Diaporthe species on Rosaceae with descriptions of D. pyracanthae sp. nov. and D. malorum sp. nov.

Abstract

The family Rosaceae includes a large number of species ranging from herbaceous (Fragaria) to ornamental plants (Rosa and Pyracantha) and fruit trees (Malus and Pyrus). Diaporthe species have been associated with twig canker, shoot blight, dieback, wood decay and fruit rot on members of the Rosaceae. In this study a collection of isolates from several Rosaceae hosts were characterised by multi-locus sequence analyses using the internal transcribed spacer, translation elongation factor 1-alpha, beta-tubulin, histone H3 and calmodulin loci. The phylogenetic analyses of the combined five loci revealed that the isolates studied were distributed among four clades, of which two correspond to D. foeniculina and D. eres. The other two clades, closely related to D. passiflorae and D. leucospermi represent two new species, D. pyracanthae sp. nov. and D. malorum sp. nov., respectively. Further, pathogenicity assays have shown that of the four species tested, D. malorum was the most aggressive species on apple fruit and D. eres was the most aggressive species on detached pear twigs. A revision of all Diaporthe (and Phomopsis) names that have been associated with Rosaceae hosts as well as their current status as pathogens of members of this family is presented.

Full text

Submitted 24 January 2017, Accepted 3 March 2017, Published 12 March 2017
Corresponding Author: Artur Alves – e-mail – artur.alves@ua.pt 485
Diaporthe species on Rosaceae with descriptions of D. pyracanthae sp.
nov. and D. malorum sp. nov.
Santos L1, Phillips AJL2, Crous PW3 and Alves A1
1 Departamento de Biologia, CESAM, Universidade de Aveiro, 3810-193 Aveiro, Portugal
2 Biosystems and Integrative Sciences Institute, Faculty of Science, University of Lisbon, Campo Grande, 1749-016
Lisbon, Portugal
3 Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
Santos L, Phillips AJL, Crous P, Alves A 2017 – Diaporthe species on Rosaceae with descriptions
of D. pyracanthae sp. nov. and D. malorum sp. nov. Mycosphere 8(5), 485–511, Doi
10.5943/mycosphere/8/5/2
Abstract
The family Rosaceae includes a large number of species ranging from herbaceous
(Fragaria) to ornamental plants (Rosa and Pyracantha) and fruit trees (Malus and Pyrus).
Diaporthe species have been associated with twig canker, shoot blight, dieback, wood decay and
fruit rot on members of the Rosaceae. In this study a collection of isolates from several Rosaceae
hosts were characterised by multi-locus sequence analyses using the internal transcribed spacer,
translation elongation factor 1-alpha, beta-tubulin, histone H3 and calmodulin loci. The
phylogenetic analyses of the combined five loci revealed that the isolates studied were distributed
among four clades, of which two correspond to D. foeniculina and D. eres. The other two clades,
closely related to D. passiflorae and D. leucospermi represent two new species, D. pyracanthae sp.
nov. and D. malorum sp. nov., respectively. Further, pathogenicity assays have shown that of the
four species tested, D. malorum was the most aggressive species on apple fruit and D. eres was the
most aggressive species on detached pear twigs. A revision of all Diaporthe (and Phomopsis)
names that have been associated with Rosaceae hosts as well as their current status as pathogens of
members of this family is presented.
Key words – Malus – Pathogenicity – Phylogeny – Pyracantha – Pyrus
Introduction
The family Rosaceae is a large family of flowering plants that includes approximately 3000
species and 90 genera of herbs, shrubs and trees (Potter et al. 2007). This family includes
herbaceous (Fragaria), medicinal (Agrimonia, Crataegus, Filipendula) and ornamental plants
(Rosa, Pyracantha), shrubs (Rubus) and fruit trees (Eriobotrya, Cydonia, Hesperomeles, Malus,
Prunus, Pyrus). Some of the species are cultivated worldwide and are economically important such
as Fragaria (strawberry), Malus (apple), Prunus (cherry, almond, peach, and plum), Pyrus (pear)
and Rubus (blackberry and raspberry) (Hummer & Janick 2009).
Diaporthe species are saprobes, endophytes, or plant pathogens (Webber & Gibbs 1984,
Boddy & Griffith 1989, Udayanga et al. 2011). Some species of Diaporthe have been associated
with twig canker, bud and shoot blight, dieback, wood decay and fruit rot of almond (Adaskaveg et
al. 1999, Diogo et al. 2010, Gramaje et al. 2012); canker, shoot dieback, bud and shoot blight of
Mycosphere 8(5) 485–511 (2017) www.mycosphere.org ISSN 2077 7019
Article (Special Issue)
Doi 10.5943/mycosphere/8/5/2
Copyright © Guizhou Academy of Agricultural Sciences

486
peach (Latham et al. 1992, Ogawa et al. 1995, Smit et al. 1996, Uddin et al. 1997, 1998, Farr et al.
1999, Thomidis & Michailides 2009); cankers and shoot blight of apple (Roberts 1913, Fujita et al.
1988, Smit et al. 1996, Abreo et al. 2012); dieback and canker of pear and plum (Sakuma et al.
1982, Nakatani et al 1984, Kobayashi & Sakuma 1982, Ogawa et al. 1995, Uddin et al. 1998).
Identification of Diaporthe species was originally based on an approach that combined
morphological features, cultural characteristics, and host affiliation (Udayanga et al. 2011). This
resulted in an unnecessary inflation in the number of Diaporthe species names, which currently
stands at 977 and 1099 for Diaporthe and 980 and 1047 for Phomopsis (asexual synonym of
Diaporthe) in Index Fungorum and MycoBank, respectively (both accessed 14 November 2016).
Thus, there was an urgent need to reformulate species delimitation in the genus Diaporthe because
accurate species identification is essential for understanding epidemiology, controlling plant
diseases, and to provide correct advice in the implementation of phytosanitary measures (Santos &
Phillips 2009, Udayanga et al. 2011).
Over the last years, multi-loci phylogenetic analyses have routinely been used for species
reassessment in Diaporthe (Santos & Phillips 2009, Thompson et al. 2011, Baumgartner et al.
2013, Gomes et al. 2013, Huang et al. 2013, Tan et al. 2013, Gao et al. 2014, Udayanga et al.
2014a, 2014b). The sequences most frequently used are the internal transcribed spacer (ITS) of the
ribosomal DNA, translation elongation factor 1-α (TEF1), ß-tubulin (TUB), histone (HIS),
calmodulin (CAL), actin and DNA-lyase (Gomes et al. 2013, Huang et al. 2013, Gao et al. 2014,
Udayanga et al. 2014a, 2014b, Wang et al. 2014). In general, these studies show that multi-loci
phylogenies provide higher resolution for Diaporthe species than single locus phylogenies
(Udayanga et al. 2012a, 2012b, Huang et al. 2013).
In this study a set of isolates obtained from different Rosaceae hosts was characterised based
on morphology, pathogenicity and multi-loci sequence data (ITS, TEF1, HIS, TUB and CAL). In
addition, a review of Diaporthe species occurring on Rosaceae and their current status as pathogens
of members in this plant family is presented.
Materials & Methods
Fungal isolation and morphological characterisations
Diaporthe species were isolated, between 2007 and 2014, from the following Rosaceae hosts:
Malus domestica fruits, collected in a local orchard, with post-harvest fruit rot; Pyrus communis,
and Pyracantha coccinea with twig cankers in Portugal and Prunus cerasus with twig cankers in
Russia (Table 1). Single spore isolates were obtained as described previously (Santos & Phillips
2009). In addition, isolations were made by directly plating out pieces of surface sterilized diseased
tissue (5–10 mm2) on potato dextrose agar (PDA) (Merck, Germany). Plant tissue was surface
sterilised in 5 % sodium hypochlorite for 1 minute followed by 96 % ethanol for 1 minute and
rinsed in sterile water for 1 minute. The plates were incubated at room temperature and checked
regularly for fungal growth. All Diaporthe isolates were transferred to half strength potato dextrose
agar (½ PDA) (Merck, Germany) and pure cultures were established.
Isolates were induced to sporulate by plating them on 2 % water agar (Merck, Germany)
containing sterilised fennel twigs or pine needles and incubating at room temperature (about 20–25
°C) where they received diffused daylight. Pycnidia were mounted in 100 % lactic acid and
morphological characters of the conidia and mode of conidiogenesis observed with a Nikon 80i
compound microscope (Nikon, Japan) and photographed with a Nikon Digital Sight DS-Ri1 camera
(Nikon, Japan).
Temperature growth studies
One plate of ½ PDA per strain of each novel species described was inoculated and incubated
for 7 days at 25 °C. From these cultures, a 5-mm diam. plug for each strain was placed in the centre
of PDA plates. Three replicate plates per strain were incubated at 5, 15, 20, 25, 30, 35 and 40 °C.

