Cytospora elaeagnicola sp. nov. Associated with Narrow-leaved Oleaster Canker Disease in China.

Cytospora is a genus including important phytopathogens causing severe dieback and canker diseases distributed worldwide with a wide host range. However, identification of Cytospora species is difficult since the currently available DNA sequence data are insufficient. Aside the limited availability of ex-type sequence data, most of the genetic work is only based on the ITS region DNA marker which lacks the resolution to delineate to the species level in Cytospora. In this study, three fresh strains were isolated from the symptomatic branches of Elaeagnus angustifolia in Xinjiang Uygur Autonomous Region, China. Morphological observation and multi-locus phylogenetic analyses (ITS, LSU, ACT and RPB2) support these specimens are best accommodated as a distinct novel species of Cytospora. Cytospora elaeagnicola sp. nov. is introduced, having discoid, nearly flat, pycnidial conidiomata with hyaline, allantoid conidia, and differs from its relatives genetically and by host association.

Introduction

The genus Cytospora contains important phytopathogens causing dieback and stem canker disease on multiple woody plants [1,2]. It was introduced by Ehrenberg in 1818 [3] and belonged to the family Cytosporaceae in Diaporthales [4]. This disease has globally caused great losses on ecologically and commercially important woody plants. Cytospora is characterized by the diaporthalean-like perithecial ascoma, clavate to elongate obovoid asci with allantoid, hyaline, aseptate ascospores in sexual state; and the single or labyrinthine locules, filamentous conidiophores, phialidic conidiogenous cells with allantoid, hyaline, aseptate conidia in the asexual state [2,5]. The asexual name Cytospora (1818) is an older name than all of the sexual synonyms Valsa (1849), Leucocytospora (1917), Leucostoma (1917), Valsella (1870) and Valseutypella (1919), and thus has the priority in nomenclature [2,6-8]. More than 610 species named Cytospora are listed at present in Index Fungorum (2019). However, the amount of species in Cytospora was with 110 estimated species [9]. Species criteria of Cytospora were previously based on host affiliations and morphology in China, however these bases are unreliable due to the uninformative illustrations and descriptions, weak host specificity and overlapping morphological characteristics [10-12]. Recent studies have reported updated phylograms for the genus Cytospora on the basis of multigene phylogenetic analyses using ex-type or reference strains [6,7,13-15]. However, because availability of the ex-type sequence data is limited to few species, identification of a strain to species level is very difficult. Recently, only 14 new species were included to this genus [16].

Elaeagnus angustifolia is a drought-resistant tree that is grown as a major biomass energy source [17], and has high medicinal and ecological value as well [18]. Furthermore, during an investigation of phytopathogens in north of China, most E. angustifolia trees were observed to suffer from dieback and stem canker caused by Cytospora species. In the current study, three representative Cytospora strains were collected from Elaeagnus angustifolia in Xinjiang Uygur Autonomous Region, China. Multilocus phylogenetic analyses using combination of ITS, LSU, ACT and RPB2 sequences confirmed finding of a new species in Cytospora. In this paper, C. elaeagnicola sp. nov. is introduced, accompanied with descriptions, illustrations and comparison with other species in the genus.

Materials and methods

Sample collection and isolation

Fresh samples of Cytospora were collected from infected branches and stems of E. angustifolia during investigations of phytopathogens in Xinjiang Uygur Autonomous Region, China. The samples placed in paper bags were brought to the laboratory for processing and experimental purpose using the same methodology as in Fan et al. [14,15]. Single conidia were isolated by taking fruiting bodies and suspend the mucoid spore mass removed from conidiomata or ascomata in a drop of sterile water. The spore suspension from each sample was then spread over the surface of 1.8% potato dextrose agar (PDA) medium in a petri-dish and incubated at 25 °C. After 24 h, a single germinating conidium was transferred to a fresh PDA plate. Samples and isolates of the new species were deposited in the Museum of Beijing Forestry University (BJFC) and single-spore cultures in the China Forestry Culture Collection Center (CFCC).

