New species and records of Cytospora (Cytosporaceae, Diaporthales) from tree branches in Hebei Province, China

187 New species and records of Cytospora (Cytosporaceae, Diaporthales) from tree branches in Hebei Province, China Tingqian Pei1, Dianguang Xiong1, Yingmei Liang1,2 1 The Key Laboratory for Silviculture and Conservation of the Ministry of Education, Beijing Forestry University, Beijing 100083, China 2 Museum of Beijing Forestry University, Beijing Forestry University, Beijing 100083, China Corresponding author: Yingmei Liang ([email protected]) Copyright: © Tingqian Pei et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Species of Cytospora have been commonly reported as plant pathogens with wide host ranges and geographic distributions. In this study, ten strains of this genus were isolated from branches collected in Hebei Province, China. They were identified based on a multi-locus phylogeny of ITS, act, rpb2, tef1-α, and tub2 genes, along with morphological characters. As a result, they were identified as six species, including five known species (C. ampla, C. pseudochrysosperma, C. sophoricola, C. sorbariae, and C. yinchuanensis) and one new species (C. hebeiensis). Among the known species, C. ampla, C. sorbariae, and C. yinchuanensis were newly discovered on Malus spectabilis; C. pseudochrysosperma was newly discovered on Salix matsudana; and C. sophoricola was newly discovered on Caragana microphylla. The results enrich the diversity of Cytospora species associated with tree canker and dieback diseases in Hebei Province, China. Key words: Canker disease, Cytosporaceae, multi-gene phylogeny, new species, new records Introduction Cytospora (Cytosporaceae, Diaporthales) was established by Ehrenberg with four species: C. betulina, C. epimyces, C. resinae, and C. ribis (Ehrenberg 1818). Subsequently, C. chrysosperma, which had been reported on Populus nigra, was designated as the type species (Donk 1964). Species of Cytospora can cause canker diseases in many woody plants, which can lead to weakness in growth or death of host plants (Sinclair et al. 1987; Adams et al. 2005; Fan et al. 2020; Stewart et al. 2022; Ilyukhin et al. 2023; Lin et al. 2023a, b). Cytospora and its related sexual morphs, Leucostoma, Valsa, Valsella, and Valseutypella, were initially listed by early fungal literature for their identification (Fries 1823; Saccardo 1884; Kobayashi 1970; Barr 1978; Sutton 1980; Gvritishvili 1982; Spielman 1983, 1985). It was not until 2005 that Adams et al. proposed that Valsa was the only sexual type genus of Cytospora, and the other genera were treated as synonyms of Valsa (Adams et al. 2005). With the end of dual nomenclature for pleomorphic fungi (Wingfield et al. 2012; Crous et al. 2015), the single-name system, which employs the earliest published or most commonly used name, led to the retention of the older genus Cytospora over its Academic editor: Ning Jiang Received: 20 October 2025 Accepted: 3 December 2025 Published: 19 December 2025 Citation: Pei T, Xiong D, Liang Y (2025) New species and records of Cytospora (Cytosporaceae, Diaporthales) from tree branches in Hebei Province, China. MycoKeys 126: 187–212. https://doi.org/10.3897/ mycokeys.126.175474 MycoKeys 126: 187–212 (2025) DOI: 10.3897/mycokeys.126.175474 188 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora sexual morph, Valsa (1849), on the list of protected fungi (McNeill et al. 2012; Fan et al. 2015a, b; Rossman et al. 2015). Previously, morphological characteristics were employed to classify the sexual morph of Cytospora. Saccardo (1884) classified the genus Valsa into Macrosporae and Microsporae based on the size of ascospores. However, researchers suggested that stromata features are highly variable and have failed to provide reliable criteria to distinguish species of Cytospora (Urban 1958; Spielman 1985). Therefore, a method combining phylogenetics and morphology was introduced for related species identification. For instance, Adams et al. (2005, 2006) identified 62 Cytospora species from various hosts (e.g., Eucalyptus, Malus, Pinus) in South Africa using morphological and ITS-rDNA phylogenetic analyses, providing an identification key. Currently, researchers use morphology combined with multi-gene phylogenetic analyses to define species of Cytospora, and this approach has been progressively refined. Following the initial discovery of 14 new species by Norphanphoun et al. (2017) using four loci (ITS, LSU, rpb2, and act), Fan et al. (2020) enhanced phylogenetic resolution with the addition of tef1-α and tub2, clarifying Cytospora diversity and proposing thirteen new species and a new combination. The methodology was further validated by Jia et al. (2023) in a limited geographical area (Fengtai, Beijing). Subsequently, Lin et al. (2024) established a taxonomic framework