Mycobiology ISSN: 1229-8093 (Print) 2092-9323 (Online) Journal homepage: https://www.tandfonline.com/loi/tmyb20 Cytospora elaeagnicola sp. nov. Associated with Narrow-leaved Oleaster Canker Disease in China Linxuan Zhang, Lourdes V. Alvarez, Guido Bonthond, Chengming Tian & Xinlei Fan To cite this article: Linxuan Zhang, Lourdes V. Alvarez, Guido Bonthond, Chengming Tian & Xinlei Fan (2019): Cytospora�elaeagnicola sp. nov. Associated with Narrow-leaved Oleaster Canker Disease in China, Mycobiology, DOI: 10.1080/12298093.2019.1633902 To link to this article: https://doi.org/10.1080/12298093.2019.1633902 © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology. Published online: 05 Jul 2019. Submit your article to this journal View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tmyb20 MYCOBIOLOGY https://doi.org/10.1080/12298093.2019.1633902 RESEARCH ARTICLE Cytospora elaeagnicola sp. nov. Associated with Narrow-leaved Oleaster Canker Disease in China Linxuan Zhanga , Lourdes V. Alvarezb , Guido Bonthondc , Chengming Tiana and Xinlei Fana a The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing, China; Department of Biology, College of Science, Polytechnic University of the Philippines, Manila, Philippines; cGEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel, Germany b ABSTRACT ARTICLE HISTORY 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. Received 5 December 2018 Revised 5 June 2019 Accepted 13 June 2019 1. 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 CONTACT Xinlei Fan KEYWORDS Cytosporaceae; molecular phylogeny; new species; taxonomy 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 extype 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. xinleifan@bjfu.edu.cn ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/Licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 L. ZHANG ET AL. 2. Materials and methods 2.1. 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). 2.2. 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]. 2.3. 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). 2.4. 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 þ G þ 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 þ I þ 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. 3. Results 3.1. Phylogeny The ITS sequences of the three isolates of Cytospora from E. angustifolia were aligned with MYCOBIOLOGY 3 Table 1. Isolates and GenBank accession numbers used in this study. GenBank accession numbers Species C. abyssinica C. abyssinica C. acaciae C. ampulliformis C. ampulliformis C. atrocirrhata C. atrocirrhata C. austromontana C. berberidis C. berberidis C. berkeleyi C. berkeleyi C. brevispora C. brevispora C. carbonacea C. carpobroti C. cedri C. centrivillosa C. centrivillosa C. chrysosperma C. chrysosperma C. chrysosperma C. cinerostroma C. cincta C. cotini C. curvata C. davidiana C. davidiana C. diatrypelloidea C. disciformis C. disciformis C. donetzica C. donetzica C. elaeagni C. elaeagni C. elaeagnicola C. elaeagnicola C. elaeagnicola C. eriobotryae C. erumpens C. eucalypti C. eucalypticola C. eucalypticola C. eucalyptina C. eugeniae C. eugeniae C. fraxinigena C. fraxinigena C. friesii C. fugax C. fugax C. germanica C. gigaspora C. gigaspora C. gigaspora C. gigaspora C. hippopha€es C. hippopha€es C. japonica C. junipericola C. junipericola C. kantschavelii C. kantschavelii C. kunzei C. leucosperma C. leucosperma C. leucostoma C. leucostoma C. longiostiolata C. mali C. mali C. melnikii C. melnikii C. melnikii C. mougeotii C. multicollis Strain Host ITS LSU ACT RPB2 CMW 10181T CMW 10178 CBS 468.69 MFLUCC 16-0583T MFLUCC 16-0629 CFCC 89615 CFCC 89616 CMW 6735T CFCC 89927T CFCC 89933 StanfordT3T UCBTwig3 CBS 116829 CBS 116811T CFCC 89947 CMW 48981T CBS 196.50 MFLUCC 16-1206T MFLUCC 17-1660 CFCC 89629 CFCC 89981 CFCC 89982 CMW 5700T ATCC 32673 MFLUCC 14-1050T MFLUCC 15-0865T CXY 1350T CXY 1374 CMW 8549T CMW 6509T CMW 6750 