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Submitted 21 May 2017, Accepted 6 June 2017, Published 12 June 2017 853
Corresponding Author: Xinghong Li – e-mail – lixinghong1962@163.com
Molecular phylogenetic analysis reveals seven new Diaporthe species
from Italy
Dissanayake AJ1,2, Camporesi E3, Hyde KD2, Zhang Wei1, Yan JY1 and Li XH1*
1 Beijing Key Laboratory of Environmental Friendly Management on Fruit diseases and Pests in North China, Institute of
Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, People’s
Republic of China
2 Center of Excellence in Fungal Research, School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
3 A.M.B. Gruppo Micologico Forlivese “Antonio Cicognani”, Via Roma 18, Forlì, Italy
Dissanayake AJ, Camporesi E, Hyde KD, Zhang Wei, Yan JY, Li XH 2017 – Molecular phylogenetic
analysis reveals seven new Diaporthe species from Italy. Mycosphere 8(5), 853–877, Doi
10.5943/mycosphere/8/5/4
Abstract
Seven new species of Diaporthe, D. acericola on Acer negundo, D. cichorii on Cichorium
intybus, D. dorycnii on Dorycnium hirsutum, D. lonicerae on Lonicera sp., Laurus nobilis and Torilis
arvensis, D. pseudotsugae on Pseudotsuga menziesii, D. schoeni on Schoenus nigricans, Carduus sp.
and Plantago sp. and D. torilicola on Torilis arvensis from Italy are described and illustrated based on
morphological characteristics and molecular analyses. In addition to the new species, eight known
species of Diaporthe, D. eres, D. foeniculina, D. gulyae, D. novem, D. ravennica, D. rhusicola, D.
rudis and D. sterilis were identified. Phylogenetic relationships of the new species with other
Diaporthe species were revealed by DNA sequence analyses based on the internal transcribed spacer
(ITS) region, translation elongation factor 1-alpha (TEF), partial regions of the β-tubulin (BT) and
calmodulin (CAL). Among 44 isolates, D. eres was the dominant species, accounting for 27% of the
frequency of occurrence. Our study revealed a high diversity of undescribed Diaporthe species from
various hosts in Italy.
Key words – Diaporthales – Hosts – Morphology – Sordariomycetes – Taxonomy
Introduction
Diaporthe (including the Phomopsis asexual morph) belongs to family Diaporthaceae, order
Diaporthales, and class Sordariomycetes (Hyde et al. 2014, Maharachchikumbura et al. 2015, 2016)
and its species are found worldwide on a diverse range of host plants as endophytes, pathogens and
saprobes (Gomes et al. 2013). Many Diaporthe species that are morphologically similar have proven to
be genetically distinct (van Rensburg et al. 2006), and several isolates that were formerly identified
based on their host, were shown to represent different taxa (Hyde et al. 2014). Diaporthe represents a
highly complex genus containing numerous cryptic species. In recent studies, species of Diaporthe were
distinguished mainly by their molecular phylogenies, and the best five gene regions to conduct a multi-
gene phylogenetic analysis are ITS, TEF, ACT CAL and HIS (van Rensburg et al. 2006, Santos et al.
Mycosphere 8(5): 853–877 (2017) www.mycosphere.org ISSN 2077 7019
Article
Doi 10.5943/mycosphere/8/5/4
Copyright © Guizhou Academy of Agricultural Sciences
854
2010, Udayanga et al. 2011, 2012, Gomes et al. 2013). Although ex-type/ex-epitype/ex-isotype/ex-
neotype strains are available for 150 species of Diaporthe (Dissanayake et al. 2017b), only 13 have been
reported associated with hosts in Italy (Table 1). During the last three years, a collection of Diaporthe
isolates was obtained from branches and stems of various woody hosts in Arezzo, Forlì-Cesena and
Ravenna Provinces in Italy. The aim of this study was to identify the species and reveal the distribution
of species on the hosts. Isolates were characterized in terms of morphology and their phylogenetic
position within Diaporthe.
Table 1 Diaporthe species associated with hosts in Italy
Species
Disease symptoms & hosts
References
Diaporthe alnea
Dieback of Alnus glutinosa
Moricca 2002
D. ambigua
Dieback of Platanus acerifolia
Gomes et al. 2013
D. eres
Cane blight of Vitis vinifera
Cinelli et al. 2016
D. foeniculina
Decline and mortality of Eucalyptus camaldulensis
Stem and shoot cankers on Castanea sativa
Branch cankers and stem-end rot of Persea americana
Deidda et al. 2016
Annesi et al. 2016
Guarnaccia et al. 2016
D. helianthi
Stem canker of Helianthus annuus
Pecchia et al. 2004
D. melonis
Black rot of Cucumis melo
Bertetti et al. 2011
D. ravennica
Tamarix sp.
Thambugala et al. 2017
D. sclerotioides
Black root rot of Cucumis sativus
Cappelli et al. 2004
Phomopsis endogena
Brown rot on nuts of Castanea sativa
Maresi et al. 2013
P. quercina
Endophyte in Quercus sp.
Ragazzi et al. 2003
Endophyte in Quercus robur
Gonthier et al. 2006
Phomopsis sp.
Dieback of Pinus nigra seedlings
Nicosia et al. 2015
Post-harvest fruit rot of Actinidia sp.
Luongo et al. 2011
Symptomatic twigs of Olea europaea
Frisullo et al. 2015
Materials & methods
Sample collection, specimen examination and isolations
During 2014 to 2016, 44 isolates were collected from woody branches and stems of 42 hosts
belonging to 26 host families (Adoxaceae, Apiaceae, Asteraceae, Betulaceae, Brassicaceae,
Caprifoliaceae, Caprifoliaceae, Cornaceae, Cupressaceae, Cyperaceae, Dioscoreaceae, Fabaceae,
Hemerocallidoideae, Juglandaceae, Lamiaceae, Lauraceae, Pinaceae, Plantaginaceae, Platanaceae,
Poaceae, Rhamnaceae, Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Simaroubaceae) from three
provinces of Italy: Arezzo, Forlì-Cesena and Ravenna (Fig. 1). Specimens were observed and examined
with a Motic SMZ 168 stereomicroscope. Micro-morphological characters were determined with a
Nikon ECLIPSE 80i compound microscope and images were captured with a Canon EOS 550D digital
camera. Observations and photographs were made from materials mounted in water. Measurements
were made with the Tarosoft (R) Image Frame Work and images used for figures were processed with
Adobe Photoshop CS3 Extended version 10.0. Single spore isolations were prepared following the
method of Chomnunti et al. (2014). Spore germination on water agar (WA) was examined after 24 h
and germinating spores were transferred to potato dextrose agar (PDA) media. Cultures were incubated
at 18°C in the dark and colony color was examined according to Rayner (1970) after 15 d of growth on
PDA at 25 °C in the dark. Herbarium specimens are deposited in Mae Fah Luang University
Herbarium (MFLU) while, ex-type living cultures are deposited at the Mae Fah Luang University
Culture Collection (MFLUCC) in Thailand (Table 2).
855
Table 2 Diaporthe species studied in this study (Fig. 2). Details of ex-type species introduced in this study are in bold.
