Access to this full-text is provided by Pensoft Publishers.
Content available from Mycokeys
This content is subject to copyright. Terms and conditions apply.
New species and records of Diaporthe
from Jiangxi Province, China
Qin Yang1,2,3, Ning Jiang1, Cheng-Ming Tian1
1e Key Laboratory for Silviculture and Conservation of the Ministry of Education, Beijing Forestry Uni-
versity, Beijing 100083, China 2Forestry Biotechnology Hunan Key Laboratories, Central South University
of Forestry and Technology, Changsha 410004, China 3e Key Laboratory for Non-Wood Forest Cultivation
and Conservation of the Ministry of Education, Central South University of Forestry and Technology, Changsha
410004, China
Corresponding author: Cheng-Ming Tian (chengmt@bjfu.edu.cn)
Academic editor: A. Miller|Received 25 October 2020|Accepted 30 December 2020|Published 14 January 2021
Citation: Yang Q, Jiang N, Tian C-M (2021) New species and records of Diaporthe from Jiangxi Province, China.
MycoKeys 77: 41–64. https://doi.org/10.3897/mycokeys.77.59999
Abstract
Diaporthe species have often been reported as important plant pathogens, saprobes and endophytes on a
wide range of plant hosts. Although several Diaporthe species have been recorded, little is known about
species able to infect forest trees in Jiangxi Province. Hence, extensive surveys were recently conducted in
Jiangxi Province, China. A total of 24 isolates were identied and analysed using comparisons of DNA
sequence data for the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3
(his3), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions, as well as their
morphological features. Results revealed ve novel taxa, D. bauhiniae, D. ganzhouensis, D. schimae, D. ver-
niciicola, D. xunwuensis spp. nov. and three known species, D. apiculatum, D. citri and D. multigutullata.
Keywords
DNA phylogeny, ve new taxa, forest trees, systematics, taxonomy
Introduction
e genus Diaporthe Nitschke (Sordariomycetes, Diaporthales) represents a cosmopol-
itan group of fungi occupying diverse ecological behaviour as plant pathogens, endo-
phytes and saprobes (Muralli et al. 2006; Rossman et al. 2007; Udayanga et al.2014,
MycoKeys 77: 41–64 (2021)
doi: 10.3897/mycokeys.77.59999
https://mycokeys.pensoft.net
Copyright Qin Yang et al.. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
RESEARCH ARTICLE
A peer-reviewed open-access journal
Launched to accelerate biodiversity research
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
42
2015; Fan et al. 2015, 2018; Guarnaccia and Crous 2017; Guarnaccia et al. 2018;
Yang et al. 2018, 2020; Manawasinghe et al. 2019; Marin-Felix et al. 2019). Diaporthe
species are responsible for diseases on a wide range of plant hosts, including agricul-
tural crops, forest trees and ornamentals, some of which are economically important.
Several symptoms, such as root and fruit rots, dieback, stem cankers, leaf spots, leaf
and pod blights and seed decay are caused by Diaporthe spp. (Uecker 1988; Rehner
and Uecker 1994; Mostert et al. 2001; Santos et al. 2011; ompson et al. 2011;
Udayanga et al. 2011).
Diaporthe was historically considered as monophyletic, based on its typical sex-
ual morph and Phomopsis asexual morph (Gomes et al. 2013). However, Gao et al.
(2017) recently revealed its paraphyletic nature, showing that Mazzantia (Wehmeyer
1926), Ophiodiaporthe (Fu et al. 2013), Pustulomyces (Dai et al. 2014), Phaeocytos-
troma and Stenocarpella (Lamprecht et al. 2011) are embedded in Diaporthe s. lat.
Furthermore, Senanayake et al. (2017) recently included additional two genera in
Diaporthe s. lat., namely Paradiaporthe and Chiangraiomyces.
Species identication criteria in Diaporthe were originally based on host as-
sociation, morphology and culture characteristics (Mostert et al. 2001; Santos and
Phillips 2009; Udayanga et al. 2011), which led to the description of over 200
species (Hyde et al. 2020). Some species of Diaporthe were reported to colonise
a single host plant, while other species were found to be associated with dierent
host plants (Santos and Phillips 2009; Diogo et al. 2010; Santos et al. 2011; Gomes
et al. 2013). In addition, considerable variability of the phenotypic characters was
found to be present within a species (Rehner and Uecker 1994; Mostert et al. 2001;
Santos et al. 2010; Udayanga et al. 2011). During the past decade, a polyphasic
approach, based on multi-locus DNA data, morphology and ecology, has been
employed for species boundaries in the genus Diaporthe (Crous et al. 2012; Huang
et al. 2015; Gao et al. 2016, 2017; Guarnaccia and Crous 2017; Guarnaccia et al.
2018; Yang et al. 2018, 2020). e classication of Diaporthe has been progress-
ing and the basis for the species identication is a combination of morphologi-
cal, cultural, phytopathological and phylogenetical analyses (Gomes et al. 2013;
Udayanga et al. 2014, 2015; Fan et al. 2015; Huang et al. 2015; Gao et al. 2016,
2017; Guarnaccia and Crous 2017; Guarnaccia et al. 2018; Yang et al. 2018, 2020;
Manawasinghe et al. 2019).
In Jiangxi Province, China, some forest trees were observed to be infected with
fungal pathogens that cause dieback and leaf spots. Cankered branches and leaves
with typical Diaporthe fruiting bodies were also found in the area. However, we
found that only limited research had been undertaken regarding the fungal patho-
gens isolated from forest trees in Jiangxi Province. Hence, the present study was con-
ducted to identify Diaporthe species that cause dieback and leaf spots disease in the
forest trees in Jiangxi Province through morphological and multi-locus phylogenetic
analyses, based on modern taxonomic concepts.
Diaporthe species from cankered branches and leaves 43
Materials and methods
Isolates
Fresh specimens of Diaporthe were isolated from the collected branches and leaves of six
host plants during the collection trips conducted in Jiangxi Province (Table 1). A total of
24 isolates were established by removing a mucoid conidia mass from conidiomata, spread-
ing the suspension on the surface of 1.8% potato dextrose agar (PDA) and incubating at
25 °C for up to 24 h. A single germinating conidium was plated on to fresh PDA plates.
Specimens were deposited at the Museum of the Beijing Forestry University (BJFC). Ax-
enic cultures were maintained at the China Forestry Culture Collection Centre (CFCC).
Morphological observation
Agar plugs (6 mm diam.) were taken from the edge of actively-growing cultures on
PDA and transferred on to the centre of 9 cm diam. Petri dishes containing 2% tap
water agar, supplemented with sterile pine needles (PNA; Smith et al. 1996) and potato
dextrose agar (PDA) and incubated at 25 °C under a 12 h near-ultraviolet light/12 h
dark cycle to induce sporulation, as described in recent studies (Gomes et al. 2013;
Lombard et al. 2014). Colony characters and pigment production on PNA and PDA
were noted in the 10-day culture. Colony features were rated according to the colour
charts of Rayner (1970). Cultures were examined periodically for the development of
conidiomata. e microscopic examination was based on the morphological features of
conidiomata obtained from the fungal growth, mounted in clear lactic acid. At least 30
conidia were measured to calculate the mean size/length. Micro-morphological obser-
vations were done at 1000× magnication using a Leica compound microscope (DM
2500) with interference contrast (DIC) optics. Descriptions, nomenclature and illus-
trations of taxonomic novelties were deposited at MycoBank (www.MycoBank.org).
DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from colonies grown on cellophane-covered PDA, using
a CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle 1990). DNA
was estimated by electrophoresis in 1% agarose gel and the yield was measured using
the NanoDrop 2000 (ermo Scientic, Waltham, MA, USA), following the user
manual (Desjardins et al. 2009). e PCR amplications were performed in the DNA
Engine Peltier ermal Cycler (PTC-200; Bio-Rad Laboratories, Hercules, CA, USA).
e primer set ITS1/ITS4 (White et al. 1990) was used to amplify the ITS region. e
primer pair CAL228F/CAL737R (Carbone and Kohn 1999) was used to amplify the
calmodulin gene (cal) and the primer pair CYLH4F (Crous et al. 2004) and H3-1b
(Glass and Donaldson 1995) were used to amplify part of the histone H3 (his3) gene.
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
44
Table 1. Reference sequences included in molecular phylogenetic analyses of Diaporthe.