based on five genetic loci (ITS, act, rpb2, tef1-α, tub2) and constructed a new morphological grouping system: three asexual morphological groups (including thirteen types, a1–a13) and three sexual morphological groups (including eight types, s1–s8). Recently, Jiang et al. (2025a) classified Cytospora into ten species complexes and twelve singletons, providing valuable information for future research. This study delineates the taxonomic status of ten Cytospora isolates from diseased branches in Hebei Province, China, with comprehensive descriptions, microscopic photographs, and updated phylogenetic trees, along with one new species and five new host records. Materials and methods Sample collection and isolation Ten specimens were collected from diseased branches of woody hosts distributed in Hebei Province. Sampled trees expressed general symptoms and signs of canker diseases, including elongated, slightly sunken, and discolored areas in the bark; several prominent, dark conidiomata and ascomata immersed in the bark; and erumpent fruiting bodies breaking through the bark surface when mature. The bark appeared yellow, brown, reddish brown, gray, or black, becoming watery or odorous as the tissues deteriorated (Fig. 1). A total of ten isolates were obtained by removing mucoid spore mass from conidiomata and ascomata, spreading the suspension on the surface of potato dextrose agar (potato, 200 g; glucose, 20 g; agar, 20 g; distilled water to complete 1000 mL) in a Petri dish, and incubating at 25 °C in the dark. After discrete colonies grew on the plate, a small piece of PDA block was cut from the edge of 189 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora Figure 1. Disease symptoms from different tree branches. A. Caragana microphylla; B, C. Malus pumila; D, E. M. spectabilis F. Salix matsudana. a well-isolated single colony and transferred to the center of a fresh PDA plate to obtain a pure culture. All specimens are deposited in the Museum of the Beijing Forestry University (BJFC), and the cultures are maintained in the China Forestry Culture Collection Center (CFCC; https://cfcc.caf.ac.cn/). Morphological observation The study of Cytospora involved morphological observations of fruiting bodies growing on tree bark surfaces. Macromorphological features were photographed using a Leica stereomicroscope (M205 FA) (Leica Microsystems, Wetzlar, Germany), including the arrangement and size of stromata; the presence or absence of a conceptacle; the size, color, and shape of discs; and the diameter of ostioles. Micromorphological features were photographed using a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan), including the size and shape of conidiophores, asci, and conidia/ascospores. We measured at least ten conidiostromata/ascostromata, 30 asci, and 50 conidia/ascospores to calculate the mean size. Colony diameters were measured, and the colony colors were described according to the color charts of Rayner (1970). 190 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora DNA extraction, PCR amplification, and sequencing Colonies used for DNA extraction were grown on PDA for five days and obtained from the surface by scraping. The genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). DNA products were stored at −20 °C. The PCR mixture volume was 20 µL, which consisted of 10 µL Mix (Promega), 7 µL double deionized water, 1 µL each preand post-primer, and 1 µL DNA template. Five loci (ITS, act, rpb2, tef1-α, and tub2) were used for comparison-based phylogenetic analyses to determine the identities of the isolates. The primers and PCR conditions used in the current study are listed in Table 1. Amplified PCR products were sent to a commercial sequencing provider (Tsingke Biotechnology Co. Ltd., Beijing, China). The forward and reverse sequences were edited and assembled using SeqMan v.7.1.0 software. The sequences obtained in this study were deposited in GenBank (http://www.ncbi.nlm.nih.gov). Phylogenetic tree The phylogenetic analysis was performed based on a combined dataset of sequences to compare Cytospora species from the current study with other sequences obtained from GenBank (Table 2). The sequence datasets used in this study were based on Jiang et al. (2025a), and outgroup taxa were set as follows: C. brunnea (CFCC 71082) and C. viticola (CBS 141605) for the Chrysosperma SC, and C. donglingensis (CFCC 53159) and C. viticola (CBS 141605) for the Ribis SC. Table 2. Strains of Cytospora used in the molecular analyses in Chrysosperma and Ribis SCs. NA: not applicable. Strains in this study are marked in bold, and * indicates ex-type strains. Species Strain numbers Host GenBank accession numbers References ITS act rpb2 tef1-α tub2 Cytospora ailanthicola CFCC 89970* Ailanthus altissima MH933618 MH933526 MH933592 MH933494 MH933565 Jiang et