Additions to wild seed and fruit fungi 1: The sexual morph of Diaporthe rosae on Magnolia champaca and Senna siamea fruits in Thailand

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Submitted 25 February 2018, Accepted 10 March 2018, Published 9 April 2018
Corresponding Author: Zuo-Yi Liu – e-mail – gzliuzuoyi@163.com 256
Additions to wild seed and fruit fungi 1: The sexual morph of
Diaporthe rosae on Magnolia champaca and Senna siamea fruits in
Thailand
Perera RH1,2, Hyde KD2,3,4, Peršoh D5, Jones EBG6, Liu JK1 and Liu ZY1*
1Guizhou Key Laboratory of Agricultural Biotechnology, Guizhou Academy of Agricultural Sciences, Guiyang,
Guizhou Province 550006, P.R. China.
2Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand.
3Key Laboratory for Plant Biodiversity and Biogeography of East Asia (KLPB), Kunming Institute of Botany, Chinese
Academy of Science, Kunming 650201, Yunnan, China.
4World Agroforestry Centre, East and Central Asia, 132 Lanhei Road, Kunming 650201, P.R. China.
5Ruhr-Universität Bochum, AG Geobotanik, Gebäude ND 03/170, Universitätsstraße 150, 44801 Bochum, Germany.
6Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box: 2455, Riyadh, 1145,
Saudi Arabia.
Perera RH, Hyde KD, Peršoh D, Jones EBG, Liu JK, Liu ZY 2018 – Additions to wild seed and
fruit fungi 1: The sexual morph of Diaporthe rosae on Magnolia champaca and Senna siamea
fruits in Thailand. Mycosphere 9(2), 256–270, Doi 10.5943/mycosphere/9/2/7
Abstract
We are studying seed and fruit fungi in Thailand and made several sexual morph collections
of Diaporthe rosae from dried fruits of Magnolia champaca and Senna siamea. The sexual morphs
were linked to the asexual morphs based on molecular data, and morphological similarity with the
asexual morph produced on PDA. The asexual morph of D. rosae was previously reported from a
dead pedicel of Rosa sp. (Rosaceae) from the same location as its sexual morph. The sexual morph
is characterized by 34–46 × 6.7–9 µm asci and 10–12.5 × 2.8–3.6 µm, ellipsoidal ascospores. We
also provide LSU, ITS, tub2 and tef sequence data of D. rosae strains which are deposited in
GenBank. The molecular analyses were performed in the ARB software environment and the
pipeline is provided as supplementary data.
Key words – ARB analysis – Diaporthaceae – saprobes – seed/fruit fungi
Introduction
Diaporthe is a well-known plant pathogenic genus, but species may also occur as endophytes
or saprobes (Udayanga et al. 2011, 2012, 2014b, 2015, Gao et al. 2014, Dissanayake et al. 2017a, b,
c). Diaporthe species can be found worldwide on a wide range of host plants (Gao et al. 2014,
Dissanayake et al. 2017a, b, c). Dissanayake et al. (2017b) provided an account of species in the
genus Diaporthe, listing 171 species with associated molecular data. Thirteen Diaporthe species,
including an epitype, have been collected in Thailand, from different hosts and substrates
(Udayanga et al. 2012, 2015, Liu et al. 2015, Doilom et al. 2016, Hyde et al. 2016, Dissanayake et
al. 2017b, Perera et al. 2018, Wanasinghe et al. 2018).
Phenotypic plasticity, cryptic diversification, and a vast range of host associations of
Diaporthe species have resulted in complications with the accurate identification of species within
Mycosphere 9(2): 256–270 (2018) www.mycosphere.org ISSN 2077 7019
Article
Doi 10.5943/mycosphere/9/2/7
Copyright © Guizhou Academy of Agricultural Sciences

257
the genus (Udayanga et al. 2014a). Recently, the systematic accounts of Diaporthe have
progressively used molecular data for delineating and characterising species (Santos & Phillips
2009, Diogo et al. 2010, Udayanga et al. 2012, 2014b, Gao et al. 2014, Dissanayake et al. 2017a, b,
c). However, Udayanga et al. (2014b) revealed the importance of detailed study of existing names
and type specimens in Diaporthe, before introducing a new name, to avoid introducing superfluous
names.