Species Isolate Host Location GenBank accession numbers
ITS cal his3 tef1 tub2
D. acericola MFLUCC 17-0956 Acer negundo Italy KY964224 KY964137 NA KY964180 KY964074
D. acerigena CFCC 52554 Acer tataricum China MH121489 MH121413 MH121449 MH121531 NA
D. acutispora CGMCC 3.18285 Coea sp. China KX986764 KX999274 NA KX999155 KX999195
D. alangii CFCC 52556 Alangium kurzii China MH121491 MH121415 MH121451 MH121533 MH121573
D. alnea CBS 146.46 Alnus sp. Netherlands KC343008 KC343250 KC343492 KC343734 KC343976
D. ampelina STEU2660 Vitis vinifera France AF230751 AY745026 NA AY745056 JX275452
D. amygdali CBS 126679 Prunus dulcis Portugal KC343022 KC343264 KC343506 AY343748 KC343990
D. angelicae CBS 111592 Heracleum
sphondylium
Austria KC343027 KC343269 KC343511 KC343753 KC343995
D. apiculatum CGMCC 3.17533 Camellia sinensis China KP267896 NA NA KP267970 KP293476
CFCC 53068 Rhus chinensis China MK432651 MK442973 MK442998 MK578127 MK578054
CFCC 53069 Rhus chinensis China MK432652 MK442974 MK442999 MK578128 MK578055
CFCC 53070 Rhus chinensis China MK432653 MK442975 MK443000 MK578129 MK578056
D. arctii CBS 139280 Arctium lappa Austria KJ590736 KJ612133 KJ659218 KJ590776 KJ610891
D. arecae CBS 161.64 Areca catechu India KC343032 KC343274 KC343516 KC343758 KC344000
D. arengae CBS 114979 Arenga enngleri Hong Kong KC343034 KC343276 KC343518 KC343760 KC344002
D. aseana MFLUCC 12-0299a Unknown dead
leaf
ailand KT459414 KT459464 NA KT459448 KT459432
D. bauhiniae CFCC 53071 Bauhinia
purpurea
China MK432648 MK442970 MK442995 MK578124 MK578051
CFCC 53072 China MK432649 MK442971 MK442996 MK578125 MK578052
CFCC 53073 China MK432650 MK442972 MK442997 MK578126 MK578053
D. beilharziae BRIP 54792 Indigofera
australis
Australia JX862529 NA NA JX862535 KF170921
D. betulicola CFCC 51128 Betula albo-
sinensis
China KX024653 KX024659 KX024661 KX024655 KX024657
D. biconispora CGMCC 3.17252 Citrus grandis China KJ490597 KJ490539 KJ490539 KJ490476 KJ490418
D. biguttulata CGMCC 3.17248 Citrus limon China KJ490582 NA KJ490524 KJ490461 KJ490403
CFCC 52584 Juglans regia China MH121519 MH121437 MH121477 MH121561 MH121598
D. bohemiae CPC 28222 Vitis vinifera Czech
Republic
MG281015 MG281710 MG281361 MG281536 MG281188
D. brasiliensis CBS 133183 Aspidosperma
tomentosum
Brazil KC343042 KC343284 KC343526 KC343768 KC344010
D. caatingaensis CBS 141542 Tacinga
inamoena
Brazil KY085927 NA NA KY115603 KY115600
D. caryae CFCC 52563 Carya illinoensis China MH121498 MH121422 MH121458 MH121540 MH121580
D. celeris CPC 28262 Vitis vinifera Czech
Republic
MG281017 MG281712 MG281363 MG281538 MG281190
D. celastrina CBS 139.27 Celastrus sp. USA KC343047 KC343289 KC343531 KC343773 KC344015
D. cercidis CFCC 52565 Cercis chinensis China MH121500 MH121424 MH121460 MH121542 MH121582
D. charlesworthii BRIP 54884m Rapistrum
rugostrum
Australia KJ197288 NA NA KJ197250 KJ197268
D. cinnamomi CFCC 52569 Cinnamomum
sp.
China MH121504 NA MH121464 MH121546 MH121586
D. citri AR 3405 Citrus sp. USA KC843311 KC843157 NA KC843071 KC843187
CFCC 53079 Citrus sinensis China MK573940 MK574579 MK574595 MK574615 MK574635
CFCC 53080 Citrus sinensis China MK573941 MK574580 MK574596 MK574616 MK574636
CFCC 53081 Citrus sinensis China MK573942 MK574581 MK574597 MK574617 MK574637
CFCC 53082 Citrus sinensis China MK573943 MK574582 MK574598 MK574618 MK574638
D. citriasiana CGMCC 3.15224 Citrus unshiu China JQ954645 KC357491 KJ490515 JQ954663 KC357459
D. citrichinensis CGMCC 3.15225 Citrus sp. China JQ954648 KC357494 NA JQ954666 NA
D. collariana MFLU 17-2770 Magnolia
champaca
ailand MG806115 MG783042 NA MG783040 MG783041
D. conica CFCC 52571 Alangium
chinense
China MH121506 MH121428 MH121466 MH121548 MH121588
D. cucurbitae CBS 136.25 Arctium sp. Unknown KC343031 KC343273 KC343515 KC343757 KC343999
D. cuppatea CBS 117499 Aspalathus
linearis
South Africa KC343057 KC343299 KC343541 KC343783 KC344025
D. discoidispora ZJUD89 Citrus unshiu China KJ490624 NA KJ490566 KJ490503 KJ490445
D. endophytica CBS 133811 Schinus
terebinthifolius
Brazil KC343065 KC343307 KC343549 KC343791 KC343065
D. eres AR5193 Ulmus sp. Germany KJ210529 KJ434999 KJ420850 KJ210550 KJ420799
Diaporthe species from cankered branches and leaves 45
Species Isolate Host Location GenBank accession numbers
ITS cal his3 tef1 tub2
D. fraxini-
angustifoliae
BRIP 54781 Fraxinus
angustifolia
Australia JX862528 NA NA JX862534 KF170920
D. fraxinicola CFCC 52582 Fraxinus
chinensis
China MH121517 MH121435 NA MH121559 NA
D. fructicola MAFF 246408 Passiora edulis
× P. edulis f.
avicarpa
Japan LC342734 LC342738 LC342737 LC342735 LC342736
D. fukushii MAFF 625034 Pyrus pyrifolia Japan JQ807469 NA NA JQ807418 NA
D. fusicola CGMCC 3.17087 Lithocarpus
glabra
China KF576281 KF576233 NA KF576256 KF576305
D. ganjae CBS 180.91 Cannabis sativa USA KC343112 KC343354 KC343596 KC343838 KC344080
D. ganzhouensis CFCC 53087 Unknown dead
wood
China MK432665 MK442985 MK443010 MK578139 MK578065
CFCC 53088 Unknown dead
wood
China MK432666 MK442986 MK443011 MK578140 MK578066
D. garethjonesii MFLUCC 12-0542a Unknown dead
leaf
ailand KT459423 KT459470 NA KT459457 KT459441
D. guangxiensis JZB320094 Vitis vinifera China MK335772 MK736727 NA MK523566 MK500168
D. gulyae BRIP 54025 Helianthus
annuus
Australia JF431299 NA NA KJ197271 JN645803
D. helicis AR5211 Hedera helix France KJ210538 KJ435043 KJ420875 KJ210559 KJ420828
D. heterophyllae CBS 143769 Acacia
heterohpylla
France MG600222 MG600218 MG600220 MG600224 MG600226
D. hispaniae CPC 30321 Vitis vinifera Spain MG281123 MG281820 MG281471 MG281644 MG281296
D. hubeiensis JZB320123 Vitis vinifera China MK335809 MK500235 NA MK523570 MK500148
D. incompleta CGMCC 3.18288 Camellia sinensis China KX986794 KX999289 KX999265 KX999186 KX999226
D. infecunda CBS 133812 Schinus
terebinthifolius
Brazil KC343126 KC343368 KC343610 KC343852 KC344094
D. juglandicola CFCC 51134 Juglans
mandshurica
China KU985101 KX024616 KX024622 KX024628 KX024634
D. kadsurae CFCC 52586 Kadsura
longipedunculata
China MH121521 MH121439 MH121479 MH121563 MH121600
D. kochmanii BRIP 54033 Helianthus
annuus
Australia JF431295 NA NA JN645809 NA
D. kongii BRIP 54031 Portulaca
grandiora
Australia JF431301 NA NA JN645797 KJ197272
D. litchicola BRIP 54900 Litchi chinensis Australia JX862533 NA NA JX862539 KF170925
D. lithocarpus CGMCC 3.15175 Lithocarpus
glabra
China KC153104 KF576235 NA KC153095 KF576311
D. lonicerae MFLUCC 17-0963 Lonicera sp. Italy KY964190 KY964116 NA KY964146 KY964073
D. lusitanicae CBS 123212 Foeniculum
vulgare
Portugal KC343136 KC343378 KC343620 KC343862 KC344104
D. masirevicii BRIP 57892a Helianthus
annuus
Australia KJ197277 NA NA KJ197239 KJ197257
D. middletonii BRIP 54884e Rapistrum
rugostrum
Australia KJ197286 NA NA KJ197248 KJ197266
D. miriciae BRIP 54736j Helianthus
annuus
Australia KJ197282 NA NA KJ197244 KJ197262
D. momicola MFLUCC 16-0113 Prunus persica China KU557563 KU557611 NA KU557631 KU55758
D. multigutullata ZJUD98 Citrus grandis China KJ490633 NA KJ490575 KJ490512 KJ490454
