Thailand	KU712449	KU749356	N/A^a	KU749369	KU743988
D. nobilis	CBS 113470	Castanea sativa	Korea	KC343146	KC343388	KC343630	KC343872	KC344114
D. nothofagi	BRIP 54801	Nothofagus cunninghamii	Australia	JX862530	N/A^a	N/A^a	JX862536	KF170922
D. novem	CBS 127270	Glycine max	Croatia	KC343155	KC343397	KC343640	KC343881	KC344123
D. ocoteae	CBS 141330	Ocotea obtusata	France	KX228293	N/A^a	N/A^a	N/A^a	KX228388
D. oraccinii	CGMCC 3.17531	Camellia sinensis	China	KP267863	N/A^a	KP293517	KP267937	KP293443
D. ovalispora	ICMP20659	Citrus limon	China	KJ490628	N/A^a	KJ490570	KJ490507	KJ490449
D. ovoicicola	CGMCC 3.17093	Citrus sp.	China	KF576265	KF576223	N/A^a	KF576240	KF576289
D. oxe	CBS 133186	Maytenus ilicifolia	Brazil	KC343164	KC343406	KC343648	KC343890	KC344132
D. padina	CFCC 52590	Padus racemosa	China	MH121525	MH121443	MH121483	MH121567	MH121604
D. padina	CFCC 52591	Padus racemosa	China	MH121526	MH121444	MH121484	MH121568	MH121605
D. pandanicola	MFLU 18-0006	Pandanus sp.	Thailand	MG646974	N/A^a	N/A^a	N/A^a	MG646930
D. paranensis	CBS 133184	Maytenus ilicifolia	Brazil	KC343171	KC343413	KC343655	KC343897	KC344139
D. parapterocarpi	CPC 22729	Pterocarpus brenanii	Zambia	KJ869138	N/A^a	N/A^a	N/A^a	KJ869248
D. pascoei	BRIP 54847	Persea americana	Australia	JX862532	N/A^a	N/A^a	JX862538	KF170924
D. passiflorae	CBS 132527	Passiflora edulis	South America	JX069860	N/A^a	KY435654	N/A^a	N/A^a
D. passifloricola	CBS 141329	Passiflora foetida	Malaysia	KX228292	N/A^a	KX228367	N/A^a	KX228387
D. penetriteum	CGMCC 3.17532	Camellia sinensis	China	KP714505	N/A^a	KP714493	KP714517	KP714529
D. perjuncta	CBS 109745	Ulmus glabra	Austria	KC343172	KC343414	KC343656	KC343898	KC344140
D. perseae	CBS 151.73	Persea gratissima	Netherlands	KC343173	KC343415	KC343657	KC343899	KC344141
D. pescicola	MFLUCC 16-0105	Prunus persica	China	KU557555	KU557603	N/A^a	KU557623	KU557579
D. phaseolorum	AR4203	Phaseolus vulgaris	USA	KJ590738	N/A^a	KJ659220	N/A^a	KP004507
D. podocarpi-macrophylli	CGMCC 3.18281	Podocarpus macrophyllus	China	KX986774	KX999278	KX999246	KX999167	KX999207
D. pseudomangiferae	CBS 101339	Mangifera indica	Dominican Republic	KC343181	KC343423	KC343665	KC343907	KC344149
D. pseudophoenicicola	CBS 462.69	Phoenix dactylifera	Spain	KC343184	KC343426	KC343668	KC343910	KC344152
D. pseudotsugae	MFLU 15-3228	Pseudotsuga menziesii	Italy	KY964225	KY964138	N/A^a	KY964181	KY964108
D. psoraleae	CBS 136412	Psoralea pinnata	South Africa	KF777158	N/A^a	N/A^a	KF777245	KF777251
D. psoraleae-pinnatae	CBS 136413	Psoralea pinnata	South Africa	KF777159	N/A^a	N/A^a	N/A^a	KF777252
D. pterocarpi	MFLUCC 10-0571	Pterocarpus indicus	Thailand	JQ619899	JX197451	N/A^a	JX275416	JX275460
D. pterocarpicola	MFLUCC 10-0580a	Pterocarpus indicus	Thailand	JQ619887	JX197433	N/A^a	JX275403	JX275441
D. pulla	CBS 338.89	Hedera helix	Yugoslavia	KC343152	KC343394	KC343636	KC343878	KC344120
D. pyracanthae	CAA483	Pyracantha coccinea	Portugal	KY435635	KY435656	KY435645	KY435625	KY435666
D. racemosae	CBS 143770	Euclea racemosa	South Africa	MG600223	MG600219	MG600221	MG600225	MG600227
D. raonikayaporum	CBS 133182	Spondias mombin	Brazil	KC343188	KC343430	KC343672	KC343914	KC344156
D. ravennica	MFLUCC 15-0479	Tamarix sp.	Italy	KU900335	N/A^a	N/A^a	KX365197	KX432254
D. rhusicola	CBS 129528	Rhus pendulina	South Africa	JF951146	KC843124	N/A^a	KC843100	KC843205
D. rosae	MFLU 17-1550	Rosa sp.	Thailand	MG828894	N/A^a	N/A^a	N/A^a	MG843878
D. rosicola	MFLU 17-0646	Rosa sp.	UK	MG828895	N/A^a	N/A^a	MG829270	MG843877
D. rostrata	CFCC 50062	Juglans mandshurica	China	KP208847	KP208849	KP208851	KP208853	KP208855
D. rostrata	CFCC 50063	Juglans mandshurica	China	KP208848	KP208850	KP208852	KP208854	KP208856
D. rudis	AR3422	Laburnum anagyroides	Austria	KC843331	KC843146	N/A^a	KC843090	KC843177
D. saccarata	CBS 116311	Protea repens	South Africa	KC343190	KC343432	KC343674	KC343916	KC344158
D. sackstonii	BRIP 54669b	Helianthus annuus	Australia	KJ197287	N/A^a	N/A^a	KJ197249	KJ197267
D. salicicola	BRIP 54825	Salix purpurea	Australia	JX862531	N/A^a	N/A^a	JX862537	JX862531
D. sambucusii	CFCC 51986	Sambucus williamsii	China	KY852495	KY852499	KY852503	KY852507	KY852511
D. sambucusii	CFCC 51987	Sambucus williamsii	China	KY852496	KY852500	KY852504	KY852508	KY852512
D. schini	CBS 133181	Schinus terebinthifolius	Brazil	KC343191	KC343433	KC343675	KC343917	KC344159
D. schisandrae	CFCC 51988	Schisandra chinensis	China	KY852497	KY852501	KY852505	KY852509	KY852513
D. schisandrae	CFCC 51989	Schisandra chinensis	China	KY852498	KY852502	KY852506	KY852510	KY852514
D. schoeni	MFLU 15-1279	Schoenus nigricans	Italy	KY964226	KY964139	N/A^a	KY964182	KY964109
D. sclerotioides	CBS 296.67	Cucumis sativus	Netherlands	KC343193	KC343435	KC343677	KC343919	KC344161
D. sennae	CFCC 51636	Senna bicapsularis	China	KY203724	KY228875	N/A^a	KY228885	KY228891
D. sennae	CFCC 51637	Senna bicapsularis	China	KY203725	KY228876	N/Aa	KY228886	KY228892
D. sennicola	CFCC 51634	Senna bicapsularis	China	KY203722	KY228873	KY228879	KY228883	KY228889
D. sennicola	CFCC 51635	Senna bicapsularis	China	KY203723	KY228874	KY228880	KY228884	KY228890
D. serafiniae	BRIP 55665a	Helianthus annuus	Australia	KJ197274	N/A^a	N/A^a	KJ197236	KJ197254
D. siamensis	MFLUCC 10-573a	Dasymaschalon sp.	Thailand	JQ619879	N/A^a	N/A^a	JX275393	JX275429
D. sojae	FAU635	Glycine max	USA	KJ590719	KJ612116	KJ659208	KJ590762	KJ610875
D. spartinicola	CBS 140003	Spartium junceum	Spain	KR611879	N/A^a	KR857696	N/A^a	KR857695
D. sterilis	CBS 136969	Vaccinium corymbosum	Italy	KJ160579	KJ160548	MF418350	KJ160611	KJ160528
D. stictica	CBS 370.54	Buxus sampervirens	Italy	KC343212	KC343454	KC343696	KC343938	KC344180
D. subclavata	ICMP20663	Citrus unshiu	China	KJ490587	N/A^a	KJ490529	KJ490466	KJ490408
D. subcylindrospora	MFLU 17-1195	Salix sp.	China	MG746629	N/A^a	N/A^a	MG746630	MG746631
D. subellipicola	MFLU 17-1197	on dead wood	China	MG746632	N/A^a	N/A^a	MG746633	MG746634
D. subordinaria	CBS 464.90	Plantago lanceolata	New Zealand	KC343214	KC343456	KC343698	KC343940	KC344182
D. taoicola	MFLUCC 16-0117	Prunus persica	China	KU557567	N/Aa	N/A^a	KU557635	KU557591
D. tectonae	MFLUCC 12-0777	Tectona grandis	China	KU712430	KU749345	N/A^a	KU749359	KU743977
D. tectonendophytica	MFLUCC 13-0471	Tectona grandis	China	KU712439	KU749354	N/A^a	KU749367	KU749354
D. tectonigena	MFLUCC 12-0767	Tectona grandis	China	KU712429	KU749358	N/A^a	KU749371	KU743976
D. terebinthifolii	CBS 133180	Schinus terebinthifolius	Brazil	KC343216	KC343458	KC343700	KC343942	KC344184
D. thunbergii	MFLUCC 10-576a	Thunbergia laurifolia	Thailand	JQ619893	JX197440	N/A^a	JX275409	JX275449
D. thunbergiicola	MFLUCC 12-0033	Thunbergia laurifolia	Thailand	KP715097	N/A^a	N/A^a	KP715098	N/A^a
D. tibetensis	CFCC 51999	Juglandis regia	China	MF279843	MF279888	MF279828	MF279858	MF279873
D. tibetensis	CFCC 52000	Juglandis regia	China	MF279844	MF279889	MF279829	MF279859	MF279874
D. torilicola	MFLUCC 17-1051	Torilis arvensis	Italy	KY964212	KY964127	N/A^a	KY964168	KY964096
D. toxica	CBS 534.93	Lupinus angustifolius	Australia	KC343220	KC343462	C343704	KC343946	KC344188
D. tulliensis	BRIP 62248a	Theobromacacao fruit	Australia	KR936130	N/A^a	N/A^a	KR936133	KR936132
D. ueckerae	FAU656	Cucumis melo	USA	KJ590726	KJ612122	KJ659215	KJ590747	KJ610881
D. ukurunduensis	CFCC 52592	Acer ukurunduense	China	MH121527	MH121445	MH121485	MH121569	N/A^a
D. ukurunduensis	CFCC 52593	Acer ukurunduense	China	MH121528	MH121446	MH121486	MH121570	N/A^a
D. undulata	CGMCC 3.18293	Leaf of unknown host	China-Laos border	KX986798	N/A^a	KX999269	KX999190	KX999230
D. unshiuensis	CGMCC 3.17569	Citrus unshiu	China	KJ490587	N/A^a	KJ490529	KJ490408	KJ490466
	CFCC 52594	Carya illinoensis	China	MH121529	MH121447	MH121487	MH121571	MH121606
	CFCC 52595	Carya illinoensis	China	MH121530	MH121448	MH121488	MH121572	MH121607
D. vaccinii	CBS 160.32	Oxycoccus macrocarpos	USA	KC343228	KC343470	KC343712	KC343954	KC344196
D. vangueriae	CPC 22703	Vangueria infausta	Zambia	KJ869137	N/A^a	N/A^a	N/A^a	KJ869247
D. vawdreyi	BRIP 57887a	Psidium guajava	Australia	KR936126	N/A^a	N/A^a	KR936129	KR936128
D. velutina	CGMCC 3.18286	Neolitsea sp.	China	KX986790	N/A^a	KX999261	KX999182	KX999223
D. virgiliae	CMW40748	Virgilia oroboides	South Africa	KP247566	N/A^a	N/A^a	N/A^a	KP247575
D. xishuangbanica	CGMCC 3.18282	Camellia sinensis	China	KX986783	N/A^a	KX999255	KX999175	KX999216
D. yunnanensis	CGMCC 3.18289	Coffea sp.	China	KX986796	KX999290	KX999267	KX999188	KX999228
Diaporthella corylina	CBS 121124	Corylus sp.	China	KC343004	KC343246	KC343488	KC343730	KC343972