MFLUCC 16-0574T MFLUCC 15-0864 CFCC 89632 CFCC 89633 CFCC 52882T CFCC 52883 CFCC 52884 IMI 136523T MFLUCC 16-0580T LSEQ ATCC 96150T CMW 5309 CMW 5882 CMW 7029 CMW 8648 BBH 42442 MFLUCC 14-0868T CBS 194.42 CXY1371 CXY1381 CXY1322 CFCC 89620T CFCC 89621 CFCC 50014 CFCC 89634T CFCC 89639 CFCC 89640 CBS 375.29 BBH 42444 MFLU 17-0882T CXY1383 CXY1386 CBS 118556 CFCC 89622 CFCC 89894 CFCC 50016 CFCC 50015 MFLUCC 16-0628T CFCC 50031 CFCC 50044 CFCC 89984 MFLUCC 15-0851T MFLUCC 16-0635 ATCC 44994 CBS 105.89T Eucalyptus globulus Eucalyptus globulus Ceratonia siliqua Sorbus intermedia Acer platanoides Juglans regia Juglans regia Eucalyptus pauciflora Berberis dasystachya Berberis dasystachya Eucalyptus globulus Eucalyptus globulus Eucalyptus grandis Eucalyptus grandis  tereticornis Ulmus pumila Carpobrotus edulis – Sorbus domestica Sorbus domestica Salix psammophila Populus alba subsp. pyramidalis Ulmus pumila Eucalyptus globulus – Cotinus coggygria Salix alba Populus davidiana Populus davidiana Eucalyptus globulus Eucalyptus grandis Eucalyptus globulus Rosa sp. Crataegus monogyna Elaeagnus angustifolia Elaeagnus angustifolia Elaeagnus angustifolia Elaeagnus angustifolia Elaeagnus angustifolia Eriobotrya japonica Salix  fragilis Sequoia sempervirens Eucalyptus nitens Eucalyptus grandis Eucalyptus grandis Tibouchina sp. Eugenia sp. Fraxinus ornus Fraxinus ornus Abies alba Populus simonii Populus ussuriensis Elaeagnus oxycarpa Juglans regia Juglans regia Juniperus procumbens Salix psammophila Hippophae rhamnoides Hippophae rhamnoides Prunus persicae Juniperus communis Juniperus communis Populus maximowiczii Populus maximowiczii Pinus radiata Pyrus bretschneideri Pyrus bretschneideri Sorbus aucuparia Sorbus pohuashanensis Salix  fragilis Crataegus sp. Malus baccata Rhus typhina Malus domestica Populus nigra Picea abies Quercus ilex subsp. rotundifolia AY347353 AY347354 DQ243804 KY417726 KY417727 KR045618 KR045619 AY347361 KR045620 KR045621 AY347350 AY347349 AF192321 AF192315 KR045622 MH382812 AF192311 MF190122 MF190123 KF765673 MH933625 KP281261 AY347377 DQ996041 KX430142 KY417728 KM034870 KM034869 AY347368 AY347374 AY347359 KY417731 KY417729 KR045626 KF765677 MK732341 MK732342 MK732343 AY347327 KY417733 AY347340 AY347358 AF260266 AY347375 AY347364 AY347344 MF190134 MF190133 AY347328 KM034852 KM034853 JQ086563 KR045628 KR045629 KR045630 KF765671 KR045632 KF765682 AF191185 MF190126 MF190125 KM034867 KM034867 DQ243791 KR045616 KR045617 MH820400 KR045634 KY417734 KR045636 KR045637 MH933644 KY417735 KY417736 AY347318 DQ243803 – – – KY417760 KY417761 KR045700 KR045701 – KR045702 KR045703 – – – – KP310812 MH411216 – MF190068 MF190069 KF765689 MH933660 KP310805 – – KX430143 KY417762 – – – – – KY417764 KY417763 KR045706 KF765693 MK732338 MK732339 MK732340 – KY417767 – – – – – – MF190079 MF190078 – – – JX524617 KR045708 KR045709 KR045710 KF765687 KR045712 KF765698 – MF190071 MF190072 – – – KR045698 KR045699 MH820393 KR045714 KY417768 KR045716 KR045717 MH933678 KY417769 KY417770 – – – – – KY417692 KY417693 KF498673 KF498674 – KU710990 KU710991 – – – – KP310842 – – – – KF765721 MH933533 KP310835 – – – KY417694 – – – – – KY417696 KY417695 KU710995 KU710996 MK732344 MK732345 MK732346 – KY417699 – – – – – – – – – – – – KU710997 KU710998 KU710999. KU711000 KU711001 KF765730 – – – – – – KU710988 KU710989 MH820408 KU711002 KY417700 KU711004 KU711005 MH933551 KY417701 KY417702 – – – – – KY417794 KY417795 KU710946 KU710947 – KU710948 KU710949 – – – – KU710950 – – MF377600 MF377601 KF765705 MH933597 KU710952 – – KX430144 KY417796 – – – – – KY417798 KY417797 KU710955 KU710956 MK732347 MK732348 MK732349 – KY417801 – – – – – – – – – – – – KU710957 KU710958 KU710959 KU710960 KU710961 KU710962 – – – – – – KU710944 KU710945 – – KY417802 KU710965 KU710966 MH933609 KY417803 KY417804 – – (continued) 4 L. ZHANG ET AL. Table 1. Continued. GenBank accession numbers Species C. myrtagena C. nivea C. nivea