D. multigutullata CFCC 53095 Citrus maxima China MK432645 MK442967 MK442992 MK578121 MK578048
CFCC 53096 Citrus maxima China MK432646 MK442968 MK442993 MK578122 MK578049
CFCC 53097 Citrus maxima China MK432647 MK442969 MK442994 MK578123 MK578050
D. musigena CBS 129519 Musa sp. Australia KC343143 KC343385 KC343627 KC343869 KC344111
D. neilliae CBS 144.27 Spiraea sp. USA KC343144 KC343386 KC343628 KC343870 KC344112
D. neoarctii CBS 109490 Ambrosia trida USA KC343145 KC343387 KC343629 KC343871 KC344113
D. oraccinii CGMCC 3.17531 Camellia sinensis China KP267863 NA KP293517 KP267937 KP293443
D. ovoicicola CGMCC 3.17093 Citrus sp. China KF576265 KF576223 NA KF576240 KF576289
D. pandanicola MFLU 18-0006 Pandanus sp. ailand MG646974 NA NA NA MG646930
D. pascoei BRIP 54847 Persea americana Australia JX862532 NA NA JX862538 KF170924
D. passioricola CBS 141329 Passiora foetida Malaysia KX228292 NA KX228367 NA KX228387
D. penetriteum CGMCC 3.17532 Camellia sinensis China KP714505 NA KP714493 KP714517 KP714529
D. perjuncta CBS 109745 Ulmus glabra Austria KC343172 KC343414 KC343656 KC343898 KC344140
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
46
Species Isolate Host Location GenBank accession numbers
ITS cal his3 tef1 tub2
D. perseae CBS 151.73 Persea gratissima Netherlands KC343173 KC343415 KC343657 KC343899 KC344141
D. pescicola MFLUCC 16-0105 Prunus persica China KU557555 KU557603 NA KU557623 KU557579
D. podocarpi-
macrophylli
CGMCC 3.18281 Podocarpus
macrophyllus
China KX986774 KX999278 KX999246 KX999167 KX999207
D. pseudomangiferae CBS 101339 Mangifera indica Dominican
Republic
KC343181 KC343423 KC343665 KC343907 KC344149
D. pseudophoe-
nicicola
CBS 462.69 Phoenix
dactylifera
Spain KC343184 KC343426 KC343668 KC343910 KC344152
D. psoraleae-pinnatae CBS 136413 Psoralea pinnata South Africa KF777159 NA NA NA KF777252
D. pterocarpicola MFLUCC 10-0580a Pterocarpus
indicus
ailand JQ619887 JX197433 NA JX275403 JX275441
D. pulla CBS 338.89 Hedera helix Yugoslavia KC343152 KC343394 KC343636 KC343878 KC344120
D. pyracanthae CAA483 Pyracantha
coccinea
Portugal KY435635 KY435656 KY435645 KY435625 KY435666
D. racemosae CBS 143770 Euclea racemosa South Africa MG600223 MG600219 MG600221 MG600225 MG600227
D. rostrata CFCC 50062 Juglans
mandshurica
China KP208847 KP208849 KP208851 KP208853 KP208855
D. sackstonii BRIP 54669b Helianthus
annuus
Australia KJ197287 NA NA KJ197249 KJ197267
D. sambucusii CFCC 51986 Sambucus
williamsii
China KY852495 KY852499 KY852503 KY852507 KY852511
D. schimae CFCC 53103 Schima superba China MK432640 MK442962 MK442987 MK578116 MK578043
CFCC 53104 Schima superba China MK432641 MK442963 MK442988 MK578117 MK578044
CFCC 53105 Schima superba China MK432642 MK442964 MK442989 MK578118 MK578045
D. schini CBS 133181 Schinus
terebinthifolius
Brazil KC343191 KC343433 KC343675 KC343917 KC344159
D. schisandrae CFCC 51988 Schisandra
chinensis
China KY852497 KY852501 KY852505 KY852509 KY852513
D. schoeni MFLU 15-1279 Schoenus
nigricans
Italy KY964226 KY964139 NA KY964182 KY964109
D. sennae CFCC 51636 Senna
bicapsularis
China KY203724 KY228875 NA KY228885 KY228891
D. seraniae BRIP 55665a Helianthus
annuus
Australia KJ197274 NA NA KJ197236 KJ197254
D. siamensis MFLUCC 10-573a Dasymaschalon
sp.
ailand JQ619879 NA NA JX275393 JX275429
D. sojae FAU635 Glycine max USA KJ590719 KJ612116 KJ659208 KJ590762 KJ610875
D. sterilis CBS 136969 Vaccinium
corymbosum
Italy KJ160579 KJ160548 MF418350 KJ160611 KJ160528
D. subclavata ICMP20663 Citrus unshiu China KJ490587 NA KJ490529 KJ490466 KJ490408
D. subellipicola MFLU 17-1197 Dead wood China MG746632 NA NA MG746633 MG746634
D. subordinaria CBS 464.90 Plantago
lanceolata
New
Zealand
KC343214 KC343456 KC343698 KC343940 KC344182
D. taoicola MFLUCC 16-0117 Prunus persica China KU557567 NA NA KU557635 KU557591
D. tectonae MFLUCC 12-0777 Tectona grandis China KU712430 KU749345 NA KU749359 KU743977
D. tectonendophytica MFLUCC 13-0471 Tectona grandis China KU712439 KU749354 NA KU749367 KU749354
D. tectonigena MFLUCC 12-0767 Tectona grandis China KU712429 KU749358 NA KU749371 KU743976
D. terebinthifolii CBS 133180 Schinus
terebinthifolius
Brazil KC343216 KC343458 KC343700 KC343942 KC344184
D. ternstroemia CGMCC 3.15183 Ternstroemia
gymnanthera
China KC153098 NA NA KC153089 NA
D. thunbergii MFLUCC 10-576a unbergia
laurifolia
ailand JQ619893 JX197440 NA JX275409 JX275449
D. tibetensis CFCC 51999 Juglandis regia China MF279843 MF279888 MF279828 MF279858 MF279873
D. tulliensis BRIP 62248a eobroma cacao Australia KR936130 NA NA KR936133 KR936132
D. ukurunduensis CFCC 52592 Acer
ukurunduense
China MH121527 MH121445 MH121485 MH121569 NA
D. unshiuensis CGMCC 3.17569 Citrus unshiu China KJ490587 NA KJ490529 KJ490408 KJ490466
CFCC 52594 Carya illinoensis China MH121529 MH121447 MH121487 MH121571 MH121606
D. undulata CGMCC 3.18293 Leaf of
unknown host
China-Laos
border
KX986798 NA KX999269 KX999190 KX999230
D. vawdreyi BRIP 57887a Psidium guajava Australia KR936126 NA NA KR936129 KR936128
Diaporthe species from cankered branches and leaves 47
Species Isolate Host Location GenBank accession numbers
ITS cal his3 tef1 tub2
D. verniciicola CFCC 53109 Vernicia
montana
China MK573944 MK574583 MK574599 MK574619 MK574639
CFCC 53110 Vernicia
montana
China MK573945 MK574584 MK574600 MK574620 MK574640
CFCC 53111 Vernicia
montana
China MK573946 MK574585 MK574601 MK574621 MK574641
CFCC 53112 Vernicia
montana
China MK573947 MK574586 MK574602 MK574622 MK574642
D. viniferae JZB320071 Vitis vinifera China MK341551 MK500107 MK500119 MK500112
D. virgiliae CMW40748 Virgilia oroboides South Africa KP247566 NA NA NA KP247575
D. xishuangbanica CGMCC 3.18282 Camellia sinensis China KX986783 NA KX999255 KX999175 KX999216
D. xunwuensis CFCC 53085 Unknown dead
wood
China MK432663 MK442983 MK443008 MK578137 MK578063
CFCC 53086 Unknown dead
wood
China MK432664 MK442984 MK443009 MK578138 MK578064
D. yunnanensis CGMCC 3.18289 Coea sp. China KX986796 KX999290 KX999267 KX999188 KX999228
Diaporthella corylina CBS 121124 Corylus sp. China KC343004 KC343246 KC343488 KC343730 KC343972
Newly sequenced material is indicated in bold type. NA, not applicable.
e primer pair EF1-728F/EF1-986R (Carbone and Kohn 1999) was used to am-
plify a partial fragment of the translation elongation factor 1-α gene (tef1). e primer
sets T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) were
used to amplify the beta-tubulin gene (tub2); the additional combination of Bt2a/
Bt2b (Glass and Donaldson 1995) was used in case of amplication failure of the T1/
Bt2b primer pair. e PCR amplications of the genomic DNA with the phylogenetic
markers were done using the same primer pairs and conditions as in Yang et al. (2018).
e PCR products were assayed via electrophoresis in 2% agarose gels, while the DNA
sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a Big-
Dye Terminater Kit v.3.1 (Inv-itrogen, USA) at the Shanghai Invitrogen Biological
Technology Company Limited (Beijing, China).
