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. 2024 Feb 9;10(2):139. doi: 10.3390/jof10020139

Morphology and Phylogeny Reveal Three New Species of Cytospora Associated with Tree Cankers in China

Shuo Wang 1, Ning Jiang 2, Rong Ma 1,*
Editor: Xiuguo Zhang
PMCID: PMC10890096  PMID: 38392811

Abstract

Cytospora (Cytosporaceae, Diaporthales) is a fungal genus that usually inhabits plants as endophytes, saprobes, as well as pathogens. Species of this genus are characterized by possessing allantoid hyaline conidia and ascospores. Samples with typical Cytospora canker symptoms on Prunus davidiana, P. padus and Salix sp. were collected in Tibet and Xinjiang, China. Species were identified using both morphological and molecular approaches of combined loci of internal transcribed spacer region rDNA (ITS), the partial actin (act) region, RNA polymerase II second largest subunit (rpb2), the translation elongation factor 1-alpha (tef1) gene and the partial be-ta-tubulin (tub2) gene. Six isolates in the present study formed three distinct clades from previously known species. Cytospora hejingensis sp. nov. from Salix sp., C. jilongensis sp. nov. from P. davidiana and C. kunsensis from P. padus were proposed herein. The current study improves the understanding of species concept in Cytospora.

Keywords: Diaporthales, plant disease, systematics, taxonomy

1. Introduction

Cytospora is a species-rich genus in family Cytosporaceae (order Diaporthales) and commonly inhabits plant tissues [1,2,3,4]. This genus was proposed in 1818 with four species, namely C. betulina, C. epimyces, C. resinae and C. ribis [5]. Another species C. chrysosperma was subsequently introduced [6] and later selected as the type species of this genus [7]. Cytospora can be different from the other diaporthalean genera by having allantoid hyaline conidia and ascospores [1,4,8,9,10].

Species of Cytospora were primarily identified and distinguished by their morphology and host [5,6,7]. However, recent studies employing molecular phylogeny revealed many cryptic species with similar morphology on the same host of known species of this genus [11,12,13,14,15]. For example, up to 28 Cytospora species were discovered from Eucalyptus spp. in South Africa with the help of DNA sequence evidence [2], eight from willow (Salix spp.) trees in China [16], six from Castanea mollissima in China [17], six from Populus hosts in China [18] and six from apple trees in Iran [19]. The taxonomy of Cytospora is currently more dependent on combined evidence of DNA sequence data, morphological features and ecology than species morphology and host associations [1,20].

Several species of Cytospora are reported to cause plant diseases including canker, wilt and dieback [21,22,23,24]. For example, C. carpobroti causes Carpobrotus edulis wilt disease in South Africa [21]; C. oleicola and C. olivarum are pathogenic to olives in the USA [22]; C. parasitica results in apple cankers in China [23]; and Cytospora pistaciae causes dieback and canker disease of pistachios in Italy [24]. There are still many cryptic species of Cytospora pathogenic to plants waiting for description.

In the present study, Cytospora canker symptoms were found from different tree hosts named Prunus davidiana, P. padus and Salix sp. in Tibet and Xinjiang, China. The aims of the present study were to identify the casual agents of the lesions, to introduce and describe new Cytospora species using both molecular and morphological approaches, and to discuss the species differences based on newly collected specimens.

2. Materials and Methods

2.1. Specimens and Strains

Investigations to collect fungal specimens were conducted in Tibet and Xinjiang during 2021 and 2022. During the surveys, dead and dying twigs and branches of tree hosts were checked manually, and then twigs and branches with obvious fungal fruiting bodies were recorded and collected. Samples were packed in paper bags and posted back for isolation.

Ascomata on branches of Prunus padus and Salix sp., and conidiomata on branches of P. davidiana were sectioned using sterile blades, and mucoid spore masses were removed and placed onto the surface of potato dextrose agar (PDA; potato, 200 g; glucose, 20 g; agar, 20 g; distilled water, to complete 1000 mL) media using sterile insect needles. Then, plates were incubated at 25 °C in darkness until spores germinated. Pieces of mycelium were cut and removed and placed onto a new PDA plate under a stereomicroscope to obtain the pure strains. Specimens and isolates were preserved in the China Forestry Culture Collection Center (CFCC; http://cfcc.caf.ac.cn/ (accessed on 2 January 2024)).

2.2. Morphological Observations

The Cytospora species observations were based on ascomata and conidiomata naturally formed on twigs and branches of Prunus davidiana, P. padus and Salix sp. The sexual and asexual fruiting bodies were sectioned using sterile blades and photographed using the Leica stereomicroscope (M205) (Leica Microsystems, Wetzlar, Germany). The asci, ascospores, conidiophores, conidiogenous cells and conidia were measured and photographed by a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan). The colony characteristics were observed and recorded on PDA plates at 25 °C in darkness.