Diaporthe species associated with economically important crops and ornamentals, and post-
harvest diseases are well-studied (Farr et al. 2002a, b, Luongo et al. 2011, Udayanga et al. 2011,
2012, 2014b, Thomidis et al. 2013, Dissanayake et al. 2017a, b, c), while knowledge of those on
wild fruits and seeds is limited. The aim of this study is to identify the species of Diaporthe on
fruits of Magnolia champaca in Thailand. Here we introduce the sexual morph of D. rosae based
on molecular data, and similarities in asexual morph characters between our collection and the
holotype.
Materials and Methods
Fruits of Magnolia champaca (and Senna siamea) were collected from Phayao and Chiang
Rai Provinces, Thailand during August 2017. Macroscopic and microscopic characters of the
specimens were observed in the laboratory. Fungal structures were observed using a Motic
dissecting microscope (SMZ 168) and a Nikon ECLIPSE 80i compound microscope. Free hand
sections of fungal fruiting bodies were taken and mounted in water and Congo red for microscopic
study. Photomicrography was carried out using a Canon 450D digital camera fitted to the
microscope. Measurements were made with Tarosoft (R) Image Frame Work software. The images
used for illustrating the fungus were processed with Adobe Photoshop CS5 v. 12.0 (Adobe
Systems, USA). Single spore colonies were established as described in Chomnunti et al. (2014).
Pure cultures were obtained on Potato Dextrose Agar (PDA) and incubated at room temperature of
25 °C. To induce sporulation, cultures were incubated in the dark at 25 °C.
Herbarium specimens were deposited in the Mae Fah Luang University (MFLU) herbarium,
Chiang Rai, Thailand. Living cultures were deposited in the Culture Collection at Mae Fah Luang
University (MFLUCC). Facesoffungi and Index Fungorum numbers were registered as detailed in
Jayasiri et al. (2015) and Index Fungorum (2018).
DNA isolation, amplification and analyses
Mycelia for DNA extraction were grown on PDA at 25 °C. Genomic DNA was extracted
using the Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux®, P.R. China)
following the manufacturer’s protocol. Partial gene sequences were amplified for the 28S large
subunit rRNA gene (LSU), the internal transcribed spacer (ITS), beta-tubulin (tub2) and translation
elongation factor 1-alpha gene (tef). The primers and PCR conditions are listed in Table 1. PCR
was performed in a 25 μl reaction volume containing, 12.5 μl of 2 × PCR Master Mix (TIANGEN
Co., China), 9.5 μl ddH2O, 5–10 ng DNA and 1 μl of each primer (10 μM). PCR products were
purified and sequenced at Shanghai Sangon Biological Engineering Technology & Services Co.,
China. Both directions of the PCR products were sequenced using the same primer pairs as used in
PCR amplification. A consensus sequence for each gene region was assembled in ContigExpress
(Vector NTI Suite 6.0). Sequences were deposited in GenBank under accession numbers
MG906794 (LSU), MG906792 (ITS), MG968953 (tef) and MG968951 (tub2) for D. rosae
(MFLUCC 18-0354), and under accession numbers MG906795 (LSU), MG906793 (ITS),
MG968952 (tub2) and MG968954 (tef) for D. rosae (MFLUCC 17-2574).
The sequences generated in this study were supplemented with additional sequences
downloaded from GenBank (Table 2). Based on BLAST results and preliminary analysis sequences
of all strains named as D. miriciae, D. passifloricola and D. ueckerae and related species were
incorporated into the final analysis. All alignments were produced with MAFFT v.7.055b (using
the E-INS-i alignment strategy, Katoh & Standley 2013), integrated in ARB program package (v.
6.0.6) (Ludwig et al. 2004), checked and refined where necessary. Maximum likelihood analyses of

258
single gene regions: ITS, tef and tub2 were carried out for selected Diaporthe species to compare
the topology of the trees and clade stability. Only reliably alignable positions were used for
phylogenetic analyses. This included those corresponding to base pairs 25–51, 58–63, 72–104,
105–194, 197–209, and 212–306 of sequence KJ590747 (D. ueckerae) for tef. Of the tub2
alignment, positions 75–174, 180–322, and 328–436 according to sequence KJ610881 were
considered. ITS gene trees were based on positions 17–64, 67–92, 98–170, 172–387, and 390–506
according to KJ590726. A combined gene analysis was carried out for concatenated alignment of
tef and tub2 sequences.
A maximum likelihood (ML) analysis was performed using RAxML (v.7.7.2, Stamatakis
2006) as implemented in ARB. Support from 1000 bootstrap replicates was mapped on the most
likely tree topology, which was found using the GTRGAMMA model of nucleotide substitution.