487
Table 1 Diaporthe isolates from Rosaceae used in this study.
Species
Strain
Host
Symptoms
Country
Accession Number
Mating genes
ITS
TEF1
TUB
HIS
CAL
MAT1
MAT2
D. foeniculina
CAA133
Pyrus communis
branch canker
Portugal
KY435634
KY435624
KY435665
KY435645
KY435655
-
+
CAA135
Pyrus communis
branch canker
Portugal
-
+
CAA136
Pyrus communis
branch canker
Portugal
-
+
CAA137
Pyrus communis
branch canker
Portugal
-
+
CAA737
Malus domestica
fruit rot
Portugal
KY435641
KY435628
KY435669
KY435649
KY435659
+
-
CAA738
Malus domestica
fruit rot
Portugal
+
-
CAA739
Malus domestica
fruit rot
Portugal
+
-
D. pyracanthae
CAA481
Pyracantha coccinea
branch canker
Portugal
-
+
CAA482
Pyracantha coccinea
branch canker
Portugal
-
+
CAA483
Pyracantha coccinea
branch canker
Portugal
KY435635
KY435625
KY435666
KY435645
KY435656
-
+
CAA484
Pyracantha coccinea
branch canker
Portugal
-
+
CAA485
Pyracantha coccinea
branch canker
Portugal
-
+
CAA486
Pyracantha coccinea
branch canker
Portugal
-
+
CAA487
Pyracantha coccinea
branch canker
Portugal
KY435636
KY435626
KY435667
KY435647
KY435657
-
+
CAA488
Pyracantha coccinea
branch canker
Portugal
KY435637
-
+
D. malorum
CAA734
Malus domestica
fruit rot
Portugal
KY435638
KY435627
KY435668
KY435648
KY435658
-
+
CAA735
Malus domestica
fruit rot
Portugal
KY435639
-
+
CAA736
Malus domestica
fruit rot
Portugal
KY435640
-
+
CAA740
Malus domestica
fruit rot
Portugal
KY435642
KY435629
KY435670
KY435650
KY435660
-
+
CAA752
Malus domestica
fruit rot
Portugal
KY435643
KY435630
KY435671
KY435651
KY435661
-
+
CAA753
Malus domestica
fruit rot
Portugal
-
+
CAA754
Malus domestica
fruit rot
Portugal
-
+
D. eres
CAA801
Prunus cerasus
branch canker
Russia
KY435644
KY435631
KY435672
KY435652
KY435662
-
+
Petri plates were examined daily for 14 days and colony diameters were measured with a caliper in two directions at right angles to each other until the
colony reached the edge of the plate.
DNA extraction and PCR fingerprinting
Isolates were grown on ½ strength PDA for 5 days at 25ºC. DNA was extracted according to Möller et al. (1992). PCR fingerprinting of the
isolates was performed using primer BOXA1R as described previously (Alves et al. 2007).

488
PCR amplification and sequencing
For this study 5 loci (ITS, TEF1, HIS, TUB and CAL) were amplified and sequenced. The
primers ITS5 and NL4 (White et al. 1990, Vilgalys & Hester 1990) were used to amplify ITS with
PCR conditions of 5 min at 95 ºC, followed by 30 cycles of 94 ºC for 30 s, 55 ºC for 30 s, 72 ºC for
1.5 min, and a final elongation step at 72 ºC for 10 min. TEF1 was amplified with the primers EF1-
688F and EF1-1251R (Alves et al. 2008). The primers T1 and Bt2b (Glass & Donaldson 1995,
O’Donnell & Cigelnik 1997) were used to sequenced part of the TUB gene, while CYLH3F and
H3-1b (Glass & Donaldson 1995, Crous et al. 2004) were used to amplify the HIS gene and CAL-
228F and CAL-737R (Carbone & Kohn 1999) were used to amplify part of the CAL gene.
All PCR reactions were carried out with NZYtaq 2× green Master Mix from Nzytech
(Lisbon, Portugal), in a Bio-Rad C1000 touch thermal cycler (Hercules, CA, USA). PCRs were
performed in 25 µl reaction mixtures containing 6.25 µl Master Mix, 15.75 µl purified water, 1 µl
of each primer (10 pmol) and 1 µl of purified template DNA. The PCR conditions for TEF, TUB,
HIS and CAL were 5 min at 95°C; followed by 30 cycles at 94°C for 30 s, 52°C, 60ºC and 53º C
for 30 s (for TEF/TUB, HIS and CAL, respectively), 72°C for 1 min; and then a final elongation
step at 72 ºC for 10 min.
Amplicons were purified with DNA Clean & ConcentrorTM 5 (Zymo Research, Irvine,
USA) following the manufacturer’s instructions. The amplicons were sequenced by GATC Biotech
(Germany). The new sequences obtained in this study were deposited in GenBank (Table 1).
Mating-type assay
The mating strategy of all isolates (Table 1) (heterothallic or homothallic) was determined by
a PCR-based mating type assay using the primers DiaMAT1F/DiaMAT1R for MAT1-1 and
DiaMAT2F/DiaMAT2R for MAT1-2 developed by Santos et al. (2010). Part of the alpha box
domain of the MAT1-1-1 gene and part of the HMG domain from the MAT1-2-1 gene were
amplified as described previously (Santos et al. 2010).
Phylogenetic analysis
A multi-locus phylogenetic analysis based on combined sequences of 5 genes (ITS, TEF1,
HIS, TUB and CAL) was performed. This analysis included all Diaporthe species found on
Rosaceae for which there were sequences available for the 5 loci as well as D. leucospermi and D.
passiflorae which were closely related to some of our isolates based on a BLASTn seach (Table 2).
Sequences were aligned with ClustalX v. 2.1 (Larkin et al. 2007) using the following parameters:
pairwise alignment parameters (gap opening = 10, gap extension = 0.1) and multiple alignment
parameters (gap opening = 10, gap extension = 0.2, transition weight = 0.5, delay divergent
sequences = 25%). The alignments were optimized manually with BioEdit (Hall 1999). MEGA v. 6
(Tamura et al. 2013) was used to create and analyse Maximum Likelihood (ML) phylogenetic trees
for these alignments (Li 1997). MEGA v. 6 was also used to determine the best substitution model
to be used to build the ML tree. ML analysis was performed on a NJ starting tree automatically
generated by the software. Nearest-Neighbour-Interchange (NNI) was used as the heuristic method
for tree inference with 1,000 bootstrap replicates. Diaporthe toxica was used as outgroup for the
multi-locus phylogenetic analysis. Alignments and trees were deposited in TreeBase (Study
Accession: S20345).
Pathogenicity tests
One representative isolate of each Diaporthe species identified (CAA487 – D. pyracanthae,
CAA737 – D. foeniculina, CAA740 – D. malorum and CAA801 – D. eres) were used for
pathogenicity assays on detached twigs of Pyrus communis and fruits of Malus domestica. For
inoculum preparation, fungi were grown on PDA ½ plates for 7 days at 25 °C.

