Three new Diaporthe species from Shaanxi Province, China.

Diaporthe species (Sordariomycetes, Diaporthales) are often reported as important plant pathogens, saprobes and endophytes on a wide range of plant hosts. In this study, Diaporthe specimens were collected from symptomatic twigs and branches at the Huoditang Forest Farm in Shaanxi Province, China. Identification was done using a combination of morphology and comparison of DNA sequence data of the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions. Three new Diaporthe species are proposed: D. albosinensis, D. coryli and D. shaanxiensis. All species are illustrated and their morphology and phylogenetic relationships with other Diaporthe species are discussed. Qin Yang, Ning Jiang, Cheng-Ming Tian.

Introduction

species (, ) are associated with a wide range of plant hosts as pathogens, endophytes or saprobes of crops, ornamentals and forest trees (Murali et al. 2006, Rossman et al. 2007, Garcia-Reyne et al. 2011, Gomes et al. 2013, Udayanga et al. 2015, Dissanayake et al. 2017, Guarnaccia and Crous 2017, 2018, Wijayawardene et al. 2017, Yang et al. 2017a, b, 2018, Fan et al. 2018, Guarnaccia et al. 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, 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, Gomes et al. 2013).

Species identification in has traditionally been based on host association, morphology and culture characteristics (Mostert et al. 2001, Santos and Phillips 2009, Udayanga et al. 2011), resulting in the description of over 200 species (Hyde et al. 2020). Multiple species of can colonise a single host and 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 within-species variability of phenotypic characters has been reported (Rehner and Uecker 1994, Mostert et al. 2001, Udayanga et al. 2011). Thus, a polyphasic taxonomic approach, based on multi-locus DNA data, morphology and ecology, has been increasingly employed for species boundaries in the genus (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, Guarnaccia et al. 2018, Long et al. 2019).

Huoditang is located in the middle part of the southern slope of the Qinling Mountains at 33°18'~33°28'N, 108°21'~108°29'E. It belongs to the transitional zone of the northern subtropical and warm temperate zone in China. The terrain is complex and the climate is changeable (Zhang and Cao 2007). The plant communities are complex and, as a result, species diversity of fungi in the forest area is high (Zhang and Cao 2007). During trips to collect forest pathogens causing dieback in Shaanxi Province, cankered branches with typical fruiting bodies were investigated and sampled. The aim of the present study was to identify these fungi, based on modern polyphasic taxonomic concepts.