Newly sequenced material is indicated in bold type.

Isolates and GenBank accession numbers used in the phylogenetic analyses of . Newly sequenced material is indicated in bold type.

Morphological analysis

Agar plugs (6 mm diam.) were taken from the edge of actively growing cultures on PDA and transferred on to the centre of 9 cm diam Petri dishes containing 2% tap water agar supplemented with sterile pine needles (PNA; Smith et al. 1996) and potato dextrose agar (PDA) and incubated at 20–21 °C under a 12 h near-ultraviolet light/12 h dark cycle to induce sporulation as described in recent studies (Gomes et al. 2013, Lombard et al. 2014). Colony characters and pigment production on PNA and PDA were noted after 10 d. Colony colours were rated according to Rayner (1970). Cultures were examined periodically for the development of ascomata and conidiomata. The morphological characteristics were examined by mounting fungal structures in clear lactic acid and 30 measurements at 1000× magnification were determined for each isolate using a Leica compound microscope (DM 2500) with interference contrast (DIC) optics. Descriptions, nomenclature and illustrations of taxonomic novelties are deposited in MycoBank (www.MycoBank.org; Crous et al. 2004b).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from colonies grown on cellophane-covered PDA using a modified CTAB [cetyltrimethylammonium bromide] method (Doyle and Doyle 1990). DNA was estimated by electrophoresis in 1% agarose gel and the quality was measured using the NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), following the user manual (Desjardins et al. 2009). PCR amplifications were performed in a DNA Engine Peltier Thermal Cycler (PTC-200; Bio-Rad Laboratories, Hercules, CA, USA). The primer sets ITS1/ITS4 (White et al. 1990) were used to amplify the ITS region. The primer pair CAL228F/CAL737R (Carbone and Kohn 1999) were used to amplify the calmodulin gene (cal) and the primer pair CYLH4F (Crous et al. 2004a) and H3-1b (Glass and Donaldson 1995) were used to amplify part of the histone H3 (his3) gene. The primer pair EF1-728F/EF1-986R (Carbone and Kohn 1999) were used to amplify a partial fragment of the translation elongation factor 1-α gene (tef1). The primer sets T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) were used to amplify the beta-tubulin gene (tub2); the additional combination of Bt2a/Bt2b (Glass and Donaldson 1995) was used in case of amplification failure of the T1/Bt2b primer pair. Amplifications of different loci were performed under different conditions (Table 2). PCR amplification products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM® 3730XL DNA Analyser with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Table 2.

Genes used in this study with PCR primers, process and references.

Gene	PCR primers (forward/reverse)	PCR: thermal cycles: (Annealing temp. in bold)	References of primers used
ITS	ITS1/ITS4	(95 °C: 30 s, 51 °C: 30 s, 72 °C: 1 min) × 35 cycles	White et al. 1990
cal	CAL228F/CAL737R	(95 °C: 15 s, 55 °C: 20 s, 72 °C: 1 min) × 35 cycles	Carbone and Kohn 1999
his3	CYLH4F/H3-1b	(95 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 35 cycles	Glass and Donaldson 1995, Crous et al. 2004a
tef1	EF1-728F/EF1-986R	(9 °C: 15 s, 55 °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 and Donaldson 1995, Glass and Donaldson 1995

Genes used in this study with PCR primers, process and references.

Phylogenetic analyses

DNA generated sequences were used to obtain consensus sequences using SeqMan v.7.1.0 DNASTAR Lasergene Core Suite software programme (DNASTAR Inc., Madison, WI, USA). Sequences were aligned using MAFFT v.6 (Katoh and Toh 2010) and edited manually using MEGA6 (Tamura et al. 2013). Two different datasets were employed to estimate two phylogenetic analyses: one for species and one for complex. The first analysis was undertaken to infer the interspecific relationships in . All the isolates recovered from samples collected during this study and additional reference sequences of species were included in the dataset of combined ITS, cal, his3, tef1, and tub2 regions (Table 1), with (CBS 121124) as outgroup. The second analysis focused on the complex based on cal, tef1 and tub2 loci (Table 3) according to recent publications (Gao et al. 2014, 2015, 2016, Udayanga et al. 2014b, Tanney et al. 2016, Fan et al. 2018), with (AR3405) as outgroup. Maximum Parsimony analysis was performed by a heuristic search option of 1000 random-addition sequences with a tree bisection and reconnection (TBR) algorithm. Maxtrees were set to 5000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). Maximum Likelihood analysis was performed with a GTR site substitution model (Guindon et al. 2010). Branch support was evaluated with a bootstrapping (BS) method of 1000 replicates (Hillis and Bull 1993).