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Table 2 Diaporthe isolates used in multi-locus sequence analysis. In bold are ex-type or ex-epitype or isotype isolates.
Species
Strain
Host
Host Family
Country
Gen Bank Accession Number
ITS
TEF1
TUB
HIS
CAL
Diaporthe ambigua
CBS 114015
Pyrus communis
Rosaceae
South Africa
KC343010
KC343736
KC343978
KC343494
KC343252
Diaporthe amygdali
CBS 115620
Prunus persica
Rosaceae
USA
KC343020
KC343746
KC343988
KC343504
KC343262
CBS 120840
Prunus salicina
Rosaceae
South Africa
KC343021
KC343747
KC343989
KC343505
KC343263
CBS 126679
Prunus dulcis
Rosaceae
Portugal
KC343022
KC343748
KC343990
KC343506
KC343264
CBS 126680
Prunus dulcis
Rosaceae
Portugal
KC343023
KC343749
KC343991
KC343507
KC343265
Diaporthe crataegi
CBS 114435
Crataegus oxyacantha
Rosaceae
Sweden
KC343055
KC343781
KC344023
KC343539
KC343297
Diaporthe eres
AR3669
Pyrus pyrifolia
Rosaceae
Japan
JQ807466
JQ807415
KJ420808
KJ420859
KJ435002
AR3670
Pyrus pyrifolia
Rosaceae
Japan
JQ807467
JQ807416
KJ420807
KJ420858
KJ435001
AR3671
Pyrus pyrifolia
Rosaceae
Japan
JQ807468
JQ807417
KJ420814
KJ420865
KJ435017
AR3672
Pyrus pyrifolia
Rosaceae
Japan
JQ807469
JQ807418
KJ420819
KJ420868
KJ435023
AR3723
Rubus fruticosus
Rosaceae
Austria
JQ807428
JQ807354
KJ420793
KJ420843
KJ435024
AR4346
Prunus mume
Rosaceae
Korea
JQ807429
JQ807355
KJ420823
KJ420872
KJ435003
AR4348
Prunus persici
Rosaceae
Korea
JQ807431
JQ807357
KJ420811
KJ420862
KJ435004
AR4355
Prunus sp.
Rosaceae
Korea
JQ807433
JQ807359
KJ420797
KJ420848
KJ435035
AR4363
Malus sp.
Rosaceae
Korea
JQ807436
JQ807362
KJ420809
KJ420860
KJ435033
AR4367
Prunus sp.
Rosaceae
Korea
JQ807438
JQ807364
KJ420824
KJ420873
KJ435019
AR4369
Pyrus pyrifolia
Rosaceae
Korea
JQ807440
JQ807366
KJ420813
KJ420864
KJ435005
AR4371
Malus pumila
Rosaceae
Korea
JQ807441
JQ807367
KJ420796
KJ420847
KJ435034
CBS 287.74
Sorbus aucuparia
Rosaceae
Netherlands
KC343084
KC343810
KC344052
KC343568
KC343326
CBS 375.61
Malus sylvestris
Rosaceae
-
KC343088
KC343814
KC344056
KC343572
KC343330
CBS 439.82
Cotoneaster sp.
Rosaceae
UK
KC343090
KC343816
KC344058
KC343574
KC343332
CBS 138594
Ulmus laevis
Ulmaceae
Germany
KJ210529
KJ210550
KJ420799
KJ420850
KJ434999
DNP128
Castaneae mollissimae
Fagaceae
China
JF957786
KJ210561
KJ420801
KJ420852
KJ435040
DP0177
Pyrus pyrifolia
Rosaceae
New Zealand
JQ807450
JQ807381
KJ420820
KJ420869
KJ435041
DP0179
Pyrus pyrifolia
Rosaceae
New Zealand
JQ807452
JQ807383
KJ420803
KJ420854
KJ435028
DP0180
Pyrus pyrifolia
Rosaceae
New Zealand
JQ807453
JQ807384
KJ420804
KJ420855
KJ435029
DP0590
Pyrus pyrifolia
Rosaceae
New Zealand
JQ807464
JQ807394
KJ420810
KJ420861
KJ435037
DP0591
Pyrus pyrifolia
Rosaceae
New Zealand
JQ807465
JQ807395
KJ420821
KJ420870
KJ435018
FAU483
Malus sp.
Rosaceae
Netherlands
KJ210537
JQ807422
KJ420827
KJ420874
KJ435022
CBS 116953
Pyrus pyrifolia
Rosaceae
New Zealand
KC343147
KC343873
KC344115
KC343631
KC343389
CBS 116954
Pyrus pyrifolia
Rosaceae
New Zealand
KC343148
KC343874
KC344116
KC343632
KC343390
CBS 124030
Malus pumila
Rosaceae
New Zealand
KC343149
KC343875
KC344117
KC343633
KC343391
Diaporthe foeniculina
CBS 123208
Foeniculum vulgare
Apiaceae
Portugal
KC343104
KC343830
KC344072
KC343588
KC343346

490
CBS 123209
Foeniculum vulgare
Apiaceae
Portugal
KC343105
KC343831
KC344073
KC343589
KC343347
CBS 187.27
Camellia sinensis
Theaceae
Italy
KC343107
KC343833
KC344075
KC343591
KC343349
CBS 116957
Pyrus pyrifolia
Rosaceae
New Zealand
KC343103
KC343829
KC344071
KC343587
KC343345
CBS 171.78
Prunus amygdalus
Rosaceae
Italy
KC343106
KC343832
KC344074
KC343590
KC343348
Diaporthe impulsa
CBS 114434
Sorbus aucuparia
Rosaceae
Sweden
KC343121
KC343847
KC344089
KC343605
KC343363
CBS 141.27
Sorbus americana
Rosaceae
-
KC343122
KC343848
KC344090
KC343606
KC343364
Diaporthe leucospermi
CBS 111980
Leucospermum sp.
Proteaceae
Australia
JN712460
KY435632
KY435673
KY435653
KY435663
Diaporthe neilliae
CBS 144.27
Spiraea sp.
Rosaceae
USA
KC343144
KC343870
KC344112
KC343628
KC343386
Diaporthe padi var. padi
CBS 114200
Prunus padus
Rosaceae
Sweden
KC343169
KC343895
KC344137
KC343653
KC343411
Diaporthe passiflorae
CBS 132527
Passiflora edulis
Passifloraceae
South America
JX069860
KY435633
KY435674
KY435654
KY435664
Diaporthe pustulata
CBS 109784
Prunus padus
Rosaceae
Austria
KC343187
KC343913
KC344155
KC343671
KC343429
Diaporthe rudis
CBS 266.85
Rosa rugosa
Rosaceae
Netherlands
KC343237
KC343963
KC344205
KC343721
KC343479
CBS 113201
Vitis vinifera
Vitaceae
Portugal
KC343234
KC343960
KC344202
KC343718
KC343476
Diaporthe toxica
CBS 534.93
Lupinus angustifolius
Fabaceae
Australia
KC343220
KC343946
KC344188
KC343704
KC343462
Pathogenicity tests on fruits
Granny Smith apples were washed with water and surface disinfected with 70% ethanol prior to inoculation. A 5-mm-diameter piece of fruit
tissue was removed with a cork borer and replaced with a plug of mycelium-colonized agar. Plugs of uninoculated PDA ½ were used as negative
controls and the inoculation points were sealed with masking tape. Five replicate fruits for each isolate and control were incubated at room temperature
for 14 days and lesion diameters were measured after 7 and 14 days. A one-way analysis of variance (ANOVA) followed by a Student test was used to
evaluate the pathogenicity of isolates. Analyses were made with JMP®8.0.1 (SAS Institute Inc., NC, USA).
Pathogenicity tests on twigs
Healthy twigs of Pyrus communis were surface disinfected with 70% ethanol and inoculated by making a hole with a 5-mm-diameter cork
borer exposing the cambium. A mycelial plug was applied, with the mycelium side facing inward, and sealed with Parafilm®. Five replicate twigs per
isolate and controls were incubated at room temperature in a humid chamber for 28 days. Plugs of uninoculated ½ PDA were used as negative
controls. Lesion lengths were measured after 28 days. The normality of the data was checked with the Shapiro-Wilk test. A one-way analysis of
variance (ANOVA) followed by a Student test was used to determine the significance of differences between means. Analyses were done with
JMP®8.0.1 (SAS Institute Inc., NC, USA).
Fungal isolation
Ten isolates were obtained from 10 apple fruits exhibiting post-harvest rot, and 10 isolates from shoot cankers, namely 1 isolate from Prunus
cerasus, 1 isolate from Pyrus communis and 8 isolates from Pyracantha coccinea. From BOX-PCR fingerprinting analysis 8 isolates representative of
the overall genetic diversity were selected for further molecular identification by sequencing five loci (ITS, TEF1, HIS, TUB and CAL).

491
Results
Figure 1 – ML tree built using the five loci ITS-TEF1-TUB- HIS-CAL for the Diaporthe species
found in Rosaceae. Bootstrap values are shown next to the branches. Ex-type, ex-epitype, or
isotype isolates are given in bold. The studied isolates are shown in green. The tree was rooted to
D. toxica (CBS 534.93).

492
Figure 2 – Lesion size in apple fruit after 7 and 14 days. The vertical lines indicate standard
deviations. Bars with the same letter are not significantly different.
Figure 3 – Lesion lengths on pear twigs after 28 days. The vertical lines indicate standard
deviations. Bars with the same letter are not significantly different.
Phylogenetic analysis
For the multi-loci (ITS, TEF1, HIS, TUB and CAL) phylogenetic analysis, apart from our
isolates we considered 10 Diaporthe species that have been found in Rosaceae and for which
sequences from all the five loci were available. Additionally, two Diaporthe species relevant for
this study (D. leucospermi and D. passiflorae) were also included (Tables 1 and 2). ML analysis
was based on the Tamura-Nei’s model assuming a gamma distribution (Tamura & Nei 1993) as
determined by MEGA6. Fig. 1 shows the ML tree for the 5 concatenated loci.

