Print
Identification of six Cytospora species on Chinese chestnut in China
expand article infoNing Jiang, Qin Yang, Xin-Lei Fan, Cheng-Ming Tian
Open Access

Abstract

Chinese chestnut (Castanea mollissima) is an important crop tree species in China. In the present study, Cytospora specimens were collected from Chinese chestnut trees and identified using molecular data of combined ITS, LSU, ACT and RPB2 loci, as well as morphological features. As a result, two new Cytospora species and four new host records were confirmed, viz. C. kuanchengensis sp. nov., C. xinglongensis sp. nov., C. ceratospermopsis, C. leucostoma, C. myrtagena and C. schulzeri.

Keywords

Castanea mollissima, Cytosporaceae, Diaporthales, systematics, taxonomy

Introduction

Chinese chestnut (Castanea mollissima) is a widely cultivated crop tree species in China, producing nutritious and delicious nuts for humans (Lu and Guo 2016). However, Cryphonectria parasitica and several fungi are causing severe chestnut diseases worldwide, which reduce the nut production, even killing the hosts. (Aghayeva et al. 2017, Shuttleworth and Guest 2017, Jiang et al. 2018a, Rigling and Prospero 2018). Recently, several diaporthalean species were described from Chinese chestnut trees for the clear taxonomic concepts of families, genera and species in Diaporthales (Rossman et al. 2007, Senanayake et al. 2017, 2018), including species of Aurantiosacculus, Coryneum, Cryphonectria, Dendrostoma, Endothia, Gnomoniopsis, Neopseudomelanconis and Ophiognomonia (Gong et al. 2017, Jiang et al. 2018b, 2018c, 2019a, 2019b, Jiang and Tian 2019).

Cytospora (Cytosporaceae, Diaporthales) is a widely distributed genus worldwide, occurring on a broad range of hosts (Sarma and Hyde 2001, Yang et al. 2015, Lawrence et al. 2017, Norphanphoun et al. 2017, 2018, Wijayawardene et al. 2018, Jayawardena et al. 2019, Phookamsak et al. 2019, Fan et al. 2020). Some species can cause severe canker diseases on woody trees, such as Cytospora chrysosperma, which is a commom pathogen on the commercial tree genera, Populus and Salix (Fan et al. 2014b, Zhang et al. 2014, Kepley et al. 2015, Wang et al. 2015). Host affiliation was considerd as the main evidence for separating species in Cytospora before DNA sequences were used; however, morphology combined with phylogeny has revealed many cryptic species. For example, 28 Cytospora species were discovered from Eucalyptus from South Africa (Adams et al. 2005) and six from apple trees in Iran (Mehrabi et al. 2011), three from Chinese scholar tree (Fan et al. 2014a), four from walnut tree (Fan et al. 2015a), six from anti-desertification plants in China (Fan et al. 2015b) and two from grapevine in North America (Lawrence et al. 2017). Several recent studies discovered new species of Cytospora using multiphasic analyses (Lawrence et al. 2018, Norphanphoun et al. 2017, 2018, Senanayake et al. 2017, 2018, Pan et al. 2018, Zhu et al. 2018, Zhang et al. 2019).

During our investigations of chestnut disease in China from 2016 to 2019, diseased branches with typical Cytospora fruiting bodies were discovered and collected (Fig. 1). In the present study, Cytospora species from Castanea mollissima were identified using a combined method of morphology and phylogeny.

Figure 1. 

Canker symptoms on Castanea mollissima caused by Cytospora spp.

Materials and methods

Sample collections and isolations

Chinese chestnut has a wide distribution in China. In the present study, we surveyed Hebei, Shaanxi and Shandong Provinces from 2016 to 2019. Dead and dying branches with typical Cytospora fruiting bodies were collected and packed in paper bags. Isolates were obtained by removing the ascospores or conidial masses from the fruiting bodies on to clean PDA plates and incubating at 25 °C until spores germinated. Single germinated spores were transferred on to the new PDA plates and incubated at 25 °C in the dark. Specimens were deposited in the Museum of the Beijing Forestry University (BJFC) and axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Morphological analysis

Observation and description of Cytospora species from Castanea mollissima was based on fruiting bodies formed on tree barks. Ascomata and conidiomata from tree barks were sectioned by hand using a double-edged blade and strctures were observed under a dissecting microscope. At least 10 conidiostromata/ascostromata, 10 asci and 50 conidia/ascospores were measured to calculate the mean size and standard deviation. Measurements are reported as maximum and minimum in parentheses and the range representing the mean plus and minus the standard deviation of the number of measurements is given in parentheses (Voglmayr et al. 2017). Microscopy photographs were captured with a Nikon Eclipse 80i compound microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast illumination. Introduction of the new species, based on molecular data, follow the recommendations of Jeewon and Hyde (2016).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from young mycelium growing on PDA plates following Doyle and Doyle (1990). PCR amplifications were performed in a DNA Engine Peltier Thermal Cycler (PTC-200; Bio-Rad Laboratories, Hercules, CA, USA). The primer pair ITS1/ITS4 (White et al. 1990) was used to amplify the ITS region. The primer pair LR0R/LR5 (Vilgalys and Hester 1990) was used to amplify the LSU region. The primer pair ACT512F/ACT783R (Carbone and Kohn 1999) was used to amplify ACT gene. The primer pair dRPB2-5f/dRPB2-7r (Voglmayr et al. 2016) was used to amplify the RPB2 gene. The polymerase chain reaction (PCR) assay was conducted as described in Fan et al. (2020). PCR amplification products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyzer with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses

The preliminary identities of the isolates sequenced were obtained by conducting a standard nucleotide BLAST search using ITS, LSU, ACT and RPB2. Then all Cytospora isolates were selected to conduct phylogenetic analyses, based on sequence datasets from Fan et al. (2020). Diaporthe vaccinia (CBS 160.32) in Diaporthaceae was selected as the outgroup taxon. All sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and edited manually using MEGA v. 6 (Tamura et al. 2013). Phylogenetic analyses were performed using PAUP v. 4.0b10 for Maximum Parsimony (MP) analysis (Swofford 2003) and PhyML v. 3.0 for Maximum Likelihood (ML) analysis (Guindon et al. 2010).

MP analysis was run using a heuristic search option of 1000 search replicates with random-additions of sequences with a tree bisection and reconnection algorithm. Maxtrees were set to 5000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). ML analysis was performed using a GTR site substitution model including a gamma-distributed rate heterogeneity and a proportion of invariant sites (Guindon et al. 2010). The branch support was evaluated using a bootstrapping method of 1000 replicates (Hillis and Bull 1993). Phylograms were shown using FigTree v. 1.4.3 (Rambaut 2016). Novel sequences, generated in the current study, were deposited in GenBank (Table 1) and the aligned matrices used for phylogenetic analyses in TreeBASE (accession number: S25160).

Strains used in the phylogenetic tree and their culture accession and GenBank numbers. Strains from this study are in bold and ex-strains are marked with *.