Phylogenetic analyses
e quality of the amplied nucleotide sequences was checked and combined using
SeqMan v.7.1.0 and reference sequences were retrieved from the National Center for
Biotechnology Information (NCBI), based on recent publications on the genus Dia-
porthe (Guarnaccia et al. 2018; Yang et al. 2018, 2020). Sequences were aligned using
MAFFT v. 6 (Katoh and Toh 2010) and corrected manually using Bioedit 7.0.9.0 (Hall
1999). e best-t nucleotide substitution models for each gene were selected using
jModelTest v. 2.1.7 (Darriba et al. 2012) under the Akaike Information Criterion.
e phylogenetic analyses of the combined gene regions were performed using
Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML was conducted
using PhyML v. 3.0 (Guindon et al. 2010), with 1000 bootstrap replicates while BI
was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes
v. 3.0 (Ronquist et al. 2003). Two MCMC chains, started from random trees for
1,000,000 generations and trees, were sampled every 100th generation, resulting in a
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
48
total of 10,000 trees. e rst 25% of trees were discarded as burn-in of each analysis.
Branches with signicant Bayesian Posterior Probabilities (BPP) were estimated in the
remaining 7500 trees. Phylogenetic trees were viewed with FigTree v.1.3.1 (Rambaut
and Drummond 2010) and processed by Adobe Illustrator CS5. Sequence alignment
and phylogenetic trees were deposited in TreeBASE (submission ID: S25213). e
nucleotide sequence data of the new taxa were deposited in GenBank (Table 1).
Results
e phylogenetic position of the 24 isolates of Diaporthe was determined by the phylo-
genetic analysis of the combined ITS, cal, his3, tef1 and tub2 sequences data. Reference
sequences of the representative species used in the analysis were selected from Yang
et al. (2018) and supplemented with sequences from GenBank. e ITS, cal, his3,
tef1 tub2 and combined data matrices contained 522, 541, 529, 520, 535 and 2 659
characters with gaps, respectively. e alignment comprised of 142 strains together
with Diaporthella corylina (culture CBS 121124) which was selected as the outgroup.
e best nucleotide substitution model used for the analysis of ITS, his3 and tub2 was
TrN+I+G, while HKY+I+G was used for cal and tef1. e topologies resulting from
ML and BI analyses of the concatenated dataset were congruent (Fig. 1) and the se-
quences from the 24 Diaporthe isolates formed eight distinct clades as shown in Fig. 1,
representing ve undescribed species and three known species.
Taxonomy
Diaporthe apiculatum Y.H. Gao & L. Cai, in Gao, Liu & Cai, Syst. Biodiv. 14:
106. 2016.
Figure 2
Description. Conidiomata pycnidial, discoid, immersed in bark, scattered, slightly
erumpent through bark surface, with a solitary undivided locule. Ectostromatic disc
yellowish to grey, one ostiole per disc, (300–)305–357(–368) µm diam. Ostiole medi-
um black, up to level of disc. Locule undivided, (338–)357–450(–464) µm diam. Co-
nidiophores reduced to conidiogenous cells. Conidiogenous cells cylindrical, hyaline,
densely aggregated, phiailidic, unbranched, straight or slightly curved. Beta conidia
hyaline, aseptate, liform, hamate, eguttulate, base subtruncate, tapering towards one
apex, (26.5–)30–39.5(–43) × 1.5–2 µm. Alpha conidia not observed.
Culture characters. Colony originally at with white uy aerial mycelium, be-
coming yellowish to pale green mycelium with age, marginal area irregular, conidi-
omata absent.
Specimens examined. C. Jiangxi Province: Ganzhou City, Fengshan Forest
Park, on branches of Rhus chinensis, 25°45'12"N, 115°00'41"E, 23 Jul 2018, Q. Yang,
Y. Liu, Y.M. Liang & C.M. Tian (BJFC-S1680; living culture: CFCC 53068, CFCC
53069 and CFCC 53070).
Diaporthe species from cankered branches and leaves 49
Figure 1. Phylogram of Diaporthe from a Maximum Likelihood analysis based on combined ITS, cal,
his3, tef1 and tub2. Values above the branches indicate Maximum Likelihood bootstrap (left, ML BP
≥ 50%) and Bayesian probabilities (right, BI PP ≥ 0.90). e tree is rooted with Diaporthella corylina.
Strains in current study are in blue font and the ex-type cultures are in bold font.
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
50
Figure 1. Continued.
Figure 2. Diaporthe apiculatum on Rhus chinensis (BJFC-S1680) a, b habit of conidiomata in wood
ctransverse section of conidiomata d longitudinal section through conidiomata e conidiogenous cells
attached with beta conidia f the colony on PDA. Scale bars: 200 µm (b–d); 10 µm (e).
Diaporthe species from cankered branches and leaves 51
Notes. Diaporthe apiculatum was originally described as an endophyte from healthy
leaves of Camellia sinensis in Jiangxi Province, China (Gao et al. 2015). In the present
study, three isolates (CFCC 53068, CFCC 53069 and CFCC 53070) from sympto-
matic branches of Rhus chinensis were found congruent with D. apiculatum, based on
DNA sequence and morphological data (Fig. 1). e clade was, therefore, conrmed
to be D. apiculatum and was found to be both an endophyte and a pathogen.
Diaporthe bauhiniae C.M. Tian & Q. Yang, sp. nov.
MycoBank No: 829519
Figure 3
Diagnosis. Distinguished from the phylogenetically closely-related species D. psorale-
ae-pinnatae in alpha and beta conidia.
Etymology. Named after Bauhinia, the host genus where the fungus was isolated.
Description. Conidiomata pycnidial, immersed in bark, scattered, slightly
erumpent through bark surface, nearly at, discoid, with a solitary undivided locule.
Ectostromatic disc grey to brown, one ostiole per disc. Locule circular, undivided,
(180–)200–290(–300) µm diam. Conidiophores reduced to conidiogenous cells. Co-
nidiogenous cells hyaline, cylindrical, unbranched, straight, tapering towards the apex.
Alpha conidia hyaline, aseptate, ellipsoidal to fusiform, biguttulate to multi-guttu-
late, (7.5–)9–13(–14) × (1.5–)2–2.5(–3) µm. Beta conidia hyaline, aseptate, liform,
straight to sinuous, eguttulate, (25–)28.5–40(–43) × 1 µm.
Culture characters. Colony at rst white, becoming wine-red in the centre with
age. Aerial mycelium white, dense, uy, conidiomata absent.
Specimens examined. C. Jiangxi Province: Ganzhou City, on branches of
Bauhinia purpurea, 25°52'21"N, 114°56'44"E, 11 May 2018, Q. Yang, Y. Liu & Y.M.
Liang (holotype BJFC-S1621; ex-type living culture: CFCC 53071; living culture:
CFCC 53072 and CFCC 53073).
Notes. ree isolates representing D. bauhiniae cluster in a well-supported clade
and appear most closely related to D. psoraleae-pinnatae. Diaporthe bauhiniae can be
distinguished from D. psoraleae-pinnatae, based on ITS and tub2 (38/458 in ITS and
11/418 in tub2). Morphologically, D. bauhiniae diers from D. psoraleae-pinnatae in
having narrower alpha conidia (2–2.5 vs. 2.5–3 µm) and the beta conidia of D. psorale-
ae-pinnatae were not observed (Crous et al. 2013).
Diaporthe citri (H.S. Fawc.) F.A. Wolf, J. Agric. Res., Washington 33(7): 625, 1926.
Figure 4
Description. Leaf spots subcircular to irregular, pale brown, with dark brown at mar-
gin. Pycnidia solitary, scattered on the leaf surface. Pycnidial conidiomata in culture,
globose, erumpent, single or clustered in groups of 3–5 pycnidia, coated with hyphae,
cream to yellowish translucent conidial droplets exuded from ostioles. Conidiophores
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
52
Figure 3. Diaporthe bauhiniae on Bauhinia purpurea (BJFC-S1621) a habit of conidiomata in wood
btransverse section of conidiomata c longitudinal section through conidiomata d the colony on PDA
econidiogenous cells attached with alpha conidia f Alpha conidia g Beta conidia. Scale bars: 100 µm (b, c );
10 µm (e–h).
Figure 4. Diaporthe citri on Citrus sinensis (BJFC-S1658) a, b symptoms on leaves of host plant c culture
on PDA (30d) d conidiomata e alpha conidia f conidiophores and alpha conidia. Scale bars: 10 µm (e, f).
reduced to conidiogenous cells. Conidiogenous cells hyaline, unbranched, septate,
straight, slightly tapering towards the apex, 14.5–25 × 2–3 µm. Alpha conidia hyaline,
aseptate, rounded at one end, apex at the other end, usually with two large guttulate,
(9.5–)10.5–12 × 3.5–4.5 µm. Beta conidia not observed.