493
In the ML phylogenetic tree 15 clades could be identified of which 13 correspond to known
Diaporthe species: D. ambigua, D. amygdali, D. crataegi, D. eres, D. foeniculina, D. impulsa, D.
leucospermi, D. neilliae, D. padi var. padi, D. passiflorae, D. pustulata, D. rudis and D. toxica. The
remaining two clades include isolates obtained in this study and represent previously undescribed
species, closely related to D. leucospermi (CAA 483 and CAA487) and D. passiflorae (CAA734,
CAA740 and CAA752) which are here described as D. pyracanthae sp. nov. and D. malorum sp.
nov. respectively. The other isolates obtained in this study clustered within the clades
corresponding to D. eres (CAA801) and D. foeniculina (CAA 133 and CAA 737). Isolates CBS
116953, CBS 116954 and CBS 124030 were initially identified as belonging in the Diaporthe
nobilis complex by Gomes et al. (2013), but in this study, we show them to reside within the D.
eres clade.
Pathogenicity test
All isolates tested caused apple rot (Fig. 2). At day 14, isolate CAA740 (isolated from Malus
domestica) produced significantly larger lesions than the other isolates tested (F3,20 = 6.508, p <
0.003), almost completely rotting the entire fruit and with partial liquefaction. Regarding the
pathogenicity assay on detached pear twigs isolate CAA801 (D. eres isolated from Prunus cerasus)
produced lesions significantly longer than the other isolates tested (F3,8 = 4.6713, p < 0.036) (Fig.
3).
Mating-type test
The mating strategy was determined for all 20 isolates (Table 1). All the tested isolates were
heterothallic. Within D. foeniculina isolates both mating types were identified, namely MAT1-2-1
(CAA133) and others with MAT1-1-1 genes (CAA737, CAA738 and CAA739). For D.
pyracanthae, D. malorum and D. eres isolates only MAT1-2-1 gene was detected.
Taxonomy
Diaporthe pyracanthae L. Santos & A. Alves, sp. nov. Fig. 4
MycoBank MB820224
Etymology – named for the host it was first isolated from, namely Pyracantha coccinea.
Conidiomata pycnidial, dark brown, superficial, solitary to aggregated, opening via a central
ostiole, exuding a creamy to white conidial cirrhus. Conidiophores lining the inner cavity,
subcylindrical, hyaline, smooth, reduced to conidiogenous cells. Conidiogenous cells phialidic,
hyaline, smooth and subcylindrical with apical taper. Alpha conidia hyaline, aseptate, smooth,
fusiform, frequently biguttulate, ellipsoid, rounded apex, and obtuse to truncate at base, on pine
needles (5.2)–6.7– (8.8) × (1.6)–2.4– (3.0) µm (mean ± S.D. = 6.7 ± 0.6 × 2.4 ± 0.2 µm, n = 100),
on fennel twigs (6.0)–6.8– (7.9) × (1.6)–2.2–(2.9) µm (mean ± S.D. = 6.8 ± 0.4 × 2.2 ± 0.2 µm, n =
100). Beta conidia hyaline, aseptate, smooth, filiform, frequently hooked in apical part, apex acute,
base truncate, on pine needles (20.8)–30.0– (36.8) × (0.8)–1.3–(1.9) µm (mean ± S.D. = 30.0 ± 2.7
× 1.3 ± 0.8 µm, n = 100), on fennel twigs (15.8)–26.8–(33.6) × (0.8)–1.3–(2.0) µm (mean ± S.D. =
26.8 ± 4.2 × 1.3 ± 0.2 µm, n = 100). Gamma conidia infrequent, aseptate, hyaline, smooth, fusoid,
apex acutely rounded, base subtruncate.
Culture characteristics – Colonies spreading, flat, with sparse to moderate aerial mycelium,
covering a Petri dish in 7 days at 25ºC; on PDA growing with concentric zones, pale brown to
smoke-grey, reverse pale brown to smoke-grey; optimal growth rate between 5 and 9 mm/day
(p<0.05), maximum temperature for growth between 37 and 40ºC (p<0.05), minimum temperature
for growth between 4 and 9 ºC (p<0.05) and optimum temperature between 21 and 27 º C (p<0.05).
Sexual morph – not observed

494
Figure 4 – Diaporthe pyracanthae. A. Upper culture surface on PDA, 25ºC and 7 days. B. Reverse
culture surface on PDA, 25 ºC and 7 days. C. Conidiogenous cells. D. Alpha, beta and gamma
conidia. Scale bar: C–D = 10 μm.
Known distribution – Portugal.
Material examined – Portugal, Aveiro, from branch canker of Pyracantha coccinea, March
2012, A. Alves, (LISE 96313 holotype), a dried culture sporulating on pine needles, ex-type living
culture, CBS142384 = CAA483. Other isolates studied are listed in Table 1.
Notes – Diaporthe pyracanthae is phylogenetically closely related but distinct from D.
leucospermi. Although conidial dimensions of both species are similar they differ in several
nucleotide positions in the following loci: ITS (3 nt), TEF1 (1 nt), TUB (8 nt), and HIS (2 nt)
(Table 3).
Diaporthe malorum L. Santos & A. Alves, sp. nov. Fig. 5
MycoBank MB820226
Etymology – named for the host it was first isolated from, namely Malus domestica.
Conidiomata pycnidial, dark brown, superficial, solitary or more frequently aggregated,
opening via a central ostiole, exuding a creamy to white conidial cirrhus. Conidiophores lining the
inner cavity, subcylindrical, hyaline, smooth, reduced to conidiogenous cells. Conidiogenous cells
phialidic, hyaline, and smooth, subcylindrical with apical taper Alpha conidia hyaline, aseptate,

495
Table 3 Nucleotide differences between D. leucospermi and D. pyracanthae (CAA483 and
CAA487).
Locus
Isolates
Diaporthe leucospermi
CAA483
CAA487
ITS
(537 bp)
61
C
T
T
450
T
C
C
467
T
C
C
TEF1
(332 bp)
16
C
T
T
TUB
(497 bp)
27
T
C
C
45
A
G
G
89
T
C
C
161
T
C
C
298
A
C
C
339
T
C
C
347
T
C
C
452
T
C
C
HIS
(457 bp)
188
G
A
A
189
G
A
A
CAL
(492 bp)
-
smooth, fusiform, rarely biguttulate, ellipsoid, rounded apex and obtuse to truncate base, on pine
needles (5.0)–6.3–(7.5) × (1.5)–2.2–(3.2) µm (mean ± S.D. = 6.3 ± 0.5 × 2.2 ± 0.3 µm, n = 100), on
fennel twigs (5.6)–7.0–(8.7) × 2.2–3.4 µm (mean ± S.D. = 7.0 ± 0.6 × 2.8 ± 0.3 µm, n = 100).
Gamma conidia infrequent, aseptate, hyaline, smooth, fusoid, apex acutely rounded, base
subtruncate, on pine needles (7.1)–9.7–(12.4) × (1.3)–1.8–(2.3) µm (mean ± S.D. = 9.7 ± 1.3 × 1.8
± 0.2 µm, n = 40), on fennel twigs (7.2)–10.6–(17.0) × (1.2)–1.9–(2.6) µm (mean ± S.D. = 10.6 ±
1.8 × 1.9 ± 0.3 µm, n = 100). Beta conidia infrequent, hyaline, aseptate, smooth, filiform,
frequently hooked in apical part, apex acute, base truncate, on pine needles very infrequent, on
fennel twigs (17.4)–21.5–(26.6) × (0.8)–1.3–(2.0) µm (mean ± S.D. = 21.5 ± 2.1 × 1.3 ± 0.3 µm, n
= 50).
Culture characteristics – Colonies spreading, flat, with sparse to moderate aerial mycelium,
not covering a Petri dish in 7 days at 25ºC, sometimes with a reddish exudate; on PDA growing
with pale brown to brown, reverse pale brown to dark reddish brown mycelia at 14 days; optimal
growth rate between 3 and 7 mm/day (p<0.05), maximum temperature between 34 and 40ºC
(p<0.05), minimum temperature between 2 and 6 ºC (p<0.05) and optimum temperature between
13 and 20 ºC (p<0.05).
Sexual morph – not observed
Known distribution – Portugal.
Material examined – Portugal, Felgueiras, from Malus domestica fruit with rot symptoms,
January 2014, A. Alves, (LISE 96314 holotype), a dried culture sporulating on pine needles, ex-
type living culture, CBS142383 = CAA734. Other isolates studied are listed in Table 1.
Notes – Diaporthe malorum is phylogenetically closely related but distinct from D.
passiflorae. Although conidial sizes of both species are similar they differ in several nucleotide
positions in the following loci: ITS (5 nt), TEF1 (21 nt), TUB (12 nt), HIS (10 nt), and CAL (13 nt)
(Table 4).