al. (2025a) C. ailanthicola CFCC 58712 Salix matsudana PQ778497 PV454710 PV461889 PV467118 PV467242 Jiang et al. (2025a) C. ailanthicola CFCC 58713 Salix chaenomeloides PQ778498 PV454711 PV461890 PV467119 PV467243 Jiang et al. (2025a) C. ailanthicola CFCC 71105 Salix sp. PQ778508 PV454719 PV461900 PV467129 PV467253 Jiang et al. (2025a) C. ampla CFCC 71189* Rubus biflorus PQ778511 PV454722 PV461903 PV467132 PV467256 Jiang et al. (2025a) C. ampla CFCC 71044* Salix sp. PQ778509 PV454720 PV461901 PV467130 PV467254 Jiang et al. (2025a) C. ample CFCC 72627 Salix matsudana PV715411 PV702606 PV714133 PV766892 PV745444 This study C. ample CFCC 72603* Malus spectabilis PV715414 PV702609 NA PV766894 NA This study C. ample CFCC 72604 Malus spectabilis PV715416 NA NA PV766896 NA This study C. annulata CBS 118089 Acer rubrum PP988738 PQ074598 PQ074911 PQ074273 PQ075228 Lin et al. (2024) C. auerswaldii CBS 153.29 unknown PP988740 PQ074600 PQ074913 PQ074275 PQ075230 Lin et al. (2024) C. betulae CBS 141622* Betula papyrifera PP988752 PQ074610 PQ074922 PQ074284 PQ075236 Lin et al. (2024) Table 1. Genes used in this study with PCR primers. Locus PCR primers Thermal cycles References ITS ITS1/ITS4 (95 °C: 30 s, 51 °C: 30 s, 72 °C: 1min) × 35 cycles White et al. (1990) act ACT-512F/ACT-783R (95 °C: 15 s, 55 °C: 20 s, 72 °C: 1min) × 35 cycles Carbone and Kohn (1999) rpb2 RPB2-5F/RPB2-7cR (95 °C: 30 s, 55 °C: 30 s, 72 °C: 1min) × 35 cycles Liu et al. (1999) tef1-α EF1-728F/EF1-1567R (95 °C: 45 s, 55 °C: 45 s, 72 °C: 1min) × 35 cycles Rehner et al. (2005) tub2 Bt2a/Bt2b (95 °C: 30 s, 52 °C: 60 s, 72 °C:1 min) × 35 cycles Glass and Donaldson (1995) 191 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora Species Strain numbers Host GenBank accession numbers References ITS act rpb2 tef1-α tub2 C. brunnea CFCC 71082* Prunus mira PQ778532 PV454738 PV461923 PV467153 PV467277 Jiang et al. (2025a) C. carbonacea CFCC 89947 Ulmus pumila KR045622 KP310842 KU710950 KP310855 KP310825 Fan et al. (2020) C. chrysosperma CBS 120 Populus balsamifera PP988773 PQ074626 PQ074942 PQ074304 PQ075255 Lin et al. (2024) C. chrysosperma CBS 197.50* Populus tremula PP988777 PQ074630 PQ074946 PQ074308 PQ075259 Lin et al. (2024) C. chungenii CFCC 71027* Hippophae rhamnoides PQ778534 PV454740 PV461925 PV467155 PV467279 Jiang et al. (2025a) C. chungenii CFCC 71310* Hippophae rhamnoides PQ778535 PV454741 PV461926 PV467156 PV467280 Jiang et al. (2025a) C. coryli CFCC 53162* Corylus mandshurica MN854450 NA MN850751 MN850758 MN861120 Zhu et al. (2020) C. crataegina CFCC 56027* Crataegus pinnatifida PP988791 PQ074642 PQ074958 PQ074321 PQ075271 Lin et al. (2024) C. crataegina CFCC 56029 Crataegus pinnatifida PP988793 PQ074644 PQ074960 PQ074323 PQ075273 Lin et al. (2024) C. diminuta CFCC 71034* Sophora moorcroftiana PQ778540 PV454746 PV461931 PV467161 PV467285 Jiang et al. (2025a) C. diminuta CFCC 71312* Sophora moorcroftiana PQ778541 PV454747 PV461932 PV467162 PV467286 Jiang et al. (2025a) C. donglingensis CFCC 53159* Platycladus orientalis MW418412 MW422903 MW422915 MW422927 MW422939 Pan et al. (2018) C. eastringensis CFCC 58222* Populus adenopoda PP988818 NA PQ074980 PQ074346 NA Lin et al. (2024) C. elaeagni CFCC 58241 Elaeagnus angustifolia PP988819 PQ074661 PQ074981 PQ074347 NA Lin et al. (2024) C. euonymicola CFCC 50499* Euonymus kiautschovicus MH933628 MH933535 MH933598 MH933503 MH933570 Fan et al. (2020) C. fengtaiensis CFCC 59449* Acer palmatum OR826167 OR832000 OR832022 OR832044 OR832064 Jia et al. (2023) C. fugax CBS 203.42* Salix sp. PP988848 PQ074686 PQ075006 NA PQ075318 Adams et al. (2006) C. fugax CFCC 71307 Salix takasagoalpina PQ778546 PV454752 PV461937 PV467167 PV467291 Jiang et al. (2025a) C. gigaloculata CFCC 89620* Juglans regia KR045628 KU710997 KU710957 KU710920 KR045669 Fan et al. (2020) C. gigaloculata CFCC 71097 Rosa multiflor PQ778547 PV454753 PV461938 PV467168 PV467292 Jiang et al. (2025a) C. gigaloculata CFCC 71098 Rosa multiflor PQ778548 PV454754 PV461939 PV467169 PV467293 Jiang et al. (2025a) C. globosa CBS 118976 Abies alba PP988851 PQ074689 PQ075009 PQ074376 PQ075321 Lin et al. (2024) C. globosa MFLUCC 161153* Abies alba MT177935 NA MT432212 MT454016 NA Li et al. (2020) C. guyuanensis CFCC 55855* Salix sp. PP988853 NA PQ075011 PQ074378 PQ075323 Lin et al. (2024) C. haidianensis CFCC 54057* Euonymus alatus MT360042 MT363979 MT363988 MT363998 MT364008 Zhou et al. (2020) C. hebeiensis CFCC 72601* Malus pumila PV715410 PV702605 PV714132 PV766891 PV745443 This study C. hejingensis CFCC 59571* Salix sp. PP060455 PP059657 PP059663 PP059667 PP059673 Wang et al. (2024) C. hippophaes CBS 259.88 Hippophae rhamnoides PP988856 NA PQ075014 PQ074381 PQ075326 Lin et al. (2024) C. hippophaes CFCC 58943 Hippophae rhamnoides PP988858 PQ074692 PQ075016 PQ074383 PQ075327 Lin et al. (2024) C. hippophaes CFCC 71064 Hippophae