2.3. DNA Extraction and Amplification

The total genomic DNA of Cytospora species were obtained from colonies growing on PDA plates by using the CTAB method [25]. The internal transcribed spacer region rDNA (ITS), the partial actin (act) region, RNA polymerase II second largest subunit (rpb2), the translation elongation factor 1-alpha (tef1) gene and the partial be-ta-tubulin (tub2) gene were amplified using primer pairs ITS1/ITS4, ACT512F/ACT783R, fRPB2-5f/fRPB2-7cR, 983F/2218R, Bt2a/Bt2b, respectively [26,27,28,29,30]. These regions were amplified as follows: an initial denaturation step of 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 50 s at 52 °C (ITS), 54°C (tef1 and tub2), 55 °C (rpb2) or 58 °C (act), and 1 min at 72 °C, and a final elongation step of 7 min at 72 °C. The polymerase chain reaction products were sequenced using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminator Kit v.3.1 (Invitrogen, Waltham, MA, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

2.4. Molecular Phylogeny

Sequences obtained in the present study were preliminarily identified by the BLAST search to confirm their classification. The referenced sequences of Cytospora were collected from recent publications (Table 1) and downloaded [1,24,25]. Strain CBS 160.32 (species Diaporthe vaccinii) was selected as the outgroup taxon. The five individual loci ITS, act, rpb2, tef1 and tub2 were aligned using MAFFT v. 6.0 and manually adjusted using MEGA v. 6.0 [31,32]. Then, five loci were combined and analyzed based on maximum likelihood (ML) and Bayes methods in the CIPRES Science Gateway platform [33]. The GTR substitution model was employed and 1000 non-parametric bootstrap replicates were set for ML phylogenic analysis. Four simultaneous Markov Chain runs for 1,000,000 generations were set during Bayesian analysis. The resulting trees were visualized in FigTree v. 1.4.0 and edited using Adobe Illustrator 2020.

Table 1.

Strains and their GenBank accession numbers used in this study.