Species
Strain
Host
Habit
Locality
Collector
Colle. date
GenBank Accession numbers
ITS
TEF
BT
CAL
D. acericola
MFLUCC 17-0956
Acer negundo (Sapindaceae)
Dead branch, samaras
Forlì-Cesena, Italy
E. Camporesi
22.01.2015
KY964224
KY964180
KY964074
KY964137
D. cichorii
MFLUCC 17-1023
Cichorium intybus (Asteraceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
17.07.2016
KY964220
KY964176
KY964104
KY964133
D. dorycnii
MFLUCC 17-1015
Dorycnium hirsutum (Fabaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
02.05.2016
KY964215
KY964171
KY964099
No
D. eres
MFLUCC 17-0957
Sambucus nigra (Adoxaceae)
Dead aerial branch
Forlì-Cesena, Italy
E. Camporesi
07.02.2015
KY964187
KY964143
KY964070
KY964114
D. eres
MFLUCC 17-0965
Lonicera sp. (Caprifoliaceae)
Dead aerial branch
Forlì-Cesena, Italy
E. Camporesi
28.02.2015
KY964189
KY964145
KY964072
KY964115
D. eres
MFLUCC 17-0964
Sonchus oleraceus (Asteraceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
06.05.2015
KY964192
KY964148
KY964076
KY964117
D. eres
MFLUCC 17-0971
Salix caprea (Salicaceae)
Dead aerial branch
Arezzo, Italy
E. Camporesi
19.06.2015
KY964194
KY964150
KY964078
KY964119
D. eres
MFLUCC 17-0993
Picea excels (Pinaceae)
Dead land cone
Forlì-Cesena, Italy
E. Camporesi
18.01.2016
KY964200
KY964156
KY964084
KY964123
D. eres
MFLUCC 17-0997
Juglans regia (Juglandaceae)
Dead land branch
Forlì-Cesena, Italy
E. Camporesi
22.02.2016
KY964202
KY964158
KY964086
KY964124
D. eres
MFLUCC 17-0999
Populus nigra (Salicaceae)
Dead aerial branch
Forlì-Cesena, Italy
-
-
KY964203
KY964159
KY964087
KY964125
D. eres
MFLUCC 17-1012
Sanguisorba minor (Rosaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
11.04.2016
KY964213
KY964169
KY964097
KY964128
D. eres
MFLUCC 17-1016
Pinus pinaster (Pinaceae)
Dead land cone
Forlì-Cesena, Italy
E. Camporesi
03.05.2016
KY964216
KY964172
KY964100
KY964129
D. eres
MFLUCC 17-1017
Ostrya carpinifolia (Betulaceae)
Dead aerial branch
Forlì-Cesena, Italy
E. Camporesi
07.05.2016
KY964217
KY964173
KY964101
KY964130
D. eres
MFLUCC 17-1021
Galega officinalis (Fabaceae)
Dead aerial stem
Arezzo, Italy
E. Camporesi
07.07.2016
KY964219
KY964175
KY964103
KY964132
D. eres
MFLUCC 17-1025
Rhamnus alpinus (Rhamnaceae)
Dead aerial branch
Forlì-Cesena, Italy
E. Camporesi
14.08.2016
KY964221
KY964177
KY964105
KY964134
D. foeniculina
MFLUCC 17-1068
Ailanthus altissima (Simaroubaceae)
Dead land stem-leaf
Forlì-Cesena, Italy
E. Camporesi
07.02.2015
KY964188
KY964144
KY964071
-
D. foeniculina
MFLUCC 17-0974
Melilotus officinalis (Fabaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
07.09.2015
KY964196
KY964152
KY964080
-
D. foeniculina
MFLUCC 17-0995
Hemerocallis fulva (Hemerocallidoideae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
10.02.2016
KY964201
KY964157
KY964085
-
D. foeniculina
MFLUCC 17-1003
Achillea millefolium (Asteraceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
16.03.2016
KY964205
KY964161
KY964089
-
D. foeniculina
MFLUCC 17-1005
Arctium minus (Asteraceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
08.03.2016
KY964207
KY964163
KY964091
-
D. foeniculina
MFLUCC 17-1006
Wisteria sinensis (Fabaceae)
Dead aerial stems
Forlì-Cesena, Italy
E. Camporesi
09.03.2016
KY964208
KY964164
KY964092
-
D. foeniculina
MFLUCC 17-1008
Lunaria rediviva (Brassicaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
16.03.2016
KY964209
KY964165
KY964093
-
D. foeniculina
MFLUCC 17-1009
Cupressus sepervirens (Cupressaceae)
Dead land cone
Forlì-Cesena, Italy
E. Camporesi
21.03.2016
KY964210
KY964166
KY964094
-
D. foeniculina
MFLUCC 17-1020
Vicia sp. (Fabaceae)
Dead aerial stem
Arezzo, Italy
E. Camporesi
19.06.2016
KY964218
KY964174
KY964102
KY964131
D. gulyae
MFLUCC 17-1026
Heracleum sphondylium (Apiaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
28.08.2016
KY964223
KY964179
KY964107
KY964136
D. lonicerae
MFLUCC 17-0963
Lonicera sp. (Caprifoliaceae)
Dead aerial branch
Forlì-Cesena, Italy
E. Camporesi
28.02.2015
KY964190
KY964146
KY964073
KY964116
D. lonicerae
MFLUCC 17-0976
Laurus nobilis (Lauraceae)
Dead aerial branch
Forlì-Cesena, Italy
E. Camporesi
15.09.2015
KY964197
KY964153
KY964081
KY964121
D. lonicerae
MFLUCC 17-0978
Torilis arvensis (Apiaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
23.09.2015
KY964198
KY964154
KY964082
KY964122
D. novem
MFLUCC 17-1028
Galium sp. (Rubiaceae)
Dead aerial stem
Arezzo, Italy
E. Camporesi
19.06.2015
KY964195
KY964151
KY964079
KY964120
D. pseudotsugae
MFLU 15-3228
Pseudotsuga menziesii (Pinaceae)
Dead land cones
Forlì-Cesena, Italy
E. Camporesi
10.04.2015
KY964225
KY964181
KY964108
KY964138
D. ravennica
MFLUCC 17-1029
Salvia sp. (Lamiaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
21.04.2015
KY964191
KY964147
KY964075
-
D. rhusicola
MFLUCC 17-0987
Amorpha fruticosa (Fabaceae)
Dead aerial branch
Forlì-Cesena, Italy
E. Camporesi
17.11.2015
KY964199
KY964155
KY964083
-
D. rhusicola
MFLUCC 17-1001
Angelica sylvestris (Apiaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
29.02.2016
KY964204
KY964160
KY964088
-
D. rhusicola
MFLUCC 17-1004
Rubus sp. (Rosaceae)
Dead aerial branch
Forlì-Cesena, Italy
E. Camporesi
14.03.2016
KY964206
KY964162
KY964090
-
D. rhusicola
MFLUCC 17-1014
Platanus hybrida (Platanaceae)
Dead aerial branch
Forlì-Cesena, Italy
E. Camporesi
27.04.2016
KY964214
KY964170
KY964098
-
D. rudis
MFLUCC 17-1030
Cornus sp. (Cornaceae)
Dead aerial branch
Forlì-Cesena, Italy
E. Camporesi
10.11.2014
KY964186
KY964142
KY964069
KY964113
D. rudis
MFLU 15-1264
Anthoxanthum odoratum (Poaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
21.05.2015
KY964227
KY964183
KY964110
KY964140
D. rudis
MFLUCC 17-0969
Carlina vulgaris (Asteraceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
22.04.2015
KY964193
KY964149
KY964077
KY964118
D. rudis
MFLUCC 17-1073
Dioscorea communis (Dioscoreaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
28.08.2016
KY964222
KY964178
KY964106
KY964135
D. schoeni
MFLU 15-1279
Schoenus nigricans (Cyperaceae)
Dead aerial stem
Ravenna, Italy
E. Camporesi
01.05.2015
KY964226
KY964182
KY964109
KY964139
D. schoeni
MFLU 15-2266
Carduus sp. (Asteraceae)
Dead aerial stem
Arezzo, Italy
E. Camporesi
26.06.2015
KY964228
KY964184
KY964111
-
D. schoeni
MFLU 15-2609
Plantago sp. (Plantaginaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
25.08.2015
KY964229
KY964185
KY964112
KY964141
D. sterilis
MFLUCC 17-1011
Cytisus sp. (Fabaceae)
Dead aerial branch
Forlì-Cesena, Italy
E. Camporesi
04.04.2016
KY964211
KY964167
KY964095
KY964126
D. torilicola
MFLUCC 17-1051
Torilis arvensis (Apiaceae)
Dead aerial stem
Forlì-Cesena, Italy
E. Camporesi
21.04.2016
KY964212
KY964168
KY964096
KY964127
856
Table 3 Isolates from GenBank used in phylogenetic analyses (Fig. 2). Ex-type isolates are in bold.