Culture characters. Colony originally at with white uy aerial mycelium, becoming
greyish mycelium with age, with yellowish-cream conidial drops exuding from the ostioles.
Specimens examined. C. Jiangxi Province: Ganzhou City, on leaves of
Citrus sinensis, 24°59'44"N, 115°31'01"E, 13 May 2018, Q. Yang, Y. Liu & Y.M.
Diaporthe species from cankered branches and leaves 53
Liang (BJFC-S1658; living culture: CFCC 53079 and CFCC 53080); 24°59'45"N,
115°31'02"E, 13 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1659; living
culture: CFCC 53081 and CFCC 53082).
Notes. Diaporthe citri is a widely distributed species in citrus-growing regions.
In the present study, four isolates (CFCC 53079, CFCC 53080, CFCC 53081 and
CFCC 53082) from symptomatic leaves of Citrus sinensis were congruent with D. citri,
based on DNA sequence and morphological data (Fig. 1). e clade was, therefore,
conrmed to be D. citri.
Diaporthe ganzhouensis C.M. Tian & Q. Yang, sp. nov.
MycoBank No: 829522
Figure 5
Diagnosis. Distinguished from the phylogenetically closely-related species D. vaw-
dreyi in having longer conidiophores and wider alpha conidia.
Etymology. Named after Ganzhou City where the species was rst collected.
Description. On PDA: Conidiomata pycnidial, subglobose, solitary, deeply em-
bedded in the medium, erumpent, dark brown to black. Pale yellow conidial drops
exuding from ostioles. Conidiophores (12–)15.5–21 × 1.5–2 µm, cylindrical, hyaline,
phiailidic, branched, straight or slightly curved. Alpha conidia 6.5–8.5(–9) × 2–2.5(–3)
µm, aseptate, hyaline, ellipsoidal to fusiform, rounded at one end, slightly apex at the
other end, biguttulate. Beta conidia hyaline, aseptate, liform, sinuous at one end, egut-
tulate, (21.5–)25.5–31(–33) × 1 µm.
Culture characters. Colony at rst white, becoming yellowish with age. Aerial
mycelium white, dense, uy, with visible solitary conidiomata at maturity.
Specimens examined. C. Jiangxi Province: Ganzhou City, unknown dead
wood, 25°45'17"N, 115°00'41"E, 23 Jul 2018, Q. Yang, Y. Liu, Y.M. Liang & C.M. Tian
(holotype BJFC-C004; ex-type culture: CFCC 53087; living culture: CFCC 53088).
Notes. Diaporthe ganzhouensis comprises the isolates CFCC 53087 and CFCC
53088, revealed to be closely related to D. vawdreyi in the combined phylogenetic tree
(Fig. 1). Diaporthe ganzhouensis can be distinguished, based on ITS, tef1-α and tub2 loci
from D. vawdreyi (6/456 in ITS, 63/357 in tef1-α and 40/469 in tub2). Diaporthe gan-
zhouensis diers morphologically from D. vawdreyi in having longer conidiopores (15.5–
21 vs. 6–15 µm) and wider alpha conidia (2–2.5 vs. 1.5–2 µm) (Crous et al. 2015).
Diaporthe multiguttulata F. Huang, K.D. Hyde & Hong Y. Li, in Huang et al.,
Fungal Biology 119(5): 343. 2015.
Figure 6
Description. Conidiomata pycnidial, 692–750(–800) µm diam., solitary and with single
necks erumpent through host bark. Tissue around neck is cylindrical. Locule circular, un-
divided, 450–565(–600) µm diam. Conidiophores reduced to conidiogenous cells. Con-
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
54
Figure 5. Diaporthe ganzhouensis on unknown host (BJFC-S1678) a the colony on PDA and conidi-
omata b alpha and beta conidia c conidiogenous cells and alpha conidia. Scale bars: 10 µm (b, c).
Figure 6. Diaporthe multiguttulata on Citrus maxima (BJFC-S1614) a, b habit of conidiomata on twig
c conidiomata on PDA d transverse section through conidiomata e longitudinal section through conidi-
omata f conidiogenous cells attached with alpha conidia g alpha conidia h the colony on PDA. Scale bars:
200 µm (b, d, e ); 10 µm (f, g).
idiogenous cells unbranched, straight or slightly curved, apical or base sometimes swelling,
(8.5–)9–10.5(–11) × 1.5–2 µm. Alpha conidia hyaline, aseptate, ellipsoidal, biguttulate or
with one large guttulate, rounded at one end, slightly apex at the other end, occasionally
submedian constriction, (7.5–)8–9(–10.5) × 4–5(–5.5) µm. Beta conidia not observed.
Diaporthe species from cankered branches and leaves 55
Culture characters. Colony originally at with white felty aerial mycelium, be-
coming pale green mycelium with age, margin area irregularly, with visible solitary
conidiomata at maturity.
Specimens examined. C. Jiangxi Province: Ganzhou City, on branches of
Citrus maxima, 25°51'28"N, 114°55'19"E, 11 May 2018, Q. Yang, Y. Liu & Y.M.
Liang (BJFC-S1614; living culture: CFCC 53095, CFCC 53096 and CFCC 53097).
Notes. Diaporthe multiguttulata was originally described as an endophyte from a
healthy branch of Citrus grandis in Fujian Province, China (Huang et al. 2015). In the
present study, three isolates (CFCC 53095, CFCC 53096 and CFCC 53097) from
symptomatic branches of Citrus maxima were congruent with D. multigutullata, based
on DNA sequence data and conrmed from the morphological analysis (Fig. 1). e
clade, therefore, was veried as D. multigutullata which could exist both as an endo-
phyte and a pathogen.
Diaporthe schimae C.M. Tian & Q. Yang, sp. nov.
MycoBank No: 829526
Figure 7
Diagnosis. Distinguished from the phylogenetically closely-related species D. sennae
in having larger alpha conidia and longer beta conidia.
Etymology. Named after the host genus Schima on which the fungus was isolated.
Description. Leaf spots subcircular to irregular, pale brown, with dark brown
at margin. Pycnidia solitary, scattered on the leaf surface. Pycnidial conidiomata in
culture, globose, (150–)173–357(–373) µm in its widest diam., erumpent, single or
clustered in groups of 3–5 pycnidia, coated with hyphae, cream to yellowish translu-
cent conidial droplets exuded from ostioles. Conidiophores reduced to conidiogenous
cells. Conidiogenous cells hyaline, unbranched, septate, straight, slightly tapering to-
wards the apex. Alpha conidia scarce, hyaline, aseptate, ellipsoidal to spindle-shaped,
four small guttulate, (7.5–)8–8.5(–9) × 2.5–3 µm. Beta conidia abundant, hyaline,
aseptate, liform, straight to sinuous at one end, eguttulate, (25–)27.5–38.5(–40.5) ×
1–1.5 µm.
Culture characters. Colony entirely white, with uy aerial mycelium, concentric
zonation, margin mbricate, reverse slightly yellowish.
Specimens examined. C. Jiangxi Province: Ganzhou City, Fengshan For-
est Park, on leaves of Schima superba, 25°44'22"N, 114°59'40"E, 15 May 2018, Q.
Yang, Y. Liu & Y.M. Liang (holotype BJFC-S1661; ex-type culture: CFCC 53103);
24°40'51"N, 115°34'36"E, 15 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1662;
living culture: CFCC 53104); 24°40'52"N, 115°34'54"E, 15 May 2018, Q. Yang, Y.
Liu & Y.M. Liang (BJFC-S1663; living culture: CFCC 53105).
Notes. Diaporthe schimae occurs in an independent clade (Fig. 1) and was revealed
to be phylogenetically distinct from D. sennae. Diaporhe schimae can be distinguished
with D. sennae by 41 nucleotides in concatenated alignment, in which three were
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
56
Figure 7. Diaporthe schimae on Schima superba (BJFC-S1661) a symptoms on leaves of host plant b the
colony on PDA c conidiomata on PDA d conidiophores cells attached with beta conidia e Alpha conidia.
Scale bars: 10 µm (d, e).
distinct in the ITS region, 20 in the tef1-α region and 18 in the tub2 region. Dia-
porthe schimae diers morphologically from D. sennae in having larger alpha conidia
and longer beta conidia (8–8.5 × 2.5–3 vs. 5.5–6.3 × 1.5–1.7 µm in alpha conidia;
27.5–38.5 vs. 18.4–20 µm in beta conidia) (Yang et al. 2017a).
Diaporthe verniciicola C.M. Tian & Q. Yang, sp. nov.
MycoBank No: 832921
Figure 8
Diagnosis. Distinguished from the phylogenetically closely-related species D. rostrata in
having smaller alpha conidia; and from D. juglandicola in having wider alpha conidia.