Species Strain GenBank Accession Numbers
ITS act rpb2 tef1 tub2
Cytospora ailanthicola CFCC 89970 MH933618 MH933526 MH933592 MH933494 MH933565
Cytospora albodisca CFCC 53161 MW418406 MW422899 MW422909 MW422921 MW422933
Cytospora albodisca CFCC 54373 MW418407 MW422900 MW422910 MW422922 MW422934
Cytospora alba CFCC 55462T NR182387 OK303457 OK303516 OK303577 OK303644
Cytospora alba CFCC 55463 MZ702596 OK303458 OK303517 OK303578 OK303645
Cytospora ampulliformis MFLUCC 16-0583T KY417726 KY417692 KY417794
Cytospora ampulliformis MFLUCC 16-0629 KY417727 KY417693 KY417795
Cytospora amygdali CBS 144233T MG971853 MG972002 MG971659
Cytospora atrocirrhata CFCC 89615 KR045618 KF498673 KU710946 KP310858 KR045659
Cytospora atrocirrhata CFCC 89616 KR045619 KF498674 KU710947 KP310859 KR045660
Cytospora beilinensis CFCC 50493T MH933619 MH933527 MH933495 MH933561
Cytospora beilinensis CFCC 50494 MH933620 MH933528 MH933496 MH933562
Cytospora berberidis CFCC 89927T KR045620 KU710990 KU710948 KU710913 KR045661
Cytospora berberidis CFCC 89933 KR045621 KU710991 KU710949 KU710914 KR045662
Cytospora bungeana CFCC 50495T MH933621 MH933529 MH933593 MH933497 MH933563
Cytospora bungeana CFCC 50496 MH933622 MH933530 MH933594 MH933498 MH933564
Cytospora californica CBS 144234T MG971935 MG972083 MG971645
Cytospora carbonacea CFCC 89947 KR045622 KP310842 KU710950 KP310855 KP310825
Cytospora carpobroti CMW48981T MH382812 MH411212 MH411207
Cytospora celtidicola CFCC 50497T MH933623 MH933531 MH933595 MH933499 MH933566
Cytospora celtidicola CFCC 50498 MH933624 MH933532 MH933596 MH933500 MH933567
Cytospora centrivillosa MFLUCC 16-1206T MF190122 MF377600
Cytospora centrivillosa MFLUCC 17-1660 MF190123 MF377601
Cytospora ceratosperma CFCC 89624 KR045645 KU710976 KP310860 KR045686
Cytospora ceratosperma CFCC 89625 KR045646 KU710977 KP310861 KR045687
Cytospora ceratospermopsis CFCC 89626T KR045647 KU711011 KU710978 KU710934 KR045688
Cytospora ceratospermopsis CFCC 89627 KR045648 KU711012 KU710979 KU710935 KR045689
Cytospora chrysosperma CFCC 89629 KF765673 KF765705
Cytospora chrysosperma CFCC 89981 MH933625 MH933533 MH933597 MH933501 MH933568
Cytospora chrysosperma CFCC 89982 KP281261 KP310835 KP310848 KP310818
Cytospora cinnamomea CFCC 53178T MK673054 MK673024 MK672970
Cytospora coryli CFCC 53162T MN854450 MN850751 MN850758 MN861120
Cytospora corylina CFCC 54684T MW839861 MW815937 MW815951 MW815886 MW883969
Cytospora corylina CFCC 54685 MW839862 MW815938 MW815952 MW815887 MW883970
Cytospora cotini MFLUCC 14-1050T KX430142 KX430144
Cytospora cotoneastricola CF 20197027 MK673072 MK673042 MK673012 MK672958 MK672988
Cytospora cotoneastricola CF 20197028 MK673073 MK673043 MK673013 MK672959 MK672989
Cytospora curvata MFLUCC 15-0865T KY417728 KY417694
Cytospora curvispora CFCC 54000T MW839851 MW815931 MW815945 MW815880 MW883963
Cytospora curvispora CFCC 54001 MW839853 MW815932 MW815946 MW815881 MW883964
Cytospora davidiana CXY 1350T KM034870
Cytospora diopuiensis CFCC 55479 OQ344753 OQ410625 OQ398735 OQ398762 OQ398791
Cytospora diopuiensis CFCC 55527 OQ344754 OQ410626 OQ398736 OQ398763 OQ398792
Cytospora discotoma CFCC 53137T MW418404 MW422897 MW422907 MW422919 MW422931
Cytospora discotoma CFCC 54368 MW418405 MW422898 MW422908 MW422920 MW422932
Cytospora donetzica MFLUCC 15-0864 KY417729 KY417695 KY417797
Cytospora donetzica MFLUCC 16-0574T KY417731 KY417697 KY417799
Cytospora donglingensis CFCC 53159T MW418412 MW422903 MW422915 MW422927 MW422939
Cytospora donglingensis CFCC 53160 MW418414 MW422905 MW422917 MW422929 MW422941
Cytospora elaeagni CFCC 89632 KR045626 KU710995 KU710955 KU710918 KR045667
Cytospora elaeagni CFCC 89633 KF765677 KU710996 KU710956 KU710919 KR045668
Cytospora elaeagnicola CFCC 52882T MK732341 MK732344 MK732347
Cytospora elaeagnicola CFCC 52883 MK732342 MK732345 MK732348
Cytospora erumpens CFCC 50022 MH933627 MH933534 MH933502 MH933569
Cytospora erumpens CFCC 53163 MK673059 MK673029 MK673000 MK672948 MK672975
Cytospora eucalypti CBS 144241 MG971907 MG972056 MG971617
Cytospora euonymicola CFCC 50499T MH933628 MH933535 MH933598 MH933503 MH933570
Cytospora euonymicola CFCC 50500 MH933629 MH933536 MH933599 MH933504 MH933571
Cytospora euonymina CFCC 89993T MH933630 MH933537 MH933600 MH933505 MH933590
Cytospora euonymina CFCC 89999 MH933631 MH933538 MH933601 MH933506 MH933591
Cytospora fraxinigena MFLU 17-0880T NR154859
Cytospora fugax CXY 1371 KM034852 KM034891
Cytospora fugax CXY 1381 KM034853 KM034890
Cytospora fusispora NFCCI 4372 MN227694
Cytospora galegicola MFLUCC 18-1199T MK912128 MN685810 MN685820
Cytospora gigalocus CFCC 89620T KR045628 KU710997 KU710957 KU710920 KR045669
Cytospora gigalocus CFCC 89621 KR045629 KU710998 KU710958 KU710921 KR045670