Species
Isolate
Host
ITS
BT
TEF
CAL
D. acaciigena
CBS 129521
Acacia retinodes
KC343005
KC343973
KC343731
KC343247
D. alleghaniensis
CBS 495.72
Betula alleghaniensis
KC343007
KC343975
KC343733
KC343249
D. alnea
CBS 146.46
Alnus sp.
KC343008
KC343976
KC343734
KC343250
CBS 159.47
Alnus sp.
KC343009
KC343977
KC343735
KC343251
D. ampelina
CBS 114016
Vitis vinifera
AF230751
JX275452
AY745056
AY230751
CBS 267.80
Vitis vinifera
KC343018
KC343986
KC343744
KC343260
D. amygdali
CBS 126679
Prunus dulcis
KC343022
KC343990
AY343748
KC343264
CBS 111811
Vitis vinifera
KC343019
KC343987
KC343745
KC343261
D. arctii
DP0482
Arctium lappa
KJ590736
KJ610891
KJ590776
KJ612133
D. asheicola
CBS 136967
Vaccinium ashei
KJ160562
KJ160518
KJ160594
KJ160542
CBS 136968
Vaccinium ashei
KJ160563
KJ160519
KJ160595
KJ160543
D. australafricana
CBS 111886
Vitis vinifera
KC343038
KC344006
KC343764
KC343280
CBS 113487
Vitis vinifera
KC343039
KC344007
KC343765
KC343281
D. baccae
CBS 136972
Vaccinium corymbosum
KJ160565
No
KJ160597
No
CPC 20585
Vaccinium corymbosum
KJ160564
No
KJ160596
No
D. betulae
CFCC 50469
Betula platyphylla
KT732950
KT733020
KT733016
KT732997
CFCC 50470
Betula platyphylla
KT732951
KT733021
KT733017
KT732998
D. bicincta
CBS 121004
Juglans sp.
KC343134
KC344102
KC343860
KC343376
D. biguttusis
CGMCC 3.17081
Lithocarpus glabra
KF576282
KF576306
KF576257
No
CGMCC 3.17082
Lithocarpus glabra
KF576283
KF576307
KF576258
No
D. canthii
CBS 132533
Canthium inerme
JX069864
KC843230
KC843120
KC843174
D. cassines
CPC 21916
Cassine peragua
KF777155
No
KF777244
No
D. celastrina
CBS 139.27
Celastrus scandens
KC343047
KC344015
KC343773
KC343289
D. chamaeropis
CBS 454.81
Chamaerops humilis
KC343048
KC344016
KC343774
KC343290
CBS 753.70
Spartium junceum
KC343049
KC344017
KC343775
KC343291
D. cucurbitae
DAOM42078
Cucumis sativus
KM453210
KP118848
KM45321
1
No
CBS 136.25
Arctium sp.
KC343031
KC343999
KC343757
KC343273
D. cynaroidis
CBS 122676
Protea cynaroides
KC343058
KC344026
KC343784
KC343300
D. cytosporella
FAU461
Citrus limon
KC843307
KC843221
KC843116
KC843141
AR5149
Citrus sinensis
KC843309
KC843222
KC843118
KC843287
D. diospyricola
CPC 21169
Diospyros whyteana
KF777156
No
No
No
D. ellipicola
CGMCC 3.17084
Lithocarpus glabra
KF576270
KF576291
KF576245
No
CGMCC 3.17085
Lithocarpus glabra
KF576271
KF576292
KF576246
No
D. eres
AR519
Ulmus sp.
KJ210529
KJ420799
KJ210550
KJ434999
CBS 138598
Ulmus sp.
KJ210521
KJ420787
KJ210545
KJ435027
CBS 439.82
Cotoneaster sp.
FJ889450
JX275437
GQ250341
JX197429
DLR12A
Vitis vinifera
KJ210518
KJ420783
KJ210542
KJ434996
CBS 587.79
Pinus pantepella
KC343153
KC344121
KC343879
KC343395
857
D. foeniculina
CBS 111553
Foeniculum vulgare
KC343101
KC344069
KC343827
KC343343
FAU460
Citrus limon
KC843304
KC843218
KC843113
KC843138
ICMP 12285
Juglans regia
KC145853
No
KC145937
No
AR5151
Citrus latifolia
KC843303
KC843217
KC843112
KC843137
CBS 187.27
Camellia sinesis
DQ286287
JX275463
DQ286261
KC843122
CBS 123208
Foeniculum valgare
EU814480
JX275464
GQ250315
KC843125
D. fusicola
CGMCC 3.17087
Lithocarpus glabra
KF576281
KF576305
KF576256
KF576233
CGMCC 3.17088
Lithocarpus glabra
KF576263
KF576287
KF576238
No
D. garethjonesii
MFLUCC 12-
0542a
Unknown dead leaf
KT459423
KT459441
KT459457
KT459470
D. gulyae
BRIP 54025
Helianthus annuus
JF431299
No
JN645803
No
BRIP 53158
Helianthus annuus
JF431284
No
JN645799
No
D. helicis
AR5211
Hedera helix
KJ210538
KJ420828
KJ210559
KJ435043
D. hickoriae
CBS 145.26
Carya glabra
KC343118
KC344086
KC343844
KC343360
D. longicicola
CGMCC 3.17089
Lithocarpus glabra
KF576267
KF576291
KF576242
No
CGMCC 3.17090
Lithocarpus glabra
KF576268
KF576292
KF576243
No
D. mahothocarpus
CGMCC 3.15181
Lithocarpus glabra
KC153096
KF576312
KC153087
No
CGMCC 3.15182
Lithocarpus glabra
KC153097
No
KC153088
No
D. maritima
DAOMC 250563
Picea rubens
No
KU574616
No
No
D. neilliae
CBS 144. 27
Spiraea sp.
KC343144
KC344112
KC343870
KC343386
D. neoarctii
CBS 109490
Ambrosia trifida
KC343145
KC344113
KC343871
KC343387
D. nothofagi
BRIP 54801
Nothofagus
cunninghamii
JX862530
KF170922
JX862536
No
D. novem
CBS 127270
Glycine max
KC343155
KC344123
KC343881
KC343397
CBS 127271
Glycine max
KC343157
KC344125
KC343883
KC343399
D. ovoicicola
CGMCC 3.17093
Citrus sp.
KF576265
KF576289
KF576240
KF576223
CGMCC 3.17092
Citrus sp.