Species Strain Host Origin GenBank accession numbers
ITS LSU ACT RPB2
Cytospora ailanthicola CFCC 89970* Ailanthus altissima China MH933618 MH933653 MH933526 MH933592
Cytospora abyssinica CMW 10181* Eucalyptus globulus Ethiopia AY347353 NA NA NA
Cytospora acaciae CBS 468.69 Ceratonia siliqua Spain DQ243804 NA NA NA
Cytospora ampulliformis MFLUCC 16-0583* Sorbus intermedia Russia KY417726 KY417760 KY417692 KY417794
MFLUCC 16-0629 Acer platanoides Russia KY417727 KY417761 KY417693 KY417795
Cytospora amygdali CBS 144233* Prunus dulcis USA MG971853 NA MG972002 NA
CFCC 89615 Juglans regia China KR045618 KR045700 KF498673 KU710946
CFCC 89616 Juglans regia China KR045619 KR045701 KF498674 KU710947
Cytospora atrocirrhata CFCC 89615 Juglans regia China KR045618 KR045700 KF498673 KU710946
Cytospora austromontana CMW 6735* Eucalyptus pauciflora Australia AY347361 NA NA NA
Cytospora beilinensis CFCC 50493* Pinus armandii China MH933619 MH933654 MH933527 NA
CFCC 50494 Pinus armandii China MH933620 MH933655 MH933528 NA
Cytospora berberidis CFCC 89927* Berberis dasystachya China KR045620 KR045702 KU710990 KU710948
CFCC 89933 Berberis dasystachya China KR045621 KR045703 KU710991 KU710949
Cytospora berkeleyi StanfordT3* Eucalyptus globulus USA AY347350 NA NA NA
UCBTwig3 Eucalyptus globulus USA AY347349 NA NA NA
Cytospora brevispora CBS 116811* Eucalyptus grandis × tereticornis Congo AF192315 NA NA NA
CBS 116829 Eucalyptus grandis Venezuela AF192321 NA NA NA
Cytospora bungeanae CFCC 50495* Pinus bungeana China MH933621 MH933656 MH933529 MH933593
CFCC 50496 Pinus bungeana China MH933622 MH933657 MH933530 MH933594
Cytospora californica CBS 144234* Juglans regia USA MG971935 NA MG972083 NA
Cytospora carbonacea CFCC 89947 Ulmus pumila China KR045622 KP310812 KP310842 KU710950
Cytospora carpobroti CMW 48981* Carpobrotus edulis South Africa MH382812 MH411216 NA NA
Cytospora castaneae AUCCT/DBT 183* Castanea sativa India KC963921 NA NA NA
Cytospora cedri CBS 196.50 NA Italy AF192311 NA NA NA
Cytospora celtidicola CFCC 50497* Celtis sinensis China MH933623 MH933658 MH933531 MH933595
CFCC 50498 Celtis sinensis China MH933624 MH933659 MH933532 MH933596
Cytospora centrivillosa MFLUCC 16-1206* Sorbus domestica Italy MF190122 MF190068 NA MF377601
MFLU 17-0887 Sorbus domestica Italy MF190123 MF190069 NA NA
MFLUCC 17-1660 Sorbus domestica Italy MF190124 MF190070 NA MF377600
Cytospora ceratosperma CFCC 89624 Juglans regia China KR045645 KR045724 NA KU710976
CFCC 89625 Juglans regia China KR045646 KR045725 NA KU710977
Cytospora ceratospermopsis CFCC 89626* Juglans regia China KR045647 KR045726 KU711011 KU710978
CFCC 89627 Juglans regia China KR045648 KR045727 KU711012 KU710979
CFCC 52471 Castanea mollissima China MK432629 MK429899 MK442953 MK578087
CFCC 52472 Castanea mollissima China MK432630 MK429900 MK442954 MK578088
Cytospora chrysosperma CFCC 89629 Salix psammophila China KF765673 KF765689 NA KF765705
CFCC 89981 Populus alba subsp. pyramidalis China MH933625 MH933660 MH933533 MH933597
CFCC 89982 Ulmus pumila China KP281261 KP310805 KP310835 NA
Cytospora cinerostroma CMW 5700* Eucalyptus globulus Chile AY347377 NA NA NA
Cytospora cotini MFLUCC 14-1050* Cotinus coggygria Russia KX430142 KX430143 NA KX430144
Cytospora curvata MFLUCC 15-0865* Salix alba Russia KY417728 KY417762 KY417694 KY417796
Cytospora davidiana CXY 1350* Populus davidiana China KM034870 NA NA NA
CXY 1374 Populus davidiana China KM034869 NA NA NA
Cytospora diatrypelloidea CMW 8549* Eucalyptus globulus Australia AY347368 NA NA NA
Cytospora disciformis CMW 6509* Eucalyptus grandis Uruguay AY347374 NA NA NA
CMW 6750 Eucalyptus globulus Australia AY347359 NA NA NA
Cytospora donetzica MFLUCC 15-0864 NA NA KY417729 KY417763 KY417695 KY417797
MFLUCC 16-0574* Rosa sp. Russia KY417731 KY417764 KY417696 KY417798
Cytospora elaeagni CFCC 89632 Elaeagnus angustifolia China KR045626 KR045706 KU710995 KU710955
CFCC 89633 Elaeagnus angustifolia China KF765677 KF765693 KU710996 KU710956
Cytospora eriobotryae IMI 136523* Eriobotrya japonica India AY347327 NA NA NA
Cytospora erumpens CFCC 50022 Prunus padus China MH933627 MH933661 MH933534 NA
MFLUCC 16-0580* Salix × fragilis Russia KY417733 KY417767 KY417699 KY417801
Cytospora eucalypti CBS 144241 Eucalyptus globulus USA MG971907 NA MG972056 NA
LSEQ Sequoia sempervirens USA AY347340 NA NA NA
Cytospora eucalypticola ATCC 96150* Eucalyptus nitens Australia AY347358 NA NA NA
CMW 5309 Eucalyptus grandis Uganda AF260266 NA NA NA
Cytospora eucalyptina CMW 5882 Eucalyptus grandis Columbia AY347375 NA NA NA
Cytospora eugeniae CMW 7029 Tibouchina sp. Australia AY347364 NA NA NA
CMW 8648 Eugenia sp. Indonesia AY347344 NA NA NA
Cytospora euonymicola CFCC 50499* Euonymus kiautschovicus China MH933628 MH933662 MH933535 MH933598
CFCC 50500 Euonymus kiautschovicus China MH933629 MH933663 MH933536 MH933599
Cytospora euonymina CFCC 89993* Euonymus kiautschovicus China MH933630 MH933664 MH933537 MH933600
CFCC 89999 Euonymus kiautschovicus China MH933631 MH933665 MH933538 MH933601
Cytospora fraxinigena MFLUCC 14-0868* Euonymus kiautschovicus China MH933631 MH933665 MH933538 MH933601
Cytospora friesii CBS 194.42 Abies alba Switzerland AY347328 NA NA NA