Cytospora gigaspora CFCC 50014 KR045630 KU710999 KU710959 KU710922 KR045671
Cytospora gigaspora CFCC 89634T KF765671 KU711000 KU710960 KU710923 KR045672
Cytospora globosa MFLU 16-2054T MT177935 MT432212 MT454016
Cytospora granati CBS 144237T MG971799 MG971949 MG971514
Cytospora haidianensis CFCC 54056 MT360041 MT363978 MT363987 MT363997 MT364007
Cytospora haidianensis CFCC 54057T MT360042 MT363979 MT363988 MT363998 MT364008
Cytospora hejingensis CFCC 59571T PP060455 PP059657 PP059663 PP059667 PP059673
Cytospora hejingensis C3479 PP060456 PP059658 PP059664 PP059668 PP059674
Cytospora hippophaës CFCC 89639 KR045632 KU711001 KU710961 KU710924 KR045673
Cytospora hippophaës CFCC 89640 KF765682 KF765730 KU710962 KP310865 KR045674
Cytospora japonica CFCC 89956 KR045624 KU710993 KU710953 KU710916 KR045665
Cytospora japonica CFCC 89960 KR045625 KU710994 KU710954 KU710917 KR045666
Cytospora jilongensis CFCC 59569T PP060457 PP059659 PP059669 PP059675
Cytospora jilongensis XZ083 PP060458 PP059660 PP059670 PP059676
Cytospora joaquinensis CBS 144235 MG971895 MG972044 MG971605
Cytospora junipericola MFLU 17-0882T MF190125 MF377580
Cytospora juniperina CFCC 50501T MH933632 MH933539 MH933602 MH933507
Cytospora juniperina CFCC 50502 MH933633 MH933540 MH933603 MH933508 MH933572
Cytospora kantschavelii CXY 1383 KM034867
Cytospora kuanchengensis CFCC 52464T MK432616 MK442940 MK578076
Cytospora kuanchengensis CFCC 52465 MK432617 MK442941 MK578077
Cytospora kunsensis CFCC 59570T PP060459 PP059661 PP059665 PP059671 PP059677
Cytospora kunsensis C3488 PP060460 PP059662 PP059666 PP059672 PP059678
Cytospora leucosperma CFCC 89622 KR045616 KU710988 KU710944 KU710911 KR045657
Cytospora leucosperma CFCC 89894 KR045617 KU710989 KU710945 KU710912 KR045658
Cytospora longispora CBS 144236T MG971905 MG972054 NA MG971615 NA
Cytospora longistiolata MFLUCC 16-0628 KY417734 KY417700 KY417802 NA NA
Cytospora lumnitzericola MFLUCC 17-0508T MG975778 MH253457 MH253461 NA NA
Cytospora mali CFCC 50028 MH933641 MH933548 MH933606 MH933513 MH933577
Cytospora mali CFCC 50029 MH933642 MH933549 MH933607 MH933514 MH933578
Cytospora mali-spectabilis CFCC 53181T MK673066 MK673036 MK673006 MK672953 MK672982
Cytospora melnikii CFCC 89984 MH933644 MH933551 MH933609 MH933515 MH933580
Cytospora myrtagena CFCC 52454 MK432614 MK442938 MK578074
Cytospora myrtagena CFCC 52455 MK432615 MK442939 MK578075
Cytospora nivea MFLUCC 15-0860 KY417737 KY417703 KY417805
Cytospora nivea CFCC 89641 KF765683 KU711006 KU710967 KU710929 KR045679
Cytospora notastroma NE_TFR5 JX438632 JX438543
Cytospora notastroma NE_TFR8 JX438633 JX438542
Cytospora ochracea CFCC 53164T MK673060 MK673030 MK673001 MK672949 MK672976
Cytospora oleicola CBS 144248T MG971944 MG972098 MG971660
Cytospora olivacea CFCC 53174 MK673058 MK673028 MK672999 MK672974
Cytospora olivacea CFCC 53175 MK673062 MK673032 MK673003 MK672978
Cytospora palm CXY 1276 JN402990 KJ781296
Cytospora palm CXY 1280T JN411939 KJ781297
Cytospora paracinnamomea CFCC 55453T MZ702594 OK303456 OK303515 OK303576 OK303643
Cytospora paracinnamomea CFCC 55455T MZ702598 OK303460 OK303519 OK303580 OK303647
Cytospora parakantschavelii MFLUCC 15-0857T KY417738 KY417704 KY417806
Cytospora parapistaciae CBS 144506T MG971804 MG971954 MG971519
Cytospora paraplurivora FDS-439 OL640182 OL631586 OL631589
Cytospora paraplurivora FDS-564T OL640183 OL631587 OL631590
Cytospora parasitica CFCC 53173 MK673070 MK673040 MK673010 MK672957 MK672986
Cytospora paratranslucens MFLUCC 15-0506T KY417741 KY417707 KY417809
Cytospora paratranslucens MFLUCC 16-0627 KY417742 KY417708 KY417810
Cytospora phialidica MFLUCC 17-2498 MT177932 MT432209 MT454014
Cytospora piceae CFCC 52841T MH820398 MH820406 MH820395 MH820402 MH820387
Cytospora piceae CFCC 52842 MH820399 MH820407 MH820396 MH820403 MH820388
Cytospora pingbianensis MFLUCC 18-1204T MK912135 MN685817 MN685826
Cytospora pistaciae CBS 144238T MG971802 MG971952 MG971517
Cytospora platycladi CFCC 50504T MH933645 MH933552 MH933610 MH933516 MH933581
Cytospora platycladi CFCC 50505 MH933646 MH933553 MH933611 MH933517 MH933582
Cytospora platycladicola CFCC 50038T KT222840 MH933555 MH933613 MH933519 MH933584
Cytospora platycladicola CFCC 50039 KR045642 KU711008 KU710973 KU710931 KR045683
Cytospora plurivora CBS 144239T MG971861 MG972010 MG971572
Cytospora populi CFCC 55472T MZ702609 OK303471 OK303530 OK303591 OK303658
Cytospora populi CFCC 55473 MZ702610 OK303472 OK303531 OK303592 OK303659
Cytospora populicola CBS 144240 MG971891 MG972040 MG971601
Cytospora populina CFCC 89644T KF765686 KU711007 KU710969 KU710930 KR045681
Cytospora populinopsis CFCC 50032T MH933648 MH933556 MH933614 MH933520 MH933585