Materials and methods

Isolates

Fresh specimens of were collected from symptomatic twigs or branches in Shaanxi Province (Table 1). 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 9 cm diam. Petri dish. Petri dishes were incubated at 25 °C until spores germinated. Single germinating conidia were transferred on to new PDA plates, which were kept at 25 °C in the dark. Specimens are 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
				ITS	cal	his3	tef1	tub2
D. acericola	MFLUCC 17-0956	Acer negundo	Italy	KY964224	KY964137	NA	KY964180	KY964074
D. acerigena	CFCC 52554	Acer tataricum	China	MH121489	MH121413	MH121449	MH121531	NA
D. albosinensis	CFCC 53066	Betula albosinensis	China	MK432659	MK442979	MK443004	MK578133	MK578059
	CFCC 53067	Betula albosinensis	China	MK432660	MK442980	MK443005	MK578134	MK578060
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. 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	NA	NA	KP267970	KP293476
D. aquatica	IFRDCC 3051	Aquatic habitat	China	JQ797437	NA	NA	NA	NA
D. arctii	CBS 139280	Arctium lappa	Austria	KJ590736	KJ612133	KJ659218	KJ590776	KJ610891
D. aseana	MFLUCC 12-0299a	Unknown dead leaf	Thailand	KT459414	KT459464	NA	KT459448	KT459432
D. asheicola	CBS 136967	Vaccinium ashei	Chile	KJ160562	KJ160542	NA	KJ160594	KJ160518
D. baccae	CBS 136972	Vaccinium corymbosum	Italy	KJ160565	NA	MF418264	KJ160597	NA
D. beilharziae	BRIP 54792	Indigofera australis	Australia	JX862529	NA	NA	JX862535	KF170921
D. benedicti	BPI 893190	Salix sp.	USA	KM669929	KM669862	NA	KM669785	NA
D. betulae	CFCC 50469	Betula platyphylla	China	KT732950	KT732997	KT732999	KT733016	KT733020
D. betulina	CFCC 52560	Betula albo-sinensis	China	MH121495	MH121419	MH121455	MH121537	MH121577
D. bicincta	CBS 121004	Juglans sp.	USA	KC343134	KC343376	KC343618	KC343860	KC344102
D. caryae	CFCC 52563	Carya illinoensis	China	MH121498	MH121422	MH121458	MH121540	MH121580
D. cassines	CPC 21916	Cassine peragua	South Africa	KF777155	NA	NA	KF777244	NA
D. celeris	CPC 28262	Vitis vinifera	Czech Republic	MG281017	MG281712	MG281363	MG281538	MG281190
D. cercidis	CFCC 52565	Cercis chinensis	China	MH121500	MH121424	MH121460	MH121542	MH121582
D. chamaeropis	CBS 454.81	Chamaerops humilis	Greece	KC343048	KC343290	KC343532	KC343774	KC344016
D. charlesworthii	BRIP 54884m	Rapistrum rugostrum	Australia	KJ197288	NA	NA	KJ197250	KJ197268
D. chensiensis	CFCC 52567	Abies chensiensis	China	MH121502	MH121426	MH121462	MH121544	MH121584
D. cichorii	MFLUCC 17-1023	Cichorium intybus	Italy	KY964220	KY964133	NA	KY964176	KY964104
D. cinnamomi	CFCC 52569	Cinnamomum sp.	China	MH121504	NA	MH121464	MH121546	MH121586
D. citriasiana	CGMCC 3.15224	Citrus unshiu	China	JQ954645	KC357491	KJ490515	JQ954663	KC357459
D. citrichinensis	CGMCC 3.15225	Citrus sp.	China	JQ954648	KC357494	NA	JQ954666	NA
D. compactum	CGMCC 3.17536	Camellia sinensis	China	KP267854	NA	KP293508	KP267928	KP293434
D. conica	CFCC 52571	Alangium chinense	China	MH121506	MH121428	MH121466	MH121548	MH121588
D. coryli	CFCC 53083	Corylus mandshurica	China	MK432661	MK442981	MK443006	MK578135	MK578061
	CFCC 53084	Corylus mandshurica	China	MK432662	MK442982	MK443007	MK578136	MK578062
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	NA	KC843116	KC843221
D. discoidispora	ZJUD89	Citrus unshiu	China	KJ490624	NA	KJ490566	KJ490503	KJ490445