Cytospora fugax CXY 1381 NA NA KM034853 NA NA NA
Cytospora germanica CXY 1322 Elaeagnus oxycarpa China JQ086563 JX524617 NA NA
Cytospora gigalocus CFCC 89620* Juglans regia China KR045628 KR045708 KU710997 KU710957
CFCC 89621 Juglans regia China KR045629 KR045709 KU710998 KU710958
Cytospora gigaspora CFCC 50014 Juniperus procumbens China KR045630 KR045710 KU710999 KU710959
CFCC 89634* Salix psammophila China KF765671 KF765687 KU711000 KU710960
Cytospora granati CBS 144237* Punica granatum USA MG971799 NA MG971949 NA
Cytospora hippophaës CFCC 89639 Hippophaë rhamnoides China KR045632 KR045712 KU711001 KU710961
CFCC 89640 Hippophaë rhamnoides China KF765682 KF765698 KF765730 KU710962
Cytospora japonica CFCC 89956 Prunus cerasifera China KR045624 KR045704 KU710993 KU710953
CFCC 89960 Prunus cerasifera China KR045625 KR045705 KU710994 KU710954
Cytospora joaquinensis CBS 144235* Populus deltoides USA MG971895 NA MG972044 NA
Cytospora junipericola MFLU 17-0882* Juniperus communis Italy MF190125 MF190072 NA NA
Cytospora juniperina CFCC 50501* Juniperus przewalskii China MH933632 MH933666 MH933539 MH933602
CFCC 50503 Juniperus przewalskii China MH933634 MH933668 MH933541 MH933604
Cytospora kantschavelii CXY 1383 Populus maximowiczii China KM034867 NA NA NA
Cytospora kuanchengensis CFCC 52464* Castanea mollissima China MK432616 MK429886 MK442940 MK578076
CFCC 52465 Castanea mollissima China MK432617 MK429887 MK442941 MK578077
Cytospora kunzei CBS 118556 Pinus radiata South Africa DQ243791 NA NA NA
Cytospora leucosperma CFCC 89622 Pyrus bretschneideri China KR045616 KR045698 KU710988 KU710944
CFCC 89894 Pyrus bretschneideri China KR045617 KR045699 KU710989 KU710945
Cytospora leucostoma CFCC 50018 Prunus serrulata China MH933636 MH933670 MH933543 NA
CFCC 50019 Rosa helenae China MH933637 MH933671 MH933544 NA
CFCC 50021 Prunus salicina China MH933639 MH933673 MH933546 NA
CFCC 50023 Cornus alba China KR045635 KR045715 KU711003 KU710964
CFCC 52461 Castanea mollissima China MK432624 MK429894 MK442948 NA
CFCC 52462 Castanea mollissima China MK432625 MK429895 MK442949 NA
Cytospora longiostiolata MFLUCC 16-0628* Salix × fragilis Russia KY417734 KY417768 KY417700 KY417802
Cytospora longispora CBS 144236* Prunus domestica USA MG971905 NA MG972054 NA
Cytospora lumnitzericola MFLUCC 17-0508* Lumnitzera racernosa Tailand MG975778 MH253461 MH253457 MH253453
Cytospora mali CFCC 50028 Malus pumila China MH933641 MH933675 MH933548 MH933606
CFCC 50029 Malus pumila China MH933642 MH933676 MH933549 MH933607
Cytospora melnikii MFLUCC 15-0851* Malus domestica Russia KY417735 KY417769 KY417701 KY417803
MFLUCC 16-0635 Populus nigra var. italica Russia KY417736 KY417770 KY417702 KY417804
Cytospora mougeotii ATCC 44994 Picea abies Norway AY347329 NA NA NA
Cytospora multicollis CBS 105.89T Quercus ilex subsp. rotundifolia Spain DQ243803 NA NA NA
Cytospora myrtagena CBS 116843* Tibouchiina urvilleana USA AY347363 NA NA NA
CFCC 52454 Castanea mollissima China MK432614 MK429884 MK442938 MK578074
CFCC 52455 Castanea mollissima China MK432615 MK429885 MK442939 MK578075
Cytospora nitschkii CMW 10180* Eucalyptus globulus Ethiopia AY347356 NA NA NA
CMW 10184 Eucalyptus globulus Ethiopia AY347355 NA NA NA
Cytospora nivea CFCC 89641 Elaeagnus angustifolia China KF765683 KF765699 KU711006 KU710967
CFCC 89643 Salix psammophila China KF765685 KF765701 NA KU710968
Cytospora oleicola CBS 144248* Olea europaea USA MG971944 NA MG972098 NA
Cytospora palm CXY 1280* Cotinus coggygria China JN411939 NA NA NA
Cytospora parakantschavelii MFLUCC 15-0857* Populus × sibirica Russia KY417738 KY417772 KY417704 KY417806
MFLUCC 16-0575 Pyrus pyraster Russia KY417739 KY417773 KY417705 KY417807
Cytospora parapersoonii T28.1* Prunus persica USA AF191181 NA NA NA
Cytospora parapistaciae CBS 144506* Pistacia vera USA MG971804 NA MG971954 NA
Cytospora parasitica MFLUCC 15-0507* Malus domestica Russia KY417740 KY417774 KY417706 KY417808
Cytospora paratranslucens MFLUCC 15-0506* Populus alba var. bolleana Russia KY417741 KY417775 KY417707 KY417809
MFLUCC 16-0627 Populus alba Russia KY417742 KY417776 KY417708 KY417810
Cytospora pini CBS 197.42 Pinus sylvestris Switzerland AY347332 NA NA NA
CBS 224.52* Pinus strobus USA AY347316 NA NA NA
Cytospora pistaciae CBS 144238* Pistacia vera USA MG971802 NA MG971952 NA
Cytospora platanicola MFLU 17-0327* Platanus hybrida Italy MH253451 MH253452 MH253449 MH253450
Cytospora platycladi CFCC 50504* Platycladus orientalis China MH933645 MH933679 MH933552 MH933610
CFCC 50505 Platycladus orientalis China MH933646 MH933680 MH933553 MH933611
CFCC 50506 Platycladus orientalis China MH933647 MH933681 MH933554 MH933612
Cytospora platycladicola CFCC 50038* Platycladus orientalis China KT222840 MH933682 MH933555 MH933613
CFCC 50039 Platycladus orientalis China KR045642 KR045721 KU711008 KU710973
Cytospora plurivora CBS 144239* Olea europaea USA MG971861 NA MG972010 NA
Cytospora populicola CBS 144240* Populus deltoides USA MG971891 NA MG972040 NA
Cytospora populina CFCC 89644* Salix psammophila China KF765686 KF765702 KU711007 KU710969
Cytospora populinopsis CFCC 50032* Sorbus aucuparia China MH933648 MH933683 MH933556 MH933614