Table 3.

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

Species	Isolate/culture collection	Host	Location	GenBank accession numbers
Species	Isolate/culture collection	Host	Location	CAL	TEF1-α	TUB
D. alleghaniensis	CBS 495.72	Betula alleghaniensis	Canada	KC343249	GQ250298	KC843228
D. alnea	CBS 146.46	Alnus sp.	Netherlands	KC343250	KC343734	KC343976
	CBS 159.47	Alnus sp.	Netherlands	KC343251	KC343735	KC343977
	LCM22b.02a	Alnus sp.	USA	KJ435020	KJ210557	KJ420825
	LCM22b.02b	Alnus sp.	USA	KJ435021	KJ210558	KJ420826
D. betulina	CFCC 52560	Betula albo-sinensis	China	MH121419	MH121537	MH121577
	CFCC 52561	Betula costata	China	MH121420	MH121538	MH121578
	CFCC 52562	Betula platyphylla	China	MH121421	MH121539	MH121579
D. bicincta	CBS 121004	Juglans sp.	USA	KC343376	KC343860	KC344102
D. biguttusis	CGMCC 3.17081	Lithocarpus glabra	China	N/A^a	KF576257	KF576306
D. camptothecicola	CFCC 51632	Camptotheca acuminata	China	KY228881	KY228887	KY228893
D. celastrina	CBS 139.27	Celastrus sp.	USA	KC343289	KC343773	KC344015
D. chensiensis	CFCC 52567	Abies chensiensis	China	MH121426	MH121544	MH121584
D. chensiensis	CFCC 52568	Abies chensiensis	China	MH121427	MH121545	MH121585
D. citri	AR3405	Citrus sp.	USA	KC843157	KC843071	KC843187
D. citrichinensis	ZJUD034	Citrus sp.	China	KC843234	KC843071	KC843187
D. citrichinensis	ZJUD034B	Citrus sp.	China	KJ435042	KJ210562	KJ420829
D. ellipicola	CGMCC 3.17084	Lithocarpus glabra	China	N/A^a	KF576245	KF576291
D. eres	AR5193	Ulmus laevis	Germany	KJ434999	KJ210550	KJ420799
	AR5196	Ulmus laevis	Germany	KJ435006	KJ210554	KJ420817
	DP0438	Ulmus minor	Austria	KJ435016	KJ210553	KJ420816
	LCM114.01a	Ulmus sp.	USA	KJ435027	KJ210545	KJ420787
	LCM114.01b	Ulmus sp.	USA	KJ435026	KJ210544	KJ420786
	FAU483	Malus sp.	Netherlands	KJ435022	JQ807422	KJ420827
	DAN001A	Daphne laureola	France	KJ434994	KJ210540	KJ420781
	DAN001B	Daphne laureola	France	KJ434995	KJ210541	KJ420782
	AR5197	Rhododendron sp.	Germany	KJ435014	KJ210552	KJ420812
	CBS 439.82	Cotoneaster sp.	UK	JX197429	GQ250341	JX275437
	AR3519	Corylus avellana	Austria	KJ435008	KJ210547	KJ420789
	FAU506	Cornus florida	USA	KJ435012	JQ807403	KJ420792
	FAU570	Oxydendrum arboreum	USA	KJ435025	JQ807410	KJ420794
	AR3723	Rubus fruticosus	Austria	KJ435024	JQ807354	KJ420793
	FAU522	Sassafras albida	USA	KJ435010	JQ807406	KJ420791
	DP0666	Juglans cinerea	USA	KJ435007	KJ210546	KJ420788
	DP0667	Juglans cinerea	USA	KC843155	KC843121	KC843229
	AR3560	Viburnum sp.	Austria	KJ435011	JQ807351	KJ420795
	AR5224	Hedera helix	Germany	KJ435036	KJ210551	KJ420802
	AR5231	Hedera helix	Germany	KJ435038	KJ210555	KJ420818
	AR5223	Acer nugundo	Germany	KJ435000	KJ210549	KJ420830
	CBS 109767	Acer sp.	Austria	KC343317	KC343801	KC344043
	DLR12a	Vitis vinifera	France	KJ434996	KJ210542	KJ420783
	DLR12b	Vitis vinifera	France	KJ434997	KJ210543	KJ420784
	AR4347	Vitis vinifera	Korea	KJ435030	JQ807356	KJ420805
	AR4355	Prunus sp.	Korea	KJ435035	JQ807359	KJ420797
	AR4367	Prunus sp.	Korea	KJ435019	JQ807364	KJ420824
	AR4346	Prunus mume	Korea	KJ435003	JQ807355	KJ420823
	AR4348	Prunus persici	Korea	KJ435004	JQ807357	JQ807357
	AR3669	Pyrus pyrifolia	Japan	KJ435002	JQ807415	KJ420808
D. eres	AR3670	Pyrus pyrifolia	Japan	KJ435001	JQ807416	KJ420807
	AR3671	Pyrus pyrifolia	Japan	KJ435017	JQ807417	KJ420814
	AR3672	Pyrus pyrifolia	Japan	KJ435023	JQ807418	KJ420819
	DP0591	Pyrus pyrifolia	New Zealand	KJ435018	JQ807395	KJ420821
	AR4369	Pyrus pyrifolia	Korea	KJ435005	JQ807366	KJ420813
	DP0180	Pyrus pyrifolia	New Zealand	KJ435029	JQ807384	KJ420804
	DP0179	Pyrus pyrifolia	New Zealand	KJ435028	JQ807383	KJ420803
	DP0590	Pyrus pyrifolia	New Zealand	KJ435037	JQ807394	KJ420810
	AR4373	Ziziphus jujuba	Korea	KJ435013	JQ807368	KJ420798
	AR4374	Ziziphus jujuba	Korea	KJ434998	JQ807369	KJ420785
	AR4357	Ziziphus jujuba	Korea	KJ435031	JQ807360	KJ420806
	AR4371	Malus pumila	Korea	KJ435034	JQ807367	KJ420796
	FAU532	Chamaecyparis thyoides	USA	KJ435015	JQ807408	KJ435015
	CBS 113470	Castanea sativa	Australia	KC343388	KC343872	KC344114
	AR4349	Vitis vinifera	Korea	KJ435032	JQ807358	KJ420822
	AR4363	Malus sp.	Korea	KJ435033	JQ807362	KJ420809
	CFCC 52575	Castanea mollissima	China	N/A^a	MH121552	MH121592
	CFCC 52576	Castanea mollissima	China	MH121432	MH121553	MH121593
	CFCC 52577	Acanthopanax senticosus	China	MH121433	MH121554	MH121594
	CFCC 52578	Sorbus sp.	China	MH121434	MH121555	MH121595
	CFCC 52579	Juglans regia	China	N/A^a	MH121556	N/A^a
	CFCC 52580	Melia azedarace	China	N/A^a	MH121557	MH121596
	CFCC 52581	Rhododendron simsii	China	N/A^a	MH121558	MH121597
D. helicis	AR5211	Hedera helix	France	KJ435043	KJ210559	KJ420828
D. longicicola	CGMCC 3.17089	Lithocarpus glabra	China	N/A^a	KF576242	KF576291
D. mahothocarpus	CGMCC 3.15181	Lithocarpus glabra	China	N/A^a	KC153087	KF576312
D. maritima	DAOMC 250563	Picea rubens	Canada	N/A^a	N/A^a	KU574616
D. momicola	MFLUCC 16-0113	Prunus persica	China	N/A^a	KU557631	KU55758
D. neilliae	CBS 144. 27	Spiraea sp.	USA	KC343386	KC343870	KC344112
D. padina	CFCC 52590	Padus racemosa	China	MH121443	MH121567	MH121604
D. padina	CFCC 52591	Padus racemosa	China	MH121444	MH121568	MH121605
D. phragmitis	CBS 138897	Phragmites australis	China	N/A^a	N/A^a	KP004507
D. pulla	CBS 338.89	Hedera helix	Yugoslavia	KC343394	KC343878	KC344120
D. vaccinii	DF5032	Vaccinium corymbosum	USA	KC849457	JQ807380	KC843225
	FAU633	Vaccinium macrocarpon	USA	KC849456	JQ807413	KC843226
	FAU446	Vaccinium macrocarpon	USA	KC849455	JQ807398	KC843224
	CBS 160.32	Vaccinium macrocarpon	USA	KC343470	GQ250326	JX270436
	FAU 468	Vaccinium macrocarpon	USA	KC849458	JQ807399	KC843227

Newly sequenced material is indicated in bold type.