D. dorycnii	MFLUCC 17-1015	Dorycnium hirsutum	Italy	KY964215	NA	NA	KY964171	KY964099
D. elaeagni-glabrae	CGMCC 3.18287	Elaeagnus glabra	China	KX986779	KX999281	KX999251	KX999171	KX999212
D. endophytica	CBS 133811	Schinus terebinthifolius	Brazil	KC343065	KC343307	KC343549	KC343791	KC343065
D. eres	AR5193	Ulmus sp.	Germany	KJ210529	KJ434999	KJ420850	KJ210550	KJ420799
D. eucalyptorum	CBS 132525	Eucalyptus sp.	Australia	NR120157	NA	NA	NA	NA
D. foeniculacea	CBS 123208	Foeniculum vulgare	Portugal	KC343104	KC343346	KC343588	KC343830	KC344072
D. fraxini-angustifoliae	BRIP 54781	Fraxinus angustifolia	Australia	JX862528	NA	NA	JX862534	KF170920
D. fraxinicola	CFCC 52582	Fraxinus chinensis	China	MH121517	MH121435	NA	MH121559	NA
D. fructicola	MAFF 246408	Passiflora edulis × P. edulis f. flavicarpa	Japan	LC342734	LC342738	LC342737	LC342735	LC342736
D. fusicola	CGMCC 3.17087	Lithocarpus glabra	China	KF576281	KF576233	NA	KF576256	KF576305
D. garethjonesii	MFLUCC 12-0542a	Unknown dead leaf	Thailand	KT459423	KT459470	NA	KT459457	KT459441
D. guangxiensis	JZB320094	Vitis vinifera	China	MK335772	MK736727	NA	MK523566	MK500168
D. helicis	AR5211	Hedera helix	France	KJ210538	KJ435043	KJ420875	KJ210559	KJ420828
D. heterophyllae	CBS 143769	Acacia heterohpylla	France	MG600222	MG600218	MG600220	MG600224	MG600226
D. hubeiensis	JZB320123	Vitis vinifera	China	MK335809	MK500235	NA	MK523570	MK500148
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. juglandicola	CFCC 51134	Juglans mandshurica	China	KU985101	KX024616	KX024622	KX024628	KX024634
D. kadsurae	CFCC 52586	Kadsura longipedunculata	China	MH121521	MH121439	MH121479	MH121563	MH121600
D. litchicola	BRIP 54900	Litchi chinensis	Australia	JX862533	NA	NA	JX862539	KF170925
D. lusitanicae	CBS 123212	Foeniculum vulgare	Portugal	KC343136	KC343378	KC343620	KC343862	KC344104
D. masirevicii	BRIP 57892a	Helianthus annuus	Australia	KJ197277	NA	NA	KJ197239	KJ197257
D. middletonii	BRIP 54884e	Rapistrum rugostrum	Australia	KJ197286	NA	NA	KJ197248	KJ197266
D. millettiae	GUCC9167	Millettia reticulata	China	MK398674	MK502086	NA	MK480609	MK502089
D. miriciae	BRIP 54736j	Helianthus annuus	Australia	KJ197282	NA	NA	KJ197244	KJ197262
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. nothofagi	BRIP 54801	Nothofagus cunninghamii	Australia	JX862530	NA	NA	JX862536	KF170922
D. novem	CBS 127270	Glycine max	Croatia	KC343155	KC343397	KC343640	KC343881	KC344123
D. oraccinii	CGMCC 3.17531	Camellia sinensis	China	KP267863	NA	KP293517	KP267937	KP293443
D. ovalispora	ICMP20659	Citrus limon	China	KJ490628	NA	KJ490570	KJ490507	KJ490449
D. ovoicicola	CGMCC 3.17093	Citrus sp.	China	KF576265	KF576223	NA	KF576240	KF576289
D. osmanthi	GUCC9165	Osmanthus fragrans	China	MK398675	MK502087	NA	MK480610	MK502090
D. padina	CFCC 52590	Padus racemosa	China	MH121525	MH121443	MH121483	MH121567	MH121604
D. pandanicola	MFLU 18-0006	Pandanus sp.	Thailand	MG646974	NA	NA	NA	MG646930
D. pascoei	BRIP 54847	Persea americana	Australia	JX862532	NA	NA	JX862538	KF170924
D. passifloricola	CBS 141329	Passiflora foetida	Malaysia	KX228292	NA	KX228367	NA	KX228387
D. perseae	CBS 151.73	Persea gratissima	Netherlands	KC343173	KC343415	KC343657	KC343899	KC344141
D. pescicola	MFLUCC 16-0105	Prunus persica	China	KU557555	KU557603	NA	KU557623	KU557579