496
Figure 5 – Diaporthe malorum. A. Upper culture surface on PDA, 20 ºC and 9 days. B. Reverse
culture surface on PDA, 20 ºC and 9 days. C. Gamma conidia. D. alpha and beta conidia. – Scale
bars: C = 2 μm, D = 10 μm.
Table 4 Nucleotide differences between D. passiflorae and D. malorum.
Locus
Isolates
Diaporthe passiflorae
CAA734
CAA740
CAA752
ITS
(542 bp)
92
G
A
A
A
383
C
G
G
G
384
G
-
-
-
385
C
-
-
-
388
G
A
A
A
TEF1
(346 bp)
27
G
A
A
A
50
A
-
-
-
51
C
-
-
-
93
C
A
A
A
96
A
G
G
G
103
-
C
C
C
172
T
G
G
G
212
C
T
T
T
236
C
T
T
T
238
G
-
-
-
239
C
-
-
-
240
A
-
-
-
241
C
-
-
-
242
C
-
-
-
243
A
-
-
-

497
244
T
-
-
-
245
C
-
-
-
246
A
-
-
-
247
C
-
-
-
248
C
-
-
-
249
A
-
-
-
TUB
(502 bp)
12
A
G
G
G
18
G
A
A
A
41
G
T
T
T
48
A
G
G
G
84
C
T
T
T
86
A
C
C
C
88
C
T
T
T
90
C
T
T
T
201
C
G
G
G
292
C
T
T
T
298
G
A
A
A
421
C
T
T
T
HIS
(425 bp)
62
C
G
G
G
150
A
G
G
G
158
A
C
C
C
164
T
G
G
G
175
A
G
G
G
181
C
G
G
G
191
C
T
T
T
376
C
T
T
T
409
T
C
C
C
421
C
T
T
T
CAL
(486 bp)
69
C
G
G
G
143
C
A
A
A
184
T
G
G
G
191
A
T
T
T
210
G
A
A
A
226
A
C
C
C
229
T
A
A
A
293
A
C
C
C
323
C
T
T
T
385
G
C
C
C
419
G
T
T
T
421
G
C
C
C
458
C
T
T
T
Review of Diaporthe names reported from Rosaceae
A search of the Systematic Mycology and Microbiology Laboratory Fungus-Host Database
(Farr & Rossman 2016) revealed 91 species of Diaporthe/Phomopsis associated with hosts in the
family Rosaceae. These names were verified against the Index Fungorum and MycoBank databases
as well as the available published literature, especially the most recent treatments of the genus
Diaporthe (e.g. Gomes et al. 2013. Udayanga et al. 2014a, 2014b), which reduced the number to 53
Diaporthe species. Table 5 lists all current names of the Diaporthe/Phomopsis species associated
with Rosaceae, their currently accepted synonymies and respective hosts.

498
Table 5 – List of Diaporthe and Phomopsis names associated with Rosaceae.
Species
Synonyms
Host
Country
Reference
Diaporthe actinidiae N.F.
Sommer & Beraha
Malus domestica
New Zealand
Farr & Rossman 2016
Diaporthe ambigua Nitschke
Phoma ambigua (Nitschke) Sacc.
Phomopsis ambigua Traverso
Malus domestica
South Africa
Farr & Rossman, 2016
Malus sylvestris
Netherlands
South Africa
Murali et al. 2006
Farr & Rossman 2016
Malus sp.
Armenia
United
Kingdom
Farr & Rossman 2016
Prunus salicina
South Africa
Farr & Rossman 2016
Prunus sp.
South Africa
Farr & Rossman 2016
van Niekerk et al. 2005
Pyrus communis
Canada
Cuba
Germany
South Africa
USA
Farr & Rossman 2016
Gomes et al. 2013
Pyrus ussuriensis
China
Farr & Rossman 2016
Diaporthe amygdali (Delacr.)
Udayanga, Crous & K.D.
Hyde
Fusicoccum amygdali Delacr.
Phomopsis amygdali (Delacr.) J.J. Tuset & M.T.
Portilla
Phomopsis amygdalina Canonaco
Amygdalus persica
Japan
Farr & Rossman 2016
Prunus amygdalus
China
Farr & Rossman 2016
Prunus armeniaca
China
Farr & Rossman 2016
Prunus dulcis
Italy
Portugal
USA
World wide
Farr & Rossman 2016
Santos et al. 2010
Diogo et al. 2010
Gomes et al. 2013
Prunus persica
China
France
Greece
Japan
Portugal
South Africa
USA
World Wide
Farr & Rossman 2016
Gomes et al. 2013
Prunus persica var. vulgaris
Japan
Farr & Rossman 2016
Prunus salicina
China
South Africa
Farr & Rossman 2016
Gomes et al. 2013
Prunus salicina var. corlata
China
Farr & Rossman 2016
Prunus sp.
USA
Murali et al. 2006
Diaporthe australafricana
Crous & Van Niekerk
Prunus dulcis
USA
Farr & Rossman 2016

499
Diaporthe beckhausii
Nitschke
Lophiosphaera beckhausii (Nitschke) Berl. &
Voglino
Lophiostoma beckhausii Nitschke
Valsa beckhausii (Nitschke) Cooke
Phomopsis beckhausii (Cooke) Traverso
Cydonia japonica
Czech Republic
Farr & Rossman 2016
Diaporthe cerasi Fuckel
Cerasus avium
Denmark
Farr & Rossman 2016
Diaporthe congesta Ellis &
Everh.
Pyrus americana
USA
Farr & Rossman 2016
Diaporthe crataegi (Curr.)
Fuckel
Valsa crataegi Curr.
Crataegus chrysocarpa
Canada
Farr & Rossman 2016
Crataegus laevigata
Poland
Farr & Rossman 2016
Crataegus oxyacantha
Austria
United Kindom
France
Germany
Italy
Poland
Sweden
Farr & Rossman 2016
Gomes et al. 2013
Crataegus sp.
Bulgaria
Denmark
Poland
Sweden
United
Kingdom
Farr & Rossman 2016
Diaporthe decorticans (Lib.)
Sacc. & Roum.
Diaporthe padi G.H. Otth
Diaporthe padi var. padi G.H. Otth
Diaporthe padi var. patria (Speg.) Wehm.
Diaporthe patria Speg.
Sphaeria decorticans Lib.
Phomopsis padina (Sacc.) Dietel
Cerasus padus
Denmark
Farr & Rossman 2016
Laurocerasus officinalis
Ukraine
Farr & Rossman 2016
Laurocerasus officinalis var. zabeliana
Ukraine
Farr & Rossman 2016
Malus sieboldii
Japan
Farr & Rossman 2016
Padus avium
Poland
Russia
USA
Farr & Rossman 2016
Prunus cerasus
United
Kingdom
USA
Farr & Rossman 2016
Prunus hortulana
USA
Farr & Rossman 2016
Prunus munsoniana
USA
Farr & Rossman 2016
Prunus padus
Austria
Germany
Poland
United
Kingdom
Farr & Rossman 2016
Sweden
Gomes et al. 2013, Farr

500
& Rossman 2016
Prunus persica
World Wide
Farr & Rossman 2016
Sorbus aria
Germany
Farr & Rossman 2016
Diaporthe eres Nitschke
Phoma oblonga Desm.
Phomopsis oblonga (Desm.) Traverso
Phomopsis cotoneastri Punith.
Diaporthe cotoneastri (Punith.) Udayanga, Crous
& K.D. Hyde
Phomopsis castaneae-mollisimae S.X. Jiang &
H.B. Ma
Diaporthe castaneae-mollisimae (S.X, Jiang &
H.B. Ma) Udayanga, Crous & K.D. Hyde
Phomopsis fukushii Tanaka & S. Endô
Chaenomeles speciosa
Ukraine
Farr & Rossman 2016
Cotoneaster adpressus
Poland
Ukraine
Farr & Rossman 2016
Cotoneaster buxifolius
Ukraine
Farr & Rossman 2016
Cotoneaster dammeri
Ukraine
Farr & Rossman 2016
Cotoneaster divaricatus
Poland
Ukraine
Farr & Rossman 2016
Cotoneaster foveolatus
Ukraine
Farr & Rossman 2016
Cotoneaster franchetii
Ukraine
Farr & Rossman 2016
Cotoneaster glaucophyllus
Ukraine
Farr & Rossman 2016
Cotoneaster microphyllus
Ukraine
Farr & Rossman 2016
Cotoneaster moupinensis
Ukraine
Farr & Rossman 2016
Cotoneaster praecox
Ukraine
Farr & Rossman 2016
Cotoneaster rhytidophyllus
Ukraine
Farr & Rossman 2016
Cotoneaster simonsii
Ukraine
Farr & Rossman 2016
Cotoneaster sp.
United
Kingdom
Farr & Rossman 2016
Udayanga et al. 2014b
Crataegus oxyacantha
Canada
Czech Republic
Germany
Farr & Rossman 2016
Crataegus pojarkovae
Ukraine
Farr & Rossman 2016
Crataegus sp.
Canada
Farr & Rossman 2016
Kerria japonica
Germany
Japan
Farr & Rossman 2016
Malus domestica
New Zealand
Uruguay
USA
Farr & Rossman 2016
Malus sylvestris
Zimbawe
Farr & Rossman 2016
-
Gomes et al. 2013
Malus pumila
Korea
Udayanga et al. 2014b
Malus pumila var. domestica
China
Farr & Rossman 2016
Malus sp.
Korea
Netherlands
Udayanga et al. 2014b
Physocarpus opulifolius
USA
Farr & Rossman 2016
Physocarpus spp.
USA
Farr & Rossman 2016
Prunus avium
China
Japan
Farr & Rossman 2016
Prunus cerasus
Bulgaria
Farr & Rossman 2016