Isolates and GenBank accession numbers used in the phylogenetic analyses of complex. Newly sequenced material is indicated in bold type. Bayesian inference (BI) analysis, employing a Markov chain Monte Carlo (MCMC) algorithm, was performed (Rannala and Yang 1996). MrModeltest v. 2.3 was used to estimate the best-fit model of nucleotide substitution model settings for each gene (Posada and Crandall 1998). Two MCMC chains started from random trees for 1,000,000 generations and trees were sampled every 100th generation, resulting in a total of 10,000 trees. The first 25% of trees were discarded as the burn-in phase of each analysis. Branches with significant Bayesian posterior probabilities (BPP) were estimated in the remaining 7500 trees. Sequences data were deposited in GenBank (Table 1). The multilocus sequence alignments were deposited in TreeBASE (www.treebase.org) as accession S22702 and S22703. The taxonomic novelties were deposited in MycoBank (Crous et al. 2004b).

Results

Collection of strains

Forty-two representative strains were isolated from 16 different host genera (Table 1) collected from six Provinces (Beijing, Heilongjiang, Jiangsu, Jiangxi, Shaanxi and Zhejiang) in China. All of these strains were isolated from symptomatic or non-symptomatic branches or twigs and preserved in the China Forestry Culture Collection Centre (CFCC). The first sequences dataset for the ITS, cal, his3, tef1, and tub2 was analysed in combination to infer the interspecific relationships within . The combined species phylogeny of the isolates consisted of 236 sequences, including the outgroup sequences of (culture CBS 121124). A total of 2948 characters including gaps (516 for ITS, 568 for cal, 520 for his3, 486 for tef1 and 858 for tub2) were included in the phylogenetic analysis. The maximum likelihood tree, conducted by the GTR model, confirmed the tree topology and posterior probabilities of the Bayesian consensus tree. For the Bayesian analyses, MrModeltest suggested that all partitions should be analysed with dirichlet state frequency distributions. The following models were recommended by MrModeltest and used: GTR+I+G for ITS, cal and his3, HKY+I+G for tef1 and tub2. The topology and branching order of ML were similar to BI analyses (Fig. 1). Based on the multi-locus phylogeny and morphology, 42 strains were assigned to 15 species, including 12 taxa which we describe here as new (Fig. 1).

Figure 1.

Phylogram of from a maximum likelihood analysis based on combined ITS, cal, his3, tef1 and tub2. Values above the branches indicate maximum likelihood bootstrap (left, ML BP ≥ 50%) and bayesian probabilities (right, BI PP ≥ 0.70). The tree is rooted with . Strains in the current study are in blue. The second dataset with cal, tef1 and tub2 sequences were analysed to focus on the complex. The alignment included 86 taxa, including the outgroup sequences of (Table 3). The aligned three-locus datasets included 1148 characters. Of these, 881 characters were constant, 105 variable characters were parsimony-uninformative and 162 characters were parsimony informative. The heuristic search using maximum parsimony (MP) generated 105 parsimonious trees (TL = 438, CI = 0.669, RI = 0.883, RC = 0.591), from which one was selected (Fig. 2). Based on the multi-locus phylogeny and morphology, seven strains were identified as , seven strains formed three distinct clades embedded in the complex, i.e. , and . MP and ML bootstrap support values above 50% are shown as first and second position, respectively. The branches with significant Bayesian posterior probability (≥ 0.70) in Bayesian analyses were thickened in the phylogenetic tree. The current results, based on the three genes (cal, tef1 and tub2), suggest that clade could be separated from other species in this complex (Fig. 2). However, (CGMCC 3.17081), (CFCC 51632), (CGMCC 3.17084), (CGMCC 3.17089), (CGMCC 3.15181) and (MFLUCC 16-0113) were clustered in clade and thus treated as the synonyms of in the current study.

Figure 2.

Phylogram of complex based on combined cal, tef1 and tub2. Values above the branches indicate maximum parsimony bootstrap (left, MP BP ≥ 50%) and maximum likelihood bootstrap (right, ML BP ≥ 50%). Values below branches represent posterior probabilities (BI PP ≥ 0.70) from Bayesian inference. The tree is rooted with . Strains in the current study are in blue. The ex-type/ex-epitype culture is in bold.

Taxonomy

C.M. Tian & Q. Yang sp. nov. MB824703 Figure 3

Figure 3.

(CFCC 52554) A Alpha conidia B–C Conidiophores D Culture on PDA and conidiomata. Scale bars: 20 μm (A–C), 200 μm (D).

Diagnosis.

can be distinguished from the phylogenetically closely related species in larger alpha conidia.

Holotype.

CHINA. Shaanxi Province: Qinling Mountain, on symptomatic twigs of , 27 June 2017, N. Jiang (holotype: BJFC-S1466; ex-type culture: CFCC 52554).

Etymology.

Named after the host genus on which it was collected, .

Description.

On PDA: Conidiomata pycnidial, globose, solitary or aggregated, deeply embedded in the medium, erumpent, dark brown to black, 185–270 μm diam, whitish translucent to cream conidial drops exuding from the ostioles. Conidiophores 14.5–17 × 1.4–2.9 μm, cylindrical, hyaline, phiailidic, branched, straight to sinuous. Alpha conidia 7–10 × 2.1–2.9 μm (av. = 8.6 × 2.5 μm, n = 30), aseptate, hyaline, ellipsoidal, rounded at one end, slightly apex at the other end, usually with two-guttulate. Beta conidia not observed. (CFCC 52554) A Alpha conidia B–C Conidiophores D Culture on PDA and conidiomata. Scale bars: 20 μm (A–C), 200 μm (D).

Culture characters.

Cultures incubated on PDA at 25 °C in darkness. Colony at first white, becoming dark brown in the centre with age. Aerial mycelium white, dense, fluffy, with cream conidial drops exuding from the ostioles.

Additional specimens examined.

CHINA. Shaanxi Province: Qinling Mountain, on symptomatic twigs of , 27 June 2017, N. Jiang, living culture CFCC 52555 (BJFC-S1467).

Notes.

Two strains representing cluster in a well-supported clade and appear most closely related to . can be distinguished from based on ITS, his3, tef1 and tub2 loci (5/469 in ITS, 8/429 in his3, 8/326 in tef1 and 5/358 in tub2). Morphologically, differs from in the longer and larger alpha conidia (8.6 × 2.5 vs. 6.6 × 1.9 μm) (Gao et al. 2016). C.M. Tian & Q. Yang sp. nov. MB824704 Figure 4

Figure 4.

(CFCC 52556) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B–C), 10 μm (D–E).

can be distinguished from the phylogenetically closely related species and by the size of conidiophores and alpha conidia. CHINA. Zhejiang Province: Tianmu Mountain, on symptomatic branches of , 19 Apr. 2017, Q. Yang (holotype: BJFC-S1468; ex-type culture: CFCC 52556). Named after the host genus on which it was collected, . Conidiomata pycnidial, immersed in bark, scattered, erumpent through the bark surface, discoid, with a solitary undivided locule. Ectostromatic disc black, one ostiole per disc, 135–330 μm diam. Locule circular, undivided, 290–445 μm diam. Conidiophores 6–12 × 1.4–2 μm, cylindrical, hyaline, phiailidic, unbranched, straight. Alpha conidia 6.5–8 × 2 μm (av. = 7 × 2 μm, n = 30), aseptate, hyaline, ellipsoidal, biguttulate, mostly with one end obtuse and the other acute, occasionally submedian constriction. Beta conidia not observed. (CFCC 52556) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B–C), 10 μm (D–E). Cultures incubated on PDA at 25 °C in darkness. Colony initially white, producing beige pigment after 7–10 d. The colony is flat, felty with a thick texture at the centre and marginal area, with thin texture in the middle, lacking aerial mycelium, conidiomata absent. CHINA. Zhejiang Province: Tianmu Mountain, on symptomatic branches of , 19 Apr. 2017, Q. Yang, living culture CFCC 52557 (BJFC-S1469); ibid. living culture CFCC 52558 (BJFC-S1470); ibid. living culture CFCC 52559 (BJFC-S1471). Four isolates clustered in a clade distinct from its closest phylogenetic neighbour, and can be distinguished from in cal, tef1 and tub2 loci (6/458 in cal, 4/308 in tef1 and 11/407 in tub2); from in ITS, tef1 and tub2 loci (6/462 in ITS, 8/308 in tef1 and 10/701 in tub2). Morphologically, differs from in shorter conidiophores (6–12 vs. 11–18 μm) and longer alpha conidia (6.5–8 vs. 5.5–6 μm); from in shorter conidiophores (6–12 vs. 15–20 μm) (Crous et al. 2015, Doilom et al. 2017). C.M. Tian & Q. Yang sp. nov. MB824705 Figure 5

Figure 5.