KF576264
KF576288
KF576239
KF576222
D. penetriteum
CGMCC 3.17532
Camellia sinensis
KP267879
KP293459
KP267953
No
D. phaseolorum
AR4203
Phaseolus vulgaris
KJ590738
KJ610893
KJ590739
KJ612135
D. phragmitis
CBS 138897
Phragmites australis
KP004445
KP004507
No
No
D. pulla
CBS 338.89
Hedera helix
KC343152
KC344120
KC343878
KC343394
D. ravennica
MFLUCC 15–
0479
Tamarix sp.
KU900335
KX432254
KX365197
No
MFLUCC 15–
0480
Tamarix sp.
KU900336
KX377688
KX426703
No
D. rhusicola
CBS 129528
Rhus pendulina
JF951146
No
No
No
D. rudis
AR3422
Laburnum anagyroides
KC843331
KC843177
KC843090
KC843146
AR3654
Rosa canina
KC843338
KC843184
KC843097
KC843153
ICMP 16419
Castanea sativa
KC145904
No
KC145976
No
DA244
Brugmansia sp.
KC843334
KC843180
KC843093
KC843149
CBS 113201
Vitis vinifera
AY485750
JX275454
GQ250327
JX197445
858
D. saccarata
CBS 116311
Protearepens
KC343190
KC344158
KC343916
KC343432
D. salicicola
BRIP 54825
Salix purpurea
JX862531
JX862531
JX862537
No
D. sojae
FAU635
Glycine max
KJ590719
KJ610875
KJ590762
KJ612116
CBS 116019
Caperonia palustris
KC343175
KC344143
KC343901
KC343417
FAU455
Stokesia laevis
KJ590712
KJ610868
KJ590755
KJ612109
DP0601
Glycine max
KJ590706
KJ610862
KJ590749
KJ612103
MAFF 410444
Cucumis melo
KJ590714
KJ610870
KJ590757
KJ612111
BRIP 54033
Helianthus annuus
JF431295
No
JN645809
No
D. spartinicola
CBS 140003
Spartium junceum
KR611879
No
No
No
D. sterilis
CBS 136969
Vaccinium corymbosum
KJ160579
KJ160528
KJ160611
KJ160548
CPC 20580
Vaccinium corymbosum
KJ160582
KJ160531
KJ160614
KJ160551
D. subclavata
ZJUD95
Citrus sp.
KJ490630
KJ490451
KJ490509
No
CGMCC 3.17253
Citrus grandis
KJ490618
KJ490439
KJ490497
No
D. ternstroemia
CGMCC 3.15183
Ternstroemia
gymnanthera
KC153098
No
KC153089
No
CGMCC 3.15184
Ternstroemia
gymnanthera
KC153099
No
KC153090
No
D. toxica
CBS 534.93
Lupinus angustifolius
KC343220
KC344188
KC343946
KC343462
CBS 546.93
Lupinus sp.
KC343222
KC344190
KC343948
KC343464
D. vaccinii
CBS 160.32
Vaccinium macrocarpon
AF317578
JX270436
GQ250326
KC343470
CBS 122116
Vaccinium corymbosum
KC343227
KC344195
KC343953
KC343469
CBS 135436
Vaccinium corymbosum
AF317570
KC843225
JQ807380
KC849456
D. virgiliae
CMW40748
Virgilia oroboides
KP247566
KP247575
No
No
Diaporthella
corylina
CBS 121124
Corylus sp.
KC343004
KC343972
KC343730
KC343246
Molecular based amplification
Total DNA was extracted from aerial mycelium of 7 day old cultures grown on PDA at 25 C following the modified cetyltrimethyl ammonium
bromide (CTAB) method described by Udayanga et al. (2012). Under circumstances where fungi failed to grow in culture, DNA was extracted directly
from fruiting bodies using aseptic techniques. For the identification of Diaporthe, rDNA internal transcribed spacer (ITS) region was amplified and
sequenced for all 44 isolates. The translation elongation factor 1-α (TEF), a portion of the β-tubulin (BT) gene and the calmodulin (CAL) gene were
employed to support species identification based on ITS gene sequence data. The rDNA ITS region was amplified using universal primers ITS1 and
ITS4 (White et al. 1990). The target region of the TEF gene was amplified using primer pairs EF-728F and EF-986R (Carbone & Kohn 1999). A
portion of the BT gene was amplified using the primers BT2a and BT2b (Glass & Donaldson 1995), while the primer pair CAL228F and CAL737R
(Carbone & Kohn 1999) was used to amplify the CAL. The PCR reactions were performed in a BIORAD 1000TM thermal cycler in a total volume of
25 l. The PCR mixture contained TaKaRa Ex-Taq DNA polymerase 0.3 l, 12.5 l of 2 × PCR buffer with 2.5 l of dNTPs, 1 l of each primer, 9.2
l of double-distilled water and 100–500 ng of DNA template. DNA samples were detected by electrophoresis and ethidium bromide (EB) staining
and were used as templates for PCR amplification. DNA sequencing was performed by Sunbiotech Company, Beijing, China.
859
Sequence alignment and phylogenetic analyses
All new sequences generated in this study were checked manually and nucleotides at
ambiguous positions were clarified with sequences from both strands and aligned with sequences
retrieved from GenBank based on recent publications (Liu et al. 2015, Hyde et al. 2016). Combined
datasets were aligned using MAFFT (Katoh & Toh 2010, http://mafft.cbrc.jp/alignment/server/)
and were manually optimized with BioEdit (Hall 2006) to allow maximum alignment. Maximum
Parsimony analysis (MP) was performed with PAUP (Phylogenetic Analysis Using Parsimony) v.
4.0b10 (Swofford 2003). Gaps were treated as missing data, and the ambiguously aligned regions
were excluded. Trees were inferred using the heuristic search option with Tree Bisection
Reconnection branch swapping and 1000 random sequence additions. Maxtrees was set at 1000,
branches of zero length were collapsed, and all multiple parsimonious trees were saved. Descriptive
tree statistics for parsimony (tree length, consistency index, retention index, rescaled consistency
index, and homoplasy index) were calculated for trees generated under different optimality criteria.
The best model of evolution for each gene region was determined with MRMODELTEST
v. 2.2 (Nylander 2004), and maximum likelihood analyses were performed in RAXML GUI v.
0.9b2 (Silvestro & Michalak 2010). The RAxML analyses were run with a rapid bootstrap analysis
of a random starting tree and 1000 ML bootstrap replicates. The search strategy was set to rapid
bootstrapping with one thousand non-parametric bootstrapping iterations using the general time
reversible model (GTR) with a discrete gamma distribution. The best scoring trees were selected
with final likelihood values. Posterior probabilities (PP) were determined by Markov Chain Monte
Carlo sampling (BMCMC) in MrBayes v. 3.0b4 (Ronquist & Huelsenbeck 2003). MrModeltest v.
2.3 (Nylander 2004) was used to perform statistical selection of the best-fit model of nucleotide
substitution and was incorporated into the analysis. Six simultaneous Markov chains were run for
1,000,000 generations, and the trees were sampled every 100th generation. The 2000 trees
representing the burn-in phase of the analyses were discarded, and the remaining 8000 trees were
used for calculating PP in the majority rule consensus tree. The fungal strains isolated in this study
are listed in Table 2 with details of the type cultures and sequence data. Novel sequence data were
deposited in GenBank (Table 2), alignments in TreeBASE (www.treebase.org, submission no.
S20936), and taxonomic novelties in the Faces of Fungi database (Jayasiri et al. 2015) and Index
Fungorum (Index Fungorum 2016).