CFCC 50033 Sorbus aucuparia China MH933649 MH933684 MH933557 MH933615
Cytospora predappioensis MFLUCC 17-2458* Platanus hybrida Italy MG873484 MG873480 NA NA
Cytospora prunicola MFLU 17-0995* Prunus sp. Italy MG742350 MG742351 MG742353 MG742352
Cytospora pruinopsis CFCC 50034* Ulmus pumila China KP281259 KP310806 KP310836 KU710970
CFCC 50035 Ulmus pumila China KP281260 KP310807 KP310837 KU710971
Cytospora pruinosa CFCC 50036 Syringa oblata China KP310800 KP310802 KP310832 NA
CFCC 50037 Syringa oblata China MH933650 MH933685 MH933558 NA
Cytospora prunicola MFLU 17-0995* Prunus sp. Italy MG742350 MG742351 MG742353 MG742352
Cytospora punicae CBS 144244 Punica granatum USA MG971943 NA MG972091 NA
Cytospora quercicola MFLU 17-0881 Quercus sp. Italy MF190129 MF190074 NA NA
MFLUCC 14-0867* Quercus sp. Italy MF190128 MF190073 NA NA
Cytospora rhizophorae MUCC302 Eucalyptus grandis Australia EU301057 NA NA NA
Cytospora ribis CFCC 50026 Ulmus pumila China KP281267 KP310813 KP310843 KU710972
CFCC 50027 Ulmus pumila China KP281268 KP310814 KP310844 NA
Cytospora rosae MFLU 17-0885 Rosa canina Italy MF190131 MF190076 NA NA
Cytospora rostrata CFCC 89909* Salix cupularis China KR045643 KR045722 KU711009 KU710974
CFCC 89910 Salix cupularis China KR045644 KR045723 KU711010 KU710975
Cytospora rusanovii MFLUCC 15-0853 Populus × sibirica Russia KY417743 KY417777 KY417709 KY417811
MFLUCC 15-0854* Salix babylonica Russia KY417744 KY417778 KY417710 KY417812
Cytospora salicacearum MFLUCC 16-0576 dead aerial branch Russia KY417747 KY417781 KY417713 KY417815
MFLUCC 15-0509* Salix alba Russia KY417746 KY417780 KY417712 KY417814
MFLUCC 15-0861 Salix × fragilis Russia KY417745 KY417779 KY417711 KY417813
MFLUCC 16-0587 NA NA KY417748 KY417782 KY417714 KY417816
Cytospora salicicola MFLUCC 14-1052* Salix alba Russia KU982636 KU982635 KU982637 NA
MFLUCC 15-0866 Salix alba Russia KY417749 KY417783 KY417715 KY417817
Cytospora salicina MFLUCC 15-0862* Salix alba Russia KY417750 KY417784 KY417716 KY417818
MFLUCC 16-0637 Salix × fragilis Russia KY417751 KY417785 KY417717 KY417819
Cytospora schulzeri CFCC 50040 Malus domestica China KR045649 KR045728 KU711013 KU710980
CFCC 50042 Malus asiatica China KR045650 KR045729 KU711014 KU710981
CFCC 52468 Castanea mollissima China MK432626 MK429896 MK442950 MK578084
CFCC 52469 Castanea mollissima China MK432627 MK429897 MK442951 MK578085
CFCC 52470 Castanea mollissima China MK432628 MK429898 MK442952 MK578086
Cytospora sibiraeae CFCC 50045* Sibiraea angustata China KR045651 KR045730 KU711015 KU710982
CFCC 50046 Sibiraea angustata China KR045652 KR045731 KU711015 KU710983
Cytospora sophorae CFCC 50048 Magnolia grandiflora China MH820401 MH820394 MH820409 MH820397
CFCC 89598 Styphnolobium japonicum China KR045654 KR045733 KU711018 KU710985
Cytospora sophoricola CFCC 89595* Styphnolobium japonicum var. pendula China KR045655 KR045734 KU711019 KU710986
CFCC 89596 Styphnolobium japonicum var. pendula China KR045656 KR045735 KU711020 KU710987
Cytospora sophoriopsis CFCC 89600* Styphnolobium japonicum China KR045623 KP310804 KU710992 KU710951
Cytospora sorbi MFLUCC 16-0631* Sorbus aucuparia Russia KY417752 KY417786 KY417718 KY417820
Cytospora sorbicola MFLUCC 16-0584* Acer pseudoplatanus Russia KY417755 KY417789 KY417721 KY417823
MFLUCC 16-0633 Cotoneaster melanocarpus Russia KY417758 KY417792 KY417724 KY417826
Cytospora spiraeae CFCC 50049* Spiraea salicifolia China MG707859 MG707643 MG708196 MG708199
CFCC 50050 Spiraea salicifolia China MG707860 MG707644 MG708197 MG708200
Cytospora tamaricicola CFCC 50507 Rosa multifolora China MH933651 MH933686 MH933559 MH933616
CFCC 50508* Tamarix chinensis China MH933652 MH933687 MH933560 MH933617
Cytospora tanaitica MFLUCC 14-1057* Betula pubescens Russia KT459411 KT459412 KT459413 NA
Cytospora thailandica MFLUCC 17-0262* Xylocarpus moluccensis Thailand MG975776 MH253463 MH253459 MH253455
MFLUCC 17-0263 Xylocarpus moluccensis Thailand MG975777 MH253464 MH253460 MH253456
Cytospora tibouchinae CPC 26333* Tibouchina semidecandra France KX228284 KX228335 NA NA
Cytospora translucens CXY 1351 Populus davidiana China KM034874 NA NA NA
Cytospora ulmi MFLUCC 15-0863* Ulmus minor Russia KY417759 NA NA NA
Cytospora ulmicola MFLUCC 18-1227* Ulmus pumila Russia MH940220 MH940218 MH940216 NA
Cytospora valsoidea CMW 4309* Eucalyptus grandis Indonesia AF192312 NA NA NA
CMW 4310 Eucalyptus grandis Indonesia AF192312 NA NA NA
Cytospora variostromatica CBS 118086 Eucalyptus grandis South Africa AF260264 NA NA NA
CMW 1240 Eucalyptus grandis South Africa AF260263 NA NA NA
CMW 6766* Eucalyptus globulus Australia AY347366 NA NA NA
Cytospora vinacea CBS 141585* Vitis interspecific hybrid ‘Vidal’ USA KX256256 NA NA NA
Cytospora viticola CBS 141586* Vitis vinifera ’Cabernet Franc’ USA KX256239 NA NA NA
Cytospora xinglongensis CFCC 52458* Castanea mollissima China MK432622 MK429892 MK442946 MK578082
CFCC 52459 Castanea mollissima China MK432623 MK429893 MK442947 MK578083
Cytospora xylocarpi MFLUCC 17-0251* Xylocarpus granatum Thailand MG975775 MH253462 MH253458 MH253454
Diaporthe vaccinii CBS 160.32 Vaccinium macrocarpon USA KC343228 NA JQ807297 NA