(CFCC 52562) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Conidiophores E Alpha conidia F Beta conidia G Culture on PDA and conidiomata. Scale bars: 500 μm (A–C), 10 μm (D–F).

can be distinguished from the phylogenetically closely related species in smaller locule and wider alpha conidia. CHINA. Heilongjiang Province: Yichun city, on symptomatic branches of , 27 July 2016, Q. Yang (holotype: BJFC-S1472; ex-type culture: CFCC 52562). Named after the host genus on which it was collected, . Conidiomata pycnidial, conical, immersed in bark, scattered, erumpent through the bark surface, with a solitary undivided locule. Ectostromatic disc brown to black, one ostiole per disc, 290–645 μm diam. Ostiole medium black, up to the level of disc. Locule undivided, 670–905 μm diam. Conidiophores 12.5–17.5 × 1.5–2 μm, cylindrical, hyaline, phiailidic, branched, straight or slightly curved. Alpha conidia hyaline, aseptate, ellipsoidal to fusiform, 0–2-guttulate, sometimes acute at both ends, 8–10 × 2.5–3 μm (av. = 9 × 2.6 μm, n = 30). Beta conidia hyaline, aseptate, filiform, straight or hamate, eguttulate, base subtruncate, tapering towards one apex, 26–32.5 × 1 µm (av. = 30 × 1 µm, n = 30). (CFCC 52562) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Conidiophores E Alpha conidia F Beta conidia G Culture on PDA and conidiomata. Scale bars: 500 μm (A–C), 10 μm (D–F). Cultures incubated on PDA at 25 °C in darkness. Colony flat with white felty aerial mycelium, turning white to dark brown aerial mycelium, conidiomata irregularly distributed on the agar surface. CHINA. Heilongjiang Province: Yichun city, on symptomatic branches of , 27 July 2016, Q. Yang, living culture CFCC 52560 (BJFC-S1473); on symptomatic branches of , 27 July 2016, Q. Yang, living culture CFCC 52561 (BJFC-S1474). was isolated from spp. cankers in Heilongjiang Province. Three strains representing cluster in a well-supported clade and appear most closely related to , which was also isolated from in Sichuang Province (Du et al. 2016). can be distinguished based on ITS, his3, tef1 and tub2 loci from (11/461 in ITS, 9/453 in his3, 12/336 in tef1 and 7/695 in tub2). Morphologically, differs from in smaller locule (470–945 vs. 600–1250 μm) and wider alpha conidia (3–4 vs. 2.5–3 μm) (Du et al. 2016). F. Huang, K.D. Hyde & H.Y. Li, 2015 Figure 6

Figure 6.

(CFCC 52584) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B–C), 10 μm (D–E).

Conidiomata pycnidial, immersed in bark, scattered, erumpent through the bark surface, discoid, with a single locule. Ectostromatic disc dark brown, one ostiole per disc, 160–320 μm diam. Locule undivided, 235–350 μm diam. Conidiophores 8.5–11 × 1.5 μm, cylindrical, hyaline, branched, straight or slightly curved, tapering towards the apex. Alpha conidia hyaline, aseptate, ellipsoidal to oval, 2-guttulate, usually rounded at both ends, occasionally with one end acute, 7–8.5 × 1.5–2 μm (av. = 6.5 × 2.6 μm, n = 30). Beta conidia not observed. (CFCC 52584) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B–C), 10 μm (D–E). Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming pale grey, with dense aerial mycelium in the centre and sparse aerial mycelium at the marginal area, conidiomata absent.

Specimens examined.

CHINA. Zhejiang Province: Tianmu Mountain, on symptomatic branches of , 20 Apr. 2017, Q. Yang, living culture CFCC 52584 and CFCC 52585 (BJFC-S1504). was originally described from a healthy branch of in Yunnan Province, China (Huang et al. 2015). In the present study, two isolates (CFCC 52584 and CFCC 52585) from symptomatic branches of were congruent with based on morphology and DNA sequences data (Fig. 1). We therefore describe as a known species for this clade. C.M. Tian & Q. Yang sp. nov. MB824706 Figure 7

Figure 7.

(CFCC 52563) A Transverse section of conidioma B Longitudinal section of conidioma C Culture on PDAD Alpha conidia E Conidiophores F Beta conidia. Scale bars: 200 μm (A), 100 μm (B), 10 μm (D, F), 20 μm (E).

differs from its closest phylogenetic neighbour, and , in ITS, tef1 and tub2 loci based on the alignments deposited in TreeBASE. CHINA. Jiangsu Province: Nanjing city, on symptomatic twigs of , 10 Nov. 2015, Q. Yang (holotype: BJFC-S1476; ex-type culture: CFCC 52563). Named after the host genus on which it was collected, . Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, nearly flat, discoid, with a solitary undivided locule. Ectostromatic disc brown to black, one ostiole per disc. Locule undivided, 310–325 μm diam. Conidiophores 7–11 × 1.4–2.2 μm, cylindrical, phialidic, unbranched, sometimes inflated. Alpha conidia hyaline, aseptate, ellipsoidal or fusiform, eguttulate, obtuse at both ends, 7–8.5 × 2.1–2.5 μm (av. = 8 × 2.3 μm, n = 30). Beta conidia hyaline, aseptate, filiform, straight or hamate, eguttulate, base subtruncate, tapering towards one apex, 15.5–34 × 1.1–1.4 µm (av. = 27.5 × 1.2 µm, n = 30). (CFCC 52563) A Transverse section of conidioma B Longitudinal section of conidioma C Culture on PDAD Alpha conidia E Conidiophores F Beta conidia. Scale bars: 200 μm (A), 100 μm (B), 10 μm (D, F), 20 μm (E). Cultures incubated on PDA at 25 °C in darkness. Colony at first flat with white felty mycelium, becoming black in the centre and black at the marginal area with age, conidiomata not observed. CHINA. Jiangsu Province: Nanjing city, on symptomatic twigs of , 10 Nov. 2015, Q. Yang, living culture CFCC 52564 (BJFC-S1477). Two strains representing cluster in a well-supported clade and appear closely related to and can be distinguished based on ITS, tef1 and tub2 loci from (50/468 in ITS, 107/338 in tef1 and 90/707 in tub2); from (4/440 in ITS, 13/340 in tef1 and 23/701 in tub2). Morphologically, can be distinguished from by its shorter conidiophores (7–11 vs. 15–35 μm); from by its longer alpha conidia (7–8.5 vs. 6–7 μm) (Thompson et al. 2015). C.M. Tian & Q. Yang sp. nov. MB824707 Figure 8

Figure 8.