Bayesian inference analysis (BI) was performed using MrBayes (v.3.2.1, Ronquist et al. 2012) as
implemented in ARB. GTR+I+G was selected as evolutionary model for phylogenetic analyses of
tef and tub2 gene regions. Two parallel analyses, each consisting of six Markov Chain Monte Carlo
(MCMC) chains, run from random starting trees for 4 000 000 generations were sampled every 100
generations; resulting in 10 000 total trees. The first 2 500 trees, representing the burn in phase of
the analyses were discarded from each run. The remaining trees were used to calculate posterior
probabilities (PP) of the branches in the majority rule consensus tree. Trees were viewed by Xfig
v.3.2 patchlevel 5c (Protocol 3.2), and edited using Microsoft PowerPoint 2010.
The ARB database including all phylogenetic trees and corresponding alignments (with
information on reliably alignable positions) is freely accessible on the SILVA project website
https://www.arb-silva.de/no_cache/download/archive/publications/diaporthe/
Table 1 PCR protocols applied for the analysed loci.
Locus
Primers (Reference)
PCR Conditions
LSU
LR5/LR0R (Vilgalys & Hester 1990, Rehner &
Samuels 1994)
a94 °C: 30 s, 55 °C: 1 min.,
72 °C: 1 min. (37 cycles)b
ITS
ITS5/ITS4 (White et al. 1990)
a94 °C: 30 s, 55 °C: 1 min.,
72 °C: 1 min. (37 cycles)b
tef
EF1-728F/ EF1-986R (Carbone & Kohn 1999)
a94 °C: 30 s, 48 °C: 30 s,
72 °C: 1.30 min. (35 cycles)b
tub2
Bt2a/Bt2b (Glass & Donaldson 1995)
a94 °C: 30 s, 55 °C: 50 s,
72 °C: 1 min. (35 cycles)b
aInitiation step of 94 °C: 3 min
bFinal elongation step of 72 °C: 7 min. and final hold at 4 °C applied to all PCR thermal cycles
Table 2 GenBank accession numbers of strains included in the study.
Species
Culture collection no.
GenBank no.
ITS
tub2
tef
D. batatas
CBS 122.21 (T)
KC343040
KC344008
KC343766
D. convolvuli
CBS 124654 (T)
KC343054
KC344022
KC343780
D. endophytica
CBS 133811 (T)
KC343065
KC344033
KC343791
D. endophytica
LGMF911
KC343066
KC344034
KC343792
D. helianthi
CBS 592.81 (T)
KC343115
KC344083
KC343841
D. helianthi
CBS 344.94
KC343114
KC344082
KC343840
D. hordei
CBS 481.92 (T)
KC343120
KC344088
KC343846
D. kochmani
BRIP 54033 (T)
JF431295
-
JN645809
D. kochmani
BRIP 54034
JF431296
-
JN645810
D. kongi
BRIP 54031 (T)
JF431301
-
JN645797
D. kongi
BRIP 54032
JF431300
-
JN645798

259
Table 2 Continued.
Species
Culture collection no.
GenBank no.
ITS
tub2
tef
D. longicolla
ATCC 60325 (T)
KJ590728
KJ610883
KJ590767
D. longicolla
FAU644
KJ590730
KJ610885
KJ590769
D. masirevici
BRIP 57892a (T)
KJ197277
KJ197257
KJ197239
D. masirevici
BRIP 54256
KJ197276
KJ197256
KJ197238
D. melonis
CBS 507.78 (T)
KC343141
KC344109
KC343867
D. melonis
FAU640
KJ590702
KJ610858
KJ590741
D. miriciae
BRIP 54736j (T)
KJ197282
KJ197262
KJ197244
D. miriciae
BRIP 56918a
KJ197284
KJ197264
KJ197246
D. miriciae
BRIP 55662c
KJ197283
KJ197263
KJ197245
D. ovalispora
ZJUD93 (T)
KJ490628
KJ490449
KJ490507
D. passifloricola
CBS 141329 (T)
KX228292
KX228387
-
D. phaseolorum
CBS 116019
KC343175
KC344143
KC343901
D. rosae
MFLUCC 17-2658 (T)
MG828894
MG843878
-
D. rosae
MFLUCC 17-2574
MG906793
MG968952
MG968954
D. rosae
MFLUCC 18-0354
MG906792
MG968951
MG968953
D. schini
CBS 133181 (T)
KC343191
KC344159
KC343917
D. schini
LGMF910
KC343192
KC344160
KC343918
D. sojae
FAU635 (T)
KJ590719
KJ610875
KJ590762
D. sojae
FAU455
KJ590712
KJ610868
KJ590755
D. sojae
DP0601
KJ590706
KJ610862
KJ590749
D. sojae
MAFF 410444
KJ590714
KJ610870
KJ590757
Diaporthe sp.