rhamnoides PQ778552 NA PV461943 PV467172 PV467297 Jiang et al. (2025a) C. iranica IRAN 4200C* Malus domestica MW295652 MZ014512 MW824359 MW394146 NA Hanifeh et al. (2022) C. joaquinensis CBS 144235 Populus deltoides MG971895 MG972044 NA MG971605 NA Lawrence et al. (2018) C. juniperina CFCC 50501* Juniperus przewalskii MH933632 MH933539 MH933602 MH933507 NA Fan et al. (2020) C. leucosperma CBS 109491 Fagus sylvatica PP988884 PQ074714 PQ075035 PQ074407 PQ075347 Lin et al. (2024) C. lhasaensis CFCC 59094* Rosa omeiensis OR769863 OR767319 OR767333 OR767359 OR767347 Li et al. (2024) C. lhasaensis CFCC 71113 Rosa sericea PQ778558 PV454762 PV461949 PV467178 PV467301 Jiang et al. (2025a) C. linzhiensis CFCC 71045* Euonymus japonicus PQ778562 PV454765 PV461952 PV467181 PV467304 Jiang et al. (2025a) C. linzhiensis CFCC 71179* Alnus nepalensis PQ778563 PV454766 PV461953 PV467182 PV467305 Jiang et al. (2025a) C. longispora CBS 144236* Prunus domestica MG971905 MG972054 NA MG971615 NA Fan et al. (2020) C. longistiolata MFLUCC 160628 Salix× fragilis KY417734 KY417700 KY417802 NA NA Norphanphoun et al. (2017) C. macropycnidia CBS 149338* Vitis vinifera OP038094 OP003977 OP095265 OP106954 OP079909 Travadon et al. (2022) C. malvicolor CFCC 56567* Corylus mandshurica PP988915 PQ074744 PQ075063 PQ074438 PQ075377 Lin et al. (2024) 192 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora Species Strain numbers Host GenBank accession numbers References ITS act rpb2 tef1-α tub2 C. malvicolor CFCC 56577* Corylus mandshurica PP988916 PQ074745 PQ075064 PQ074439 PQ075378 Lin et al. (2024) C. melnikii CFCC 89984 Rhus typhina MH933644 MH933551 MH933609 MH933515 MH933580 Fan et al. (2020) C. mougeotii CBS 198.50 Picea abies PP988918 PQ074747 PQ075066 PQ074441 PQ075380 Lin et al. (2024) C. neolhasaensis CFCC 58706* Salix wallichiana PP988902 PQ074732 PQ075052 PQ074425 PQ075365 Jiang et al. (2025a) C. neolhasaensis CFCC 58862* Salix wallichiana PQ778576 PV454777 PV461963 PV467192 PV467315 Jiang et al. (2025a) C. nobilis CFCC 58227 Laurus nobilis PP988928 PQ074756 PQ075075 PQ074449 PQ075389 Lin et al. (2024) C. nobilis CFCC 58228 Laurus nobilis PP988929 PQ074757 PQ075076 PQ074450 PQ075390 Lin et al. (2024) C. nobilis CFCC 71102 Salix takasagoalpina PQ778577 PV454778 PV461964 PV467193 PV467316 Jiang et al. (2025a) C. nobilis CFCC 71414 Salix takasagoalpina PQ778578 PV454779 PV461965 PV467194 PV467317 Jiang et al. (2025a) C. piceae CFCC 52841* Picea crassifolia MH820398 MH820406 MH820395 MH820402 MH820387 Pan et al. (2018) C. pinastri CBS 113.81 Abies alba PP988941 PQ074768 PQ075087 PQ074461 PQ075401 Lin et al. (2024) C. platycladicola CFCC 50038* Platycladus orientalis KT222840 MH933555 MH933613 MH933519 MH933584 Fan et al. (2020) C. populina CFCC 89644* Salix psammophila KF765686 KU711007 KU710969 KU710930 KR045681 Fan et al. (2020) C. populinopsis CFCC 50032* Sorbus aucuparia MH933648 MH933556 MH933614 MH933520 MH933585 Fan et al. (2020) C. prunicola MFLU 170995* Prunus sp. MG742350 MG742353 MG742352 NA NA Norphanphoun et al. (2017) C. pseudochrysosperma CFCC 54081* Populus sp. MZ702631 NA NA OK303613 OK303680 Lin et al. (2024) C. pseudochrysosperma CFCC 89981* Populus alba var. pyramidalis MH933625 MH933533 MH933597 MH933501 MH933568 Lin et al. (2024) C. pseudochrysosperma CFCC 72617 Salix babylonica PV715412 PV702607 PV714134 PV766893 PV745445 This study C. pseudochrysosperma CFCC 72613 Salix babylonica PV715413 PV702608 PV714135 NA PV745446 This study C. qinghaiensis CFCC 50026* Ulmus pumila KP281267 KP310843 KU710972 KP310856 KP310826 Lin et al. (2024) C. qingshuiensis CFCC 56268* Platycladus orientalis PP988956 PQ074782 PQ075099 NA NA Lin et al. (2024) C. qingshuiensis CFCC 56349* Platycladus orientalis PP988957 PQ074783 PQ075100 PQ074474 PQ075415 Lin et al. (2024) C. qingshuiensis ZHKUCC 230978 Malus domestica PP829299 PP850109 PP839460 NA PP839451 Lin et al. (2024) C. ribis CBS 187.36 Ribes rubrum PP988963 PQ074788 PQ075106 PQ074480 PQ075420 Adams et al. (2006) C. rosigena MFLUCC 180921 Rosa sp. MN879872 NA NA NA NA Li et al. (2020) C. rostrata CFCC 89909* Salix cupularis KR045643 KU711009 KU710974 NA NA Lin et al. (2024) C. rostrata CFCC 89910 Salix cupularis KR045644 KU711010 KU710975 KU710933 NA Lin et al. (2024) C. saccardoi CBS 109752R Juniperus communis PP988975 NA NA PQ074492 PQ075430 Lin et al. (2024) C. salicacearum MFLUCC 150509* Salix alba KY417746 KY417712 KY417814 NA NA Fan et al. (2020) C. salicina CBS 507.77 unknowm PP988981 PQ074804 PQ075122 PQ074497 PQ075435 Lin et al. (2024) C. salicina MFLUCC 150862* Salix alba KY417750 KY417716 KY417818 NA NA Fan et al. (2020) C. sanbaensis CFCC 58242* Populus adenopoda PP988983 PQ074806 PQ075124 PQ074499 PQ075437 Lin et al. (2024) C. schulzeri MFLUCC 150507* Malus domestica KY417740 KY417706 KY417808 NA NA Norphanphoun et al. (2017) C. shaanxiensis