Results

Phylogenetic analyses

The alignment based on the combined sequence dataset (ITS, LSU, ACT and RPB2) included 124 ingroup taxa and one outgroup taxon, comprising 2097 characters in the aligned matrix. Of these, 1375 characters were constant, 89 variable characters were parsimony-uninformative and 663 characters were parsimony informative. The MP analysis resulted in 14 equally most parsimonious trees and the first tree (TL = 3270, CI = 0.344, RI = 0.815, RC = 0.281) was present as in Fig. 2. The ML analysis yielded a tree with a likelihood value of ln: -18627.915604 and the following model parameters: alpha: 0.181328; Π(A): 0.246855, Π(C): 0.260898, Π(G): 0.272379 and Π(T): 0.219868. Isolates from Castanea mollissima formed six clades in Fig. 2, representing two undescribed species and four known species.

Figure 2. 

Maximum parsimony phylogram of Cytospora obtained from the combined matrix of ITS, LSU, ACT and RPB2 genes. Bootstrap value ≥ 50% for MP and ML analyses are presented at the first and second position. Scale bar = 200 nucleotide substitutions. The strains in the current study are in blue and ex-strains are marked with *.

Taxonomy

Cytospora ceratosp ermopsis C.M. Tian & X.L. Fan, Persoonia 45: 19. 2020

Figure 3

Description

Sexual morph: Ascostromata immersed in the bark, erumpent through the surface of bark, scattered, (350–)550–900(–1300) µm diam., with 15–40 perithecia arranged circularly or irregularly. Conceptacle absent. Ectostromatic disc black, usually surrounded by tightly ostiolar necks, circular to ovoid, (180–)240–410(–450) µm diam. Ostioles black, at the same level as the disc or slightly above, concentrated, dark brown to black, arranged circularly in a disc, (55–)60–85(–110) µm diam. Perithecia dark brown, flask-shaped to spherical, arranged circularly or irregularly, (255–)280–350(–420) µm diam. Asci clavate to elongate obovoid, 8-spored, (20.5–)27–35.5(–43) × (4–)4.5–6.5(–8) μm (x̄= 31.2 × 5.6 μm). Ascospores biseriate, elongate-allantoid, thin-walled, hyaline, aseptate, (5.8–)7.5–9.2(–11.5) × (3–)3.2–4.8(–5.5) μm (x̄ = 8.6 × 4.1 μm). Asexual morph: Pycnidial stromata ostiolated, immersed in bark, scattered, erumpent through the surface of bark, discoid to conical, with multiple locules. Conceptacle absent. Ectostromatic disc light brown to grey, circular to ovoid, (230–)280–360(–480) µm diam., with one ostiole per disc. Ostiole in the centre of the disc, dark grey to black, conspicuous, at the same level as the disc, (60–)75–110(–135) μm diam. Locule numerous, arranged circularly or elliptically with independent walls, (300–)350–600(–950) µm diam. Peridium comprising few layers of cells of textura angularis, with innermost layer brown, outer layer brown to dark brown. Conidiophores hyaline, branched or not, thin walled, filamentous. Conidiogenous cells enteroblastic polyphialidic, (6.5–)8.5–15.5(–18) × 1.5–2.5 μm (x̄ = 12.2 × 1.9 μm). Conidia hyaline, allantoid, smooth, aseptate, thin-wall, (4.5–)5–6.5(–7) × 1–1.5 μm (x̄ = 5.9 × 1.3 μm).

Culture characters

On PDA at 25 °C in darkness. Cultures are initially white, becoming olivaceous buff in centre after 7 d and finally olivaceous at 30 d. The colony is flat, thin with a felt and tight texture in centre. Pycnidia distributed irregularly on medium surface.

Specimens examined

China, Hebei Province, Chengde City, Xinglong County, chestnut plantation, 40°24'32"N, 117°27'56"E, on branches of Castanea mollissima, 11 October 2017, N. Jiang (BJFC-S1699, living culture CFCC 52471 from the ascospore; BJFC-S1700, living culture CFCC 52472 from the conidium).

Notes

Fresh specimens with both sexual and asexual morphs were collected from cankered branches of Castanea mollissima and two isolates were obtained from the ascospore and conidium, respectively. Phylogenically, the two isolates were close to Cytospora ceratospermopsis represented by CFCC 89626 and CFCC 89627 (Fig. 2). We compared their sequences and found no differences in LSU and RPB2, but 2 bp differences in ITS and 3 bp differences in ACT. Fan et al. (2020) reported the asexual morph of Cytospora ceratospermopsis from Juglans regia in China with conidial size in 4.5–6 × 1–1.5 μm, which is exactly matched with the asexual characters observed in the present study. Hence, we described the asexual morph of Cytospora ceratospermopsis in its sexual morph for the first time and reported a new host, Castanea mollissima.

Figure 3. 

Cytospora ceratospermopsis on Castanea mollissima (BJFC-S1699, BJFC-S1700). A, C Habit of conidiomata on branches B habit of ascomata on branches D transverse section of conidiomata E transverse section of ascomata F longitudinal section through conidiomata G longitudinal section through ascomata H, I asci J ascospores K conidiogenous cells with attached conidia L conidia. Scale bars: 500 μm (C–G), 10 μm (H–L).

Cytospora kuanchengensis C.M. Tian & N. Jiang, sp. nov.

MycoBank No: 829514
Figure 4

Diagnosis

Cytospora kuanchengensis can be distinguished from C. oleicola and C. pruinose by longer conidia.

Etymology

Named after the county where it was collected, Kuancheng County.

Description

Sexual morph: not observed. Asexual morph: Pycnidial stromata ostiolated, immersed in bark, scattered, erumpent through the surface of bark, discoid, with multiple locules. Conceptacle black, circular surrounded stromata. Ectostromatic disc black, circular to ovoid, (350–)455–540(–575) µm diam., with 1–7 ostiole per disc. Ostioles black, at the same level as the disc, (40–)60–85(–115) μm diam. Locule numerous, arranged circularly or elliptically with independent walls, (285–)355–520(–605) µm diam. Peridium comprising few layers of cells of textura angularis, with innermost layer brown, outer layer brown to dark brown. Conidiophores hyaline, unbranched, thin walled, filamentous. Conidiogenous cells enteroblastic polyphialidic, (6.5–)8.5–11(–15) × 1–1.5 μm (x̄ = 9.8 × 1.3 μm). Conidia hyaline, allantoid, smooth, aseptate, thin-walled, (5.5–)6–7.5(–8) × 1–2 μm (x̄ = 6.9 × 1.6 μm).

Culture characters

On PDA at 25 °C in darkness. Cultures are initially white, producing pale brown pigment after 10 d. The colony is flat, felt-like, with concentric circular texture. Pycnidia distributed irregularly on medium surface.

Specimens examined

China, Hebei Province, Chengde City, Kuancheng County, chestnut plantation, 40°38'37"N, 118°27'54"E, on branches of Castanea mollissima, 13 October 2017, N. Jiang (holotype BJFC-S1695, ex-type living culture CFCC 52464; paratype BJFC-S1696, living culture CFCC 52465).

Notes

Cytospora kuanchengensis is associated with canker disease of Castanea mollissima in China. Cytospora kuanchengensis differs from its phylogenetically closely species, C. pruinosa, by ITS and ACT loci (7/470 in ITS and 21/245 in ACT). Morphologically, C. kuanchengensis has slightly larger conidia than C. pruinose (5.5–8 × 1–2 μm in Cytospora kuanchengensis vs. 5–7.5 × 1–1.5 μm in C. pruinosa) (Fan et al. 2020).

Figure 4. 

Cytospora kuanchengensis on Castanea mollissima (BJFC-S1695). A, B Habit of conidiomata on branches C longitudinal section through conidiomata D transverse section of conidiomata E peridium F, G conidiogenous cells attached with conidia H conidia. Scale bars: 500 μm (B–D), 10 μm (E–G), 5 μm (H).