Results
Phylogenetic analyses
The collection of saprobic specimens from numerous woody hosts in Italy (Fig. 1) resulted
in the isolation of 44 isolates of Diaporthe (Fig. 2). The ITS, TEF, BT and CAL sequences were
determined to be approximately 530, 350, 510 and 410 bp, respectively.
The combined ITS, TEF, BT and CAL sequences of Diaporthe contained data for 144
isolates, including one outgroup taxon, and consisted of 44 isolates from this study and other
sequences originating from GenBank (Table 3). Out of a total of 1998 characters, 882 were
constant, and 295 were variable and parsimony uninformative. The remaining 821 parsimony-
informative characters resulted in 10 most parsimonious trees (TL = 4190, CI = 0.464, RI = 0.883,
RC = 0.410, HI = 0.536) and the best tree is shown in Fig. 2. The maximum parsimony (MP) and
Bayesian (BM) analyses produced trees with nearly identical topologies (Bayesian tree not shown).
The isolates obtained in this study grouped into 15 distinct clades. The majority (12 isolates)
grouped with the ex-epitype isolate of Diaporthe eres (AR5193); nine isolates clustered with the
ex-epitype of D. foeniculina (CBS 111553); four isolates clustered with D. rhusicola (CBS 129528)
and another four isolates clustered with D. rudis (AR3422). Moreover, four isolates grouped each
with D. gulyae (BRIP 54025), D. novem (CBS 127270), D. ravennica (MFLUCC 15-0479) and D.
sterilis (CBS 136969). Eleven isolates did not cluster with any known Diaporthe species and thus
seven novel species, D. acericola, D. cichorii, D. dorycnii, D. lonicerae, D. pseudotsugae, D.
schoeni and D. torilicola are introduced based on morphology and phylogenetic placement (Fig. 2).
860
Figure 1 – Habitats of Diaporthe species in Italy. a Plantago sp., b Tamus communis on Rubus sp.
and Ostrya carpinifolia. c Cupressus sempervirens. d Acer negundo. e Sambucus nigra. f Amorpha
fruticosa. g Galium aparine. h land stem-leaf under Ailanthus altissima. i Ostrya carpinifolia. j
Tamus communis on Cornus sanguinea. Photos by Erio Camporesi.
Morphology and culture characteristics
All 44 isolates identified based on the phylogenetic analyses using the combined data
comprised 15 Diaporthe species (Diaporthe acericola, D. cichorii, D. dorycnii, D. eres, D.
foeniculina, D. gulyae, D. lonicerae, D. novem, D. pseudotsugae, D. ravennica, D. rhusicola, D.
rudis, D. schoeni, D. sterilis and D. torilicola) and were further characterized on the basis of colony
morphology and conidial characteristics. Growth of all isolates was rapid on PDA, with mycelia
covering the entire surface of the Petri dishes. Aerial mycelium was initially white and turned dirty
white or greyish after 4–5 days of incubation at 25 C in the dark. For all isolates, structures of the
asexual morph appeared within 2–4 weeks of incubation. Sexual structures did not form on PDA
throughout the growth period. All species showed morphological features typical of the genus.
861
862
Figure 2 – Phylogram generated from maximum likelihood analysis of Diaporthe species isolated
in this study and their phylogenetically closely related species based on combined ITS, TEF, BT
and CAL sequence data. Parsimony bootstrap support values for ML≥70 %, MP≥70 %, are
indicated above the nodes and the branches are in bold indicate Bayesian posterior probabilities
≥0.9. The tree is rooted with Diaporthella corylina (CBS 121124). Isolate numbers of ex-types and
reference strains are in bold. Taxa isolated in this study are in red and the ex-type isolate numbers
of novel species are in bold.
The new species of Diaporthe described here are phylogenetically distinct from all previously
described species for which sequence data are available.
Taxonomy
Seven undescribed species of Diaporthe were recognized by DNA sequence analysis, together with
culture morphology, and with description of anamorphic structures. Two of the novel species, D.
pseudotsugae and D. schoeni did not grow under the conditions used in this study and we could not
obtain single conidial cultures. Therefore, DNA was extracted directly from the
conidiomata/ascomata.
863
Figure 3 – Diaporthe acericola (MFLU 15-3254, holotype). a, b Conidiomata on host surface. d
Cross section of conidioma. c,e Peridium. f, g Conidia attached to conidiogenous cells. h, i Alpha
conidia. j Germinating spore. k, l Culture on PDA after one week. Scale bars: b = 0.5 mm, c–f =
100 µm, g–i = 10 µm.
864
Diaporthe acericola Dissanayake, Camporesi & K.D. Hyde, sp. nov. Fig. 3
Index fungorum number: IF553186; Facesoffungi number: FoF 03270
Etymology – The specific epithet acericola is based on the host genus (Acer).
Holotype – MFLU 15-3254
Saprobic on aerial branch and samaras of Acer negundo L. Sexual morph: Not observed. Asexual
morph: Conidiomata up to 460 μm in diameter, 285 μm high, superficial, solitary, scattered on host,
oval, black. Peridium 65–77 μm thick, inner layer composed of light brown textura angularis, outer
layer composed of dark brown textura angularis. Conidiophores 21–35 ×1.5–2.5 μm (
x
= 27 × 2
μm), cylindrical, aseptate, densely aggregated, straight or sinuous, terminal, slightly tapered
towards the apex. Conidiogenous cells 10–15 × 2–3 μm, phialidic, cylindrical, terminal and lateral.
Alpha conidia 9.7–13.5 ×3–4.5 μm (
x
= 11 × 4 μm), hyaline, fusiform or oval, both ends obtuse.
Beta conidia not observed.
Culture characteristics – Colonies on PDA covering entire Petri dishes after seven days at
25 °C, grey, with scant aerial mycelium; reverse fuscous black. Surface dirty white with profuse
aerial mycelium, reverse umber.
Material examined – ITALY, Forlì-Cesena Province, San Colombano – Meldola, on dead
aerial branches and samaras of Acer negundo (Sapindaceae), 22 January 2015, Erio Camporesi;
(MFLU 15-3254, holotype); ex-type living culture MFLUCC 17-0956.
Notes – Diaporthe acericola forms a sister clade to D. schoeni which is also a new species
introduced in this study (Fig. 2). However, the two species differed by 62 nucleotides in the
concatenated alignment, of which 13 were distinct in the ITS region, 26 in the TEF region, 2 in the
BT region and 21 in the CAL region. Morphologically, D. acericola differs from D. schoeni in
having larger conidiomata and smaller conidia (Figs 3, 8). Conidia of D. acericola are obtuse at
both ends, while the conidia of D. schoeni are slightly acute and tapered at both ends.
Diaporthe cichorii Dissanayake, Camporesi & K.D. Hyde, sp. nov. Fig. 4
Index fungorum number: IF553187; Facesoffungi number: FoF 03271
Etymology – The specific epithet cichorii is based on the host genus (Cichorium).
Holotype – MFLU 16-2168
Saprobic on dead aerial stem of Cichorium intybus L. Sexual morph: Not observed. Asexual
morph: Conidiomata up to 540 μm in diameter, 390 μm high, superficial, solitary or aggregated,
globose to oval, dark brown to black, clustered in groups of 2-5 conidiomata. Peridium 47–58 μm
thick, inner layer composed of light brown textura angularis, outer layer composed of dark brown
textura angularis. Conidiophores 24–37 × 1.5–3 μm (
x
= 29 × 3 μm), cylindrical, aseptate, densely
aggregated, straight or sinuous, terminal, slightly tapered towards the apex. Conidiogenous cells 7–
10 × 2–3 μm, hyaline, subcylindrical, filiform, straight to curved, tapering towards the apex. Alpha
conidia 10–14 × 3–4 μm (
x
= 12 × 3 μm) hyaline, fusiform or oval, both ends obtuse. Beta conidia
not observed.