Morphology observation

Samples were observed on infected plant tissues including the structure and size of fruiting bodies. The photographs of the macro-morphological characteristics were recorded using a Leica stereomicroscope (M205 FA) while the micro-morphological observations were determined under a Leica compound microscope (DM 2500) with differential interference contrast (DIC). Over 20 fruiting bodies were sectioned, both vertically and horizontally, and 50 conidia were selected randomly to get the measurement of their length and width. Cultural characteristics, including the colony characters and the production of pigment of isolates on PDA incubated at 25 °C in the dark were recorded, after 3, 7, and 30-days growth [19].

DNA extraction, PCR amplification, and sequencing

Fungal mycelium grown on the cellophane of PDA was scraped for the extraction of genomic DNA following a modified CTAB approach [20]. The ITS region was amplified with the primers ITS1 and ITS4 [21]; the LSU region with LR0R and LR7 [22]; the partial ACT region with ACT512F and ACT783R [23] and the RPB2 region with RPB2-5F and fRPB2-7cR [24]. The PCR amplicons were estimated visually by electrophoresis in 2% agarose gels. Fragments were sequenced in both directions using the respective primers and the BigDye Terminater v.3.1 Cycle Sequencing Kit (Applied Biosystems; Foster City, CA). Sequences were joined and quality was examined with Seqman v.7.1.0 in the DNASTAR lasergene core suite software (DNASTAR Inc.; Madison, WI).

DNA sequence analysis

Sequences based on ITS region and the combined dataset (ITS, LSU, ACT and RPB2) were aligned using MAFFT v.6 [25] and edited manually using MEGA6 [26], and some characters were excluded from both ends of the alignments to approximate the size of our sequences to those included in the dataset.

MP analysis was carried out by using PAUP v.4.0b10 with a heuristic search option of 1000 random-addition sequences with a tree bisection and reconnection (TBR) as the branch swapping algorithm [27]. Zero length branches were collapsed, whereas all equally parsimonious trees were saved. Stability of the clade was assessed with a bootstrap analysis of 1000 replicates [28]. Other measures calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC) [27]. ML analysis was carried out by using RAxML v.7.2.8 with a GTR + G + I model of site substitution, including estimation of gamma-distributed rate heterogeneity and a proportion of invariant sites [29]. And the branch support from MP and ML analyses was evaluated with a bootstrapping method of 1000 replicates [28].

BI analysis employing a Markov Chain Monte Carlo (MCMC) algorithm was performed using in MrBayes v.3.1.2 with the inverse gamma rates (GTR + I + G) nucleotide substitution model, which was selected based on the AIC criterion, using MrModeltest v.2.3 [30,31]. Two MCMC chains were run from random trees for 1,000,000 generations, and trees were sampled every 100th generation, resulting in 10,000 total trees. The first 25% of trees were discarded as the burn-in phase of the analysis and the Bayesian posterior probabilities (BPP) were calculated using the remaining 7500 trees [32].

In all analyses, C. elaeagnicola was selected as a distinct and new grape. Phylograms were examined in Figtree v.1.3.1 [33]. Novel sequence data was deposited in GenBank (Table 1), the multilocus sequences alignment file was deposited in TreeBASE (www.treebase.org) accession S24181 and the taxonomic novelty was deposited in MycoBank.

Table 1.

Isolates and GenBank accession numbers used in this study.