Cytospora leucostoma (Pers.) Sacc., Michelia 2(7): 264. 1881.

Figure 5

Description

Sexual morph: not observed. Asexual morph: Pycnidial stromata ostiolated, immersed in bark, scattered, erumpent through the surface of bark, with multiple locules. Conceptacle black. Ectostromatic disc black, circular to ovoid, (150–)250–300(–375) µm diam., with one ostiole per disc. Ostioles black, at the same level as the disc, (40–)50–85(–115) μm diam. Locule numerous, arranged circularly or elliptically with independent walls, (550–)700–1200(–1350) µm diam. Peridium comprising few layers of cells of textura angularis, with innermost layer brown, outer layer brown to dark brown. Conidiophores hyaline, unbranched, thin walled, filamentous. Conidiogenous cells enteroblastic polyphialidic, (7.5–)9.5–21(–22.5) × 1–1.5 μm (x̄ = 15.2 × 1.3 μm). Conidia hyaline, allantoid, smooth, aseptate, thin-walled, (3.5–)4.5–5.5(–7) × 1–1.5 μm (x̄ = 4.9 × 1.3 μm).

Specimens examined

China, Hebei Province, Chengde City, Kuancheng County, chestnut plantation, 40°38'37"N, 118°27'5"E, on branches of Castanea mollissima, 13 October 2017, N. Jiang (BJFC-S1697, living culture CFCC 52461; BJFC-S1698, living culture CFCC 52462).

Notes

Cytospora leucostoma is a common species causing canker disease on Rosaceae in China (Teng 1963, Tai 1979, Wei 1979, Fan et al. 2020). In this study, fresh specimens were collected from diseased branches of the Chinese chestnut for the first time and identified as Cytospora leucostoma, based on strictly matched asexual morph (4–5.5 × 1–2 μm from Castanea mollissima in this study vs. 4.5–5.5 × 1–1.5 μm from multiple specimens in Fan et al. 2020) and phylogenic analysis (Fig. 2).

Figure 5. 

Cytospora leucostoma on Castanea mollissima (BJFC-S1697). A, B Habit of conidiomata on branches C transverse section of conidiomata D longitudinal section through conidiomata E conidiogenous cells attached with conidia F conidia. Scale bars: 500 μm (B–D), 10 μm (E, F).

Cytospora myrtagena (G.C. Adams & M.J. Wingf.) G.C. Adams & Rossman, IMA Fungus 6 (1): 147. 2015.

Figure 6

Description

Sexual morph: not observed. Asexual morph: Pycnidial stromata pulvinate, immersed in bark, scattered, erumpent through the surface of bark. Conceptacle absent. Ostiole dark grey to black, conspicuous, at the same level as the disc, (50–)65–75(–82) μm diam. Locules undivided, circular to ovoid, (430–)550–720(–810) µm diam. Peridium comprising few layers of cells of textura angularis, with innermost layer brown, outer layer brown to dark brown. Conidiophores hyaline, unbranched, thin-walled, filamentous. Conidiogenous cells enteroblastic polyphialidic, (6.5–)8.4–12.5(–15.3) × 0.9–1.4 μm (x̄ = 10.2 × 1.2 μm). Conidia hyaline, allantoid, smooth, aseptate, thin-walled, (3.2–)3.4–5.4(–6.2) × 1–1.5 μm (x̄ = 4.7 × 1.3 μm).

Culture characters

On PDA at 25 °C in darkness. Cultures are initially white, becoming olivaceous buff in centre after 7 d and finally olivaceous at 30 d. The colony is flat, thin with a felt and tight texture in centre. Pycnidia distributed irregularly on medium surface.

Specimens examined

China, Shaanxi Province, Ankang City, Xiangxidong forest park, 32°40'33"N, 109°18'57"E, on stem barks of Castanea mollissima, 1 July 2017, N. Jiang (BJFC-S1704, living culture CFCC 52454; BJFC-S1705, living culture CFCC 52455).

Notes

Cytospora myrtagena was introduced from Eucalyptus and Tibouchina in America and Indonesia (Adams et al. 2005). Two ITS sequences of Cytospora myrtagena were available, AY347363 from the type strain CBS 116843 and AY347380 from CBS 117013. However, there are 14 bp differences between AY347363 and AY347380. Cytospora tibouchinae was introduced as a phylogenically close species to Cytospora myrtagena (Suppl. material 1: Fig. S1), with 21 bp differences to CBS 116843 and 14 bp bp differences to CBS 117013 (Crous et al. 2016). Two isolates from Castanea mollissima in the present study were close to Cytospora myrtagena and Cytospora tibouchinae (Suppl. material 1: Fig. S1), with 22 bp differences to CBS 116843, 15 bp differences to CBS 117013 and 6 bp differences to Cytospora tibouchinae. Morphologically, they have similar conidial sizes (3.4–5.4 × 1–1.5 μm in BJFC-S1704 vs. 3–4 × 1 μm in C. myrtagena vs. 3–4 × 1.5–2 μm in C. tibouchinae) (Adams et al. 2005, Crous et al. 2016). Hence, it is hard to identify our isolates to C. myrtagena or C. tibouchinae, for the large differences between two ITS sequences in C. myrtagena provided by Adams et al. (2005) and absence of ACT and RPB2 loci in C. myrtagena and C. tibouchinae. We give the name Cytospora myrtagena to our isolates provisionally, and hope for more studies on this species.

Figure 6. 

Cytospora myrtagena on Castanea mollissima (BJFC-S1704). A, B Habit of conidiomata on branches C, E transverse section of conidiomata D longitudinal section through conidiomata F, G conidiogenous cells attached with conidia H conidia. Scale bars: 500 μm (B–D), 5 μm (E, G), 10 μm (H).

Cytospora schulzeri Sacc. & P. Syd., Syll. fung. (Abellini) 14(2): 918. 1899.

Figure 7

Description

Sexual morph: not observed. Asexual morph: Pycnidial stromata ostiolated, immersed in bark, scattered, erumpent through the surface of bark, flat, discoid, with multiple locules. Conceptacle absent. Ectostromatic disc brown, circular to ovoid, (250–)300–400(–475) µm diam., with 1–5 ostiole per disc. Ostioles black, at the same level as the disc, (40–)50–85(–115) μm diam. Locule numerous, arranged circularly with common walls, (600–)700–1500(–1750) µm diam. Peridium comprising a few layers of cells of textura angularis, with innermost layer brown, outer layer brown to dark brown. Conidiophores hyaline, unbranched, thin walled, filamentous. Conidiogenous cells enteroblastic polyphialidic, (6.5–)8.5–18.5(–21) × 1–2 μm (x̄ = 13.1 × 1.6 μm). Conidia hyaline, allantoid, smooth, aseptate, thin-walled, (3.5–)4.5–6.5(–7) × 1–1.5 μm (x̄ = 5.2 × 1.3 μm).

Specimens examined

China, Hebei Province, Chengde City, Kuancheng County, chestnut plantation, 40°38'37"N, 118°27'54"E, on branches of Castanea mollissima, 13 October 2017, N. Jiang (living culture CFCC 52468; BJFC-S1702, living culture CFCC 52469; BJFC-S1703, living culture CFCC 52470).