LGMF947/CPC 20323
KC343203
KC344171
KC343929
D. subordinaria
CBS 464.90 (T)
KC343214
KC344182
KC343940
D. tectonendophytica
MFLUCC 13-0471 (T)
KU712439
KU743986
KU749367
D. terebinthifolii
CBS 133180 (T)
KC343216
KC344184
KC343942
D. terebinthifolii
LGMF907
KC343217
KC344185
KC343943
D. thunbergiicola
MFLUCC 12-0033 (T)
KP715097
-
KP715098
D. ueckerae
FAU656/CBS 139283 (T)
KJ590726
KJ610881
KJ590747
D. ueckerae
FAU660
KJ590723
KJ610878
KJ590744
D. ueckerae
FAU659
KJ590724
KJ610879
KJ590745
D. ueckerae
FAU658
KJ590725
KJ610880
KJ590746
D. ueckerae
SLHX14
KY565426
-
-
D. ueckerae
SLHX11
KY565425
-
-
D. ueckerae
SLHX3
KY565424
-
-
D. ueckerae
K.L. Chen 034
-
LC086655
-
D. ueckerae
K.L. Chen 015
-
LC086654
-
D. unshiuensis
ZJUD52/ CGMCC
3.17569 (T)
KJ490587
KJ490408
KJ490466
D. unshiuensis
ZJUD50
KJ490585
KJ490406
KJ490464
(T) Ex-type strains
*New isolates are in bold
Results
Two species of Diaporthe were found on fruits of Magnolia. One species was unambiguously
identified as D. collariana according to morphological and molecular data (Perera et al. 2018). A

260
second species was reminiscent of D. rosae, of which only the asexual morph is currently known. It
was therefore analyzed in more detail.
Phylogenetic analyses
Forty one Diaporthe isolates including our two new strains and an outgroup taxon were
selected for the tef data analysis. The tef data set comprised 270 characters with gaps. The best
scoring RAxML tree with a final likelihood value of -2151.932507 is presented. The matrix had
178 distinct alignment patterns, with 0.63% of undetermined characters or gaps. Estimated base
frequencies were as follows; A = 0.187328, C = 0.339338, G= 0.202059, T = 0.271275;
substitution rates AC = 1.390930, AG = 4.610159, AT = 1.162314, CG = 1.485311, CT =
3.678796, GT = 1.000000; gamma distribution shape parameter α = 0.147398. The tub2 data set
comprised 41 Diaporthe isolates and 352 characters including gaps. The best scoring RAxML tree
with a final likelihood value of -1433.994346 is presented. The matrix had 105 distinct alignment
patterns, with 0.14% of undetermined characters or gaps. Estimated base frequencies were as
follows; A = 0.196503, C = 0.353247, G= 0.228837, T = 0.221413; substitution rates AC =
0.915963, AG = 3.289956, AT = 0.903206, CG = 1.558142, CT = 4.661162, GT = 1.000000;
gamma distribution shape parameter α = 0.421968. Tree topologies of the ML analyses were
similar to the BI. Our two isolates consistently grouped in a monophyletic clade in ML and BI
analyses of both tub2 and tef datasets with high support (Figs 1, 2), and moderate support in the
tub2 and tef combined tree (Appendix 2). The ITS tree was largely unresolved and the clades
lacked reasonable support (Appendix 2).
Diaporthe rosae formed a monophylum with our strains in the tub2 tree and the combined
tub2 and tef tree, while tef sequences of D. rosae were not available for the analysis. Diaporthe
miriciae, D. passifloricola and D. ueckerae clustered in close relationship to D. rosae. However,
the species differ according to their DNA sequences and morphological characters (Table 3).
Furthermore, Diaporthe sp. (LGMF947/CPC 20323), which was isolated from a seed of Glycine
max from Brazil, shows close phylogenetic affinities to D. rosae in the tef tree (Fig. 2). The two
species (Diaporthe sp. and D. rosae) showed 5 nucleotides differences in the tub2 region, and 3
different nucleotides in the ITS region, while their tef sequences were identical.