D. phaseolorum	AR4203	Phaseolus vulgaris	USA	KJ590738	NA	KJ659220	NA	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. psoraleae-pinnatae	CBS 136413	Psoralea pinnata	South Africa	KF777159	NA	NA	NA	KF777252
D. pulla	CBS 338.89	Hedera helix	Yugoslavia	KC343152	KC343394	KC343636	KC343878	KC344120
D. racemosae	CBS 143770	Euclea racemosa	South Africa	MG600223	MG600219	MG600221	MG600225	MG600227
D. ravennica	MFLUCC 15-0479	Tamarix sp.	Italy	KU900335	NA	NA	KX365197	KX432254
D. rhusicola	CBS 129528	Rhus pendulina	South Africa	JF951146	KC843124	NA	KC843100	KC843205
D. rosae	MFLU 17-1550	Rosa sp.	Thailand	MG828894	NA	NA	NA	MG843878
D. rosicola	MFLU 17-0646	Rosa sp.	UK	MG828895	NA	NA	MG829270	MG843877
D. rudis	AR3422	Laburnum anagyroides	Austria	KC843331	KC843146	NA	KC843090	KC843177
D. sackstonii	BRIP 54669b	Helianthus annuus	Australia	KJ197287	NA	NA	KJ197249	KJ197267
D. salicicola	BRIP 54825	Salix purpurea	Australia	JX862531	NA	NA	JX862537	JX862531
D. sambucusii	CFCC 51986	Sambucus williamsii	China	KY852495	KY852499	KY852503	KY852507	KY852511
D. schini	CBS 133181	Schinus terebinthifolius	Brazil	KC343191	KC343433	KC343675	KC343917	KC344159
D. schoeni	MFLU 15-1279	Schoenus nigricans	Italy	KY964226	KY964139	NA	KY964182	KY964109
D. sennicola	CFCC 51634	Senna bicapsularis	China	KY203722	KY228873	KY228879	KY228883	KY228889
D. serafiniae	BRIP 55665a	Helianthus annuus	Australia	KJ197274	NA	NA	KJ197236	KJ197254
D. shaanxiensis	CFCC 53106	on branches of liana	China	MK432654	MK442976	MK443001	MK578130	NA
	CFCC 53107	on branches of liana	China	MK432655	MK442977	MK443002	MK578131	NA
D. siamensis	MFLUCC 10-573a	Dasymaschalon sp.	Thailand	JQ619879	NA	NA	JX275393	JX275429
D. sojae	FAU635	Glycine max	USA	KJ590719	KJ612116	KJ659208	KJ590762	KJ610875
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	NA	KJ490529	KJ490466	KJ490408
D. subcylindrospora	MFLU 17-1195	Salix sp.	China	MG746629	NA	NA	MG746630	MG746631
D. subellipicola	MFLU 17-1197	on dead wood	China	MG746632	NA	NA	MG746633	MG746634
D. subordinaria	CBS 464.90	Plantago lanceolata	New Zealand	KC343214	KC343456	KC343698	KC343940	KC344182
D. tectonendophytica	MFLUCC 13-0471	Tectona grandis	China	KU712439	KU749354	NA	KU749367	KU749354
D. tectonigena	MFLUCC 12-0767	Tectona grandis	China	KU712429	KU749358	NA	KU749371	KU743976
D. terebinthifolii	CBS 133180	Schinus terebinthifolius	Brazil	KC343216	KC343458	KC343700	KC343942	KC344184
D. ternstroemia	CGMCC 3.15183	Ternstroemia gymnanthera	China	KC153098	NA	NA	KC153089	NA
D. thunbergii	MFLUCC 10-576a	Thunbergia laurifolia	Thailand	JQ619893	JX197440	NA	JX275409	JX275449
D. tibetensis	CFCC 51999	Juglandis regia	China	MF279843	MF279888	MF279828	MF279858	MF279873
D. ueckerae	FAU656	Cucumis melo	USA	KJ590726	KJ612122	KJ659215	KJ590747	KJ610881
D. ukurunduensis	CFCC 52592	Acer ukurunduense	China	MH121527	MH121445	MH121485	MH121569	NA
D. unshiuensis	CFCC 52594	Carya illinoensis	China	MH121529	MH121447	MH121487	MH121571	MH121606
D. vaccinii	CBS 160.32	Oxycoccus macrocarpos	USA	KC343228	KC343470	KC343712	KC343954	KC344196
D. velutina	CGMCC 3.18286	Neolitsea sp.	China	KX986790	NA	KX999261	KX999182	KX999223
D. viniferae	JZB320071	Vitis vinifera	China	MK341551	MK500107	NA	MK500119	MK500112
D. xishuangbanica	CGMCC 3.18282	Camellia sinensis	China	KX986783	NA	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. NA, not applicable.