Etymology. Named after the host genus Vernicia on which the fungus was isolated.
Description. Conidiomata pycnidial, 825–1050 × 445–500 µm diam., solitary
and with single necks erumpent through host bark. Tissue around neck is conical. Loc-
ule circular, undivided, 400–665 µm diam. Conidiophores reduced to conidiogenous
cells. Conidiogenous cells unbranched, straight or sinuous, 14.5–21.5 × 1–1.5 µm.
Alpha conidia hyaline, aseptate, ellipsoidal to fusiform, with 1–2-guttulate, 7–8.5 ×
3–3.5 µm. Beta conidia not observed.
Culture characters. Colony white to yellowish, with dense and felted mycelium in
the centre, lacking aerial mycelium, conidiomata absent.
Diaporthe species from cankered branches and leaves 57
Specimens examined. C. Jiangxi Province: Ganzhou City, on branches of Ve r -
nicia montana, 24°40'51"N, 115°34'52"E, 12 May 2018, Q. Yang, Y. Liu & Y.M. Liang
(holotype BJFC-S1622; ex-type culture: CFCC 53109); 24°40'52"N, 115°34'50"E, 12
May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1623; living culture: CFCC 53110);
24°45'14"N, 115°34'00"E, 12 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1624;
living culture: CFCC 53111); 25°44'15"N, 114°59'32"E, 15 May 2018, Q. Yang, Y.
Liu & Y.M. Liang (BJFC-S1624; living culture: CFCC 53112).
Notes. Two isolates of D. verniciicola clustered in a well-supported clade (ML/
BI=100/1) and appeared closely related to D. rostrata and D. juglandicola (Fig. 1).
Morphologically, D. verniciicola is similar to D. rostrata characterised by conidiomata
with single necks erumpent through the host bark. However, the new taxon can be
distinguished from D. rostrata in having smaller alpha conidia (7–8.5 × 3–3.5 vs. 8.5–
11.5 × 4–5 µm) (Fan et al. 2015) and D. verniciicola diers from D. juglandicola in
having wider alpha conidia (3–3.5 vs. 2.5–3 µm) (Yang et al. 2017b). is is the rst
discovery of a Diaporthe species isolated from infected branches or twigs on Vernicia
montana and was conrmed as a new species, based on phylogeny and morphology.
Diaporthe xunwuensis C.M. Tian & Q. Yang, sp. nov.
MycoBank No: 829521
Figure 9
Diagnosis. Distinguished from the phylogenetically closely-related species D. oraccinii
in having longer conidiophores and larger alpha conidia.
Figure 8. Diaporthe verniciicola on Vernicia montana (BJFC-S1622) a, b habit of conidiomata on twig
c transverse section through conidiomata d longitudinal section through conidiomata e alpha conidia
fconidiophores g culture on PDA (30d). Scale bars: 500 µm (b); 200 µm (c); 10 µm (e, f).
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
58
Etymology. Named after the county (Xunwu) where the species was rst collected.
Description. On PDA: Conidiomata pycnidial, globose, solitary or aggregated,
deeply embedded in the medium, erumpent, dark brown to black. Hyaline conidial
drops exuding from ostioles. Conidiophores (18.5–)21.5–30(–32.5) × 1–1.5(–2)µm, cy-
lindrical, hyaline, phiailidic, unbranched, straight to sinuous. Alpha conidia (6.5–)7–8.5
× 2–3 µm, aseptate, hyaline, ellipsoidal to fusiform, rounded at one end, slightly apex at
the other end, usually with 2-guttulate. Beta conidia not observed.
Culture characters. Colony at rst white, becoming dark brown in the centre with age.
Aerial mycelium white, dense, uy, with black conidial drops exuding from the ostioles.
Specimens examined. C. Jiangxi Province: Ganzhou City, unknown dead
wood, 25°45'17"N, 115°00'41"E, 23 Jul 2018, Q. Yang, Y. Liu, Y.M. Liang & C.M. Tian
(holotype BJFC-C003; ex-type culture: CFCC 53085; living culture: CFCC 53086).
Notes. Two isolates representing D. xunwuensis clustered in a well-supported clade
and appear most closely related to D. oraccinii. Diaporthe xunwuensis can be distin-
guished from D. oraccinii, based on ITS, his3 and tef1-α loci (5/471 in ITS, 5/432 in
his3 and 5/325 in tef1-α). Morphologically, D. xunwuensis diers from D. oraccinii
in having longer conidiopores (21.5–30 vs. 10.5–22.5 µm) and larger alpha conidia
(7–8.5 × 2–3 vs. 5.5–7.5 × 0.5–2 µm) (Gao et al. 2016).
Discussion
e current study described eight Diaporthe species from 24 strains, based on a large set
of freshly-collected specimens. It includes ve new species and three known species, which
were sampled from six host genera distributed in Jiangxi Province of China (Table 1). In
this study, 142 reference sequences (including outgroup) were selected, based on BLAST
searches of NCBIs GenBank nucleotide database and included in the phylogenetic analyses
(Table 1). Phylogenetic analyses, based on ve combined loci (ITS, cal, his3, tef1 and tub2),
as well as morphological characters, revealed the diversity of Diaporthe species in Jiangxi
Province, mainly focusing on diebacks from major ecological or economic forest trees.
Figure 9. Diaporthe xunwuensis on unknown host (BJFC-S1679) a the colony on PDA and conidiomata
b alpha conidia c conidiogenous cells attached with alpha conidia. Scale bars: 10 µm (a–c).
Diaporthe species from cankered branches and leaves 59
e identication and characterisation of novel taxa and new host records indicate
the high potential of Diaporthe to evolve rapidly. In the present study, ve species were
rst reported in China as pathogens. Amongst these species, D. bauhiniae was char-
acterised by having longer alpha conidia (9–13 × 2–2.5 µm). Diaporthe ganzhouensis
and D. xunwuensis were isolated from unknown dead wood, but D. ganzhouensis can
be distinguish from D. xunwuenesis in having beta conidia and was supported by
analysis of the sequence data. Diaporthe schimae was identied as the most widespread
species from isolates collected in Jiangxi Province. Diaporthe verniciicola have conidi-
omata with single necks erumpent through the host bark. Furthermore, two new host
records were described, D. apiculatum from Rhus chinensis and D. multiguttulata from
Citrus maxima.
Recent plant pathological studies have revealed that several Diaporthe species cause
disease, particularly to important plant hosts on a wide range of economically-signi-
cant agricultural crops, such as blueberries, citrus, grapes, oaks, sunowers, soybeans,
tea plants, tropical fruits, vegetables and various trees (van Rensburg et al. 2006; Santos
and Phillips 2009; Santos et al. 2011; ompson et al. 2011; Grasso et al. 2012; Lom-
bard et al. 2014; Huang et al. 2015; Udayanga et al. 2015; Gao et al. 2016; Guarnaccia
et al. 2018; Yang et al. 2020). For example, research conducted by Huang et al. (2015)
revealed seven endophytic Diaporthe species on Citrus; Gao et al. (2016) demonstrated
that Diaporthe isolates associated with Camellia spp. could be assigned to seven species
and two species complexes; Guarnaccia et al. (2018) explored the occurrence, diversity
and pathogenicity of Diaporthe species associated with Vitis vinifera and revealed four
new Diaporthe species; Yang et al. (2018) provided the rst molecular phylogenetic
framework of Diaporthe diversity associated with dieback diseases in China. Following
the adoption of DNA sequence-based methods, Diaporthe taxonomy is actively chang-
ing, with numerous species being described each year.
e present study is the rst evaluation of Diaporthe species, associated with die-
back diseases in Jiangxi Province using the combined morphology and molecular data
and provided useful information for evaluating the pathogenicity of various species.
Multiple strains from dierent locations should also be subjected to multi-locus phy-
logenetic analysis to determine intraspecic variation and redene species boundaries.
e descriptions and molecular data of Diaporthe species, provided in this study, rep-
resent a resource for plant pathologists, plant quarantine ocials and taxonomists for
identication of Diaporthe.
Acknowledgements
is study is nanced by the National Natural Science Foundation of China (Project
No.: 31670647). We are grateful to Chungen Piao and Minwei Guo (China Forestry
Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing) for sup-
port of strain preservation during this study.
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
60
References
Carbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in la-
mentous ascomycetes. Mycologia 3: 553–556. https://doi.org/10.1080/00275514.1999.1
2061051
Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G (2004) MycoBank: an online initiative
to launch mycology into the 21st century. Studies in Mycology 50: 19–22.