CFCC 56032* Lindera obtusiloba PP988987 PQ074810 PQ075128 PQ074502 PQ075441 Lin et al. (2024) C. sidaohensis CFCC 56042* Corylus heterophylla PP988992 PQ074815 PQ075133 PQ074507 PQ075446 Lin et al. (2024) C. sinensis CFCC 58231 Populus simonii PP988995 PQ074818 PQ075136 PQ074510 PQ075449 Lin et al. (2024) C. sinensis CFCC 58235* Populus simonii PP988997 PQ074820 PQ075138 PQ074512 PQ075451 Lin et al. (2024) C. songshanensis CFCC 56351* Platycladus orientalis PP989006 PQ074828 PQ075147 PQ074521 PQ075459 Lin et al. (2024) C. sophorae CFCC 50048 Magnolia grandiflora MH820401 MH820409 MH820397 MH820405 MH820390 Fan et al. (2020) C. sophorae CFCC 89598 Styphnolobium japonicum KR045654 KU711018 KU710985 KU710941 KR045695 Fan et al. (2020) C. sophoricola CFCC 89595* Styphnolobium japonicum KR045655 KU711019 KU710986 KU710942 KR045696 Fan et al. (2020) C. sophoricola CFCC 72610 Caragana microphylla PV715409 PV702604 PV714131 PV766890 PV745442 This study C. sophoriopsis CFCC 58464 Populus szechuanica PP989009 PQ074831 PQ075150 PQ074524 PQ075461 Lin et al. (2024) 193 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora Species Strain numbers Host GenBank accession numbers References ITS act rpb2 tef1-α tub2 C. sorbariae CFCC 56025* Corylus heterophylla PP988788 PQ074639 PQ074955 PQ074318 PQ075268 Jia et al. (2023) C. sorbariae CFCC 59445* Sorbaria sorbifolia OR826176 OR832009 OR832031 OR832053 OR832073 Jia et al. (2023) C. sorbariae CFCC 72609 Malus spectabilis PV715417 NA NA NA PV745448 This study C. suecica CBS 450.51* Populus tremula PP989015 PQ074834 PQ075156 PQ074530 PQ075467 Lin et al. (2024) C. tanaitica MFLUCC 141057* Betula pubescens KT459411 KT459413 NA NA NA Ariyawansa et al. (2015) C. tetraspora CFCC 55847* Quercus aliena PP989021 PQ074840 PQ075162 PQ074536 PQ075473 Lin et al. (2024) C. tetraspora CFCC 56279* Tilia mongolica PP989023 PQ074842 PQ075164 PQ074538 PQ075475 Lin et al. (2024) C. tritici CBS 118561 Populus simonii PP989038 PQ074856 PQ075175 PQ074549 PQ075486 Lin et al. (2024) C. tritici CBS 118563 Populus nigra cv. italica PP989039 PQ074857 PQ075176 PQ074550 PQ075487 Lin et al. (2024) C. ulmi MFLUCC 150863* Xylocarpus moluccensis KY417759 KY417725 KY417827 NA NA Norphanphoun et al. (2017) C. ulmicola MFLUCC 181227* Xylocarpus moluccensis MH940220 MH940216 NA NA NA Phookamsak et al. (2019) C. verrucosa CFCC 53157 * Platycladus orientalis MW418408 NA MW422911 MW422923 MW422935 Pan et al. (2021) C. viticola CBS 141605 Vitis vinifera PP989058 PQ074868 PQ075185 PQ074562 PQ075497 Travadon et al. (2022) C. washingtonensis CBS 141619* Crataegus sp. PP989065 PQ074874 PQ075192 PQ074569 PQ075502 Lin et al. (2024) C. xinjiangensis CFCC 53183* Rosa sp. MK673065 MK673035 MK673005 MK672952 MK672981 Pan et al. (2020) C. yakimana CBS 149297* Vitis vinifera OM976602 ON012555 ON045093 ON012569 ON086750 Travadon et al. (2022) C. yinchuanensis CFCC 50040* Malus domestica KR045649 KU711013 KU710980 KU710936 KR045690 Lin et al. (2024) C. yinchuanensis CFCC 50042* Malus asiatica KR045650 KU711014 KU710981 KU710937 KR045691 Lin et al. (2024) C. yinchuanensis CFCC 72611 Malus spectabilis PV715415 PV702610 NA PV766895 PV745447 This study C. yinchuanensis CFCC 72612 Malus pumila PV715418 NA NA NA NA This study C. yingmeiae CFCC 71203* Quercus semecarpifolia PQ778629 PV454829 PV462013 PV467238 PV467361 Jiang et al. (2025a) C. yingmeiae CFCC 71308* Quercus semecarpifolia PQ778630 PV454830 NA PV467239 NA Jiang et al. (2025a) C. zhaitangensis CFCC 56227* Euonymus japonicus OQ344750 OQ410623 OQ398733 OQ398760 OQ398789 Lin et al. (2024) C. zhaitangensis CFCC 58608 Corylus mandshurica PP989073 PQ074882 PQ075199 PQ074577 PQ075509 Lin et al. (2024) All sequences were aligned in MAFFT v. 7 on the web server (https://mafft. cbrc.jp/alignment/server/) (Katoh and Standley 2013; Katoh et al. 2019) and adjusted in MEGA v. 7.0 (Tamura et al. 2013). Alignments excluded ambiguous regions. Multi-gene phylogenetic analyses employing Maximum Likelihood (ML) analysis (Guindon et al. 2010) and Bayesian inference (BI) analysis (Ronquist et al. 2012) were conducted using PhyML v. 3.0 and MrBayes v. 3.1.2 software, respectively. The phylogenetic tree was visualized with FigTree v. 1.4.0 (http://tree. bio.ed.ac.uk/software/figtree/) and additionally edited with Adobe Illustrator CS v. 5 (Adobe Systems Inc., USA). Maximum Likelihood bootstrap values (MLBP) ≥ 75% and Bayesian posterior probabilities (BPP) ≥ 0.90 are shown for each tree. Results Phylogenetic analyses Phylogenetic analyses placed the ten isolates into two species complexes, two in the Chrysosperma SC and eight in the Ribis SC. To better resolve their relationships, separate phylogenetic trees were constructed for each complex. 