Species	Strain	Host	GenBank accession numbers
			ITS	LSU	ACT	RPB2
C. abyssinica	CMW 10181T	Eucalyptus globulus	AY347353	–	–	–
C. abyssinica	CMW 10178	Eucalyptus globulus	AY347354	–	–	–
C. acaciae	CBS 468.69	Ceratonia siliqua	DQ243804	–	–	–
C. ampulliformis	MFLUCC 16-0583T	Sorbus intermedia	KY417726	KY417760	KY417692	KY417794
C. ampulliformis	MFLUCC 16-0629	Acer platanoides	KY417727	KY417761	KY417693	KY417795
C. atrocirrhata	CFCC 89615	Juglans regia	KR045618	KR045700	KF498673	KU710946
C. atrocirrhata	CFCC 89616	Juglans regia	KR045619	KR045701	KF498674	KU710947
C. austromontana	CMW 6735T	Eucalyptus pauciflora	AY347361	–	–	–
C. berberidis	CFCC 89927T	Berberis dasystachya	KR045620	KR045702	KU710990	KU710948
C. berberidis	CFCC 89933	Berberis dasystachya	KR045621	KR045703	KU710991	KU710949
C. berkeleyi	StanfordT3T	Eucalyptus globulus	AY347350	–	–	–
C. berkeleyi	UCBTwig3	Eucalyptus globulus	AY347349	–	–	–
C. brevispora	CBS 116829	Eucalyptus grandis	AF192321	–	–	–
C. brevispora	CBS 116811T	Eucalyptus grandis × tereticornis	AF192315	–	–	–
C. carbonacea	CFCC 89947	Ulmus pumila	KR045622	KP310812	KP310842	KU710950
C. carpobroti	CMW 48981T	Carpobrotus edulis	MH382812	MH411216	–	–
C. cedri	CBS 196.50	–	AF192311	–	–	–
C. centrivillosa	MFLUCC 16-1206T	Sorbus domestica	MF190122	MF190068	–	MF377600
C. centrivillosa	MFLUCC 17-1660	Sorbus domestica	MF190123	MF190069	–	MF377601
C. chrysosperma	CFCC 89629	Salix psammophila	KF765673	KF765689	KF765721	KF765705
C. chrysosperma	CFCC 89981	Populus alba subsp. pyramidalis	MH933625	MH933660	MH933533	MH933597
C. chrysosperma	CFCC 89982	Ulmus pumila	KP281261	KP310805	KP310835	KU710952
C. cinerostroma	CMW 5700T	Eucalyptus globulus	AY347377	–	–	–
C. cincta	ATCC 32673	–	DQ996041	–	–	–
C. cotini	MFLUCC 14-1050T	Cotinus coggygria	KX430142	KX430143	–	KX430144
C. curvata	MFLUCC 15-0865T	Salix alba	KY417728	KY417762	KY417694	KY417796
C. davidiana	CXY 1350T	Populus davidiana	KM034870	–	–	–
C. davidiana	CXY 1374	Populus davidiana	KM034869	–	–	–
C. diatrypelloidea	CMW 8549T	Eucalyptus globulus	AY347368	–	–	–
C. disciformis	CMW 6509T	Eucalyptus grandis	AY347374	–	–	–
C. disciformis	CMW 6750	Eucalyptus globulus	AY347359	–	–	–
C. donetzica	MFLUCC 16-0574T	Rosa sp.	KY417731	KY417764	KY417696	KY417798
C. donetzica	MFLUCC 15-0864	Crataegus monogyna	KY417729	KY417763	KY417695	KY417797
C. elaeagni	CFCC 89632	Elaeagnus angustifolia	KR045626	KR045706	KU710995	KU710955
C. elaeagni	CFCC 89633	Elaeagnus angustifolia	KF765677	KF765693	KU710996	KU710956
C. elaeagnicola	CFCC 52882T	Elaeagnus angustifolia	MK732341	MK732338	MK732344	MK732347
C. elaeagnicola	CFCC 52883	Elaeagnus angustifolia	MK732342	MK732339	MK732345	MK732348
C. elaeagnicola	CFCC 52884	Elaeagnus angustifolia	MK732343	MK732340	MK732346	MK732349
C. eriobotryae	IMI 136523T	Eriobotrya japonica	AY347327	–	–	–
C. erumpens	MFLUCC 16-0580T	Salix × fragilis	KY417733	KY417767	KY417699	KY417801
C. eucalypti	LSEQ	Sequoia sempervirens	AY347340	–	–	–
C. eucalypticola	ATCC 96150T	Eucalyptus nitens	AY347358	–	–	–
C. eucalypticola	CMW 5309	Eucalyptus grandis	AF260266	–	–	–
C. eucalyptina	CMW 5882	Eucalyptus grandis	AY347375	–	–	–
C. eugeniae	CMW 7029	Tibouchina sp.	AY347364	–	–	–
C. eugeniae	CMW 8648	Eugenia sp.	AY347344	–	–	–
C. fraxinigena	BBH 42442	Fraxinus ornus	MF190134	MF190079	–	–