Notes

Cytospora schulzeri is a common species causing apple canker disease in China (Teng 1963, Tai 1979, Wei 1979, Zhuang 2005, Fan et al. 2020). In this study, fresh specimens were collected from diseased branches of chestnut trees and identified as Cytospora schulzeri, based on the strictly matched asexual morph (4.5–6.5 × 1–2 μm from Castanea mollissima in this study vs. 4–7 × 1–1.5 μm from multiple specimens in Fan et al. (2020)) and phylogenic analysis (Fig. 2).

Figure 7. 

Cytospora schulzeri on Castanea mollissima (BJFC-S1702). A, B Habit of conidiomata on branches C transverse section of conidiomata D longitudinal section through conidiomata E conidiogenous cells attached with conidia F conidia. Scale bars: 500 μm (B–D), 10 μm (E, F).

Cytospora xinglongensis C.M. Tian & N. Jiang, sp. nov.

MycoBank No: 829517
Figure 8

Diagnosis

Cytospora xinglongensis can be distinguished from C. californica and C. eucalypti by longer conidia.

Etymology

Named after the county where it was collected, Xinglong County.

Description

Sexual morph: not observed. Asexual morph: Pycnidial stromata immersed in bark, erumpent through the surface of bark, discoid, with a solitary undivided locule. Conceptacle black, circular surrounded stromata. Ostiole inconspicuous. Locules undivided, circular to ovoid, (480–)540–685(–755) µm diam. Conidiophores hyaline, unbranched. Peridium comprising a few layers of cells of textura angularis, with innermost layer brown, outer layer brown to dark brown. Conidiogenous cells enteroblastic polyphialidic, (4.5–)6.5–8.5(–12) × 1–1.5 μm (x̄ = 7.4 × 1.3 μm). Conidia hyaline, allantoid, eguttulate, smooth, aseptate, thin-walled, (7.5–)8.5–9.5(–10.5) × 1–1.5 μm (x̄ = 8.9 × 1.3 μm).

Culture characters

On PDA at 25 °C in darkness. Cultures are white. The colony is flat, thin with a uniform texture, lacking aerial mycelium. Pycnidia distributed uniformly on medium surface.

Specimens examined

China, Hebei Province, Chengde City, Xinglong County, chestnut plantation, 40°24'32"N, 117°28'56"E, on branches of Castanea mollissima, 11 October 2017, N. Jiang (holotype BJFC-S1706, ex-type living culture CFCC 52458; paratype BJFC-S1707, living culture CFCC 52459).

Notes

Cytospora xinglongensis is associated with canker disease of Castanea mollissima in China. Cytospora xinglongensis can be distinguished from its phylogenetically closely species C. thailandica by having much longer conidia (8.5–9.5 μm in C. xinglongensis vs. 3.3–4 μm in C. thailandica) (Norphanphoun et al. 2018). In addition, Cytospora xinglongensis differs from C. thailandica by ITS, ACT and RPB2 loci (16/470 in ITS, 22/245 in ACT and 52/726 in RPB2).

Figure 8. 

Cytospora xinglongensis on Castanea mollissima (BJFC-S1706). A Habit of conidiomata on branches B longitudinal section through conidiomata C transverse section of conidiomata D peridium E conidiogenous cells attached with conidia F, G conidia. Scale bars: 500 μm (B, C), 10 μm (E–G).

Discussion

In the present study, an important fruit tree species, Castanea mollissima was investigated and Cytospora canker was found as a commom disease in plantations in Hebei Province. Identification was conducted based on 13 isolates from fruiting bodies using both morphological and molecular methods. As a result, six Cytospora species were confirmed. Cytospora kuanchengensis and C. xinglongensis are introduced as new species, C. ceratospermopsis, C. leucostoma, C. myrtagena and C. schulzeri are firstly reported on Castanea mollissima.

These six chestnut Cytospora species can be easily distinguished using DNA sequences of single ITS sequence or combined sequences of ITS, LSU, ACT and RPB2 (Fig. 2; Suppl. material 1: Fig. S1). In addition, colonies on PDA and MEA of these six species are also different (Fig. 9). Cytospora xinglongensis never produce fruiting bodies on PDA or MEA, while the other five species form conidiomata in one month (Fig. 9). Morphologically, Cytospora xinglongensis has obviously longer conidia than others. However, the conidial dimension can hardly distinguish C. ceratospermopsis, C. kuanchengensis, C. leucostoma, C. myrtagena and C. schulzeri.

Figure 9. 

Cultures of Cytospora species from Castanea mollissima after 1 month at 25 °C. A C. myrtagena B C. kuanchengensis C C. ceratospermopsis D C. leucostoma E C. xinglongensis F C. schulzeri A1–G2 cultures on PDA A3–G4 cultures on MEA A5–G6 fruiting bodies or hyphal masses produced on cultures

Dar and Rai reported Cytospora diseases on Castanea sativa in India, causing perennial cankers on stems and branches (Dar and Rai 2014). The Cytospora isolates were identified mainly based on ITS sequence data, which were introduced as a new species named Cytospora castaneae (wrongly wrriten as Cytospora castanae in the original paper) (Dar and Rai 2014). However, further study is required to confirm the species position within the genus, including detailed morphogical features and sequences of high quality.

Cytospora canker is a common disease on chestnut trees, but there are few formal reports. In China, this disease is known amongst phytopathologists, but no-one conducted accurate identifications. Hence, this paper is the first formal report of Cytospora chestnut canker in China. From our investigations of chestnut diseases in China, Cytospora species are closely associated with canker diseases in chestnut plantations. In most cases, they infect twigs or small branches, causing necrotic lesions (Fig. 1A), finially forming fruiting bodies on dead tissues (Fig. 1D). However, Cytospora myrtagena was discovered on stems of a 15-year-old chestnut tree, causing typical Cytospora canker symptoms. More works should be conducted on the newly emerging pathogens from several aspects.

As the species concept of Cytospora has been improved a lot by using molecular data (Yang et al. 2015, Lawrence et al. 2017, Norphanphoun et al. 2017, 2018, Jayawardena et al. 2019, Fan et al. 2020), many Cytospora canker diseases and new species have been discovered and reported in recent years. Further studies are, however, now required to confirm their pathogenicity.

Acknowledgements

This study is financed by National Natural Science Foundation of China (Project No.: 31670647). We are grateful to Chungen Piao, Minwei Guo (China Forestry Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing.