Crous PW, Summerell BW, Shivas RG, Burgess TI, Decock CA, Dreyer LL, Granke LL, Guest
DI, Hardy GESTJ, Hausbeck MK, Hüberli D, Jung T, Koukol O, Lennox CL, Liew ECY,
Lombard L, McTaggart AR, Pryke JS, Roets F, Saude C, Shuttleworth LA, Stukely MJC,
Vánky K, Webster BJ, Windstam ST, Groenewald JZ (2012) Fungal Planet description
sheets: 107–127. Persoonia 28: 138–182. https://doi.org/10.3767/003158512X652633
Crous PW, Wingeld MJ, Guarro J, Cheewangkoon, R, van der Bank, M, Swart WJ, Stchigel
AM, Cano-Lira JF, Roux J, Madrid H, Damm U, Wood AR, Shuttleworth LA, Hodges
CS, Munster, M, de Jesús Yáñez-Morales M, Zúñiga-Estrada L, Cruywagen EM, De Hoog
GS, Silvera C, Najafzadeh J, Davison EM, Davison PJN, Barrett MD, Barrett RL, Man-
amgoda DS, Minnis AM, Kleczewski NM, Flory SL, Castlebury LA, Clay K, Hyde KD,
Maússe-Sitoe SND, Shuaifei C, Lechat C, Hairaud M, Lesage-Meessen L, Pawłowska J,
Wilk M, Śliwińska-Wyrzychowska A, Mętrak M, Wrzosek M, Pavlic-Zupanc D, Maleme
HM, Slippers B, Mac Cormack WP, Archuby DI, Grünwald NJ, Tellería MT, Dueñas M,
Martín MP, Marincowitz S, de Beer ZW, Perez CA, Gené J, Marin-Felix Y, Groenewald
JZ (2013) Fungal Planet description sheets: 154–213. Persoonia 31: 188–296. https://doi.
org/10.3767/003158513X675925
Crous PW, Wingeld MJ, Le Roux JJ, Richardson DM, Strasberg D, Shivas RG, Alvarado P,
Edwards J, Moreno G, Sharma R, Sonawane MS, Tan YP, Altés A, Barasubiye T, Barnes
CW, Blanchette RA, Boertmann D, Bogo A, Carlavilla JR, Cheewangkoon R, Daniel R,
de Beer ZW, Yáñez-Morales M de Jesús, Duong TA, Fernández-Vicente J, Geering ADW,
Guest DI, Held BW, Heykoop M, Hubka V, Ismail AM, Kajale SC, Khemmuk W, Kolařík
M, Kurli R, Lebeuf R, Lévesque CA, Lombard L, Magista D, Manjón JL, Marincowitz
S, Mohedano JM, Nováková A, Oberlies NH, Otto EC, Paguigan ND, Pascoe IG, Pérez-
Butrón JL, Perrone G, Rahi P, Raja HA, Rintoul T, Sanhueza RMV, Scarlett K, Shouche
YS, Shuttleworth LA, Taylor PWJ, orn RG, Vawdrey LL, Solano-Vidal R, Voitk A,
Wong PTW, Wood AR, Zamora JC, Groenewald JZ (2015) Fungal Planet description
sheets: 371–399. Persoonia 35: 264–327. https://doi.org/10.3767/003158515X690269
Dai DQ, Wijayawardene NN, Bhat DJ, Chukeatirote E, Bahkali AH, Zhao R-L, Xu J-C,
Hyde KD (2014) Pustulomyces gen. nov. accommodated in Diaporthaceae, Diaporthales,
as revealed by morphology and molecular analyses. Cryptogamie, Mycologie 35: 63–72.
https://doi.org/10.7872/crym.v35.iss1.2014.63
Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics
and parallel computing. Nature Methods 9: e772. https://doi.org/10.1038/nmeth.2109
Desjardins P, Hansen JB, Allen M (2009) Microvolume protein concentration determination
using the NanoDrop 2000c spectrophotometer. Journal of Visualized Experiments: JoVE
33: 1–3. https://doi.org/10.3791/1610
Diaporthe species from cankered branches and leaves 61
Diogo E, Santos JM, Phillips AJ (2010) Phylogeny, morphology and pathogenicity of Dia-
porthe and Phomopsis species on almond in Portugal. Fungal Diversity 44: 107–115.
https://doi.org/10.1007/s13225-010-0057-x
Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12: 13–15. https://
doi.org/10.2307/2419362
Fan XL, Hyde KD, Udayanga D, Wu XY, Tian CM (2015) Diaporthe rostrata, a novel ascomy-
cete from Juglans mandshurica associated with walnut dieback. Mycological Progress 14:
1–8. https://doi.org/10.1007/s11557-015-1104-5
Fan XL, Yang Q, Bezerra JDP, Alvarez LV, Tian CM (2018) Diaporthe from walnut tree (Juglans
regia) in China, with insight of Diaporthe eres complex. Mycological Progress 1–13. https://
doi.org/10.1007/s11557-018-1395-4
Fu CH, Hsieh HM, Chen CY, Chang TT, Huang YM, Ju YM (2013) Ophiodiaporthe cyatheae
gen. et sp. nov., a diaporthalean pathogen causing a devastating wilt disease of Cyathea
lepifera in Taiwan. Mycologia 105: 861–872. https://doi.org/10.3852/12-346
Gao YH, Liu F, Cai L (2016) Unravelling Diaporthe species associated with Camellia. Systemat-
ics and Biodiversity 14: 102–117. https://doi.org/10.1080/14772000.2015.1101027
Gao YH, Liu F, Duan W, Crous PW, Cai L (2017) Diaporthe is paraphyletic. IMA Fungus 8:
153–187. https://doi.org/10.5598/imafungus.2017.08.01.11
Gao YH, Su YY, Sun W, Cai L (2015) Diaporthe species occurring on Lithocarpus glabra in
China, with descriptions of ve new species. Fungal Biology 115: 295–309. https://doi.
org/10.1016/j.funbio.2014.06.006
Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR
to amplify conserved genes from lamentous ascomycetes. Applied and Environmental
Microbiology 61: 1323–1330. https://doi.org/10.1128/AEM.61.4.1323-1330.1995
Gomes RR, Glienke C, Videira SIR, Lombard L, Groenewald JZ, Crous PW (2013) Diaporthe:
a genus of endophytic, saprobic and plant pathogenic fungi. Persoonia 31: 1–41. https://
doi.org/10.3767/003158513X666844
Grasso FM, Marini M, Vitale A, Firrao G, Granata G (2012) Canker and dieback on Pla-
tanus × acerifolia caused by Diaporthe scabra. Forest Pathology 42: 510–513. https://doi.
org/10.1111/j.1439-0329.2012.00785.x
Guarnaccia V, Crous PW (2017) Emerging citrus diseases in Europe caused by species of Dia-
porthe. IMA Fungus 8: 317–334. https://doi.org/10.5598/imafungus.2017.08.02.07
Guarnaccia V, Groenewald JZ, Woodhall J, Armengol J, Cinelli T, Eichmeier A, Ezra D, Fon-
taine F, Gramaje D, Gutierrez-Aguirregabiria A, Kaliterna J, Kiss L, Larignon P, Luque J,
Mugnai L, Naor V, Raposo R, Sándor E, Váczy KZ, Crous PW (2018) Diaporthe diversity
and pathogenicity revealed from a broad survey of grapevine diseases in Europe. Persoonia
40: 135–153. https://doi.org/10.3767/persoonia.2018.40.06
Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algo-
rithms and methods to estimate maximum-likelihood phylogenies: assessing the perfor-
mance of PhyML 3.0. Systematic Biology 59: 307–321. https://doi.org/10.1093/sysbio/
syq010
Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis pro-
gram for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
62
Huang F, Udayanga D, Wang X, Hou X, Mei X, Fu Y, Hyde KD, Li HY (2015) Endophytic
Diaporthe associated with Citrus: A phylogenetic reassessment with seven new species from
China. Fungal Biology 119: 331–347. https://doi.org/10.1016/j.funbio.2015.02.006
Hyde KD, Dong Y, Phookamsak R, Jeewon R, Jayarama Bhat D, Gareth Jones EB, Liu N-G,
Abeywickrama PD, Mapook A, Wei D, Perera RH, Manawasinghe IS, Pem D, Bundhun D,
Karunarathna A, Ekanayaka AH, Bao D-F, Li J, Samarakoon MC, Chaiwan N, Chuan-Gen
Lin, Phutthacharoen K, Zhang S-N, Senanayake IC, Goonasekara ID, ambugala KM,
Phukhamsakda C, Tennakoon DS, Jiang H-B, Yang J, Zeng M, Huanraluek N, Liu J-K,
Wijesinghe SN, Tian Q, Tibpromma S, Brahmanage RS, Boonmee S, Huang S-K, iyaga-
raja V, Lu Y-Z, Jayawardena RS, Dong W, Yang E-F, Singh SK, Singh MS, Rana S, Lad SS,
Anand G, Devadatha B, Niranjan M, Sarma VV, Liimatainen K, Aguirre-Hudson B, Nis-
kanen T, Overall A, Alvarenga LRM, Gibertoni BT, Piegler WP, Horváth E, Imre A, Alves
LA, da Silva Santos CA, Tiago VP, Bulgakov TS, Wanasinghe DN, Bahkali AH, Doilom M,
Elgorban AM, Maharachchikumbura SSN, Rajeshkumar KC, Haelewaters D, Mortimer PE,
Zhao Q, Lumyong S, Xu J, Sheng J (2020) Fungal diversity notes 1151–1276: taxonomic
and phylogenetic contributions on genera and species of fungal taxa. Fungal Diversity 100:
5–277. https://doi.org/10.1007/s13225-020-00439-5
Katoh K, Toh H (2010) Parallelization of the MAFFT multiple sequence alignment program.