C. fraxinigena	MFLUCC 14-0868T	Fraxinus ornus	MF190133	MF190078	–	–
C. friesii	CBS 194.42	Abies alba	AY347328	–	–	–
C. fugax	CXY1371	Populus simonii	KM034852	–	–	–
C. fugax	CXY1381	Populus ussuriensis	KM034853	–	–	–
C. germanica	CXY1322	Elaeagnus oxycarpa	JQ086563	JX524617	–	–
C. gigaspora	CFCC 89620T	Juglans regia	KR045628	KR045708	KU710997	KU710957
C. gigaspora	CFCC 89621	Juglans regia	KR045629	KR045709	KU710998	KU710958
C. gigaspora	CFCC 50014	Juniperus procumbens	KR045630	KR045710	KU710999.	KU710959
C. gigaspora	CFCC 89634T	Salix psammophila	KF765671	KF765687	KU711000	KU710960
C. hippophaës	CFCC 89639	Hippophae rhamnoides	KR045632	KR045712	KU711001	KU710961
C. hippophaës	CFCC 89640	Hippophae rhamnoides	KF765682	KF765698	KF765730	KU710962
C. japonica	CBS 375.29	Prunus persicae	AF191185	–	–	–
C. junipericola	BBH 42444	Juniperus communis	MF190126	MF190071	–	–
C. junipericola	MFLU 17-0882T	Juniperus communis	MF190125	MF190072	–	–
C. kantschavelii	CXY1383	Populus maximowiczii	KM034867	–	–	–
C. kantschavelii	CXY1386	Populus maximowiczii	KM034867	–	–	–
C. kunzei	CBS 118556	Pinus radiata	DQ243791	–	–	–
C. leucosperma	CFCC 89622	Pyrus bretschneideri	KR045616	KR045698	KU710988	KU710944
C. leucosperma	CFCC 89894	Pyrus bretschneideri	KR045617	KR045699	KU710989	KU710945
C. leucostoma	CFCC 50016	Sorbus aucuparia	MH820400	MH820393	MH820408	–
C. leucostoma	CFCC 50015	Sorbus pohuashanensis	KR045634	KR045714	KU711002	–
C. longiostiolata	MFLUCC 16-0628T	Salix × fragilis	KY417734	KY417768	KY417700	KY417802
C. mali	CFCC 50031	Crataegus sp.	KR045636	KR045716	KU711004	KU710965
C. mali	CFCC 50044	Malus baccata	KR045637	KR045717	KU711005	KU710966
C. melnikii	CFCC 89984	Rhus typhina	MH933644	MH933678	MH933551	MH933609
C. melnikii	MFLUCC 15-0851T	Malus domestica	KY417735	KY417769	KY417701	KY417803
C. melnikii	MFLUCC 16-0635	Populus nigra	KY417736	KY417770	KY417702	KY417804
C. mougeotii	ATCC 44994	Picea abies	AY347318	–	–	–
C. multicollis	CBS 105.89T	Quercus ilex subsp. rotundifolia	DQ243803	–	–	–
C. myrtagena	CBS 116843T	Tibouchiina urvilleana	AY347363	–	–	–
C. nivea	MFLUCC 15-0860	Salix acutifolia	KY417737	KY417771	KY417703	KY417805
C. nivea	CFCC 89641	Elaeagnus angustifolia	KF765683	KF765699	KU711006	KU710967
C. nivea	CFCC 89643	Salix psammophila	KF765685	–	–	KU710968
C. palm	CXY1276	Cotinus coggygria	JN402990	–	–	–
C. palm	CXY1280T	Cotinus coggygria	JN411939	–	–	–
C. parakantschavelii	MFLUCC 15-0857T	Populus × sibirica	KY417738	KY417772	KY417704	KY417806
C. parakantschavelii	MFLUCC 16-0575	Pyrus pyraster	KY417739	KY417773	KY417705	KY417807
C. parapersoonii	T28.1T	Prunus persicae	AF191181	–	–	–
C. parasitica	MFLUCC 15-0507T	Malus domestica	KY417740	KY417774	KY417706	KY417808
C. paratranslucens	MFLUCC 15-0506T	Populus alba var. bolleana	KY417741	KY417775	KY417707	KY417809
C. paratranslucens	MFLUCC 16-0627	Populus alba	KY417742	KY417776	KY417708	KY417810
C. pini	CBS 197.42	Pinus Sylvestirs	AY347332	–	–	–
C. pini	CBS 224.52T	Pinus strobus	AY347316	–	–	–
C. populina	CFCC 89644T	Salix psammophila	KF765686	KF765702	KU711007	KU710969
C. predappioensis	MFLUCC 17-2458T	Platanus sp.	MG873484	MG873480	–	–
C. pruinopsis	CFCC 50034T	Ulmus pumila	KP281259	KP310806	KP310836	KU710970
C. pruinosa	CFCC 50035	Ulmus pumila	KP281260	KP310807	KP310837	KU710971