Taxonomy
Diaporthe rosae Samarakoon & K.D. Hyde Figs 3, 4
Saprobic on Rosa sp. and, dried fruits of Magnolia champaca and Senna siamea. Visible as
raised, black spots or, black necks immerging through the host surface. Sexual morph – Ascomata
260–350 µm high, 210–340 µm diam. ( = 260 × 300 µm, n = 6), immersed in the ectostroma,
immersed in the host epidermis, globose to sub-globose, solitary or occur in clusters, black,
ostiolate, papillate. Neck 190 × 435 µm diam. Ostiole periphysate. Peridium 8–22 µm wide,
comprising 4–10 layers, outer layers heavily pigmented, thin-walled, comprising dark brown cells
of textura angularis, inner layers composed of hyaline to brown thin-walled cells of textura
angularis. Paraphyses 5.4–8 µm (n = 10), 2–4-septate, wide at base, tapering towards the apex, thin
walled. Asci 34–46 × 6.7–9 µm ( = 40.5 × 7.9 µm, n = 20), 8-spored, unitunicate, clavate to
subclavate, straight to slightly curved, sessile, with a J- apical ring. Ascospores 10–12.5 × 2.8–3.6
µm ( = 11.1 × 3.2 µm, n = 30), overlapping uniseriate to biseriate, 1-septate, constricted at the
septum, often tetra-guttulate, ellipsoidal, straight, hyaline, without appendages or a mucilaginous
sheath. Asexual morph on PDA – Conidiomata pycnidial or multiloculate, scattered, globose or
irregular, black. Peridium consisting brown cells of textura angularis in surface view. Conidial
mass globose, white to pale-yellow. Paraphyses absent. Conidiophores 10–63 × 1.4–2.7 μm (
=36.4 × 2 μm), 2–3-septate, branched, densely aggregated, cylindrical, straight to sinuous rarely
reduced to conidiogenous cells, hyaline, smooth-walled. Alpha conidiogenous cells 8–23 × 0.7–3
μm ( = 15.4 × 1.8 μm) phialidic, subcylindrical, sometimes ampulliform, slightly tapering
towards the apex, hyaline, with visible periclinal thickening, and a flared collarette. Alpha conidia

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5–7 × 2–3.1 μm ( = 5.9 × 2.5 μm), enteroblstic, ovate to ellipsoidal, base obtuse to subtruncate,
aseptate, straight, bi-guttulate, hyaline, smooth-walled. Beta conidiogenous cells 4.1–22.6 × 1.3–
4.2 μm ( = 16.6 × 2 μm) phialidic, subcylindrical, tapering towards the apex, hyaline, with
periclinal thickening, and a flared collarette. Beta conidia 18–28 × 0.9–1.3 μm ( = 22.3–1.1 μm),
fusiform to hooked, aseptate, hyaline, smooth-walled. Gamma conidia not observed.
Culture characteristics – Colonies on PDA, reaching 40 mm diam. after 2 weeks at 25°C, flat,
circular, margin entire, white with radially arranged minute mycelium clots later becoming pale
yellow, reverse whitish, azonate. Odour not pronounced. Sporulated on PDA after 2 months
incubation period in dark, at 25°C.
Material examined – THAILAND, Chiang Rai Province, Mae Fah Luang University
premises, on dried fruits and pedicels of Magnolia champaca (L.) Baill. ex Pierre (Magnoliaceae),
17 August 2017, S. Boonmee, Fruit 5 (MFLU 18-0186); dry culture, MFLU 18-0515; living
culture, MFLUCC 18-0354; ibid. Phayao Province, Pong, Pha Chang Noi, dried pods of Senna
siamea (Lam.) Irwin et Barneby (Fabaceae), 04 August 2017, R.H. Perera, PH-FB 1 (MFLU 18-
0187), living culture, MFLUCC 17-2574.
Figure 1 – Phylogram generated from maximum likelihood analysis based on tub2 sequences of
selected Diaporthe species. Maximum likelihood bootstrap support (ML≥50%) and posterior
probabilities (PP≥0.90) from Bayesian inference analysis are indicated respectively near the nodes.
The ex-type strains are in bold and new isolates in blue. The scale bar indicates 0.025 nucleotide
changes per site. The tree is rooted with Diaporthe subordinaria.

262
Figure 2 – Phylogram generated from maximum likelihood analysis based on tef sequence data of
selected Diaporthe species. Maximum likelihood bootstrap support (ML≥50%) and posterior
probabilities (PP≥0.90) from Bayesian inference analysis are indicated respectively near the nodes.