Culture characteristics – Colonies on PDA flat, with an entire edge, mycelium growing in
concentric rings, cottony texture, white to smoke-grey; colonies reaching up to 64 mm diameter
after one week at 25 °C; reverse buff and isabelline.
Material examined – ITALY, Forlì-Cesena Province, Santa Sofia, on dead aerial stem of
Cichorium intybus (Asteraceae), 17 July 2016, Erio Camporesi; (MFLU 16-2168, holotype); ex-
type living culture MFLUCC 17-1023.
Notes – Diaporthe cichorii occurs in a clade separate from D. gulyae, D. cucurbitae and D.
subordinaria and differs from D. gulyae by 19 nucleotides in the concatenated alignment, in which
7 were distinct in the ITS region and 12 in the TEF region. Though the sequences of BT region and
CAL region are available for D. cichorii, the sequences of those regions are unavailable for D.
gulyae. Morphologically, the conidiomata of D. gulyae are up to 3 mm in diam, whereas in D.
cichorii they are up to 540 μm in diameter Alpha conidia of D. gulyae are smaller (6.5–9 μm)
compared to those of D. cichorii (10–14 μm).
865
Figure 4 – Diaporthe cichorii (MFLU 16-2168, holotype). a, b Conidiomata on host surface. c
Cross section of conidioma. d Peridium. e, f Conidia attached to conidiogenous cells. g Alpha
conidia. h Germinating spore. i, j Culture on PDA after one week. Scale bars: b = 1 mm, c–f = 100
µm, g = 20 µm, h = 10 µm.
866
Figure 5 – Diaporthe dorycnii (MFLU 16-1322, holotype). a, b Conidiomata on host surface. c
Cross section of conidioma. d Peridium. e, Conidia attached to conidiogenous cells. f Alpha
conidia. g Germinating conidium. h, i Culture on PDA after one week. Scale bars: b = 1 mm, c–e =
100 µm, f = 10 µm, g = 20 µm.
867
Diaporthe dorycnii Dissanayake, Camporesi & K.D. Hyde, sp. nov. Fig. 5
Index fungorum number: IF553188; Facesoffungi number: FoF 03272
Etymology – The specific epithet dorycnii is based on the host genus (Dorycnium).
Holotype – MFLU 16-1322
Saprobic on dead aerial stem of Dorycnium hirsutum L. Sexual morph: Not observed.
Asexual morph: Conidiomata up to 420 μm in diameter, 380 μm high, superficial or immersed,
solitary or gregarious, scattered on host surface, globose, dark brown to black, clustered in groups
of 2-5 conidiomata. Peridium 35–50 μm thick, inner layer composed of light brown textura
angularis, outer layer composed of dark brown textura angularis. Conidiophores 21–35 × 1.5–2.5
μm (
x
= 27 × 2 μm), cylindrical, aseptate, densely aggregated, straight or sinuous, terminal,
slightly tapered towards the apex. Conidiogenous cells 13–19 × 2–3 μm hyaline, subcylindrical and
filiform, straight, tapering towards the apex. Alpha conidia 9–13.5 × 3–4 μm (
x
= 11 × 4 μm)
hyaline, biguttulate, fusiform or oval, both ends obtuse. Beta conidia not observed.
Culture characteristics – Colonies on PDA covering entire Petri dishes after 10 days, flat,
with an entire edge, aerial mycelium forming concentric rings with cottony texture, white,
olivaceous on surface.
Material examined – ITALY, Forlì-Cesena Province, Fiumicello di Premilcuore, on dead
aerial stem of Dorycnium hirsutum (Fabaceae), 2 May 2016, Erio Camporesi; (MFLU 16-1322,
holotype); ex-type living culture MFLUCC 17-1015.
Notes – Diaporthe dorycnii occurs in a clade separate from D. diospyricola, D.
chamaeropsis and D. cytosporella with high bootstrap support (Fig. 2). Diaporthe diospyricola
differs from D. dorycnii, in the presence of beta conidia. Phylogenetically, D. diospyricola is the
closest species to D. dorycnii (Fig. 2), differing by 24 nucleotides in the ITS region. Though the
sequences of EF region, BT region and CAL region are available for D. dorycnii, the sequences of
those regions are unavailable for D. diospyricola and thus the nucleotide comparison is incomplete.
Diaporthe lonicerae Dissanayake, Camporesi & K.D. Hyde, sp. nov. Fig. 6
Index fungorum number: IF553189; Facesoffungi number: FoF 03273
Etymology – The specific epithet lonicerae is based on the host genus (Lonicera).
Holotype – MFLU 15-3511
Saprobic on dead aerial branch of Lonicera sp. Sexual morph: Not observed. Asexual
morph: Conidiomata up to 680 μm in diameter, superficial, solitary, scattered on PDA, globose,
dark brown to black, clustered in groups of 2–5 pycnidia. Peridium 15–60 μm thick, inner layer
composed of light brown textura angularis, outer layer composed of dark brown textura angularis.
Conidiophores 21–35 × 1.5–2.5 μm (
x
= 27 × 2 μm), cylindrical, aseptate, densely aggregated,
straight or sinuous, terminal, slightly tapered towards the apex.
Conidiogenous cells 8–11 × 2–3 μm hyaline, subcylindrical, straight to curved, tapering
towards the apex. Alpha conidia 12.5–16 × 3.5–4 μm (
x
= 14.5 × 4 μm) hyaline, biguttulate,
fusiform or oval, both ends obtuse. Beta conidia 32–39 × 1–1.5 μm (
x
= 36 × 1.5 μm) hyaline,
aseptate, filiform, hamate, tapering towards both ends.
Culture characteristics – Colonies on PDA covering entire Petri dishes after 10 days, flat,
with an entire edge, aerial mycelium forming irregular concentric rings with cottony texture,
olivaceous-buff, isabelline to honey on surface.
Material examined – ITALY, Forlì-Cesena Province, Predappio Alta, on dead aerial branch
of Lonicera sp. (Caprifoliaceae), 28 Febrary 2015, Erio Camporesi; (MFLU 15-3511, holotype);
ex-type living culture MFLUCC 17-0963.
Notes – Diaporthe lonicerae clusters closer to D. saccarata, D. canthi and D. hickoriae.
Phylogenetically, D. saccarata is the closest species to D. lonicerae (Fig. 2), differing by 107
nucleotides in the concatenated alignment, in which 19 were distinct in the ITS region, 34 in the
TEF region, 16 in the BT region and 38 in the CAL region. Both species possess beta conidia and
morphologically, D. saccarata differs from D. lonicerae, in having 1-septate alpha conidia (Mostert
et al. 2001).
868
Figure 6 – Diaporthe lonicerae (MFLU 15-3511, holotype). a–c Conidiomata on host surface. d
Cross section of conidioma. e Peridium. f Alpha conidium attached to conidiogenous cells. g Alpha
conidia. h Beta conidium. i Germinating conidium. j, k Culture on PDA after two weeks. Scale
bars: b, c = 1 mm, d, e = 100 µm, f, g = 15 µm.