C. pruinosa	CFCC 50036	Syzygium aromaticum	KP310800	KP310802	KP310832	–
C. pruinosa	CFCC 50037	Syzygium aromaticum	MH933650	MH933685	MH933558	–
C. prunicola	MFLU 17-0995T	Prunus sp.	MG742350	MG742351	MG742353	MG742352
C. quercicola	MFBBH 42443	Quercus sp.	MF190128	MF190074	–	–
C. quercicola	MFLUCC 14-0867T	Quercus sp.	MF190129	MF190073	–	–
C. rhizophorae	MUCC302	Eucalyptus grandis	EU301057	–	–	–
C. ribis	CFCC 50026	Ulmus pumila	KP281267	KP310813	KP310843	KU710972
C. ribis	CFCC 50027	Ulmus pumila	KP281268	KP310814	KP310844	–
C. rosae	MFLU 17-0885T	Rosa canina	MF190131	MF190075	–	–
C. rostrata	CFCC 89909T	Salix cupularis	KR045643	KR045722	KU711009	KU710974
C. rostrata	CFCC 89910	Salix cupularis	KR045644	KR045723	KU711010	KU710975
C. rusanovii	MFLUCC 15-0853	Populus × sibirica	KY417743	KY417777	KY417709	KY417811
C. rusanovii	MFLUCC 15-0854T	Salix babylonica	KY417744	KY417778	KY417710	KY417812
C. sacculus	CFCC 89624	Juglans regia	KR045645	KR045724	KM401888	KU710976
C. sacculus	CFCC 89625	Juglans regia	KF225616	KM401887	KM401889	–
C. salicacearum	MFLUCC 15-0509T	Salix alba	KY417746	KY417780	KY417712	KY417814
C. salicacearum	MFLUCC 15-0861	Salix × fragilis	KY417745	KY417779	KY417711	KY417813
C. salicacearum	MFLUCC 16-0587	Prunus cerasus	KY417748	KY417782	KY417714	KY417816
C. salicicola	MFLUCC 15-0866	Salix alba	KY417749	KY417783	KY417715	KY417817
C. salicicola	MFLUCC 14-1052T	Salix alba	KU982636	KU982635	KU982637	–
C. salicina	MFLUCC 15-0862T	Salix alba	KY417750	KY417784	KY417716	KY417818
C. salicina	MFLUCC 16-0637	Salix × fragilis	KY417751	KY417785	KY417717	KY417819
C. schulzeri	CFCC 50040	Malus domestica	KR045649	KR045728	KU711013	KU710980
C. schulzeri	CFCC 50042	Malus asiatica	KR045650	KR045729	KU711014	KU710981
C. sibiraeae	CFCC 50045T	Sibiraea angustata	KR045651	KR045730	KU711015	KU710982
C. sibiraeae	CFCC 50046	Sibiraea angustata	KR045652	KR045731	KU711015	KU710983
C. sophorae	CFCC 50047	Styphnolobium japonicum	KR045653	KR045732	KU711017	KU710984
C. sophorae	CFCC 89598	Styphnolobium japonicum	KR045654	KR045733	KU711018	KU710985
C. sophoricola	CFCC 89596	Styphnolobium japonicum	KR045656	KR045735	KU711020	KU710987
C. sophoricola	CFCC 89595T	Styphnolobium japonicum var.	KR045655	KR045734	KU711019	KU710986
C. sorbi	MFLUCC 16-0631T	Sorbus aucuparia	KY417752	KY417786	KY417718	KY417820
C. sorbicola	MFLUCC 16-0584T	Acer pseudoplatanus	KY417755	KY417789	KY417721	KY417823
C. sorbicola	MFLUCC 16-0633	Cotoneaster melanocarpus	KY417758	KY417792	KY417724	KY417826
C. spiraeae	CFCC 50049T	Spiraea salicifolia	MG707859	MG707643	MG708196	MG708199
C. spiraeae	CFCC 50050	Spiraea salicifolia	MG707860	MG707644	MG708197	MG708200
C. tanaitica	MFLUCC 14-1057T	Betula pubescens	KT459411	KT459412	KT459413	–
C. tibouchinae	CPC 26333T	Tibouchina semidecandra	KX228284	KX228335	–	–
C. translucens	CXY1351	Populus davidiana	KM034874	–	–	–
C. ulmi	MFLUCC 15-0863T	Ulmus minor	KY417759	–	–	–
C. valsoidea	CMW 4309T	Eucalyptus grandis	AF192312	–	–	–
C. valsoidea	CMW 4310	Eucalyptus grandis	AF192312	–	–	–
C. variostromatica	CMW 6766T	Eucalyptus globulus	AY347366	–	–	–
C. variostromatica	CMW 1240	Eucalyptus grandis	AF260263	–	–	–
C. vinacea	CBS 141585T	Vitis interspecific	KX256256	–	–	–
C. viticola	CBS 141586T	Vitis vinifera	KX256239	–	–	–
Diaporthe vaccinii	CBS 160.32	Vaccinium macrocarpon	KC343228	–	JQ807297	–