The ex-type strains are in bold and new isolates in blue. The scale bar indicates 0.05 nucleotide
changes per site. The tree is rooted with Diaporthe subordinaria.
Notes – Our two new isolates grouped with the ex-type strain of D. rosae (MFLUCC 17-
2658), which was collected from Rosa sp. (Rosaceae) in Chiang Rai Province, Thailand (Fig. 1)
(Wanasinghe et al. 2018). The asexual morph of one of the new strains (MFLUCC 18-0354)
produced on PDA is similar to that of the holotype of D. rosae (MFLU 17-1550). DNA sequences
of D rosae and strains (MFLUCC 18-0354 and MFLUCC 17-2574) differed in 2 positions of the
ITS region, while tub2 sequences were identical. Sequence data of the tef region were not available
for the ex-type of D. rosae for the comparison. Neither molecular nor morphological data
accordingly allow delimiting the new collection from D. rosae. It is therefore reported here as the
sexual morph of D. rosae.
Discussion
In this study, we used the ARB software environment to analyze the sequence data related to
our collection and the pipeline is provided as the Supplementary Data to this paper (Appendix 1).
Sequence heterogeneity of ITS has been observed earlier within the same species of Diaporthe,
even within the same geographic region and the same host by different authors (Farr et al. 2002a, b,
Santos et al. 2010; Udayanga et al. 2014a). The difference of 2 nucleotides in the ITS region

263
Table 3 Comparison of our new collection with holotype of Diaporthe rosae, and related species
Characters
Diaporthe rosae Samarakoon
& K.D. Hyde
Diaporthe rosae Samarakoon
& K.D. Hyde
Diaporthe ueckerae
Udayanga & Castl. 2014
Diaporthe miriciae R.G.
Shivas, S.M. Thomps. &
Y.P. Tan 2015
Diaporthe passiflorae Crous
& L. Lombard 2012
Wanasinghe et al. (2018)
This study
Udayanga et al. (2015)
Thompson et al. (2015)
Crous et al. (2012)
Lifestyle and host
Saprobic on dead pedicel of
Rosa sp./ Thailand
Saprobic on dried fruits of
Magnolia champaca and Senna
siamea/ Thailand
Cucumis melo/ USA
Helianthus annuus, Vigna
radiata, Glycine max/
Australia
Passiflora edulis/ South
America
Conidiomata
Multiloculate, scattered on
PDA
Multiloculate or pycnidial,
scattered on PDA
Pycnidial, globose, 150–200
μm diam., ostiolate with necks
Pycnidial or multilocular,
ostiolate with necks
Pycnidial, globose, 300 μm
diam.
Conidiophores
Present, sometimes reduced to
conidiogenous cells
Present, 2–3-septate, rarely
reduced to conidiogenous cells,
cylindrical, straight to sinuous
Present, unbranched,
ampulliform, long, slender
Reduced to conidiogenous
cells or 1–2-septate
Present, 2–3-septate,
branched, densely
aggregated, cylindrical,
straight to sinuous
Conidiophore
dimension (µm)
10–19 × 1.9–3.3
10–63 × 1.4–2.7
(9–)12–28(–30) × 1.5–2.5
10–20 × 1.5–3
20–30 × 2.5–4
Alpha and beta
conidiogenous cells
Phialidic, ampulliform,
slightly tapering towards the
apex with periclinal
thickening, with a flared
collarette
Phialidic, subcylindrical,
sometimes ampulliform,
slightly tapering towards the
apex, with periclinal
thickening, and a flared
collarette
Phialidic, cylindrical,
terminal, slightly tapering
towards apex
Cylindrical to obclavate
Phialidic, cylindrical,
terminal and lateral
Conidiogenous cells
dimension (µm)
7–13 × 1–2.5 (Alpha)
8–23 × 0.7–3 (Alpha)
0.5–1 (diam.)