Morphological analysis

Morphological observations of the asexual morph in the natural environment were based on features of the fruiting bodies produced on infected plant tissues and micromorphology, supplemented by cultural characteristics. Conidiomata from tree barks were sectioned by hand, using a double-edged blade and structures were observed under a dissecting microscope. The gross morphology of fruiting bodies was recorded using a Leica stereomicroscope (M205 FA). Fungal structures were mounted in clear lactic acid and micromorphological characteristics were examined at 1000× magnification using a Leica compound microscope (DM 2500) with differential interference contrast (DIC) optics. Thirty measurements of each structure were determined for each collection. Colony characters and pigment production on PDA were noted after 10 d. Colony colours were described according to Rayner (1970).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from colonies grown on cellophane-covered PDA, using the CTAB [cetyltrimethylammonium bromide] method (Doyle and Doyle 1990). PCR amplifications of phylogenetic markers were done using the same primer pairs and conditions as in Yang et al. (2018). PCR products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyzer with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses

The quality of our amplified nucleotide sequences was checked and combined by SeqMan v.7.1.0 and reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), based on recent publications on the genus (Guarnaccia et al. 2018, Yang et al. 2018, Long et al. 2019). Sequences were aligned using MAFFT v. 7.310 (http://mafft.cbrc.jp/alignment/server/index.html) (Katoh and Standley 2016) and manually corrected using Bioedit 7.0.9.0 (Hall 1999). 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.

Phylogenetic analyses of the combined gene regions were performed using Maximum-Likelihood (ML) and Bayesian Inference (BI) methods. ML was conducted using PhyML v. 3.0 (Guindon et al. 2010), with 1000 bootstrap replicates. BI was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0b4 (Ronquist and Huelsenbeck 2003). 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 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 (Rambaut and Drummond 2010) and processed by Adobe Illustrator CS5. Alignment and trees were deposited in TreeBASE (submission ID: S25522). The nucleotide sequence data of the new taxa have been deposited in GenBank (Table 1).

Results

The five-gene sequence dataset (ITS, , , and ) was analysed to infer the interspecific relationships within . The dataset consisted of 124 sequences including the outgroup, (culture CBS 121124). A total of 2555 characters including gaps (505 for ITS, 513 for , 528 for , 475 for and 522 for ) were included in the phylogenetic analysis. The best nucleotide substitution model for ITS, and was TrN+I+G, while HKY+I+G was selected for both and . The topologies resulting from ML and BI analyses of the concatenated dataset were congruent (Fig. 1). Isolates from Shaanxi Province formed three individual clades representing three undescribed species.

Figure 1.

Phylogram of resulting from a maximum likelihood analysis based on combined ITS, , , and . Numbers above the branches indicate ML bootstraps (left, ML BS ≥ 50%) and Bayesian Posterior Probabilities (right, BPP ≥ 0.90). The tree is rooted with . Isolates in current study are in blue. “-” indicates ML BS < 50% or BI PP < 0.90.

Taxonomy

C.M. Tian & Q. Yang sp. nov.

27FA9968-ECF3-56FB-9BA6-7478C45CE54B

829518

Fig. 2

Figure 2.

on (BJFC-S1670). A Habit of conidiomata in wood B transverse section of conidiomata C longitudinal section through conidiomata D conidiogenous cells attached with beta conidia E conidiogenous cells attached with alpha conidia F beta conidia. Scale bars: 200 μm (B–C); 20 μm (D, F); 10 μm (E).

Diagnosis.

Distinguished from in having shorter conidiophores and longer beta conidia.

Etymology.

Named after the host plant, , from which the holotype was collected.

Description.