All the new isolates used in this study are indicated in bold type and the strains from type materials are marked by an superscript (T).

Results

Phylogeny

The ITS sequences of the three isolates of Cytospora from E. angustifolia were aligned with available ITS sequences from related Cytospora species of published articles, resulting in an alignment containing 138 Cytospora ingroup strains and a total of 609 characters including gaps. In the alignment, 369 characters were constant, 72 variable characters were parsimony-uninformative and 168 characters were variable and parsimony-informative. MP analyses generated 145 parsimonious trees, one of which is presented in Figure 1 (TL = 927, CI = 0.409, RI = 0.830, RC = 0.339). ML and BI analyses resolved results similar to the MP tree. C. elaeagnicola represented a monophyletic clade with overall high bootstrap support values (MP/ML/BI = 99/100/1; marked in blue in Figure 1). Subsequently, phylogenetic analyses were performed based on a concatenated alignment of ITS, LSU, ACT and RPB2 from published articles, comprised of 102 Cytospora ingroup strains with a total of 2207 characters including gaps. In the alignment, 1538 characters were constant, 104 variable characters were parsimony-uninformative and 565 characters were variable and parsimony-informative. MP analysis generated 105 parsimonious trees, one of which is presented in Figure 1 (TL = 2,350, CI = 0.412, RI = 0.827, RC = 0.341). ML and BI analyses were similar to the MP tree. Cytospora elaeagnicola represented a monophyletic clade with full support values (MP/ML/BI = 100/100/1) (marked in blue in Figure 2).

Phylogram of Cytospora based on ITS gene. MP and ML bootstrap support values above 50% are shown at the first and second position. Thickened branches represent posterior probabilities above 0.95 from BI. Ex-type strains are in bold. Strains in current study are in blue.

Phylogram of Cytospora based on combined ITS, LSU, ACT and RPB2 genes. MP and ML bootstrap support values above 50% are shown at the first and second position. Thickened branches represent posterior probabilities above 0.95 from BI. Ex-type strains are in bold. Strains in current study are in blue.

Taxonomy

X.L. Fan sp. nov. Figure 3

Figure 3.

Morphology of Cytospora elaeagnicola from Elaeagnus angustifolia (CF 20175831). (A), (B) Habit of conidiomata on twig; (C) Transverse section of conidioma; (D) Longitudinal section through conidioma; (E) Conidiophores and conidiogenous cells; (F) Conidia; (G) Colonies on PDA after 3 d and 14 d (scale bars: B–C = 250 μm, D = 200 μm, E = 10 μm, F = 5 μm).

Mycobank: MB830292.