(CFCC 52565) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Beta conidia F Conidiophores G Culture on PDA and conidiomata. Scale bars: 100 μm (B–C), 10 μm (D–F).

can be distinguished from the phylogenetically closely related species in larger alpha conidia. CHINA. Jiangsu Province: Nanjing city, on twigs and branches of , 11 Nov. 2015, Q. Yang (holotype: BJFC-S1478; ex-type culture: CFCC 52565). Named after the host genus on which it was collected, . Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, nearly flat, discoid, with a solitary undivided locule. Ectostromatic disc grey to brown, one ostiole per disc. Locule circular, undivided, 135–200 μm diam. Conidiophores 7–17 × 1.4–2.1 μm, phialidic, unbranched, straight or slightly curved, tapering towards the apex. Alpha conidia hyaline, aseptate, fusiform to oval, biguttulate, 6.5–10 × 3–3.5 μm (av. = 8.6 × 3.3 μm, n = 30). Beta conidia hyaline, aseptate, filiform, straight or hamate, eguttulate, 20–28.5 × 1–1.3 µm (av. = 25.5 × 1.2 µm, n = 30). (CFCC 52565) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Beta conidia F Conidiophores G Culture on PDA and conidiomata. Scale bars: 100 μm (B–C), 10 μm (D–F). Cultures incubated on PDA at 25 °C in darkness showed colony at first white, becoming pale brown with yellowish dots with age, flat, with dense and felted mycelium, with visible solitary or aggregated conidiomata at maturity. CHINA. Jiangsu Province: Yangzhou city, on twigs and branches of , 11 Nov. 2015, N. Jiang, living culture CFCC 52566 (BJFC-S1479). is distinguished from in the ITS, cal and tef1 loci (13/458 in ITS, 47/442 in cal and 6/328 in tef1). Morphologically, differs from in shorter conidiophores (7–17 vs. 21–35 μm) and larger alpha conidia (6.5–10 × 3–3.5 vs. 6–8.5 × 2–3 μm) (Dissanayake et al. 2017a). C.M. Tian & Q. Yang sp. nov. MB824708 Figure 9

Figure 9.

(CFCC 52567) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha conidia F Beta conidia G Conidiophores H Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C–D), 10 μm (E), 20 μm (F).

differs from its closest phylogenetic neighbour, , in ITS, cal, his3 and tef1 loci based on the alignments deposited in TreeBASE. CHINA. Shaanxi Province: Ningshan County, Huoditang forest farm, on symptomatic twigs of , 5 July 2017, Q. Yang (holotype: BJFC-S1480; ex-type culture: CFCC 52567). Named after the host species on which it was collected, . Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, discoid, with a single locule. Ectostromatic disc white to brown, one ostiole per disc, 200–325 μm diam. Locule undivided, 385–540 μm diam. Conidiophores 8.5–13 × 2–3 μm, cylindrical, hyaline, phiailidic, unbranched, straight or slightly curved, tapering towards the apex. Alpha conidia hyaline, aseptate, smooth, ellipsoidal, biguttulate, rounded at both ends, 6.5–11 × 2–2.2 μm (av. = 8.5 × 2.1 μm, n = 30). Beta conidia present on the host, hyaline, eguttulate, smooth, filiform, hamate, 21–28.5 × 0.8–1.1 μm (av. = 25 × 1 μm, n = 30). (CFCC 52567) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha conidia F Beta conidia G Conidiophores H Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C–D), 10 μm (E), 20 μm (F). Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white felted aerial mycelium, becoming light brown mycelium due to pigment formation, conidiomata irregularly distributed over agar surface, with yellowish conidial drops exuding from the ostioles. CHINA. Shaanxi Province: Ningshan County, Huoditang forest farm, on symptomatic twigs of , 5 July 2017, Q. Yang, living culture CFCC 52568 (BJFC-S1481). occurs in an independent clade (Fig. 1) and is phylogenetically distinct from . can be distinguished from by 57 nucleotides in concatenated alignment, in which 14 were distinct in the ITS region, 13 in the cal region, 10 in the his3 region, 15 in the tef1 region and 15 in the tub2 region. Although this species belongs to the complex, it is, however, distinct from the known species within the complex (Fig. 2). C.M. Tian & Q. Yang sp. nov. MB824709 Figure 10

Figure 10.

(CFCC 52569) A Culture on PDAB Conidiomata C Alpha conidia D Conidiophores. Scale bars: 200 μm (B), 10 μm (C–D).

differs from its closest phylogenetic species in ITS, his3 and tef1 loci based on the alignments deposited in TreeBASE. CHINA. Zhejiang Province: Linan city, on symptomatic twigs of sp., 22 Apr. 2017, Q. Yang (holotype: BJFC-S1482; ex-type culture: CFCC 52569). Named after the host genus on which it was collected, . On PDA: Conidiomata pycnidial, globose, solitary or aggregated, deeply embedded in the substrate, erumpent, dark brown to black, 170–235 μm diam., whitish translucent to cream conidial drops exuding from the ostioles. Conidiophores 11–25 × 1.5–2 μm, cylindrical, hyaline, branched, straight or curved, tapering towards the apex. Alpha conidia hyaline, aseptate, ellipsoidal to oval, biguttulate, rounded at both ends, 5–7 × 2.5–3 μm (av. = 6 × 2.9 μm, n = 30). Beta conidia not observed. (CFCC 52569) A Culture on PDAB Conidiomata C Alpha conidia D Conidiophores. Scale bars: 200 μm (B), 10 μm (C–D). Cultures incubated on PDA at 25 °C in darkness showed colony originally flat with white felty mycelium, developing petaloid mycelium after 7–10 d and turning yellowish at the centre and brownish at the marginal area after 15 d. Conidiomata erumpent at maturity.

Additional material examined.

CHINA. Zhejiang Province: Linan city, on symptomatic twigs of sp., 22 Apr. 2017, Q. Yang, living culture CFCC 52570 (BJFC-S1483). comprises strains CFCC 52569 and CFCC 52570 closely related to in the combined phylogenetic tree (Fig. 1). can be distinguished based on ITS, his3 and tef1 loci from (4/460 in ITS, 17/448 in his3 and 38/339 in tef1). C.M. Tian & Q. Yang sp. nov. MB824710 Figure 11

Figure 11.

(CFCC 52571) A–B Habit of conidiomata on branches C Longitudinal section of conidioma D Alpha conidia E–F Conidiophores G Culture on PDA and conidiomata. Scale bars: 300 μm (B–C), 10 μm (D–F).

is phylogenetically and morphologically distinct from , in smaller locule and alpha conidia. CHINA. Zhejiang Province: Tianmu Mountain, on symptomatic branches of , 20 Apr. 2017, Q. Yang (holotype: BJFC-S1484; ex-type culture: CFCC 52571). Named after the conical conidiomata. Conidiomata pycnidial, 420–580 μm diam., solitary and with single necks erumpent through the host bark. Tissue around the neck is conical. Locule oval, undivided, 385–435 μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells unbranched, straight or sinuous, apical or base sometimes swelling, 19–23.5 × 2.8 μm. Alpha conidia hyaline, aseptate, ellipsoidal, biguttulate, 5.5–7 × 2.3–3 μm (av. = 6.5 × 2.6 μm, n = 30). Beta conidia not observed. (CFCC 52571) A–B Habit of conidiomata on branches C Longitudinal section of conidioma D Alpha conidia E–F Conidiophores G Culture on PDA and conidiomata. Scale bars: 300 μm (B–C), 10 μm (D–F). Cultures incubated on PDA at 25 °C in darkness. Colony white to yellowish, with dense and felted mycelium, lacking aerial mycelium, with maize-coloured conidial drops exuding from the ostioles. CHINA. Zhejiang Province: Tianmu Mountain, on symptomatic branches of , 20 Apr. 2017, Q. Yang, living culture CFCC 52572 (BJFC-S1485); ibid. living culture CFCC 52573 (BJFC-S1486); ibid. living culture CFCC 52574 (BJFC-S1487). Four isolates clustered in a clade distinct from further species based on DNA sequence data. Morphologically, this species is characterised by conical conidiomata, which is similar with from . However, differs from by having smaller locule and alpha conidia (310–385 vs. 620–1100 μm in locule; 5.5–7 × 2.3–3 vs. 8.5–11.5 × 4–5 μm in alpha conidia) (Fan et al. 2015). Nitschke, 1870 Figure 12

Figure 12.

(CFCC 52575) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha conidia F Conidiophores G Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C–D), 10 μm (E–F).