Conidiomata pycnidial, conical, immersed in bark, solitary to aggregated, erumpent through the bark surface, with a solitary undivided locule. Ectostromatic disc yellowish to brown, one ostiole per disc. Ostiole medium black, up to the level of disc. undivided, (280–)290–375(–380) μm diam. Conidiophores (6–)8.5–13(–14.5) × (1.5–)2–2.5 μm, hyaline, cylindrical, smooth, phialidic, unbranched, straight or slightly curved. Alpha conidia hyaline, aseptate, fusiform, 0–1-guttulate, (7–)8–10(–11) × 2.5–3 μm. Beta conidia hyaline, aseptate, filiform, straight or slightly curved, eguttulate, base subtruncate, tapering towards one apex, (24–)25.5–30(–32) × 1–1.5 µm.

Culture characters.

Cultures incubated on PDA at 25 °C in the dark. Colony originally flat with white felted aerial mycelium, becoming light brown due to pigment formation, conidiomata irregularly distributed over agar surface, with yellowish conidial drops exuding from the ostioles.

Specimens examined.

China. Shaanxi Province: Ningshan County, Huoditang Forest Farm, , on branches of , 10 July 2018, N. Jiang (holotype BJFC-S1670; ex-type living culture: CFCC 53066; living culture: CFCC 53067).

Notes.

Two isolates, representing , are retrieved in a well-supported clade (ML BS/BPP=100/1) and appear most closely related to (Fig. 1). can be distinguished from , based on and loci (3/335 in and 19/429 in ). Morphologically, differs from in having shorter conidiophores (8.5–13 vs. 10.5–17.5 μm) and longer beta conidia (25.5–30 vs. 19–29.5 μm) (Yang et al. 2018).

C.M. Tian & Q. Yang sp. nov.

0790CB77-EE63-5681-AD57-D83CB470F50D

829520

Fig. 3

Figure 3.

on (BJFC-S1671). A, B Habit of conidiomata in wood C transverse section of conidiomata D longitudinal section through conidiomata E conidiogenous cells attached with alpha conidia F alpha conidia. Scale bars: 500 μm (B–D); 10 μm (E); 20 μm (F).

Distinguished from and in having larger alpha conidia.

Named after the genus of the host plant from which the holotype was collected, .

Conidiomata pycnidial, conical to spherical, immersed in the host bark, erumpent from surface of host branches, scattered, 950–1200 × 420–650 μm diam., covered by orange discharged conidial masses at maturity, usually conspicuous. Ectostromatic disc inconspicuous. Central column beneath the disc more or less conical, bright yellow. Conidiophores reduced to conidiogenous cells. Conidiogenous cells cylindrical, hyaline, smooth, unbranched, tapering towards the apex, (8.5–)10–12(–13) × (2–)2.5–3 μm. Alpha conidia hyaline, aseptate, fusiform, multiguttulate, rarely 2-guttulate, (10.5–)11.5–13(–13.5) × 3–3.5 μm. Beta conidia not observed.

Cultures incubated on PDA at 25 °C in the dark. Colony flat, felty with thick texture at the marginal area, with thin texture in the centre, producing beige pigment after 7–10 d. Aerial mycelium white, dense, conidiomata distributed in the centre, with translucent conidial drops exuding from the ostioles.

CHINA. Shaanxi Province: Ningshan County, Huoditang Forest Farm, , on branches of , 10 July 2018, N. Jiang (holotype BJFC-S1671; ex-type living culture: CFCC 53083); , on branches of , 10 July 2018, N. Jiang (paratype BJFC-S1672; living culture: CFCC 53084).

We generated sequences for two isolates of , CFCC 53083 and CFCC 53084. This new species is phylogenetically most closely related to and (Fig. 1). can be distinguished from , based on ITS, and loci (8/467 in ITS, 1/460 in and 1/336 in ); and from based on and loci (4/335 in and 25/428 in ). Morphologically, can be distinguished from both (11.5–13 × 3–3.5 vs. 5–6 × 2–3 μm) and (11.5–13 × 3–3.5 vs. 5.5–9 × 1.5–2.5 μm) in having larger alpha conidia (Huang et al. 2013, Gao et al. 2016).

C.M. Tian & Q. Yang sp. nov.