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

Holotype: CF 20175831.

Host/Distribution: from branches of Elaeagnus angustifolia in China.

Descriptions: Asexual state: Conidiomata pycnidial, ostiolate, discoid, nearly flat, immersed in bark, scattered, producing black area on bark, erumpent through the surface of bark when mature. Locules multiple, circular to ovoid, arranged irregularly with common walls, (890–)905–1160(–1240) μm ( = 1060 ± 120 µm, n = 30) in diameter. Conceptacle absent. Ectostromatic disc iron grey to violaceous black, circular, disc dark, (160–)170–310(–350) µm ( = 240 ± 60 µm, n = 30) in diameter, with one ostiole in the centre of disc. Ostiole conspicuous, circular to ovoid, iron grey to violaceous black at the same level as the disc, (48–)51–71(–78) µm ( = 60 ± 11 µm, n = 30) in diameter. Conidiophores hyaline, branched at base or not branched, thin walled, filamentous, (12–)13.5–19.5(–20) μm ( = 16.5 ± 3 µm, n = 30). Conidiogenous cells enteroblastic, phialidic. Conidia hyaline, allantoid, eguttulate, smooth, aseptate, thin-wall, 5.5–6.5(–7) × (1–)1.5–2 µm ( = 6.1 ± 0.4 × 1.6 ± 0.1 µm, n = 50). Sexual morph: not observed.

Culture characteristics: On PDA, cultures are white. The colony is flat, felt-like with a thick texture at the center with thin surrounding texture. Pycnidia are sparse, distributed irregularly on medium surface.

Materials examined: China, Xinjiang Uygur Autonomous Region, Bole Mongol Autonomous Prefecture, Provincial Road 202, 45°06'29.50"N, 82°33'32.82"E, from branches of Elaeagnus angustifolia, July 2017, C.M. Tian & X.L. Fan, deposited by X.L. Fan, holotype CF 20175831, ex-type living culture CFCC 52882; ibid. CF 20175832, living culture CFCC 52883; CF 20175833, living culture CFCC 52884.

Notes: Cytospora elaeagnicola is associated with canker disease of Elaeagnus angustifolia. The phylogenetic inferences resolved this species as an individual clade both in ITS and combined multigene phylograms (Figures 1 and 2), which was closed to C. spiraeae from Spiraea salicifolia. Morphologically, Cytospora elaeagnicola has obvious symptoms with black area on bark, and smaller conidia (5.5–6.5 × 1.5–2 vs. 7–8 × 2–2.5 µm) as compared with C. spiraeae; the cultures of C. elaeagnicola are white, differing from the cultures of C. spiraeae which becomes fawn after 7–10 days [34]. Considering the clearly distinction between these two species based on molecular phylogenetic position and on the host affiliation, Cytospora elaeagnicola is thus described as a novel species.

Discussion

In the current study, C. elaeagnicola sp. nov. was described from infected branches and twigs of E. angustifolia in northwest region of China, an area that has undergone desertification at an alarming rate. Previously, Fan et al. [7] described C. elaeagni and C. nivea from E. angustifolia during the investigation of canker disease of three anti-desertification plants. Compared to C. elaeagnicola, C. elaeagni has smaller locules (630–920 µm) with larger conidia (6.3–9.3 × 2–2.9 µm) and dense cultures producing light brown pigment; C. nivea has obvious dark black conceptacle surrounding the conidiomata with larger conidia (6.2–9.2 × 1.7–2.4 µm), and cultures producing dark green to black pigment [7]. These morphological deviations are in line with the combined phylogenetic analyses which resolved C. elaeagnicola as a separate, highly supported clade, both in the single ITS analyses and the concatenated analyses.

Cytospora species were previously identified by host association and morphological characteristics. However, the uninformative illustrations and descriptions, overlapping morphological characteristics and low host-specificity have caused confusion in the identification of strains. Current study indicated more than one species of Cytospora are present on one host plant. In the future study, the taxonomy requires fresh collections from wide geographical ranges with comprehensive pathogenicity tests. Further studies are also needed in the clarification of the species diversity and in the understanding of their roles in plant diseases, especially for anti-desertification plants such as E. angustifolia in Northwestern China.