Cytospora populinopsis CFCC 50033 MH933649 MH933557 MH933615 MH933521 MH933586
Cytospora predappioensis MFLUCC 17-2458T MG873484
Cytospora predappioensis MFLU 17-0327 MH253451 MH253449 MH253450
Cytospora prunicola MFLU 17-0995T MG742350 MG742353 MG742352
Cytospora pruni-mume CFCC 53179 MK673057 MK673027 MK672947 MK672973
Cytospora pruni-mume CFCC 53180T MK673067 MK673037 MK673007 MK672954 MK672983
Cytospora pruinopsis CFCC 50034T KP281259 KP310836 KU710970 KP310849 KP310819
Cytospora pruinopsis CFCC 53153 MN854451 MN850763 MN850752 MN850759 MN861121
Cytospora pruinosa CFCC 50036 KP310800 KP310832 KP310845 KP310815
Cytospora pruinosa CFCC 50037 MH933650 MH933558 MH933522 MH933589
Cytospora pubescentis MFLUCC 18-1201T MK912130 MN685812 MN685821
Cytospora punicae CBS 144244 MG971943 MG972091 MG971654
Cytospora quercicola MFLU 17-0881 MF190128
Cytospora ribis CFCC 50026 KP281267 KP310843 KU710972 KP310856 KP310826
Cytospora ribis CFCC 50027 KP281268 KP310844 KP310857 KP310827
Cytospora rosae MFLU 17-0885 MF190131
Cytospora rosicola CF 20197024T MK673079 MK673049 MK673019 MK672965 MK672995
Cytospora rosigena MFLUCC 18-0921T MN879872
Cytospora rostrata CFCC 89909 KR045643 KU711009 KU710974 KU710932 KR045684
Cytospora rostrata CFCC 89910 KR045644 KU711010 KU710975 KU710933
Cytospora rusanovii MFLUCC 15-0853 KY417743 KY417709 KY417811
Cytospora rusanovii MFLUCC 15-0854T KY417744 KY417710 KY417812
Cytospora salicacearum MFLUCC 15-0509 KY417746 KY417712 KY417814
Cytospora salicacearum MFLUCC 15-0861 KY417745 KY417711 KY417813
Cytospora salicicola MFLUCC 14-1052T KU982636 KU982637
Cytospora salicicola MFLUCC 15-0866 KY417749 KY417715 KY417817
Cytospora salicina MFLUCC 15-0862 KY417750 KY417716 KY417818
Cytospora salicina MFLUCC 16-0637 KY417751 KY417717 KY417819
Cytospora schulzeri CFCC 50042 KR045650 KU711014 KU710981 KU710937 KR045691
Cytospora sibiraeae CFCC 50045T KR045651 KU711015 KU710982 KU710938 KR045692
Cytospora sibiraeae CFCC 50046 KR045652 KU711016 KU710983 KU710939 KR045693
Cytospora sophorae CFCC 50047 KR045653 KU711017 KU710984 KU710940 KR045694
Cytospora sophorae CFCC 89598 KR045654 KU711018 KU710985 KU710941 KR045695
Cytospora sophoricola CFCC 89596 KR045656 KU711020 KU710987 KU710943 KR045697
Cytospora sophoricola CFCC 89595T KR045655 KU711019 KU710986 KU710942 KR045696
Cytospora sophoriopsis CFCC 55469 MZ702583 OK303445 OK303504 OK303565 OK303632
Cytospora sophoriopsis CFCC 89600 KR045623 KU710992 KU710951 KU710915 KP310817
Cytospora sorbi MFLUCC 16-0631T KY417752 KY417718 KY417820
Cytospora sorbicola MFLUCC 16-0584T KY417755 KY417721 KY417823
Cytospora sorbicola MFLUCC 16-0633 KY417758 KY417724 KY417826
Cytospora sorbina CF 20197660T MK673052 MK673022 MK672943 MK672968
Cytospora spiraeae CFCC 50049T MG707859 MG708196 MG708199
Cytospora spiraeae CFCC 50050 MG707860 MG708197 MG708200
Cytospora spiraeicola CFCC 53138T MN854448 MN850749 MN850756 MN861118
Cytospora spiraeicola CFCC 53139 MN854449 MN850750 MN850757 MN861119
Cytospora tamaricicola CFCC 50507 MH933651 MH933559 MH933616 MH933525 MH933587
Cytospora tamaricicola CFCC 50508T MH933652 MH933560 MH933617 MH933523 MH933588
Cytospora tanaitica MFLUCC 14-1057T KT459411 KT459413
Cytospora thailandica MFLUCC 17-0262T MG975776 MH253459 MH253463
Cytospora thailandica MFLUCC 17-0263T MG975777 MH253460 MH253464
Cytospora tibetensis CF 20197026 MK673076 MK673046 MK673016 MK672962 MK672992
Cytospora tibetensis CF 20197029 MK673077 MK673047 MK673017 MK672963 MK672993
Cytospora tibouchinae CPC 26333T KX228284
Cytospora translucens CXY 1351 KM034874 KM034895
Cytospora translucens CXY 1359 KM034871 KM034894
Cytospora ulmi MFLUCC 15-0863T KY417759
Cytospora verrucosa CFCC 53157T MW418408 MW422911 MW422923 MW422935
Cytospora verrucosa CFCC 53158 MW418410 MW422901 MW422913 MW422925 MW422937
Cytospora vinacea CBS 141585T KX256256 KX256277 KX256235
Cytospora viridistroma CBS 202.36T MN172408 MN271853
Cytospora viticola Cyt2 KX256238 KX256259 KX256217
Cytospora viticola CBS 141586T KX256239 KX256260 KX256218
Cytospora xinjiangensis CFCC 53182 MK673064 MK673034 MK673004 MK672951 MK672980
Cytospora xinjiangensis CFCC 53183T MK673065 MK673035 MK673005 MK672952 MK672981
Cytospora xinglongensis CFCC 52458 MK432622 MK442946 MK578082
Cytospora xinglongensis CFCC 52459 MK432623 MK442947 MK578083
Cytospora xylocarpi MFLUCC 17-0251T MG975775 MH253458 MH253462
Cytospora zhaitangensis CFCC 56227T OQ344750 OQ410623 OQ398733 OQ398760 OQ398789
Cytospora zhaitangensis CFCC 57537 OQ344751 OQ410624 OQ398734 OQ398761 OQ398790
Diaporthe vaccinii CBS 160.32 KC343228 JQ807297 KC343954 KC344196
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Note. Ex-type strains are marked with T and isolates from the present study are in bold.