10–20 × 1.5–3
7–15 × 1.5–2.5
7.7–15 × 1.2–2.3 (Beta)
4.1–22.6 × 1.3–4.2 (Beta)
Alpha conidia
Ovate to ellipsoidal, base
subtruncate, bi- biguttulate,
aseptate, hyaline, smooth-
walled
Ovate to ellipsoidal, base
obtuse to subtruncate, bi-
guttulate, aseptate, hyaline,
smooth, smooth-walled
Abundant, aseptate, hyaline,
smooth, ellipsoidal, often
biguttulate, base subtruncate
Abundant, aseptate, hyaline,
fusiform to oval, rounded at
the apex, narrowed at the
base
Aseptate, hyaline, smooth,
multi-guttulate, fusoid to
ellipsoid, tapering
towards both ends, straight,
apex subobtuse, base
subtruncate
Alpha conidia
dimension (µm)
5.5–7.5 × 2–3
5–7 × 2–3.1
(6–)6.4–8.2(–8.6) × (2–)2.3–3
6–7.5(–9) × 2–2.5(–3)
(5.5–)6–7(–8) × (2–)2.5–3(–
3.5)
Beta conidia
Fusiform to hooked
Fusiform to hooked
Not observed
Scattered or in groups
amongst the alpha conidia,
flexuous to hamate, hyaline
Spindle-shaped, aseptate,
apex acutely rounded, base
truncate, tapering from lower
third towards Apex, curved
Beta conidia
dimension (µm)
12.5–18 × 1–2 (on natural
substrate),
12.6–21.1 × 0.7–1.2 (on PDA)
9–23 × 0.5–0.8
18–28 × 0.9–1.3 (on PDA)
-
20–35 × 1.0–1.5
(14–) 16–18(–20) × 1.5(–2)

264
Figure 3 – Diaporthe rosae (MFLU 18-0186) a Herbarium material. b, c Ascomata on host
substrate (white arrow: ascospores mass on the neck). d Section through ascoma. e Section through
the peridium. f Surface view of the peridium. g Paraphyses. h–l Asci. m–p Ascospores. q
Germinating ascospores. Bars – b, c = 200 µm, d = 100 µm, e, f = 50 µm, g–l = 20 µm, m–p = 10, q
= 20 µm.

265
Figure 4 – Diaporthe rosae (MFLU 18-0515, asexual morph on PDA) a, b Sporulation on PDA. c,
d Conidioma on PDA. e Surface view of the peridium. f Conidiophores with alpha and beta
conidia. g, h Conidiophores with alpha conidia. i Alpha conidia. j, k Beta conidia. Bars – b, c = 200
µm, d = 100 µm, e, f = 50 µm, g–l = 20 µm, m–p = 10, q = 20 µm.
between our collection and the asexual morph of D. rosae is therefore in the range of intraspecific
variability within the genus.
Diaporthe miriciae and D. ueckerae strains only form a monophyletic clade together in the
tub2, tef and combined tef and tub2 analyses (Figs 1, 2, Appendix 2), ‘ueckerae’ Clade).
Furthermore, ITS, tef and tub2 sequence data of their ex-types (BRIP 54736j and FAU656) are
almost identical except for 1 bp difference in the tef region. When considering other D. miriciae
(BRIP 56918a, BRIP 55662c) and D. ueckerae (FAU660, FAU659, FAU658) strains, a maximum
difference of 3 nucleotides is found within the clade. Diaporthe miriciae and D. ueckerae also
share similar morphological characters (Table 3). Even though those similarities are in the same
range as for D. rosae and the new collections, we refrain from describing our collection as a new
species. We agree with Udayanga et al (2014a) that such differences most likely represent

266
intraspecific variation. Another two strains putatively named as D. ueckerae (K.L. Chen 015 and
K.L. Chen 034) are unrelated to the main ‘ueckerae’ clade (Fig. 1). They showed 4 nucleotide
substitutions in tub2 region to the D. ueckerae species in the main clade. However, no other gene
regions or morphological features are available for comparison.
One problematic sequence of D. thunbergiicola (MFLUCC 12-0033) clusters with long
branches and its phylogenetic position is conflicting notably between ITS and tef trees (Fig. 2,
Appendix 2). Re-sequencing of the isolate of D. thunbergiicola would be required to finally
confirm its phylogenetic position. While we did not exclude D. thunbergiicola (MFLUCC 12-0033)
from our analysis, we do not consider our results strong evidence for the species being related to
the D. rosae clade.
Acknowledgements
The Research of Featured Microbial Resources and Diversity Investigation in Southwest
Karst area (Project No. 2014FY120100) is thanked for financial support. Kevin D. Hyde thanks the
Chinese Academy of Sciences, project number 2013T2S0030, for the award of a Visiting
Professorship for Senior International Scientists at Kunming Institute of Botany. This work was
also supported by the Thailand Research Fund, ‘The future of specialist fungi in a changing
climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron
species and Dracaena species’ (Project No. DBG6080013).