= = = = = = Conidiomata pycnidial, immersed in bark, erumpent through the bark surface, serried, with a single locule. Ectostromatic disc obviously, brown to black, with one ostiole per disc, 245–572 μm diam. Ostiole medium black, up to the level of disc. Locule circular, undivided, 335–450 μm diam. Conidiophores 10.5–19 × 1–1.5 μm, cylindrical, hyaline, unbranched, straight or slightly sinuous. Conidiogenous cells phialidic, cylindrical, terminal. Alpha conidia hyaline, aseptate, ellipsoidal to lanceolate, one guttulate at each end, 6–7.5 × 1.5–2.5 μm (av. = 6.5 × 2 μm, n = 30). Beta conidia not observed. (CFCC 52575) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha conidia F Conidiophores G Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C–D), 10 μm (E–F). Cultures on PDA incubated at 25 °C in darkness. Colony with white felty aerial mycelium, becoming white felted aerial mycelium in the centre and grey-brown mycelium at the marginal area, conidiomata irregularly distributed over agar surface. CHINA. Beijing: Pinggu district, on symptomatic branches of , 1 Nov. 2016, N. Jiang, living culture CFCC 52576 (BJFC-S1489); ibid. living culture CFCC 52577 (BJFC-S1490). Heilongjiang Province: Liangshui Nature Reserve, on symptomatic twigs of , 29 July 2016, Q. Yang, living culture CFCC 52580 (BJFC-S1493). Heilongjiang Province: Harbin city, Botanical garden, on symptomatic twigs of sp., 2 Aug. 2016, Q. Yang, living culture CFCC 52575 (BJFC-S1488). Shaanxi Province: Zhashui County, on symptomatic branches of , 29 July 2016, Q. Yang, living culture CFCC 52579 (BJFC-S1492). Zhejiang Province: Yangzhou city, on symptomatic twigs of , 8 July 2017, N. Jiang, living culture CFCC 52578 (BJFC-S1491). Zhejiang Province: Tianmu Mountain, on symptomatic twigs of , 20 Apr. 2017, Q. Yang, living culture CFCC 52581 (BJFC-S1494). , the type species of the genus, was described by Nitschke (1870) on sp. collected in Germany, which has a widespread distribution and a broad host range as a pathogen, endophyte or saprobe causing leaf spots, stem cankers and diseases of woody plants (Udayanga et al. 2014b). Fan et al. (2018) indicated that , , and should be treated as synonyms of using cal, tef1 and tub2 gene regions. In this study, we extended the work presented in Fan et al. (2018) and found seven additional strains belonging to . Additionally, the phylogenetic tree demonstrated that and should also be treated as synonyms of (Fig. 2). from and from are described and illustrated based on the combined ITS, cal, his3, tef1 and tub2 regions (Dissanayake et al. 2017a, Yang et al. 2017c). Both of the two species are embedded in the complex. However, ITS analysis resulted in an unresolved phylogenetic tree without definitive bootstrap at the internodes, highly discordant to the trees resulting from the other four genes (Udayanga et al. 2014b). Therefore, the ITS region was not used in the combined analysis in the current study. To further investigate this complex, a second set of four (cal, his3, tef1 and tub2), three (cal, tef1 and tub2), two (tef1 and tub2) and one (tef1) data matrices were performed following Santos et al. (2017) and Fan et al. (2018). The results showed that the three genes analyses (cal, tef1 and tub2) appeared to be a better species recognition (Fig. 2). When it comes to this species complex, sequences supported by Udayanga et al. (2014b) are necessary to perform a more robust phylogenetic tree, clarifying the real species boundaries in this group in the future work. C.M. Tian & Q. Yang sp. nov. MB824711 Figure 13

Figure 13.

(CFCC 52582) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha conidia F Beta conidia G Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C), 100 μm (D), 10 μm (E–F).

can be distinguished from the closely related species and (described above) based on ITS, tef1 and tub2 loci. differs from in larger alpha conidia and from in wider alpha conidia. CHINA. Shaanxi Province: Zhashui city, Niubeiliang Reserve, on symptomatic twigs of , 7 July 2017, Q. Yang (holotype: BJFC-S1495; ex-type culture: CFCC 52582). Named after the host genus on which it was collected, . Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, nearly flat, discoid, with a single locule. Ectostromatic disc grey to dark brown, circular to ovoid, one ostiole per disc, 150–325 μm diam. Locule circular, undivided, 275–480 μm diam. Conidiophores 10.5–17.5 × 2.1–3.2 μm, hyaline, branched, cylindrical to clavate, straight, tapering towards the apex. Alpha conidia hyaline, aseptate, ellipsoidal to oval, 2–3-guttulate, rounded at both ends, 7–10 × 2.9–3.2 μm (av. = 8.5 × 3 μm, n = 30). Beta conidia hyaline, filiform, straight or hamate, eguttulate, aseptate, base subtruncate, tapering towards one apex, 19–29.5 × 1.4 µm (av. = 24.5 × 1.4 µm, n = 30). (CFCC 52582) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha conidia F Beta conidia G Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C), 100 μm (D), 10 μm (E–F). Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming yellowish, dense and felted aerial mycelium with age, with visible solitary or aggregated conidiomata at maturity. CHINA. Shaanxi Province: Zhashui city, Niubeiliang Reserve, on symptomatic twigs of , 7 July 2017, Q. Yang, living culture CFCC 52583 (BJFC-S1496). This new species is introduced as molecular data, shows it to be a distinct clade with high support (ML/BI=100/1) and it appears most closely related to and . can be distinguished from by 22 nucleotides in concatenated alignment, in which 6 were distinct in the ITS region, 8 in the tef1 region and 8 in the tub2 region; from by 27 nucleotides in concatenated alignment, in which 11 were distinct in the ITS region, 3 in the tef1 region and 13 in the tub2 region. Morphologically, differs from in longer and larger alpha conidia (7–10 × 2.9–3.2 vs. 5.5–7.5 × 0.5–2 μm); differs from in larger alpha conidia (2.9–3.2 vs. 2.1–2.9 μm) (Gao et al. 2016). C.M. Tian & Q. Yang sp. nov. MB824713 Figure 14

Figure 14.

(CFCC 52586) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B–C), 10 μm (D–E).

differs from its closest phylogenetic species and in ITS, cal and tef1 loci based on the alignments deposited in TreeBASE. CHINA. Jiangxi Province: Shangrao city, Sanqing Mountain, on symptomatic branches of , 1 Apr. 2017, B. Cao, Y.M. Liang & C.M. Tian (holotype: BJFC-S1497; ex-type culture: CFCC 52586). Named after the host genus on which it was collected, . Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, nearly flat, discoid, with a single locule. Ectostromatic disc obviously, brown to black, one ostiole per disc. Locule undivided, 475–525 μm diam. Conidiophores 7–11 × 1.8–2.9 μm, cylindrical, hyaline, unbranched, straight or slightly curved, tapering towards the apex. Alpha conidia hyaline, aseptate, oval or fusoid, biguttulate, 5.5–7.5 × 2.1–2.9 μm (av. = 6.5 × 2.5 μm, n = 30). Beta conidia not observed. (CFCC 52586) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B–C), 10 μm (D–E). Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming dense and felted aerial mycelium in the centre and grey to black mycelium at the marginal area with solitary conidiomata at maturity. CHINA. Jiangxi Province: Shangrao city, Sanqing Mountain, on symptomatic branches of , 1 Apr. 2017, B. Cao, Y.M. Liang & C.M. Tian, living culture CFCC 52587 (BJFC-S1498); Yunbifeng National Forest Park, on symptomatic twigs of sp., 31 Mar. 2017, B. Cao, Y.M. Liang & C.M. Tian, living culture CFCC 52588 (BJFC-S1499); ibid. living culture CFCC 52589 (BJFC-S1500). This new species is introduced as molecular data show it to be a distinct clade with high support (ML/BI=100/1) and it appears most closely related to and . can be distinguished from by 11 nucleotides in concatenated alignment, in which 4 were distinct in the ITS region and 7 in the cal region; from by 25 nucleotides in concatenated alignment, in which 12 were distinct in the ITS region, 6 in the cal region and 7 in the tef1 region. Morphologically, differs from and in shorter conidiophores (7–11 μm in vs. 11–24.1 μm in ; 7–11 μm in vs. 14.2–23.6 μm in ) (Gao et al. 2014). C.M. Tian & Q. Yang sp. nov. MB824714 Figure 15

Figure 15.