3. Results

3.1. Phylogeny

In the phylogenetic analysis, the combined dataset of ITS, act, rpb2, tef1 and tub2 consisted of 202 strains. The final alignment comprised 2561 characters including 588 characters in ITS, 211 characters in act, 617 characters in rpb2, 536 characters in tef1 and 609 characters tub2. The final ML optimization likelihood value of the best RAxML tree was −48,006.19, and the matrix had 1534 distinct alignment patterns, with 29.35% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.242905, C = 0.286434, G = 0.242250 and T = 0.228411; substitution rates AC = 1.374316, AG = 3.693698, AT = 1.471034, CG = 0.981185, CT = 6.248472 and GT = 1.0; and gamma distribution shape parameter α = 0.321644. The topology of our phylogenetic tree is nearly identical to previous publications. The topology of isolates from the present study in the RAxML and Bayesian analyses were congruent. Isolates CFCC 59571 and C3479 formed a distinct clade to CFCC 89984 (C. melnikii), MFLUCC 15-0509 and MFLUCC 15-0861 (C. salicacearum) with high support values (BS = 100, BPP = 1). Isolates CFCC 59570 and C3488 formed a clade close to CFCC 50014 and CFCC 89634 (C. gigaspora) with full support values (BS = 100, BPP = 1). Isolates CFCC 59570 and C3488 clustered together with CFCC 89956 and CFCC 89960 (C. japonica), CFCC 53164 (C. ochracea), CF 20197660 (C. sorbina), CF 20197026 and CF 20197029 (C. tibetensis), and CFCC 53179 and CFCC 53180 (C. pruni-mume) supported by high values (BS = 100, BPP = 1). Hence, six isolates from the present study formed three new clades distinct from previously known species named Cytospora hejingensi sp. nov., C. jilongensis sp. nov. and C. kunsensis sp. nov. (Figure 1).

Figure 1.

Figure 1

Figure 1

Figure 1

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Phylogram of Cytospora resulting from a maximum likelihood analysis, based on a combined matrix of ITS, act, rpb2, tef1 and tub2. Numbers above the branches indicate ML bootstraps (left, ML BS ≥ 50%) and Bayesian posterior probabilities (right, BPP ≥ 0.90). The tree is rooted with Diaporthe vaccinii (CBS 160.32). Isolates obtained from the present study are marked in blue.

3.2. Description of Cytospora hejingensis sp. nov. from Salix sp.

Cytospora hejingensis R. Ma & Ning Jiang, sp. nov.

Figure 2.

MycoBank: MB851771

Etymology: named after the collection site of the holotype, Hejing County.

Description: Associated with branch and twig canker disease of Salix sp. Sexual morph: Ascostromata immersed in the bark, erumpent through the bark surface, scattered, (400–)650–900(–1250) μm diam., with 4–9 perithecia arranged irregularly. Conceptacle absent. Ectostromatic disc inconspicuous, usually surrounded by tightly aggregated ostiolar necks, (100–)150–250(–350) μm diam. Ostioles numerous, black, concentrated, arranged irregularly in a disc, (35–)50–65(–90) μm diam. Perithecia black, spherical, arranged circularly or irregularly, (120–)150–250(–300) μm diam. Asci free, clavate, (38–)45–70(–77) × (7–)8.5–10.5(–12.5) μm, 8-spored. Ascospores biseriate, allantoid, thin-walled, hyaline, aseptate, (6.5–)7–8(–9) × 2–2.5 μm. Asexual morph: undetermined.