References

  • Adams GC, Roux J, Wingfield MJ, Roux J (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. Australasian Plant Pathology 35: 521–548. https://doi.org/10.1071/AP06058
  • Fan XL, Hyde KD, Liu M, Liang YM, Tian CM (2015a) Cytospora species associated with walnut canker disease in China, with description of a new species C. gigalocus. Fungal Biology 119: 310–319. https://doi.org/10.1016/j.funbio.2014.12.011
  • Fan XL, Hyde KD, Yang Q, Liang YM, Ma R, Tian CM (2015b) Cytospora species associated with canker disease of three anti-desertification plants in northwestern China. Phytotaxa 197: 227–244. https://doi.org/10.11646/phytotaxa.197.4.1
  • Fan XL, Liang YM, Ma R, Ma R, Tian CM (2014a) Morphological and phylogenetic studies of Cytospora (Valsaceae, Diaporthales) isolates from Chinese scholar tree, with description of a new species. Mycoscience 55: 252–259. https://doi.org/10.1016/j.myc.2013.10.001
  • Fan XL, Tian CM, Yang Q, Liang YM, You CJ, Zhang YB (2014b) Cytospora from Salix in northern China. Mycotaxon 129: 303–315. https://doi.org/10.5248/129.303
  • Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59: 307–321. https://doi.org/10.1093/sysbio/syq010
  • Gong S, Zhang X, Jiang S, Chen C, Ma H, Nie Y (2017) A new species of Ophiognomonia from Northern China inhabiting the lesions of chestnut leaves infected with Diaporthe eres. Mycological Progress 16: 83–91. https://doi.org/10.1007/s11557-016-1255-z
  • Hillis DM, Bull JJ (1993) An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Systematic Biology 42: 182–192. https://doi.org/10.1093/sysbio/42.2.182
  • Jayawardena RS, Hyde KD, McKenzie EHC et al. (2019) One stop shop III: taxonomic update with molecular phylogeny for important phytopathogenic genera: 51–75. Fungal Diversity 98: 1–84. https://doi.org/10.1007/s13225-019-00433-6
  • Jiang N, Fan XL, Tian CM (2019a) Identification and pathogenicity of Cryphonectriaceae species associated with chestnut canker in China. Plant Pathology 68: 1132–1145. https://doi.org/10.1111/ppa.13033
  • Jiang N, Fan XL, Crous PW, Tian CM (2019b) Species of Dendrostoma (Erythrogloeaceae, Diaporthales) associated with chestnut and oak canker diseases in China. Mycokeys 48: 67–96. https://doi.org/10.3897/mycokeys.48.31715
  • Jiang N, Li J, Piao CG, Guo MW, Tian CM (2018b) Identification and characterization of chestnut branch-inhabiting melanocratic fungi in China. Mycosphere 9: 1268–1289. https://doi.org/10.5943/mycosphere/9/6/14
  • Kepley JB, Reeves FB, Jacobi WR, Adams GC (2015) Species associated with Cytospora canker on Populus tremuloides. Mycotaxon 130: 783–805. https://doi.org/10.5248/130.783
  • Lawrence DP, Holland LA, Nouri MT, Travadon R, Abramians A, Michailides TJ, Trouillas FP (2018) Molecular phylogeny of Cytospora species associated with canker diseases of fruit and nut crops in California, with the descriptions of ten new species and one new combination. IMA Fungus 9: 333–370. https://doi.org/10.5598/imafungus.2018.09.02.07
  • Lawrence DP, Travadon R, Pouzoulet J, Rolshausen PE, Wilcox WF, Baumgartner K (2017) Characterization of Cytospora isolates from wood cankers of declining grapevine in North America, with the descriptions of two new Cytospora species. Plant Pathology 66: 713–725. https://doi.org/10.1111/ppa.12621
  • Lu C, Guo SJ (2016) Analysis on the nutritional characters and comprehensive evaluation of 16 chestnut germplasm resources. Science and Technology of Food Industry 37: 357–376.
  • Mehrabi ME, Mohammadi GE, Fotouhifar KB (2011) Studies on Cytospora canker disease of apple trees in Semirom region of Iran. Journal of Agricultural Technology 7: 967–982.
  • Norphanphoun C, Doilom M, Daranagama DA, Phookamsak R, Wen TC, Bulgakov TS, Hyde KD (2017) Revisiting the genus Cytospora and allied species. Mycosphere 8: 51–97. https://doi.org/10.5943/mycosphere/8/1/7
  • Norphanphoun C, Raspé O, Jeewon R, Wen TC, Hyde KD (2018) Morphological and phylogenetic characterisation of novel Cytospora species associated with mangroves. Mycokeys 38: 93–120. https://doi.org/10.3897/mycokeys.38.28011
  • Phookamsak R, Hyde KD, Jeewon R et al. Fungal diversity notes 929–1035: taxonomic and phylogenetic contributions on genera and species of fungi. Fungal Diversity 95: 1–273. https://doi.org/10.1007/s13225-019-00421-w
  • Rambaut A (2016) FigTree, version 1.4.3. University of Edinburgh, Edinburgh.
  • Rigling D, Prospero S (2018) Cryphonectria parasitica, the causal agent of chestnut blight: invasion history, population biology and disease control. Molecular Plant Pathology 19: 7–20. https://doi.org/10.1111/mpp.12542
  • Sarma VV, Hyde KD (2001) A review on frequently occurring fungi in mangroves. Fungal Diversity 8: 1–34.
  • Senanayake IC, Jeewon R, Chomnunti P et al. (2018) Taxonomic circumscription of Diaporthales based on multigene phylogeny and morphology. Fungal Diversity 93: 241–443. https://doi.org/10.1007/s13225-018-0410-z
  • Shuttleworth LA, Guest DI (2017) The infection process of chestnut rot, an important disease caused by Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) in Oceania and Europe. Australasian Plant Pathology 46: 397–405. https://doi.org/10.1007/s13313-017-0502-3
  • Swofford DL (2003) PAUP*: Phylogenetic Analyses Using Parsimony, * and Other Methods, Version 4.0b10. Sinauer Associates, Sunderland.
  • Tai FL (1979) Sylloge fungorum sinicorum. Science Press, 1–1527.
  • Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246. https://doi.org/10.1128/JB.172.8.4238-4246.1990
  • Voglmayr H, Akulov OY, Jaklitsch WM (2016) Reassessment of Allantonectria, phylogenetic position of Thyronectroidea, and Thyronectria caraganae sp. nov. Mycological Progress 15: 921–937. https://doi.org/10.1007/s11557-016-1218-4
  • Voglmayr H, Castlebury LA, Jaklitsch WM (2017) Juglanconis gen. nov. on Juglandaceae, and the new family Juglanconidaceae (Diaporthales). Persoonia 38: 136–155. https://doi.org/10.3767/003158517X694768
  • Wei JC (1979) Identification of Fungus Handbook. Science Press, 1–780.
  • Wijayawardene NN, Hyde KD, Lumbsch HT, Liu JK, Maharachchikumbura SSN, Ekanayaka AH, Tian Q, Phookamsak R (2018) Outline of Ascomycota: 2017. Fungal Diversity 88: 167–263. https://doi.org/10.1007/s13225-018-0394-8
  • Zhang LX, Alvarez LV, Bonthond G, Tian CM, Fan XL (2019) Cytospora elaeagnicola sp. nov. associated with narrow-leaved oleaster canker disease in China. Mycobiology 47: 319–328. https://doi.org/10.1080/12298093.2019.1633902
  • Zhang YB, You CJ, Fan XL, Tian CM (2014) Taxonomy and phylogeny of Cytospora in Northeast China. Mycosystema 33: 806–818.
  • Zhuang WY (2005) Fungi of Northwestern China, New York, USA. Ithaca, Mycotaxon, Ltd, 1–430.