C. nivea C. palm C. palm C. parakantschavelii C. parakantschavelii C. parapersoonii C. parasitica C. paratranslucens C. paratranslucens C. pini C. pini C. populina C. predappioensis C. pruinopsis C. pruinosa C. pruinosa C. pruinosa C. prunicola C. quercicola C. quercicola C. rhizophorae C. ribis C. ribis C. rosae C. rostrata C. rostrata C. rusanovii C. rusanovii C. sacculus C. sacculus C. salicacearum C. salicacearum C. salicacearum C. salicicola C. salicicola C. salicina C. salicina C. schulzeri C. schulzeri C. sibiraeae C. sibiraeae C. sophorae C. sophorae C. sophoricola C. sophoricola C. sorbi C. sorbicola C. sorbicola C. spiraeae C. spiraeae C. tanaitica C. tibouchinae C. translucens C. ulmi C. valsoidea C. valsoidea C. variostromatica C. variostromatica C. vinacea C. viticola Diaporthe vaccinii Strain CBS 116843T MFLUCC 15-0860 CFCC 89641 CFCC 89643 CXY1276 CXY1280T MFLUCC 15-0857T MFLUCC 16-0575 T28.1T MFLUCC 15-0507T MFLUCC 15-0506T MFLUCC 16-0627 CBS 197.42 CBS 224.52T CFCC 89644T MFLUCC 17-2458T CFCC 50034T CFCC 50035 CFCC 50036 CFCC 50037 MFLU 17-0995T MFBBH 42443 MFLUCC 14-0867T MUCC302 CFCC 50026 CFCC 50027 MFLU 17-0885T CFCC 89909T CFCC 89910 MFLUCC 15-0853 MFLUCC 15-0854T CFCC 89624 CFCC 89625 MFLUCC 15-0509T MFLUCC 15-0861 MFLUCC 16-0587 MFLUCC 15-0866 MFLUCC 14-1052T MFLUCC 15-0862T MFLUCC 16-0637 CFCC 50040 CFCC 50042 CFCC 50045T CFCC 50046 CFCC 50047 CFCC 89598 CFCC 89596 CFCC 89595T MFLUCC 16-0631T MFLUCC 16-0584T MFLUCC 16-0633 CFCC 50049T CFCC 50050 MFLUCC 14-1057T CPC 26333T CXY1351 MFLUCC 15-0863T CMW 4309T CMW 4310 CMW 6766T CMW 1240 CBS 141585T CBS 141586T CBS 160.32 Host Tibouchiina urvilleana Salix acutifolia Elaeagnus angustifolia Salix psammophila Cotinus coggygria Cotinus coggygria Populus  sibirica Pyrus pyraster Prunus persicae Malus domestica Populus alba var. bolleana Populus alba Pinus Sylvestirs Pinus strobus Salix psammophila Platanus sp. Ulmus pumila Ulmus pumila Syzygium aromaticum Syzygium aromaticum Prunus sp. Quercus sp. Quercus sp. Eucalyptus grandis Ulmus pumila Ulmus pumila Rosa canina Salix cupularis Salix cupularis Populus  sibirica Salix babylonica Juglans regia Juglans regia Salix alba Salix  fragilis Prunus cerasus Salix alba Salix alba Salix alba Salix  fragilis Malus domestica Malus asiatica Sibiraea angustata Sibiraea angustata Styphnolobium japonicum Styphnolobium japonicum Styphnolobium japonicum Styphnolobium japonicum var. Sorbus aucuparia Acer pseudoplatanus Cotoneaster melanocarpus Spiraea salicifolia Spiraea salicifolia Betula pubescens Tibouchina semidecandra Populus davidiana Ulmus minor Eucalyptus grandis Eucalyptus grandis Eucalyptus globulus Eucalyptus grandis Vitis interspecific Vitis vinifera Vaccinium macrocarpon ITS LSU ACT RPB2 AY347363 KY417737 KF765683 KF765685 JN402990 JN411939 KY417738 KY417739 AF191181 KY417740 KY417741 KY417742 AY347332 AY347316 KF765686 MG873484 KP281259 KP281260 KP310800 MH933650 MG742350 MF190128 MF190129 EU301057 KP281267 KP281268 MF190131 KR045643 KR045644 KY417743 KY417744 KR045645 KF225616 KY417746 KY417745 KY417748 KY417749 KU982636 KY417750 KY417751 KR045649 KR045650 KR045651 KR045652 KR045653 KR045654 KR045656 KR045655 KY417752 KY417755 KY417758 MG707859 MG707860 KT459411 KX228284 KM034874 KY417759 AF192312 AF192312 AY347366 AF260263 KX256256 KX256239 KC343228 – KY417771 KF765699 – – – KY417772 KY417773 – KY417774 KY417775 KY417776 – – KF765702 MG873480 KP310806 KP310807 KP310802 MH933685 MG742351 MF190074 MF190073 – KP310813 KP310814 MF190075 KR045722 KR045723 KY417777 KY417778 KR045724 KM401887 KY417780 KY417779 KY417782 KY417783 KU982635 KY417784 KY417785 KR045728 KR045729 KR045730 KR045731 KR045732 KR045733 KR045735 KR045734 KY417786 KY417789 KY417792 MG707643 MG707644 KT459412 KX228335 – – – – – – – – – – KY417703 KU711006 – – – KY417704 KY417705 – KY417706 KY417707 KY417708 – – KU711007 – KP310836 KP310837 KP310832 MH933558 MG742353 – – – KP310843 