Culture characteristics: colonies on PDA flat, spreading, with flocculent mycelium, initially white to grey, secreting a dark green to black pigment in culture medium after 10 days, reaching a 90 mm diameter after 15 days at 25 °C in the dark.

Materials examined: China, Xinjiang Uygur Autonomous Region, Bayingolin Mongol Autonomous Prefecture, Hejing County, Kunse Forest Park, on cankered twigs and branches of Salix sp., 24 July 2021, Rong Ma (XJAU 3488, holotype); ex-type culture CFCC 59571; ibid. (culture C3488).

Notes: Cytospora hejingensis from Salix sp. in China is phylogenetically close to C. melnikii from Malus domestica in Russia and C. salicacearum from Salix alba in Russia (Figure 1). C. hejingensis is only known in sexual morph, and the other two species in asexual morph. Hence, it is impossible to compare them in morphology. However, C. hejingensis differs from C. melnikii and C. salicacearum by sequence data (22/560 in ITS, 35/211 in act, 36/617 in rpb2 and 27/306 in tef1 from C. melnikii; 25/560 in ITS, 37/211 in act and 26/617 in rpb2 from C. salicacearum) [34].

Figure 2.

Figure 2

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Morphology of Cytospora hejingensis from Salix sp. (A,B) Ascomata formed on branches. (C) Longitudinal section through the ascomata. (D) Transverse section of ascomata. (E) Asci. (F) Ascospores. Scale bars: (B) = 500 μm; (C) = 200 μm; (D) = 300 μm; (E,F) = 10 μm.

3.3. Description of Cytospora jilongensis sp. nov. from Prunus davidiana

Cytospora jilongensis R. Ma & Ning Jiang, sp. nov.

Figure 3.

MycoBank: MB851772

Etymology: named after the collection site of the holotype, Jilong County.

Description: Associated with branch canker disease of Prunus davidiana. Sexual morph: undetermined. Asexual morph: Pycnidial stromata ostiolated, semi-immersed in the host bark, scattered, pulvinate, with multiple locules. Conceptacle dark brown, circular surrounded stromata. Ectostromatic grey, circular to ovoid, (100–)180–240(–370) μm diam., with one ostiole per disc. Ostioles dark, at the same level as the disc, (30–)50–75(–95) μm diam. Locule numerous, arranged circularly or elliptically with independent walls, (250–)400–500(–750) μm diam. Peridium comprising few layers of cells of textura angularis, brown to dark brown. Conidiophores hyaline, branched, thin-walled, filamentous. Conidiogenous cells enteroblastic polyphialidic, 7.5–18.5 × 1.5–2.5 μm. Conidia hyaline, allantoid, smooth, aseptate, thin-walled, (9.3–)10.2–11.6(–12.5) × 2.6–3.2 μm.

Culture characteristics: colonies on PDA flat, spreading, with moderate flocculent mycelium, initially white, becoming orange after 10 days, reaching a 90 mm diameter after 25 days at 25 °C in the dark.

Materials examined: China, Tibet Tibetan Autonomous Region, Shigatse City, Jilong County, Jilong Town, on cankered branches of Prunus davidiana, 12 August 2022, Jin Peng, Jiang Ning and Liu Min (CAF800087, holotype); ex-type culture CFCC 59569; ibid. (culture XZ083).

Notes: Cytospora jilongensis from Prunus davidiana is phylogenetically close to C. japonica from P. cerasifera and P. persica, C. ochracea from Cotoneaster sp., C. sorbina from Sorbus tianschanica, C. tibetensis from Cotoneaster sp. and C. pruni-mume from Prunus mume (Figure 1). However, C. jilongensis (10.2–11.6 × 2.6–3.2 μm) differs from C. japonica (6.5–8.5 × 1.5–2 μm), C. ochracea (8.5–9.0 × 1.5–2.5 μm), C. sorbina (4.5–5.5 × 1–1.5 μm), C. tibetensis (5.0–5.5 × 1.5–2 μm) and C. pruni-mume (5.5–6.5 × 1.5–2 μm) in conidial size and hosts [1,14].

Figure 3.

Figure 3

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Morphology of Cytospora jilongensis from Prunus davidiana. (A) Symptoms of canker disease on the host. (B,C) Conidiomata formed on branches. (D) Transverse section through the conidioma. (E) Longitudinal section through the conidioma. (F–H) Conidiophores and conidiogenous cells. (I,J) Conidia. Scale bars: (B) = 2 mm; (C,D) = 1 mm; (E) = 800 μm; (F–J) = 10 μm.