869
Figure 7 – Diaporthe pseudotsugae (MFLU 15-3228, holotype). a, b Ascomata on host surface. c
Cross section of ascoma. d Peridium. e Immature ascus. f Immature ascus immersed in Indian ink.
g Mature ascus. h Mature ascus mounted in methylene blue. i Cluster of immature asci. j ascospore.
Scale bars: b = 1 mm, c, d = 100 µm, e–i = 30 µm, j = 20 µm.
Diaporthe pseudotsugae Dissanayake, Camporesi & K.D. Hyde, sp. nov. Fig. 7
Index fungorum number: IF553190; Facesoffungi number: FoF 03274
Etymology – The specific epithet pseudotsugae is based on the host genus (Pseudotsuga).
Holotype – MFLU 15-1274
870
Saprobic on dead land cones of Pseudotsuga menziesii (Mirb.). Sexual morph: Ascomata up
to 465 μm in diameter, 255 μm high, black, globose to oval, clustered in groups, deeply immersed
in host tissue protruding through substrata. Peridium 28–42 μm thick, inner layer composed of light
brown textura angularis, outer layer composed of dark brown textura angularis. Asci 60–85 × 21–
37 μm (
x
= 75 × 29 μm), unitunicate, 8-spored, sessile, elongate to clavate. Ascospores 19–21 × 6–
8 μm (
x
= 20 × 7 μm), hyaline, two-celled, often 4-guttulate, with larger guttules at centre and
smaller ones at the ends, elongated to elliptical. Asexual morph: Not observed.
Material examined – ITALY, Forlì-Cesena Province, Premilcuore, on dead land cones of
Pseudotsuga menziesii (Pinaceae), 10 April 2015, Erio Camporesi; (MFLU 15-1274, holotype).
Notes – We could not obtain a culture from single ascospore. Therefore, DNA was
extracted directly from the ascomata. Diaporthe pseudotsugae occurs in a clade separate from D.
salicicola, D. cynaroidis, D. cassines and D. nothofagi. Although D. pseudotsugae is a sexual
morph, none of the above mentioned species possess any sexual morph. Phylogenetically, D.
cassines is the closest species to D. pseudotsugae (Fig. 2), differing by 64 nucleotides in the
concatenated alignment, in which 34 were distinct in the ITS region, 30 in the TEF region. Though
the sequences of BT region and CAL region are available for D. pseudotsugae, the sequences of
those regions are unavailable for D. cassines.
Diaporthe schoeni Dissanayake, Camporesi & K.D. Hyde, sp. nov. Fig. 8
Index fungorum number: IF553191; Facesoffungi number: FoF 03275
Etymology – The specific epithet schoeni is based on the host genus (Schoenus).
Holotype – MFLU 15-1279
Saprobic on dead aerial stem of Schoenus nigricans L. Sexual morph: Not observed.
Asexual morph: Conidiomata up to 210 μm in diameter, 110 μm high, immersed, solitary or
gregarious, scattered on host surface, globose to oval, dark brown to black. Peridium 9–32 μm
thick, inner layer composed of light brown textura angularis, outer layer composed of dark brown
textura angularis. Conidiophores absent, Conidiogenous cells 21–35 × 1.5–2.5 μm (
x
= 27 × 2
μm), cylindrical, aseptate, densely aggregated, straight or sinuous, terminal, slightly tapered
towards the apex. Alpha conidia 11–14.5 × 2–3 μm (
x
= 13.5 × 3 μm), hyaline, fusiform or oval,
both ends slightly acute and tapered. Beta conidia 21–33 × 1–1.5 μm (
x
= 27 × 1.5 μm), rarely
found among alpha conidia, hyaline, aseptate, filiform, hamate, tapering towards both ends.
Material examined – ITALY, Ravenna Province, Lido di Dante, on dead aerial stem of
Schoenus nigricans (Cyperaceae), 1 May 2015, Erio Camporesi; (MFLU 15-1279, holotype).
Notes – We could not obtain a culture from single conidia. Therefore, fungal DNA was
extracted directly from the conidiomata. Three isolates of D. schoeni were isolated from three
different hosts, Carduus sp. (Asteraceae), Plantago sp. (Plantaginaceae) and Schoenus nigricans
(Cyperaceae). However, any of those isolates were failed to germinate. Diaporthe schoeni occurs
in a clade closer to D. acericola (Fig. 2). Both species can be differentiated by smaller conidiomata
and larger conidia of D. schoeni. Conidia of D. acericola are obtuse at both ends, while the conidia
of D. schoeni are slightly acute and tapered at both ends (Figs 3, 8). Phylogenetically, D. schoeni
differs from D. acericola by 62 nucleotides in the concatenated alignment, of which 13 were
distinct in the ITS region, 26 in the TEF region, 2 in the BT region and 21 in the CAL region.
Diaporthe torilicola Dissanayake, Camporesi & K.D. Hyde, sp. nov. Fig. 9
Index fungorum number: IF553192; Facesoffungi number: FoF 03276
Etymology – The specific epithet torilicola is based on the host genus (Torilis).
Holotype – MFLU 16-1166
Pathogenic on Torilis arvensis (Huds.). Sexual morph: Not observed. Asexual morph:
Conidiomata up to 300 μm in diameter, superficial, solitary, scattered on PDA, globose, dark brown
to black, clustered in groups of 2–5 pycnidia. Peridium 16–20 μm thick, inner layer composed of
light brown textura angularis, outer layer composed of dark brown textura angularis.
Conidiophores 21–35 × 1.5–2.5 μm (
x
= 27 × 2 μm), cylindrical, aseptate, densely aggregated,
871
Figure 8 – Diaporthe schoeni (MFLU 15-1279, holotype). a–c Conidiomata on host surface. d
Cross section of conidiomata. e Peridium. f, g Alpha conidia. h Alpha conidia with a beta
conidium. Scale bars: b, c = 1 mm, d,e = 100 µm, f–h = 15 µm.
straight or sinuous, terminal, slightly tapered towards the apex. Alpha conidia 6–8.5 × 2–3 μm (
x
=
8 × 3 μm) hyaline, biguttulate, fusiform or oval, both ends obtuse. Beta conidia 18–37 ×1–1.5 μm
(
x
= 27 × 1.5 μm) hyaline, aseptate, filiform, hamate, guttulate, tapering towards both ends.
Culture characteristics – Colonies on PDA covering entire Petri dishes after 10 days, grey,
with scant aerial mycelium; reverse fuscous black. Colonies on PDA flat, with entire edge, cottony,
olivaceous buff, with aerial mycelium in concentric rings, with olivaceous patches; colonies
reaching entire petri dish after 2 wk at 25 °C; reverse olivaceous buff and greenish olivaceous.
872
Figure 9 – Diaporthe torilicola (MFLU 16-1166, holotype). a, b Conidiomata on host surface. c
Cross section of conidiomata. d–f Conidia attached to conidiogenous cells. g Alpha conidia. h, i
Culture on PDA after one week. Scale bars: a, b = 0.5 mm, c–e = 100 µm, f, g = 10 µm.
873
Material examined – ITALY, Forlì-Cesena Province, Monte Pallareto - Meldola dead aerial
stem of Torilis arvensis (Apiaceae), 12 April 2016, Erio Camporesi; (MFLU 16-1166, holotype);
ex-type living culture MFLUCC 17-1051.
Notes – In the phylogenetic analysis, D. torilicola forms a sister clade to D. toxica.
Williamson et al. (1994) designated the name D. toxica for the sexual state of the toxicogenic
variety, P. leptostromiformis var. leptostromiformis. Phylogenetically, D. toxica differs from D.
torilicola by 92 nucleotides in the concatenated alignment, in which 26 were distinct in the ITS
region, 27 in the TEF region, 17 in the BT region and 22 in the CAL region.