Bioinformatics 26: 1899–1900. https://doi.org/10.1093/bioinformatics/btq224
Lamprecht SC, Crous PW, Groenewald JZ, Tewoldemedhin YT, Marasas WFO (2011) Dia-
porthaceae associated with root and crown rot of maize. IMA Fungus 2: 13–24. https://
doi.org/10.5598/imafungus.2011.02.01.03
Lombard L, Van Leeuwen GCM, Guarnaccia V, Polizzi G, Van Rijswick PC, Karin Rosen-
dahl KC, Gabler J, Crous PW (2014) Diaporthe species associated with Vaccinium, with
specic reference to Europe. Phytopathologia Mediterranea 53: 287–299. https://doi.
org/10.14601/Phytopathol_Mediterr-14034
Manawasinghe IS, Dissanayake A, Liu M, Liu M, Wanasinghe DN, Xu J, Zhao W, Zhang W,
Zhou Y, Hyde KD, Brooks S, Yan J (2019) High genetic diversity and species complexity
of Diaporthe associated with grapevine dieback in China. Frontiers in Microbiology 10:
e1936. https://doi.org/10.3389/fmicb.2019.01936
Marin-Felix Y, Hernández-Restrepo M, Wingeld M J, Akulov A, Carnegie AJ, Cheewang-
koon R, Gramaje D, Groenewald JZ, Guarnaccia V, Halleen F, Lombard L, Luangsa-ard
J, Marincowitz S, Moslemi A, Mostert L, Quaedvlieg W, Schumacher RK, Spies CFJ,
angavel R, Taylor PWJ, Wilson AM, Wingeld BD, Wood AR, Crous PW (2019) Gen-
era of phytopathogenic fungi: GOPHY 2. Studies in Mycology 92: 47–133. https://doi.
org/10.1016/j.simyco.2018.04.002
Mostert L, Crous PW, Kang JC, Phillips AJ (2001) Species of Phomopsis and a Libertella sp.
occurring on grapevines with specic reference to South Africa: morphological, cultural,
molecular and pathological characterization. Mycologia 93: 146–167. https://doi.org/10.
1080/00275514.2001.12061286
Muralli TS, Suryanarayanan TS, Geeta R (2006) Endophytic Phomopsis species: host range
and implications for diversity estimates. Canadian Journal of Microbiology 52: 673–680.
https://doi.org/10.1139/w06-020
Diaporthe species from cankered branches and leaves 63
O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a
monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenet-
ics and Evolution 7: 103–116. https://doi.org/10.1006/mpev.1996.0376
Rambaut A, Drummond A (2010) FigTree v.1.3.1. Institute of Evolutionary Biology, Univer-
sity of Edinburgh, Edinburgh.
Rayner RW (1970) A mycological colour chart. Commonwealth Mycological Institute, Kew,
34 pp.
Rehner SA, Uecker FA (1994) Nuclear ribosomal internal transcribed spacer phylogeny and
host diversity in the coelomycete Phomopsis. Canadian Journal of Botany 72: 1666–1674.
https://doi.org/10.1139/b94-204
Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed
models. Bioinformatics 19: 1572–1574. https://doi.org/10.1093/bioinformatics/btg180
Rossman AY, Farr DF, Castlebury LA (2007) A review of the phylogeny and biology of the
Diaporthales. Mycoscience 48: 135–144. https://doi.org/10.1007/S10267-007-0347-7
Santos JM, Correia VG, Phillips AJL (2010) Primers for mating-type diagnosis in Diaporthe
and Phomopsis, their use in teleomorph induction in vitro and biological species denition.
Fungal Biology 114: 255–270. https://doi.org/10.1016/j.funbio.2010.01.007
Santos JM, Phillips AJL (2009) Resolving the complex of Diaporthe (Phomopsis) species occur-
ring on Foeniculum vulgare in Portugal. Fungal Diversity 34: 111–125.
Santos JM, Vrandečić K, Ćosić J, Duvnjak T, Phillips AJL (2011) Resolving the Dia-
porthe species occurring on soybean in Croatia. Persoonia 27: 9–19. https://doi.
org/10.3767/003158511X603719
Senanayake IC, Crous PW, Groenewald JZ, Senanayake IC, Crous PW, Groenewald JZ, Maha-
rachchikumbura SSN, Jeewon R, Phillips AJL, Bhat JD, Perera RH, Li QR, Li WJ, Tangthi-
rasunun N, Norphanphoun C, Karunarathna SC, Camporesi E, Manawasighe IS, Al-Sadi
AM, Hyde KD (2017) Families of Diaporthales based on morphological and phylogenetic
evidence. Studies in Mycology 86: 217–296. https://doi.org/10.1016/j.simyco.2017.07.003
Smith H, Wingfeld MJ, Coutinho TA, Crous PW (1996) Sphaeropsis sapinea and Botryospha-
eria dothidea endophytic in Pinus spp. and Eucalyptus spp. in South Africa. South African
Journal of Botany 62: 86–88. https://doi.org/10.1016/S0254-6299(15)30596-2
ompson SM, Tan YP, Young AJ, Neate SM, Aitken EAB, Shivas RG (2011) Stem cankers
on sunower (Helianthus annuus) in Australia reveal a complex of pathogenic Diaporthe
(Phomopsis) species. Persoonia 27: 80–89. https://doi.org/10.3767/003158511X617110
Udayanga D, Castlebury LA, Rossman AY, Chukeatirote E, Hyde KD (2014) Insights into the
genus Diaporthe: phylogenetic species delimitation in the D. eres species complex. Fungal
Diversity 67: 203–229. https://doi.org/10.1007/s13225-014-0297-2
Udayanga D, Castlebury LA, Rossman AY, Chukeatirote E, Hyde KD (2015) e Diaporthe
sojae species complex: Phylogenetic re-assessment of pathogens associated with soybean,
cucurbits and other eld crops. Fungal Biology 119: 383–407. https://doi.org/10.1016/j.
funbio.2014.10.009
Udayanga D, Liu X, McKenzie EH, Chukeatirote E, Bahkali AH, Hyde KD (2011) e genus
Phomopsis: biology, applications, species concepts and names of common phytopathogens.
Fungal Diversity 50: 189–225. https://doi.org/10.1007/s13225-011-0126-9
Qin Yang et al. / MycoKeys 77: 41–64 (2021)
64
Uecker FA (1988) A world list of Phomopsis names with notes on nomenclature, morphology
and biology. Mycological Memoirs 13: 1–231.
van Rensburg JCJ, Lamprecht SC, Groenewald JZ, Castlebury LA, Crous PW (2006) Charac-
terization of Phomopsis spp. associated with die-back of rooibos (Aspalathus linearis) in South
Africa. Studies in Mycology 55: 65–74. https://doi.org/10.3114/sim.55.1.65
Wehmeyer LE (1926) A biologic and phylogenetic study of stromatic Sphaeriales. American
Journal of Botany 13: 575–645. https://doi.org/10.1002/j.1537-2197.1926.tb05903.x
White TJ, Bruns T, Lee S, Taylor J (1990) Amplication and direct sequencing of fungal ribo-
somal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applica-
tions 18: 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
Yang Q, Fan XL, Du Z, Tian CM (2017b) Diaporthe juglandicola sp. nov. (Diaporthales, As-
comycetes), evidenced by morphological characters and phylogenetic analysis. Mycosphere
8: 817–826. https://doi.org/10.5943/mycosphere/8/5/3
Yang Q, Fan XL, Guarnaccia V, Tian CM (2018) High diversity of Diaporthe species associated
with dieback diseases in China, with twelve new species described. MycoKeys 39: 97–149.
https://doi.org/10.3897/mycokeys.39.26914
Yang Q, Fan XL, Du Z, Tian CM (2017a) Diaporthe species occurring on Senna bicapsularis in
southern China, with descriptions of two new species. Phytotaxa 302: 145–155. https://
doi.org/10.11646/phytotaxa.302.2.4
Yang Q, Jiang N, Tian CM (2020) ree new Diaporthe species from Shaanxi Province, China.
Mycokeys 67: 1–18. https://doi.org/10.3897/mycokeys.67.49483
Available via license: CC BY 4.0
Content may be subject to copyright.
Content uploaded by Ning Jiang
Author content
All content in this area was uploaded by Ning Jiang on Jan 15, 2021
Content may be subject to copyright.