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Appendix 1: Supplementary information to manuscript
Phylogenetic analyses of multi gene alignments using ARB
1) ARB (http://www.arb-home.de/) was installed on a QIIME 2 Core VirtualBox Image (v
2017.12, https://qiime2.org/), on which libxm4 and Xfig had been installed previously.
2) A new ARB database was created using the ITS sequences downloaded from GenBank
(https://www.ncbi.nlm.nih.gov) in GenBank format.
 Sequences were imported into the alignment “ali_ITS”
 The newly created import filter, “GB_MFU.ift” (https://www.arb-
silva.de/no_cache/download/archive/imp_exp_filters/), was applied to import a
maximum of sequence associated information.
3) The newly generated sequences were imported in FASTA format (File > Import > Import
from external format).
4) The sequence accession number was preserved.
 The accession was copied to new field called “Acc_ITS”
i. Sequences with entries in the ali_ITS/data field were searched (Species >
Search and query) and the accession numbers were copied using “More
functions > Modify Fields of Listed Species” in the “SEARCH and
QUERY” window.
5) Imported ITS sequences were aligned using MAFFT (Sequences > Align Sequences > Mafft).
6) A selected sequence was copied to a new ‘species’ called ‘filter’ and used as a filter sequence
for phylogenetic analyses.
 Positions in the newly created filter sequence, which correspond to ambiguously
aligned regions were replaced by Gap symbols (“-”).
7) Successive import of sequences from other genes
 A new alignment was created (Sequence > Sequennce/Alignment Admin) for each
additional gene (ITS, tub2 and tef); ie. ‘ali_ITS’, ‘ali_tub2’ and ‘ali_tef, respectively,
and specified appropriately.
 Reference sequences were imported (File > Import > Import from external format)
in GenBank format and using the filter “GB_MFU.ift”.
 Newly obtained sequences were imported in FASTA format and using the filter
“fasta.ift”.
 Sequence Accession numbers were copied to the corresponding field, i.e.
‘Acc_ITS’, ‘Acc_tub2’ and ‘Acc_tef’, respectively.
 Newly imported sequences were aligned using MAFFT.
 A filter sequence, always called ‘filter’, was created and modified appropriately.
8) Merging of sequences
 A new field (“individual”) was created (Species > Database fields admin > create
fields…)
 Strain or specimen Ids were copied (using “More functions > Modify Fields of
Listed Species” in the “SEARCH and QUERY” window) to the field “individual”
and curated.
 Expert mode was enabled (Properties > Toggle expert mode).
 Sequence of the same individual were merged (Species > Merge Species > Create
merged species from similar species) using entries in the database field “individual”
as identifier.
 The newly created field “merged_species” was modified by adding a “1” to those
individuals (strain or specimens) which are only represented by a single sequence.

269
 Database entries with single sequences were deleted; i.e. species having no entry in
the “merged_species” field were searched (Species > Search and query) and deleted
(Delete Listed).
9) Calculating phylogenetic trees using RAxML.
 Only positions in which the filter sequence has no Gap (“-”) were considered for
phylogenetic reconstructions.
 The resulting trees were renamed.
 To assure traceability of the analyses, the alignment (including the filter sequence)
underlying the phylogenetic tree was copied to a new alignment, which was
renamed including the name of the corresponding tree.
10) Calculating multi-gene phylogenies.
 Single gene alignments (including the filter sequences) were concatenated
(Sequence > Concatenate Sequences/Alignments).
 Phylogenetic trees were calculated as detailed above based on the positions specified
by the filter sequence.
 Trees were renamed and the underlying alignment copied to a correspondingly
named alignment for documentation.
Appendix 2
Figure 1 – Phylogram generated from maximum likelihood analysis based on ITS sequences of
selected Diaporthe species. Maximum likelihood bootstrap support values (ML≥50%) are indicated
near the nodes. The ex-type strains are in bold and new isolates in blue. The scale bar indicates
0.025 nucleotide changes per site. The tree is rooted with Diaporthe subordinaria.

270
Figure 2 – Phylogram generated from maximum likelihood analysis based on combined tef and
tub2 sequence data of selected Diaporthe species. Maximum likelihood bootstrap support
(ML≥50%) is indicated near the nodes. The ex-type strains are in bold and new isolates in blue. The
scale bar indicates 0.025 nucleotide changes per site. The tree is rooted with Diaporthe
subordinaria.