Discussion
Studies on Diaporthe, dealing with the phylogenetic traits and morphology of isolates
associated with various hosts, have increased in recent years, enabling the worldwide identification
of taxa at the species level (Gomes et al. 2013, Udayanga et al. 2014a, b). In this study, seven new
species have been described in Diaporthe, on the basis of morphological and molecular
characteristics. Two of the novel species, D. pseudotsugae and D. schoeni did not grow under the
conditions of this study and single spore isolates could not be obtained. In addition to the new
species, eight known species of Diaporthe (D. eres, D. foeniculina, D. gulyae, D. novem, D.
ravennica, D. rhusicola, D. rudis and D. sterilis) were identified. Apart from D. eres, D.
foeniculina and D. ravennica; none of the other species were identified in previous studies on
Italian hosts, which probably implies an association with geographic origin and/or host species. A
phylogenetic tree derived from an alignment of ITS sequences is beneficial as a guide for
identification of isolates of Diaporthe species (Udayanga et al. 2012, Tan et al. 2013). ITS
sequences offer convincing proof for species demarcation where a limited number of taxa are
analyzed, such as species associated with the same host (Santos & Phillips 2009, Santos et al. 2011,
Thompson et al. 2011). However, confusion arises when a large number of species from an
extensive range of host species are examined. Santos et al. (2010) proposed that TEF is a superior
phylogenetic marker in Diaporthe than ITS, and has been commonly used as a secondary locus for
phylogenetic studies (Santos et al. 2011, Udayanga et al. 2012, Dissanayake et al. 2015). Gomes et
al. (2013) studied five loci from 95 species. They stated that TEF poorly distinguished species, and
recommended that histone and BT were suitable possibilities as subordinate phylogenetic markers
to accompany the authorized fungi barcode, ITS. In this study, a combined four gene analyses of
ITS, TEF, BT and CAL was used to study eight known Diaporthe species and to assist in the
introduction of seven new Diaporthe species.
Diaporthe eres was the most frequent species in the present study, comprising 27% of the
isolates, and was associated with Galega officinalis (Fabaceae), Juglans regia (Juglandaceae),
Lonicera sp. (Caprifoliaceae), Ostrya carpinifolia (Betulaceae), Picea excels (Pinaceae), Pinus
pinaster (Pinaceae), Populus nigra (Salicaceae), Rhamnus alpinus (Rhamnaceae), Salix caprea
(Salicaceae), Sambucus nigra (Adoxaceae), Sanguisorba minor (Rosaceae) and Sonchus oleraceus
(Asteraceae) in the provinces of Arezzo and Forlì-Cesena (Table 2). In all of the studies conducted
in Italy, involving gene sequencing, this species was detected as the most common, but not the
most virulent (Gomes et al. 2013, Cinelli et al. 2016, Udayanga et al. 2015). Phylogenetic studies
indicated that, as well as the aforementioned phenomenon, there is low posterior probability
support between the internal branches of the D. eres clade, indicating a large intraspecific diversity
in this species (Gomes et al. 2013, Dissanayake et al. 2017a, b). After several phylogenetic studies
of D. eres, from 2005 to the present day, including sampled plants and areas previously unexplored,
it was shown how this morphological species is complex, harbouring several cryptic species with
various hosts in different geographical locations (Crous 2005, Gao et al. 2016, Gomes et al. 2013,
Dissanayake et al. 2015, 2017a, Udayanga et al. 2014b).
Diaporthe foeniculina was the second most common species, with 20% of the isolates in
this study, and was associated with Achillea millefolium (Asteraceae), Ailanthus altissima
(Simaroubaceae), Arctium minus (Asteraceae), Cupressus sepervirens (Cupressaceae),
Hemerocallis fulva (Hemerocallidoiceae), Lunaria rediviva (Brassicaceae), Melilotus officinalis
874
(Fabaceae), Vicia sp. (Fabaceae) and Wisteria sinensis (Fabaceae) in the provinces of Arezzo and
Forlì-Cesena (Table 2). Recently, D. foeniculina was epitypified by Udayanga et al. (2014a) and
the utility of individual genes for accurate circumscription of this species was assessed. Diaporthe
foeniculina, including the synonym D. neotheicola, is recognized as a species with an extensive
host range (Udayanga et al. 2014a). Regarding D. neotheicola, this species has been reported to
cause diseases of temperate and tropical fruits in Australia, Europe and South Africa (Golzar et al.
2012, Thomidis et al. 2013).
Diaporthe rudis was isolated from Anthoxanthum odoratum (Poaceae), Carlina vulgaris
(Asteraceae), Cornus sp. (Cornaceae) and Dioscorea communis (Dioscoreaceae). Since its
description, this species has been identified around the world as being associated with numerous
hosts (Udayanga et al. 2014a, Chen et al. 2014a, b, Huang et al. 2015, Lombard et al. 2014), which
highlights its high degree of dissemination, distribution and wide host range, similar to D. eres.
Udayanga et al. (2014a) determined D. viticola to be a synonym of D. rudis, which was previously
recognized as a distinct taxon.
Diaporthe rhusicola occurred at the same frequency as D. rudis, with 9% of isolates taken
from dead aerial stem or branch in Amorpha fruticosa (Fabaceae), Angelica sylvestris (Apiaceae),
Platanus hybrida (Platanaceae) and Rubus sp. (Rosaceae). Diaporthe rhusicola was described and
first reported in South Africa as causing leaf spots of Rhus pendulina (Crous et al. 2011) and was
subsequently proved to be pathogenic in English walnut (Chen et al. 2014b) and pistachio in
California (Chen et al. 2014a). The other known Diaporthe species (D. gulyae, D. novem, D.
ravennica and D. sterilis) were isolated from Italian hosts Heracleum sphondylium (Apiaceae),
Galium sp. (Rubiaceae), Salvia sp. (Lamiaceae) and Cytisus sp. (Fabaceae) respectively.
Except for D. lonicerae and D. schoeni, all other novel species identified in this study were
associated with only one host (Table 2). Diaporthe acericola, D. cichorii, D. dorycnii, D.
pseudotsugae, and D. torilicola were isolated from Acer negundo (Sapindaceae), Cichorium
intybus (Asteraceae), Dorycnium hirsutum (Fabaceae), Pseudotsuga menziesii (Pinaceae) and
Torilis arvensis (Apiaceae) respectively. Diaporthe lonicerae was isolated from Lonicera sp.
(Caprifoliaceae), Laurus nobilis (Lauraceae) and Torilis arvensis (Apiaceae) in Forlì-Cesena
province, while D. schoeni was isolated from Schoenus nigricans (Cyperaceae), Carduus sp.
(Asteraceae) and Plantago sp. (Plantaginaceae) in Arezzo, Forlì-Cesena and Ravenna provinces.
The discovery of these species of Diaporthe on diverse hosts and in different geographical
localities in Italy as well as worldwide shows the polyphagous and cosmopolitan behavior of
species in this genus. Certainly, it is obvious that performing complementary studies based on
sequencing at least four gene regions of Diaporthe species is essential in order to support reliable
species identification. Such studies are necessary to investigate this group of fungi in different
unexploited biomes, to reveal the degree of diversity and to support more suitable control measures
to prevent their dissemination.
Acknowledgements
This work was financed by JNKYT201605, Innovation funds of IPEP, BAAFS and CARS-
30.
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