501
Prunus cornuta
Pakistan
Farr & Rossman 2016
Prunus davidiana
Japan
Farr & Rossman 2016
Prunus domestica
Bulgaria
Farr & Rossman 2016
Prunus dulcis
Portugal
Diogo et al. 2010
Prunus lannesiana f. sekiyama
Japan
Farr & Rossman 2016
Prunus mume
Korea
Udayanga et al. 2014b
Prunus persica
Australia
Greece
USA
Farr & Rossman 2016
Korea
Udayanga et al. 2014b
Prunus persica var. vulgaris
Japan
Farr & Rossman 2016
Prunus sargentii
Japan
Farr & Rossman 2016
Prunus sp.
Japan
New Zealand
USA
Farr & Rossman 2016
Korea
Udayanga et al. 2014b
Pyracantha crenatoserrata
Ukraine
Farr & Rossman 2016
Pyracantha rogersiana
Ukraine
Farr & Rossman 2016
Pyracantha sp.
Ukraine
Farr & Rossman 2016
Pyrus communis
USA
New Zealand
Farr & Rossman 2016
Pyrus pyrifolia
China
Japan
Farr & Rossman 2016
Pyrus pyrifolia var. culta
China
Farr & Rossman 2016
Pyrus serotina
Japan
Korea
Farr & Rossman 2016
Pyrus pyrifolia
Japan
Korea
New Zealand
Murali et al. 2006
Udayanga et al. 2014b
Pyrus serotina var. culta
Japan
Farr & Rossman 2016
Pyrus ussuriensis
China
Farr & Rossman 2016
Pyrus sp.
China
Farr & Rossman 2016
Rhaphiolepis indica
Ukraine
Farr & Rossman 2016
Rosa canina
Belgium
Czech Republic
United
Kingdom
USA
Germany
Farr & Rossman 2016
Rosa sp.
USA
Italy
Farr & Rossman 2016

502
New Zealand
Rubus fruticosus
Ireland
Farr & Rossman 2016
Austria
Udayanga et al. 2014b
Rubus idaeus
Germany
Farr & Rossman 2016
Rubus sp.
Croatia
France
Farr & Rossman 2016
Sorbus aucuparia
Netherlands
USA
Farr & Rossman 2016
Gomes et al. 2013
Spiraea cantoniensis
Ukraine
Farr & Rossman 2016
Spiraea chamaedryfolia
Ukraine
Farr & Rossman 2016
Spiraea sp.
Ukraine
Farr & Rossman 2016
Diaporthe fibrosa (Pers.)
Fuckel
Sphaeria fibrosa Pers.
Hercospora fibrosa (Pers.) Petr
Prunus cerasifera
Bulgaria
Farr & Rossman 2016
Prunus spinosa
Poland
Farr & Rossman 2016
Diaporthe foeniculina (Sacc.)
Udayanga & Castl.
Phoma foeniculina Sacc.
Phoma foeniculina Sacc.
Phomopsis foeniculina (Sacc.) Câmara
Phomopsis theicola Curzi
Diaporthe neotheicola A.J.L. Phillips & J.M.
Santos
Diaporthe foeniculacea Niessl,
Diaporthe theicola Curzi
Phomopsis theicola Curzi
Phomopsis californica H.S. Fawc.
Diaporthe rhusicola Crous
Malus domestica
New Zealand
Udayanga et al. 2014a
Prunus amygdalus
Italy
Gomes et al. 2013
Farr & Rossman 2016
Prunus dulcis
Portugal
Diogo et al. 2010
Farr & Rossman 2016
Prunus spinosa
Poland
Farr & Rossman 2016
Pyrus pyrifolia
New Zealand
Gomes et al. 2013
Diaporthe fuckelii J. Kunze
Spiraea ulmifolia
Sweden
Farr & Rossman 2016
Diaporthe impulsa (Cooke &
Peck) Sacc.
Valsa impulsa Cooke & Peck
Sorbus americana
-
Gomes et al. 2013
Canada
USA
Farr & Rossman 2016
Sorbus aria
Austria
Farr & Rossman 2016
Sorbus aucuparia
Austria
Czech Republic
Poland
Sweden
United
Kingdom
Gomes et al. 2013
Farr & Rossman 2016
Sorbus aucuparia subsp. glabrata
Poland
Farr & Rossman 2016
Sorbus commixta
Japan
Farr & Rossman 2016
Sorbus sitchensis
USA
Farr & Rossman 2016
Sorbus sp.
USA
Farr & Rossman 2016
Diaporthe incarcerata (Berk.
& Broome) Nitschke
Diatrype incarcerata Berk. & Broome
Phoma incarcerata (Nitschke) Sacc.
Rosa canina
Poland
Farr & Rossman 2016
Rosa indica
India
Farr & Rossman 2016

503
Sphaeropsis depressa Lév.
Phomopsis incarcerata Höhn.
Phomopsis depressa (Lév.) Traverso
Rosa sp.
Denmark
South Africa
United
Kingdom
Zimbabwe
Farr & Rossman 2016
Diaporthe insignis Fuckel.
Rubus fruticosus
Denmark
Poland
Farr & Rossman 2016
Rubus idaeus
Denmark
Farr & Rossman 2016
Diaporthe japonica Sacc.
Phoma japonica (Sacc.) Sacc., Michelia 1 (5):
521. 1879
Phomopsis japonica (Sacc.) Traverso, Flora Italica
Cryptogama. Pars 1: Fungi. Pyrenomycetae.
Xylariaceae, Valsaceae, Ceratostomataceae 1(1):
241. 1906
Kerria japonica
Poland
USA
Farr & Rossman 2016
Kerria japonica var. pleniflorae
Portugal
Farr & Rossman 2016
Diaporthe mali Bres.
Malus pumila
Japan
Farr & Rossman 2016
Diaporthe neilliae Peck
Spiraea sp.
USA
Udayanga et al., 2014b
Diaporthe nobilis complex
Malus pumila
New Zealand
Gomes et al. 2013
Farr & Rossman 2016
Pyrus pyrifolia
New Zealand
Gomes et al. 2013
Farr & Rossman 2016
Diaporthe novem J.M.
Santos, Vrandečić & A.J.L.
Phillips
Prunus dulcis
USA
Farr & Rossman 2016
Diaporthe parabolica Fuckel
Prunus spinosa
Denmark
Farr & Rossman 2016
Diaporthe pardalota (Mont.)
Nitschke ex Fuckel
Sphaeria pardalota Mont.
Phomopsis pardalota Died.
Prunus divaricata
Ukraine
Farr & Rossman 2016
Prunus laurocerasus
France
Farr & Rossman 2016
Rubus fruticosus
Germany
Farr & Rossman 2016
Diaporthe pennsylvanica
(Berk. & M.A. Curtis)
Wehm.
Valsa pennsylvanica Berk. & M.A. Curtis
Calospora pennsylvanica (Berk. & M.A. Curtis)
Sacc.
Prunus pensylvanica
USA
Farr & Rossman 2016
Prunus serotina
USA
Farr & Rossman 2016
Prunus virginiana
USA
Farr & Rossman 2016
Diaporthe perniciosa
Marchal & É.J. Marchal
Phomopsis prunorum (Cooke) Grove
Phomopsis mali Roberts
Phomopsis mali (Schulzer & Sacc.) Died.
Cydonia oblonga
Greece
Farr & Rossman 2016
Malus domestica
Brazil
Greece
Japan
New Zealand
United
Kingdom
Farr & Rossman 2016
Malus melliana
China
Farr & Rossman 2016
Malus pumila
Chile
Farr & Rossman 2016
Malus pumila var. dulcissima
Korea
Farr & Rossman 2016
Malus sylvestris
Australia
Farr & Rossman 2016