3.4. Description of Cytospora kunsensis sp. nov. from Prunus padus

Cytospora kunsensis R. Ma & Ning Jiang, sp. nov.

Figure 4.

MycoBank: MB851773

Etymology: named after the collection site of the holotype, Kunse Forest Park.

Description: Associated with branch and twig canker disease of Prunus padus. Sexual morph: Ascostromata immersed in the bark, erumpent through the bark surface, scattered, (750–)950–1100(–1350) μm diam., with 5–11 perithecia arranged circularly. Conceptacle absent. Ectostromatic disc white, surrounded by tightly aggregated ostiolar necks, (100–)150–300(–350) μm diam. Ostioles numerous, black, concentrated, arranged circularly in a disc, (40–)50–75(–90) μm diam. Perithecia black, spherical, arranged circularly or irregularly, (180–)250–350(–420) μm diam. Asci free, clavate, (38–)48–80(–86) × (7.5–)9–12(–13.5) μm, eight-spored. Ascospores biseriate, allantoid, thin-walled, hyaline, aseptate, (10–)12.5–17(–19.5) × 2–2.5 μm. Asexual morph: undetermined.

Culture characteristics: colonies on PDA flat, spreading, with flocculent mycelium, white, with a dark grey color in the center, fast growing, reaching a 90 mm diameter after 7 days and forming abundant black ascomata after 25 days at 25 °C.

Materials examined: China, Xinjiang Uygur Autonomous Region, Bayingolin Mongol Autonomous Prefecture, Hejing County, Kunse Forest Park, on cankered twigs and branches of Prunus padus, 24 July 2021, Rong Ma (XJAU 3479, holotype); ex-type culture CFCC 59570; ibid. (culture C3479).

Notes: Cytospora kunsensis from Prunus padus is phylogenetically close to C. gigaspora from Salix psammophila (Figure 1). However, C. kunsensis can be distinguished from C. gigaspora by sequence data (19/548 in ITS, 32/211 in act, 56/617 in rpb2, 36/303 in tef1 and 42/421 in tub2) [11].

Figure 4.

Figure 4

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Morphology of Cytospora kunsensis from Prunus padus. (A,B) Ascomata formed on branches. (C) Longitudinal section through the ascomata. (D) Transverse section of ascomata. (E) Asci. (F) Ascospores. Scale bars: (B) = 500 μm; (C,D) = 300 μm; (E,F) = 10 μm.

4. Discussion

In the present study, samples of Cytospora with fruiting bodies were collected from Xinjiang and Tibet, and identified based on both morphological and phylogenetical approaches of combined ITS, act, rpb2, tef1 and tub2 loci. We proposed three new species, i.e., Cytospora hejingensis sp. nov. from Salix sp., C. jilongensis sp. nov. from P. davidiana and C. kunsensis from P. padus.

Of the new species introduced in the current study, two taxa (C. jilongensis and C. kunsensis) were isolated from the plant genus Prunus. Hence, a total of nine species of Cytospora were found in host genus Prunus, where the previous seven species are C. cinnamomea, C. erumpens, C. japonica, C. leucostoma, C. olivacea, C. populinopsis and C. pruni-mume [14]. C. kunsensis is distinguished from C. populinopsis in eight-spored asci, and these two species are only known in sexual morph [1]. The other seven species are known in asexual species with similar conidial morphology but different sequence data of ITS, act, rpb2, tef1 and tub2 loci. The example of Cytospora species from Prunus implies that DNA sequence data are necessary to separate species during pathogen identifications.

Another example is the Cytospora species from the host genus Salix. Until now, over 10 species of Cytospora were discovered from the host genus Salix, including one species Cytospora hejingensis introduced in the current study [35]. Most of them are confirmed to be pathogens associated with canker diseases [35]. The new species from the present study needs a pathogenicity test to evaluate its virulence to willow trees in the future.

In the traditional classification and identification of species in Cytospora, spore morphology and host information are the most important evidence to identify Cytospora species [5,6,7]. However, by using the molecular data, many cryptic species with the same hosts and similar spore morphology were recently revealed [1,14,15,35]. The molecular classification system for Cytospora based on morphology, phylogeny and host information is more scientific than that mainly based on morphology before.

Author Contributions

Conceptualization, S.W. and R.M.; methodology, N.J.; software, S.W.; validation, S.W., N.J. and R.M.; formal analysis, S.W., N.J. and R.M.; investigation, S.W., N.J. and R.M.; resources, S.W., N.J. and R.M.; data curation, S.W., N.J. and R.M.; writing—original draft preparation, S.W., N.J. and R.M.; writing—review and editing, S.W., N.J. and R.M.; visualization, S.W., N.J. and R.M.; supervision, S.W., N.J. and R.M.; project administration, R.M.; funding acquisition, R.M. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All sequence data are available in NCBI GenBank (Table 1).

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the Natural Science Foundation of China, grant number 31960316.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All sequence data are available in NCBI GenBank (Table 1).

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