KP310844 – KU711009 KU711010 KY417709 KY417710 KM401888 KM401889 KY417712 KY417711 KY417714 KY417715 KU982637 KY417716 KY417717 KU711013 KU711014 KU711015 KU711015 KU711017 KU711018 KU711020 KU711019 KY417718 KY417721 KY417724 MG708196 MG708197 KT459413 – – – – – – – – – JQ807297 – KY417805 KU710967 KU710968 – – KY417806 KY417807 – KY417808 KY417809 KY417810 – – KU710969 – KU710970 KU710971 – – MG742352 – – – KU710972 – – KU710974 KU710975 KY417811 KY417812 KU710976 – KY417814 KY417813 KY417816 KY417817 – KY417818 KY417819 KU710980 KU710981 KU710982 KU710983 KU710984 KU710985 KU710987 KU710986 KY417820 KY417823 KY417826 MG708199 MG708200 – – – – – – – – – – – 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). 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 ¼ 927, CI ¼ 0.409, RI ¼ 0.830, RC ¼ 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 ¼ 99/100/1; marked in blue in MYCOBIOLOGY 5 Figure 1. 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. 6 L. ZHANG ET AL. Figure 1. Continued 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 parsimonyinformative. MP analysis generated 105 parsimonious trees, one of which is presented in Figure 1 (TL ¼ 2,350, CI ¼ 0.412, RI ¼ 0.827, RC ¼ 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 ¼ 100/ 100/1) (marked in blue in Figure 2). 3.2. Taxonomy Cytospora elaeagnicola X.L. Fan sp. nov. Figure 3 Mycobank: MB830292. Etymology: Named after the host genus on which it was collected, Elaeagnus. MYCOBIOLOGY 7 Figure 2. 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. 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) lm (x ¼ 1060 ± 120 mm, n ¼ 30) in diameter. Conceptacle absent. Ectostromatic disc iron grey to violaceous black, circular, disc dark, (160–)170–310(–350) mm (x ¼ 240 ± 60 mm, n ¼ 30) in diameter, with one ostiole in the centre of disc. Ostiole conspicuous, circular to ovoid, iron grey to violaceous 8 L. ZHANG ET AL. Figure 2. Continued black at the same level as the disc, (48–)51–71(–78) mm (x ¼ 60 ± 11 mm, n ¼ 30) in diameter. Conidiophores hyaline, branched at base or not branched, thin walled, filamentous, (12–)13.5–19.5(–20) lm (x ¼ 16.5 ± 3 mm, n ¼ 30). Conidiogenous cells enteroblastic, phialidic. Conidia hyaline, allantoid, eguttulate, smooth, aseptate, thin-wall, 5.5–6.5(–7)  (1–)1.5–2 mm (x ¼ 6.1 ± 0.4  1.6 ± 0.1 mm, n ¼ 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 mm) 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. 4. Discussion In the current study, C. elaeagnicola sp. nov. was described from infected branches and twigs of E. MYCOBIOLOGY 9 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 ¼ 250 lm, D ¼ 200 lm, E ¼ 10 lm, F ¼ 5 lm). 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 mm) with larger conidia (6.3–9.3  2–2.9 mm) 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 mm), 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. Disclosure statement No potential conflict of interest was reported by the authors. 10 L. ZHANG ET AL. Funding [15] This work was supported by the [National Natural Science Foundation of China] under Grant [number 31670647]. All authors want to thank the Experimental Teaching Centre (College of Forestry, Beijing Forestry University) for providing installed scientific equipments during the whole process. [16] ORCID Linxuan Zhang http://orcid.org/0000-0002-4828-882X Lourdes V. Alvarez http://orcid.org/0000-00033246-5489 Guido Bonthond http://orcid.org/0000-0002-9823-6761 Chengming Tian http://orcid.org/0000-0002-3352-7664 Xinlei Fan http://orcid.org/0000-0002-4946-4442 [17] [18] [19] [20] [21] References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] Spielman LJ. Taxonomy and biology of Valsa species on hardwoods in North America, with special reference to species on maples. New York, US: Cornell University; 1983. Adams GC, Wingfield MJ, Common R, et al. 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