194 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora In the Chrysosperma SC, the gene loci ITS, act, rpb2, tef1-α, and tub2 were combined and analyzed to infer the phylogenetic placement of two isolates. The dataset consisted of 36 sequences, including the outgroup Cytospora brunnea (CFCC 71082) and C. viticola (CBS 141605). Cytospora ingroup strains had a total of 2,846 characters, including gaps (523 characters for ITS, 267 for act, 728 for rpb2, 590 for tef1-α, and 738 for tub2). MLBP ≥ 75% and BPP ≥ 0.90 are shown above the branches. For ML analysis, the substitution model (GTR+G+I model) for each dataset was selected following recent studies (Pan et al. 2018, 2020; Fan et al. 2020). Confidence levels for the nodes were determined using 1,000 replicates of bootstrapping methods (Hillis and Bull 1993). The matrix had 757 distinct alignment patterns. Estimated base frequencies were as follows: A = 0.250457, C = 0.289689, G = 0.236963, T = 0.222891; substitution rates: AC = 1.108390, AG = 3.318151, AT = 1.367714, CG = 0.680654, CT = 6.041623, GT = 1.000000; gamma distribution shape parameter: α = 0.250954. The two isolates aggregated with C. pseudochrysosperma (Fig. 2). Figure 2. Phylogenetic tree inferred from ML analysis based on combined ITS, act, rpb2, tef1-α, and tub2 sequence data of the Chrysosperma SC. The tree was rooted with Cytospora brunnea (CFCC 71082) and C. viticola (CBS 141605). Numbers above the branches indicate MLBP ≥ 75% and BPP ≥ 0.90. Ex-type isolates are marked in bold. Isolates in this study are highlighted in green. 0.03 Cytospora ailanthicola CFCC 89970 Cytospora salicina CBS 507.77 Cytospora brunnea CFCC 71082 Cytospora nobilis CFCC 71414 Cytospora nobilis CFCC 71102 Cytospora pseudochrysosperma CFCC 89981 Cytospora pseudochrysosperma CFCC 72613 Cytospora neolhasaensis CFCC 58706 Cytospora sinensis CFCC 58231 Cytospora tritici CBS 118563 Cytospora suecica CBS 450.51 Cytospora salicacearum MFLUCC 15-0509 Cytospora ailanthicola CFCC 58712 Cytospora pseudochrysosperma CFCC 54081 Cytospora chrysosperma CBS 197.50 Cytospora salicina MFLUCC 15-0862 Cytospora viticola CBS 141605 Cytospora hejingensis CFCC 59571 Cytospora rostrata CFCC 89910 Cytospora tritici CBS 118561 Cytospora sophoriopsis CFCC 58464 Cytospora nobilis CFCC 58227 Cytospora nobilis CFCC 58228 Cytospora joaquinensis CBS 144235 Cytospora neolhasaensis CFCC 58862 Cytospora yakimana CBS 149297 Cytospora ailanthicola CFCC 71105 Cytospora pseudochrysosperma CFCC 72617 Cytospora guyuanensis CFCC 55855 Cytospora longistiolata MFLUCC 16-0628 Cytospora melnikii CFCC 89984 Cytospora rostrata CFCC 89909 Cytospora ailanthicola CFCC 58713 Cytospora sinensis CFCC 58235 Cytospora eastringensis CFCC 58222 Cytospora chrysosperma CBS 120.83 100/1.00 100/1.00 100/1.00 98/0.99. 75/0.94 -/0.97 100/1.00 100/1.00 100/1.00 100/1.00 97/1.00 100/1.00 100/1.00 100/1.00 -/0.99 92/1.00 100/1.00 100/1.00 100/1.00 -/0.99 83/- 100/1.00 -/1.00 -/0.99 100/1.00 88/1.00 86/1.00 -/0.90 2× Outgroup C. pseudochrysosperma Chrysosperma SC 195 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora In the Ribis SC, the dataset of Cytospora isolates consisted of 87 sequences, using Cytospora donglingensis (CFCC 53159) and C. viticola (CBS 141605) as the outgroup. Cytospora ingroup strains had a total of 2,951 characters, including gaps (546 characters for ITS, 320 for act, 721 for rpb2, 619 for tef1-α, and 745 for tub2). The phylogenetic tree similarly shows MLBP ≥ 75% and BPP ≥ 0.90 above the branches. The ML analysis also employed the GTR+G+I model, and confidence levels for the nodes were determined using 1,000 replicates of bootstrapping methods (Hillis and Bull 1993). The matrix had 1,367 distinct alignment patterns. Estimated base frequencies were as follows: A = 0.245854, C = 0.287010, G = 0.236346, T = 0.230790; substitution rates: AC = 1.271593, AG = 3.563785, AT = 1.403228, CG = 0.929449, CT = 6.467342, GT = 1.000000; gamma distribution shape parameter: α = 0.261475. Seven isolates were identified as known species: C. ampla, C. sophoricola, C. sorbariae, and C. yinchuanensis. One isolate formed a separate clade, representing a new species, which we named C. hebeiensis (Fig. 3). Taxonomy Cytospora ampla Ning Jiang, Persoonia 55: 389 (2025) Fig. 4 Lineage: Ribis SC. Description. Sexual morph: Stromata Group SIII (type s8), immersed in the bark, erumpent through the surface when mature, without extending to a large circular area. Conceptacle absent. Disc light grey, surrounded by ostiolar, circular to ovoid, 64–206 µm in diam, with 12–18 ostioles irregularly circularly in the disc. Ostioles umber to black when mature, arranged regularly in a disc, flask-shaped to spherical, 73–201 µm in diam. Asci hyaline, with a chitinoid, refractive ring, clavate to elongate-obovoid, 60–77 × 11–13 (av. = 46.4 ± 1.9 × 9.4 ± 0.7, n = 30) µm, 4-spored. Ascospores hyaline, elongate-allantoid, thin-walled, aseptate, 18.8–24.0 × 4.5–6.2 (av. = 