504
USA
Malus sp.
Canada
Farr & Rossman 2016
Prunus cerasus
Bulgaria
Farr & Rossman 2016
Prunus domestica
Bulgaria
Central Asia
USA
Farr & Rossman 2016
Prunus dulcis
World Wide
Farr & Rossman 2016
Prunus mahaleb
Canada
Farr & Rossman 2016
Prunus persica
USA
World Wide
Farr & Rossman 2016
Prunus sp.
Cyprus
Lithuania
New Zealand
USA
World Wide
Farr & Rossman 2016
Pyrus communis
Australia
Greece
Japan
New Zealand
Poland
USA
Farr & Rossman 2016
Pyrus malus
USA
Farr & Rossman 2016
Diaporthe pruni Ellis &
Everh.
Prunus angustifolia
USA
Farr & Rossman 2016
Prunus hortulana
USA
Farr & Rossman 2016
Prunus munsoniana
USA
Farr & Rossman 2016
Prunus serotina
USA
Farr & Rossman 2016
Prunus virginiana
Canada
USA
Farr & Rossman 2016
Prunus sp.
Canada
USA
Farr & Rossman 2016
Diaporthe prunicola (Peck)
Wehm.
Valsa prunicola Peck
Engizostoma prunicola (Peck) Kuntze
Prunus americana
USA
Farr & Rossman 2016
Prunus divaricata
Ukraine
Farr & Rossman 2016
Prunus pensylvanica
Canada
USA
Farr & Rossman 2016
Prunus serotina
Canada
USA
Farr & Rossman 2016
Prunus virginiana
Canada
Farr & Rossman 2016
Prunus sp.
Canada
USA
Farr & Rossman 2016
Diaporthe pustulata Sacc.
Prunus padus
Austria
Farr & Rossman 2016
Diaporthe rehmii Nitschke
Sorbus aucuparia
United
Farr & Rossman 2016

505
Kingdom
Diaporthe rudis (Fr.)
Nitschke
Sphaeria rudis Fr.
Rabenhorstia rudis (Fr.) Fr.
Aglaospora rudis (Fr.) Tul. & C. Tul.
Phoma rudis Sacc.
Phomopsis rudis (Sacc.) Höhn.
Diaporthe faginea Sacc.
Diaporthe medusaea Nitschke
Diaporthe viticola Nitschke
Diaporthe silvestris Sacc. & Berl.
Malus pumila var. domestica
Japan
Farr & Rossman 2016
Pyrus communis
Japan
Farr & Rossman 2016
Pyrus serotina var. culta
Japan
Farr & Rossman 2016
Pyrus ussuriensis var. sinensis
Japan
Farr & Rossman 2016
Pyrus sp.
New Zealand
Udayanga et al. 2014a
Rosa canina
Austria
Udayanga et al. 2014a
Farr & Rossman 2016
Rosa rugosa
Netherlands
Gomes et al. 2013
Farr & Rossman 2016
Spiraea sp.
USA
Murali et al. 2006
Diaporthe sorbariae Nitschke
Spiraea salicifolia
Poland
Farr & Rossman 2016
Diaporthe spiculosa (Pers.)
Nitschke
Sphaeria spiculosa Pers.
Hypoxylon spiculosum (Pers.) Westend.
Cerastoma spiculosum (Pers.) Quél.
Sorbus aucuparia
Switzerland
Farr & Rossman 2016
Diaporthe tanakae Ts.
Kobay. & Sakuma
Malus pumila var. domestica
Japan
Farr & Rossman 2016
Pyrus communis
Japan
Farr & Rossman 2016
Diaporthe vexans (Sacc. & P.
Syd.) Gratz
Phoma vexans Sacc. & P. Syd.
Phomopsis vexans (Sacc. & P. Syd.) Harter
Prunus armeniaca
Argentina
Korea
Farr & Rossman 2016
Prunus mume
Korea
Farr & Rossman 2016
Diaporthe viburni Dearn. &
Bisby, in Bisby
Diaporthe viburni var. spiraeicola Wehm.
Spiraea tomentosa
Canada
USA
Farr & Rossman 2016
Spiraea sp.
Canada
USA
Farr & Rossman 2016
Phomopsis biwa Hara
Eriobotrya japonica
Japan
Farr & Rossman 2016
Phomopsis corticis (Fuckel)
Grove
Phoma corticis Fuckel
Macrophoma corticis (Fuckel) Berl. & Voglino
Rubus sp.
Poland
Farr & Rossman 2016
Phomopsis hughesii N.D.
Sharma
Eriobotrya japonica
China
India
Farr & Rossman 2016
Phomopsis muelleri (Cooke)
Grove
Phoma muelleri Cooke
Rubus giraldianus
Poland
Farr & Rossman 2016
Rubus idaeus
Russia
Farr & Rossman 2016
Phomopsis obscurans (Ellis
& Everh.) B. Sutton
Phoma obscurans Ellis & Everh.
Sphaeropsis obscurans (Ellis & Everh.) Kuntze
Phyllosticta obscurans (Ellis & Everh.) Tassi
Dendrophoma obscurans (Ellis & Everh.) H.W.
Anderson
Fragaria ananassa
Bulgaria
Tonga
Farr & Rossman 2016
Fragaria chiloensis
USA
Farr & Rossman 2016
Fragaria vesca
Brazil
Brunei
Darussalam
Malawi
Myanmar
Farr & Rossman 2016
Fragaria × ananassa
Australia
Canada
Farr & Rossman 2016

506
China
Korea
New Zealand
USA
Fragaria sp.
Australia
Brazil
South Africa
USA
Farr & Rossman 2016
Photinia serrulata
China
Farr & Rossman 2016
Phomopsis padina (Sacc.)
Dietel
Phoma padina Sacc.
Laurocerasus officinalis
Ukraine
Farr & Rossman 2016
Laurocerasus officinalis var. zabeliana
Ukraine
Farr & Rossman 2016
Prunus avium
USA
Farr & Rossman 2016
Prunus cerasus
USA
Farr & Rossman 2016
Prunus dulcis
World Wide
Farr & Rossman 2016
Prunus padus
United
Kingdom
Farr & Rossman 2016
Prunus persica
World Wide
Farr & Rossman 2016
Phomopsis parabolica Petr.
Prunus dulcis
World Wide
Farr & Rossman 2016
Prunus persica
World Wide
Farr & Rossman 2016
Phomopsis perniciosa Grove
Cerasus avium
Poland
Farr & Rossman 2016
Crataegus sp.
Poland
Farr & Rossman 2016
Laurocerasus phaeosticta f.
ciliospinosa
China
Farr & Rossman 2016
Malus domestica
Portugal
Farr & Rossman 2016
Malus pumila
Poland
Farr & Rossman 2016
Malus purpurea
Poland
Farr & Rossman 2016
Malus sylvestris
Kenya
Farr & Rossman 2016
Malus sp.
Poland
Farr & Rossman 2016
Padus avium
Russia
Farr & Rossman 2016
Prunus dulcis
World Wide
Farr & Rossman 2016
Prunus persica
Portugal
World Wide
Farr & Rossman 2016
Prunus sp.
Canada
Lithuana
Poland
Yugoslavia
Farr & Rossman 2016
Pyrus communis
India
Farr & Rossman 2016
Pyrus malus
Southern Africa
Farr & Rossman 2016
Phomopsis pyrorum Sacc. &
Trotter
Phomopsis pyrorum Sacc. & Trotter
Pyrus pyrifolia
China
Farr & Rossman 2016
Phomopsis pruni (Ellis &
Cytospora pruni Ellis & Dearn
Prunus dulcis
World Wide
Farr & Rossman 2016