(CFCC 52590) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha and beta conidia F, I Beta conidia G–H Conidiophores J Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C–D), 10 μm (E–I).

can be distinguished from the phylogenetically closely related species in smaller conidiomata and alpha conidia. CHINA. Heilongjiang Province: Liangshui Nature Reserve, on symptomatic twigs of , 31 July 2016, Q. Yang (holotype: BJFC-S1501; ex-type culture: CFCC 52590). Named after the host genus on which it was collected, . Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, discoid, with a single locule. Ectostromatic disc light brown, one ostiole per disc, 330–520 μm diam. Locule circular, undivided, 250–550 μm diam. Conidiophores 5.5–12.5 × 1–1.5 μm, hyaline, unbranched, cylindrical, straight or slightly curved. Alpha conidia hyaline, aseptate, ellipsoidal to fusiform, eguttulate, 7–8 × 1.5–2 μm (av. = 7.5 × 1.8 μm, n = 30). Beta conidia hyaline, filiform, straight or hamate, eguttulate, aseptate, base truncate, 21–24 × 1 µm (av. = 22 × 1 µm, n = 30). (CFCC 52590) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha and beta conidia F, I Beta conidia G–H Conidiophores J Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C–D), 10 μm (E–I). Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming grey to brown in the centre, with pale grey, felted, valviform mycelium at the marginal area and aggregated conidiomata at maturity. CHINA. Heilongjiang Province: Liangshui Nature Reserve, on symptomatic twigs of , 31 July 2016, Q. Yang, living culture CFCC 52591 (BJFC-S1502). Four strains representing cluster in a well-supported clade and appear closely related to . This species is phylogenetically closely related to, but clearly differentiated from, by 40 different unique fixed alleles in ITS, cal, his3, tef1 and tub2 loci (4, 7, 10, 13 and 6 respectively) based on the alignments deposited in TreeBASE. Morphologically, differs from in smaller conidiomata and alpha conidia (250–550 vs. 600–1250 μm in conidiomata; 7–8 × 1.5–2 vs. 8.5–11 × 3–4 μm in alpha conidia) (Du et al. 2016). C.M. Tian & Q. Yang sp. nov. MB824715 Figure 16

Figure 16.

(CFCC 52592) A Habit of conidiomata on branches B Transverse section of conidioma C–D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B), 10 μm (C–E).

can be distinguished from the phylogenetically closely related species in longer conidiophores and shorter alpha conidia. CHINA. Shaanxi Province: Qinling Mountain, on symptomatic twigs of , 27 June 2017, Q. Yang (holotype: BJFC-S1503; ex-type culture: CFCC 52592). Named after the host species on which it was collected, . Conidiomata pycnidial, immersed in bark, serried, slightly erumpent through the bark surface, nearly flat, discoid, with a single locule. Ectostromatic disc dark brown to black, one ostiole per disc. Locule circular, undivided, 165–215 μm diam. Conidiophores 11.5–18 × 1.5 μm, hyaline, branched, cylindrical, straight or curved. Alpha conidia hyaline, aseptate, ellipsoidal to oval, biguttulate, 5–6 × 2.1–2.9 μm (av. = 5.5 × 2.5 μm, n = 30). Beta conidia not observed. (CFCC 52592) A Habit of conidiomata on branches B Transverse section of conidioma C–D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B), 10 μm (C–E). Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming brown to pale black in the centre, dense, felted, conidiomata not observed. CHINA. Shaanxi Province: Qinling Mountain, on symptomatic twigs of , 27 June 2017, Q. Yang, living culture CFCC 52593 (BJFC-S1503). comprises strains CFCC 52592 and CFCC 52593 closely related to in the combined phylogenetic tree (Fig. 1). can be distinguished from based on ITS and tef1 loci (10/470 in ITS and 4/336 in tef1). F. Huang, K.D. Hyde & H.Y. Li, 2015 Figure 17

Figure 17.

(CFCC 52594) A Culture on PNA B Conidiomata C Alpha conidia D Conidiophores. Scale bars: 500 μm (B), 10 μm (C–D).

On PNA: Conidiomata pycnidial, globose or rostrated, black, erumpent in tissue, erumpent at maturity, 260–500 μm diam, often with translucent conidial drops exuding from the ostioles. Conidiophores 18–28.5 × 1.4–2.1 μm, cylindrical, hyaline, branched, septate, straight or curved, tapering towards the apex. Alpha conidia abundant in culture, hyaline, aseptate, ellipsoidal to fusiform, biguttulate, sometimes with one end obtuse and the other acute, 6.5–8.5 × 2.1–2.5 μm (av. = 7.8 × 2.3 μm, n = 30). Beta conidia not observed. (CFCC 52594) A Culture on PNA B Conidiomata C Alpha conidia D Conidiophores. Scale bars: 500 μm (B), 10 μm (C–D). Cultures incubated on PNA at 25 °C in darkness. Colony entirely white at surface, reverse with pale brown pigmentation, white, fluffy aerial mycelium. CHINA. Jiangsu Province: Nanjing city, on non-symptomatic twigs of , 10 Nov. 2015, Q. Yang, living culture CFCC 52594 and CFCC 52595 (BJFC-S1476). was originally described from twigs of non-symptomatic in Zhejiang Province, China (Huang et al. 2015). In the present study, two isolates from twigs of asymptomatic were congruent with based on morphology and DNA sequences data (Fig. 1). We therefore describe as a known species for this clade.

Discussion

The current study described 15 species from 42 strains based on a large set of freshly collected specimens. It includes 12 new species and 3 known species, which were sampled from 16 host genera distributed over six Provinces of China (Table 1). In this study, 194 reference sequences (including outgroup) were selected based on BLAST searches of NCBIs GenBank nucleotide database and included in the phylogenetic analyses (Table 1). Phylogenetic analyses based on five combined loci (ITS, cal, his3, tef1 and tub2), as well as morphological characters, revealed the diversity of species in China, mainly focusing on diebacks from major ecological or economic forest trees. Several studies have been conducted associated with various hosts in China. For instance, the research conducted by Huang et al. (2015) revealed seven apparently undescribed endophytic species on . Gao et al. (2016) demonstrated that isolates, associated with spp., could be assigned to seven species and two species complexes. Recently, e has been revealed as paraphyletic by Gao et al. (2017), showing that , , and embed in s. lat. and eight new species of were introduced from leaves of several hosts. However, the identification of species associated with dieback of forest trees has rarely been studied, thus a large-scale investigation of spp. was conducted from 2015 to 2017. This study provides the first molecular phylogenetic frame of diversity associated with dieback in China, combined with morphological descriptions. , the type species of the genus, was initially described by Nitschke (1870), from sp. collected in Germany. The major problem with this generic type was the lack of an ex-type culture or ex-epitype culture, although a broad species concept has historically been associated with (Udayanga et al. 2014b). Udayanga et al. (2014b) designed strain AR5193 as the epitype of and provided the phylogram of this complex using seven loci (ITS, act, Apn2, cal, his3, FG1093, tef1 and tub2), amongst which the tef1, Apn2 and his3 genes were recognised as the best markers for defining species in the complex. Moreover, they showed that poorly supported non-monophyletic grouping was observed when ITS sequences were included in the combined analysis. In this study, although we conducted phylogenetic analysis as performed in previous studies on species (Santos et al. 2017), much confusion has, however, occurred in species separation of the complex (Fig. 1). Especially, the ITS region could lead to a confused taxonomic situation within this species complex. We found the three-gene analysis, excluding the ITS and his3 regions, resulted in a more robust tree congruent with Udayanga et al. (2014b) and resolved the species boundaries within the species complex. The isolates, clustering with in this study, occur on multiple hosts from many different geographic locations. This study revealed three new species belonging to the complex, i.e. , and . It also shows , , , , and were clustered in and should be treated as synonyms of , which is in conformity with Fan et al. (2018). The initial species concept of , based on the assumption of host-specificity, resulted in the introduction of more than 1000 taxa (http://www.indexfungorum.org/). Thus, during the past decade, a polyphasic approach, employing multi-locus DNA data together with morphology and ecology, has been employed for species boundaries in the genus (Crous et al. 2012, Udayanga et al. 2014a, b, Huang et al. 2015, Gao et al. 2016, 2017, Guarnaccia and Crous 2017, 2018, Hyde et al. 2017, 2018, Yang et al. 2017a, b, 2018, Guarnaccia et al. 2018, Jayawardena et al. 2018, Perera et al. 2018a, b, Tibpromma et al. 2018, Wanasinghe et al. 2018). Further studies are required in order to conduct an extensive collection of isolates, to resolve taxonomic questions and to redefine species boundaries. Multiple strains from different locations should also be subjected to multi-gene phylogenetic analysis to determine intraspecific variation. The descriptions and molecular data of species provided in this study represent a resource for plant pathologists, plant quarantine officials and taxonomists for identification of .

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