20.2 ± 0.4 × 5.3 ± 0.8, n = 50) µm. Asexual morph: not observed. Culture characteristics. Colonies initially white and entirely covering the 6 cm Petri dish after 5 d, becoming olivaceous buff to slight helical after 14 d. The colonies are flat, felt-like, thin with a uniform texture. Materials examined. China, Hebei Province, Saihanba, 42°23'33"N, 117°22'17"E, from branches of Malus spectabilis, 11 September 2024, C.M. Tian, T.Q. Pei & M.H. Wang (BJFC-S2549, living cultures CFCC 72603; BJFC-S2550, living culture CFCC 72604); 42°23'33"N, 117°22'17"E, from branches of Salix matsudana, 8 July 2024, C.M. Tian, T.Q. Pei & Y.Y. Wu (BJFC-S2551, living culture CFCC 72627). Notes. Cytospora ampla was initially isolated from branches of Salix sp. and Rubus biflorus in Xizang, China. This species is characterized by its sexual morphs having four-spored asci (Jiang et al. 2025a). In the present study, we obtained three new isolates (CFCC 72603, CFCC 72604, and CFCC 72627) from Malus spectabilis and Salix matsudana. These isolates clustered robustly with the ex-type isolate CFCC 71044 (Fig. 3) with high support (MLBP/BPP = 92/1.00), confirming their identification as C. ampla. Therefore, this represents a new host record (M. spectabilis) for C. ampla and its first occurrence in Hebei Province, China. 202 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora Figure 8. Cytospora sorbariae (BJFC-S2555). A, B. Habit of conidiomata on branch; C. Transverse section through conidioma; D. Longitudinal section through conidioma; E. Conidiophores and conidiogenous cells; F. Conidia. Scale bars: 100 μm (B–D); 10 μm (E, F). (MLBP/BPP = 100/1.00). Therefore, we identify this isolate as C. sorbariae. Meanwhile, C. sorbariae is reported for the first time on Malus spectabilis and in Hebei Province, China. Cytospora yinchuanensis L. Lin & X.L. Fan, Studies in Mycology. 109: 393 (2024) Fig. 9 Lineage: Ribis SC. Description. Sexual morph: not observed. Asexual morph: Conidiomata Group AII (type a6), immersed in bark, erumpent when mature, flat, discoid, flaskshaped to conical, with large multi-locules. Conceptacle absent. Disc light brown, circular to ovoid, 164–245 µm in diam, with one ostiole per disc. Ostiole circular to ovoid, isabelline to black, 35–45 µm. Locules subdivided frequently by invaginations with common walls, 149–299 µm. Conidiophores unbranched or branched at the bases, 13–20 × 1.5–2 (av. = 18 ± 2.3 × 1.7 ± 0.3, n = 30) µm. Conidia hyaline, unicellular, eguttulate, elongate-allantoid, 5.9–7.6 × 1.0–1.6 (av. = 6.3 ± 0.2 × 1.3 ± 0.1, n = 50) µm. Culture characteristics. Cultures initially white, covering the entire 6 cm Petri dish within 2 d, exhibiting radially uniform growth; no color change observed at 4 d, with mycelium becoming denser after 7 d. Materials examined. China, Hebei Province, Saihanba District, 42°23'33"N, 117°22'17"E, from branches of Malus spectabilis, 11 September 2024, C.M. Tian, T.Q. Pei & M.H. Wang (BJFC-S2556, living culture CFCC 72611); 42°23'33"N, 117°22'17"E, from branches of M. pumila, 11 September 2024, C.M. Tian, T.Q. Pei & M.H. Wang (BJFC-S2557, living culture CFCC 72612). 203 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora Notes. Cytospora yinchuanensis was initially misidentified as C. schulzeri based on its numerous ostioles and erumpent pycnidia (Fan et al. 2020) and was reported to cause canker and dieback disease. Lin et al. (2024) isolated this strain from Malus pumila in Ningxia and first described it as a new species, C. yinchuanensis, based on phylogenetic inference. In this study, two isolates (CFCC 72611 and CFCC 72612) grouped together with C. yinchuanensis (MLBP/BPP = 100/1.00), and their morphological characteristics are similar. Therefore, they are identified as C. yinchuanensis, representing a new host record on M. spectabilis and the first record in Hebei Province. Discussion This study describes and illustrates six species of Cytospora from Hebei Province, China, for the first time. These species reside in two species complexes. The Chrysosperma SC comprises a new host record, and the Ribis SC comprises four new host records and one new species, namely C. hebeiensis. Cytospora was introduced with C. chrysosperma as the type species (Ehrenberg 1818). Infected branches or twigs are characterized by elongate, slightly sunken, and discolored areas and often split along the canker margin (Fan et al. 2020). Meanwhile, the morphs of Cytospora are identified by single or labyrinthine locules (and diaporthalean-like perithecia), filamentous conidiophores (and clavate to elongate obovoid asci), and allantoid, hyaline conidia (and ascospores) (Spielman 1983, 1985; Adams et al. 2005). Early taxonomic studies of ascomycetes primarily relied on ITS single-gene phylogenetics. However, this approach often failed to distinguish between closely related species within several genera, such as Colletotrichum and Diaporthe (Weir et al. 2012; Gomes et al. 2013); thus, a multi-locus phylogenetic method was introduced to resolve this issue. In recent years, it has been widely applied to the identification of ascomycetes, and many scholars have identified pathogens on different hosts such as Rosa and Larix by combining ITS and othFigure 9. Cytospora yinchuanensis (BJFC-S2557). A, B. Habit of conidiomata on branch; C. Transverse section through conidioma; D. Longitudinal section through conidioma; E. Conidiophores and conidiogenous cells; F. Conidia. Scale bars: 100 μm (B–D); 10 μm (E, F). 