507
Dearn.) Wehm.
Prunus × yedoensis
Japan
Farr & Rossman 2016
Prunus sp.
World Wide
Farr & Rossman 2016
Phomopsis rhodophila
(Sacc.) N.F. Buchw.
Phoma rhodophila Sacc.
Rosa sp.
China
Farr & Rossman 2016
Phomopsis ribatejana Sousa
da Câmara
Prunus persica
Portugal
Sousa da Câmara 1948
Phomopsis rubiseda Fairm.
Rubus sp.
USA
Farr & Rossman 2016
Phomopsis sorbariae (Sacc.)
Höhn.
Phoma sorbariae Sacc.
Spiraea chamaedryfolia
Armenia
Farr & Rossman 2016
Phomopsis sorbicola Grove
Sorbus aucuparia
Poland
Farr & Rossman 2016
Sorbus sp.
Canada
Farr & Rossman 2016
Phomopsis spiraeae (Desm.)
Grove
Phoma spiraeae Desm.
Spiraea nipponica
Poland
Farr & Rossman 2016
Spiraea sp.
USA
Farr & Rossman 2016
Phomopsis strictosoma
Grove
Cydonia oblonga
Zimbabwe
Farr & Rossman 2016
Phomopsis truncicola Miura
Malus prunifolia
China
Farr & Rossman 2016
Malus pumila
China
Farr & Rossman 2016
Malus pumila var. domestica
Japan
Farr & Rossman 2016
Discussion
In the present study four Diaporthe species were identified from Rosaceae hosts. Of these, two were described as new (D. pyracanthae
associated with canker of firethorn and D. malorum associated with post-harvest fruit rot of apple). These two species are closely related to D.
leucospermi and D. passiflorae, respectively, but clearly distinct phylogenetically. Within D. malorum isolate CAA752 clustered on a separated
branch from CAA734 and CAA740 with high bootstrap support, but this was considered as intraspecific genetic variability. This isolate differs in 7
nucleotide positions in the sequence of one locus (CAL) but the sequences from the remaining loci are 100% identical to other isolates in the species.
We also identified D. eres from canker of Prunus cerasus in Russia and D. foeniculina from canker of pear tree and post-harvest fruit rot of apple in
Portugal.
Diaporthe eres (syn. Phomopsis oblonga) is the type species of the genus and one of the most studied species of Diaporthe. Despite this, the
delimitation of the species and its many synonyms has been complicated by the absence of ex-type cultures. Recently, Udayanga et al. (2014b)
addressed the issue of species delimitation in the D. eres complex using a multi-gene genealogical approach and clearly resolved nine distinct
phylogenetic species. Moreover, they designated epitypes for several species, including for D. eres, thus clarifying the status of D. eres and closely
related species.
Diaporthe eres is a cosmopolitan species and has been found on the following members of Rosaceae: Chaenomeles speciosa, Cotoneaster spp.,
Crataegus spp., Kerria japonica, Malus spp., Physocarpus spp., Prunus spp., Pyracantha spp., Pyrus spp., Rhaphiolepis indica, Rosa spp., Rubus spp.,
Sorbus aucuparia, and Spiraea spp. (Farr & Rossman 2016, Vrandečić et al. 2011). As far as we know D. eres has never been reported from Prunus
cerasus in Russia.

508
Although it is a well-known species there are relatively few studies on pathogenicity of D.
eres on Rosaceae, although it is known to cause shoot blight and canker in peaches (Thomidis &
Michailides 2009); cane blight in blackberry (Vrandečić et al. 2011); trunk canker and death of
young apple trees (Abreo et al. 2012) and wilting of shoots of Cotoneaster species (Frużyńska-
Jóźwick & Jerzak 2006). Vrandečić et al. (2011) showed that D. eres can produce lesions on long
green shoots of potted blackberry plants. Thomidis & Michailides (2009) showed that D. eres is
able to produce necrosis in peach and nectarine fruits, but when the fruits were stored at 10ºC or
lower the fungus was unable to cause fruit rot. They also showed that this species is aggressive
when tested on peach shoots in the field.
Here we showed that in artificial inoculation trials D. eres caused rotting of apple fruits and
lesions on detached pear twigs. In the detached pear twigs inoculation assay, it was the most
aggressive species tested and caused lesions with a mean of 6.9 cm. Surprisingly, D. eres is
considered a weak to moderate pathogen of woody plants (Udayanga et al. 2014b).
Another well-known species associated with hosts in Rosaceae, but less common than D.
eres, is D. foeniculina. This species has been found on Malus domestica, Prunus amygdalus,
Prunus dulcis, Pyrus bretschneideri and Pyrus pyrifolia (Cloete et al. 2011, Diogo et al. 2010, Farr
& Rossman 2016). The present study represents the first report of the species on Pyrus communis
and also the first report on Malus domestica in Portugal. There is only one other report from M.
domestica and that was from New Zealand (Udayanga et al. 2014b). In Portugal, until now, D.
foeniculina (as D. neotheicola) has been reported on Prunus dulcis and Prunus armeniaca (Diogo
et al. 2010) as well as several others hosts outside the Rosaceae such as Acer negundo, Euphorbia
pulcherrima, Foeniculum vulgare, and Hydrangea macrophylla (Santos & Phillips 2009, Santos et
al. 2010).
In our pathogenicity trials, D. foeniculina caused rot on apple fruits and lesions on detached
pear twigs being the second most aggressive species in both tests. However, Cloete et al. (2011)
observed that D. foeniculina (as Phomopsis theicola) did not form lesions significantly different
from controls on detached woody shoots of apple and pear. Also, Diogo et al. (2010) inoculated
detached almond twigs with D. foeniculina and considered it as a weak pathogen of Prunus dulcis.
These differences in aggressiveness may be a reflection of variation in the aggressiveness of
different isolates within the speces.
Diaporthe ambigua and D. amygdali, although not found in this study, are known pathogens
of several Rosaceae hosts with worldwide distribution. Diaporthe ambigua has been found on
Malus domestica, M. sylvestris, Prunus armeniaca, Prunus salicina, Pyrus communis and Pyrus
ussuriensis (Gomes et al. 2013, Farr & Rossman 2016). Diaporthe ambigua is an important
pathogen causing canker of apple (Malus domestica), pear (Pyrus communis) and plum (Prunus
salicina) rootstocks in South Africa (Smit et al. 1996). The species was shown to kill nursery
rootstocks quickly while mature rootstocks were killed over a longer period of time (Smit et al.
1996).
Diaporthe amygdali has been reported on Prunus armeniaca, Prunus dulcis, Prunus persica,
Prunus salicina, and Pyrus pyrifolia (Farr & Rossman 2016). This species is well known as the
causal agent of twig canker and blight of almond (Prunus dulcis) and peach (Prunus persica) in all
areas where these hosts are cultivated (Diogo et al. 2010). It has also been associated with wood
decay of almonds, fruit rot of peaches and fruit rot and branch dieback of almond (Adaskaveg et al.
1999, Kanematsu et al. 1999, Michailides & Thomidis 2006, Carlier et al. 2011, Gramaje et al.
2012). When inoculated on peach twigs and young almond twigs or apple twigs this species
produced lesions, sometimes resulting in constriction canker (Dai et al. 2012, Diogo et al. 2010).
When inoculated on mature and immature peaches, almonds and Japanese pears it caused fruit rot
(Adaskaveg et al. 1999, Kanematsu et al 1999, Michailides & Thomidis 2006).
More than 50 Diaporthe (and its asexual morph Phomopsis) species names have been
associated with hosts in the family Rosaceae. However, apart from the above-mentioned species, D.
ambigua, D. amygdali, D. eres, D. foeniculina, and the two newly described species, there is a
scarcity of information regarding the taxonomic and pathogenic status of those taxa. For most of

509
them there is no other information available apart from the original description of the species. To
complicate matters even further, often there are no ex-type cultures from which phenotypical,
phytopathological and molecular data can be obtained. In the past Diaporthe/Phomopsis species
have mostly been described assuming they were host-specific (Udayanga et al. 2011). However, it
is now clear that although some species appear to be host specific, many are not and can be found
on diverse plant hosts. Currently, the circumscription of species within Diaporthe can be
accomplished only by use of multi-gene DNA sequence data (Gomes et al. 2013, Udayanga et al.
2012b, 2014a, 2014b, 2014c). Thus, in the absence of ex-type cultures it is impossible to carry out
multi-locus phylogenetic analyses to assess the validity of these older species names and their
relationship to currently accepted species in Diaporthe.
In recent years, a revision of the genus Diaporthe has been initiated and considerable
progress has been made towards resolving species complexes and the epitypification/neotypificaton
of species (Gomes et al. 2013, Udayanga et al. 2012b, 2014a, 2014b, 2014c). However, considering
the large number of species described in Diaporthe/Phomopsis there is still much to be done.
Acknowledgements
This work was partially financed by European Funds through COMPETE and by National
Funds through the Portuguese Foundation for Science and Technology (FCT) within project
PANDORA (PTDC/AGR-FOR/3807/2012 – FCOMP-01-0124-FEDER-027979). The authors
acknowledge financing from FCT to CESAM (UID/AMB/50017/2013 – POCI-01- 0145-FEDER-
007638), Artur Alves (FCT Investigator Programme – IF/00835/2013) and Liliana Santos (post-
doctoral grant –SFRH/BPD/90684/2012). Alan JL Phillips acknowledges the support from
Biosystems and Integrative Sciences Institute (BioISI, FCT/UID/ Multi/04046/2013).
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