1B8F6C9B-59D3-5D30-B416-6175363932FD

829527

Fig. 4

Figure 4.

on liana (BJFC-S1674). A, B Habit of conidiomata on twig C transverse section through conidiomata D longitudinal section through conidiomata E conidiogenous cells attached with beta conidia F beta conidia. Scale bars: 200 μm (B–D); 10 μm (E, F).

Distinguished from and in having longer beta conidia.

Named after Province Shaanxi, where the holotype was collected.

Conidiomata pycnidial, immersed in bark, scattered, erumpent through the bark surface, discoid, with a solitary undivided locule. Ectostromatic disc yellowish to pale brown, one ostiole per disc, usually conspicuous, (485–)500–687(–695) μm diam. circular, undivided, (500–)526–765(–792) μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, cylindrical, unbranched, slightly curved, tapering towards the apex, (12.5–)14.5–17(–18) × 1–1.5(–2) μm. Alpha conidia not observed. Beta conidia hyaline, aseptate, filiform, straight to curved, eguttulate, (35.5–)37–47.5(–50) × 1 µm.

Cultures incubated on PDA at 25 °C in the dark. Colony originally flat with white fluffy aerial mycelium, becoming pale brown with pigment, with visible solitary conidiomata at maturity.

CHINA. Shaanxi Province: Ningshan County, Huoditang Forest Farm, , on branch of liana, 10 July 2018, N. Jiang (holotype BJFC-S1674; ex-type living culture: CFCC 53106); , on branch of liana, 10 July 2018, N. Jiang (Paratype BJFC-S1675; living culture: CFCC 53107).

In the combined tree, is a distinct clade with maximum support and it appears to be most closely related to and (Fig. 1). can be distinguished from by a 17 nt difference in the ITS region. For , only ITS sequences are available in NCBI GenBank (Hu et al. 2012). The new species can be distinguished from , based on ITS, , and (24/454 in ITS, 14/443 in , 66/468 in and 24/311 in ). Morphologically, differs from both (37–47.5 vs. 9–12.5 µm) and (37–47.5 vs. 19–44 µm) in having longer beta conidia (Gao et al. 2016, 2017).

Discussion

In this study, an investigation of forest pathogens from Huoditang in Shaanxi Province was carried out and canker was observed as a common disease. Identification of our collections was conducted, based on isolates from fruiting bodies using five combined loci (ITS, , , and ), as well as morphological characters. Three new species were described. These are sp. nov., sp. nov. and sp. nov.

is associated with . Thus far, six species have been reported from . These are , , , , and (Kobayashi 1970, Gomes et al. 2013, Du et al. 2016, Yang et al. 2018). Morphologically, differs from (600–1250 μm), (700–1300 μm) and (670–905 μm) in having smaller locules (Du et al. 2016, Yang et al. 2018); and from (5–8 × 1.5–2 μm) and (6.5–8.5 × 3–4 μm) in having larger alpha conidia (Arnold 1967, Anagnostakis 2007, Gomes et al. 2013). In addition, our phylogenetic reconstruction of a five-locus dataset adds support for the new species, although no sequence data are currently available for , and (Fig. 1). Interestingly, is found on different plant hosts; it was described from in London and then recorded from , and (Dearness 1926, Wehmeyer 1933, Kobayashi 1970). is characterised by the ostiole with orange discharged conidial masses and a yellow central column (Fig. 3). was found on branches of liana with an obvious ostiole per disc and characterised by hyaline, filiform beta conidia. Alpha conidia were found neither in the natural environment nor in culture for this species.

Species delimitation of has improved considerably by using a combination of morphological, cultural, phytopathological and molecular phylogenetic analyses (Udayanga et al. 2014a, b, 2015, Fan et al. 2015, Gao et al. 2017, Guarnaccia and Crous 2017, Hyde et al. 2017, 2020, Guarnaccia et al. 2018, Yang et al. 2018, Long et al. 2019). As a result, many canker diseases and new species have been discovered and reported from all over the world and also in China. The descriptions and molecular data of species represent an important resource for plant pathologists, plant quarantine officials and taxonomists.