204 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora er gene regions, which has further enriched the understanding of the diversity of fungi associated with various plants (Vu et al. 2019; Peng et al. 2022; Xiao et al. 2023; Dewing et al. 2025; Jiang et al. 2025b, c; Zhu et al. 2025). Given that Cytospora species are quite similar to each other in morphology, this method is applicable for reliable identification. Accordingly, many researchers have successfully employed a five-gene system (ITS, act, rpb2, tef1-α, and tub2) for phylogenetic analyses to achieve accurate Cytospora species discrimination. The phenomenon of a single host being infected by multiple congeneric fungal species is ubiquitous. Norphanphoun et al. (2025) reported a complex of three Colletotrichum species (C. actinidicola, C. poalesicola, and C. karsti) on Rosa chinensis. In the walnut pathosystem isolated seven Diaporthe species associated with stem blight and demonstrated via pathogenicity assays that all were pathogenic, causing blight and dieback. Likewise, Azizi et al. (2024) isolated five Cytospora species from cankered apple stems and branches. These studies collectively demonstrate that a single host can be infected by more than one species. In the present study, we summarized a total of 15 reported Cytospora species on Malus spp (Table 3), including a newly identified species in this study. This finding significantly contributes to the understanding of Cytospora diversity associated with apples in China. The discovery and description of novel species is a fundamental component of advancing our knowledge of fungal diversity (Ariyawansa et al. 2020; Crous et al. 2023a, b). Although studies on Cytospora have increased in recent years (Fan et al. 2014; Jiang et al. 2020; Jia et al. 2023; Azizi et al. 2024; Lin et al. 2024; Jiang et al. 2025a), relatively few fresh specimens have been obtained from Hebei. Up to now, only a limited number of species, such as C. microspora and C. personata, have been documented (Fan et al. 2020). The Saihanba Region in Hebei is characterized by monoculture plantations with a rich shrub understory, a combination that increases its vulnerability to economically significant disease outbreaks. While the findings of this study provide crucial data for formulating effective control measures and substantially enhance our knowledge of Cytospora diversity in this unique ecosystem, more extensive sampling remains necessary to better delineate the Cytospora taxa in this region. Acknowledgments We are grateful to Yong Li (China Forestry Culture Collection Center, Chinese Academy of Forestry, Beijing) for support of strain preservation during this study. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of AI No use of AI was reported. 205 MycoKeys 126: 187–212 (2025), DOI: 10.3897/mycokeys.126.175474 Tingqian Pei et al.: New species of Cytospora Funding This study is financed by the National Key Research and Development Program of China (Project No.: 2021YFD1400300). Author contributions Conceptualization, Yingmei Liang and Tingqian Pei; Investigation, Yingmei Liang and Tingqian Pei; Isolation and identification of strains, Tingqian Pei; Phylogenetic analysis, Yingmei Liang and Tingqian Pei; Data curation and writing – original draft, Tingqian Pei; Writing – review and editing, Tingqian Pei, Dianguang Xiong, and Yingmei Liang. All authors have read and approved the published version of the manuscript. Author ORCIDs Tingqian Pei https://orcid.org/0009-0004-0073-7608 Dianguang Xiong https://orcid.org/0000-0001-5737-1476 Yingmei Liang https://orcid.org/0000-0002-1690-5512 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References Adams GC, Roux J, Wingfield MJ, Common R (2005) Phylogenetic relationships and morphology of Cytospora species and related teleomorphs (Ascomycota, Diaporthales, Valsaceae) from Eucalyptus. Studies in Mycology 52: 1–144. Adams GC, Roux J, Wingfield MJ (2006) Cytospora species (Ascomycota, Diaporthales, Valsaceae), introduced and native pathogens of trees in South Africa. 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The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.126.175474.suppl1