Paraphoma species associated with Convolvulaceae

Abstract

Substantial difficulties in the morphological identification of phoma-like fungi, including Paraphoma spp., have resulted in poor understanding of the generic and species boundaries in this group of organisms. This study was devoted to the reidentification and taxonomic revision of phoma-like isolates derived from Convolvulaceae leaves collected from different geographical locations in Russia and territories of neighboring countries. The study was based primarily on sequencing phylogenetically informative loci (ITS, LSU, TUB, and RPB2) and on traditional morphological approaches. The resulting phylogenetic tree revealed three well-supported monophyletic clades, corresponding to three Paraphoma species. The new species Paraphoma melnikiae Gomzhina M. M. & Gasich E. L. was described, and a new taxonomic combination, Paraphoma convolvuli (Dearn. & House) Gomzhina M. M. & Gasich E. L., was established for Stagonospora convolvuli. Several isolates were preliminarily identified as Paraphoma cf. convolvuli and are likely new species of the genus Paraphoma, but this requires further verification.

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ORIGINAL ARTICLE
Paraphoma species associated with Convolvulaceae
M. M. Gomzhina
1
&E. L. Gasich
1
&L. B. Khlopunova
1
&P. B. Gannibal
1
Received: 25 July 2019 / Revised: 10 January 2020 / Accepted: 13 January 2020
#German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Substantial difficulties in the morphological identification of phoma-like fungi, including Paraphoma spp., have resulted in poor
understanding of the generic and species boundaries in this group of organisms. This study was devoted to the reidentification
and taxonomic revision of phoma-like isolates derived from Convolvulaceae leaves collected from different geographical
locations in Russia and territories of neighboring countries. The study was based primarily on sequencing phylogenetically
informative loci (ITS, LSU, TUB,andRPB2) and on traditional morphological approaches. The resulting phylogenetic tree
revealed three well-supported monophyletic clades, corresponding to three Paraphoma species. The new species Paraphoma
melnikiae Gomzhina M. M. & Gasich E. L. was described, and a new taxonomic combination, Paraphoma convolvuli (Dearn. &
House) Gomzhina M. M. & Gasich E. L., was established for Stagonospora convolvuli. Several isolates were preliminarily
identified as Paraphoma cf. convolvuli and are likely new species of the genus Paraphoma, but this requires further verification.
Keywords Phaeosphaeriaceae .Phoma-like fungi .Ta xonomy .Multilocus phylogeny .New species .New combination
Introduction
The genus Paraphoma Morgan-Jones & J. F. White
(Phaeosphaeriaceae) was established in 1983 with
Paraphoma radicina (McAlpine) Morgan-Jones & J. F.
White (≡Pyrenochaeta radicina McAlpine) as the type spe-
cies (Morgan-Jones and White 1983). Initially, it was sug-
gested that the most informative taxonomic feature for mem-
bers of this genus was setose pycnidia. However, the presence
of such pycnidia in the fungal life cycle is specific for both
Paraphoma and Pyrenochaeta De Not. species. Thus, it has
been proposed to use ultrastructural features of conidiogenesis
to distinguish the species of these two genera. According to
those data, Paraphoma species have been placed in the genus
Phoma Sacc. as a part of the appropriate section Paraphoma
(Boerema et al. 2004).
Substantial difficulties in morphological identification
have resulted in poor understanding of the generic and
species boundaries generally in coelomycetes and partic-
ularly in the genus Phoma and its sections (sensu
Boerema et al. 2004). Additionally, classification systems
based on morphological features are highly artificial and
do not represent evolutionary relationships. Molecular
phylogenetic studies based on DNA sequencing have
shown that Paraphoma is not a sister clade to phoma-
like fungi transferred to the family Didymellaceae, but it
is closely related to other genera affiliated with the fami-
lies Phaeosphaeriaceae (de Gruyter et al. 2010),
Cucurbitariaceae, and Coniothyriaceae (Chen et al.
2015). Currently, MycoBank categorizes eight species in
the genus Paraphoma, and this group of organisms is
actively being investigated. In the last 6 years, at least
four new Paraphoma species have been described
(Quaedvlieg et al. 2013; Crous et al. 2017;Moslemi
et al. 2017).
Convolvulus arvensis and Calystegia sepium are perennial,
soboliferous plants and two of the most harmful weeds.
Controlling these weeds requires intense tillage and the use
of a considerable amount of herbicides (Stetsov and
Sadovnikova 2012; Nadtochiy 2008). Consequently, the po-
tential application of biological weed control alternatives, par-
ticularly phytopathogenic fungi, has been studied more inten-
sively in recent years. Several members of Phaeosphaeriaceae,
including Paraphoma species, can be applied as living
Section Editor: Gerhard Rambold
*M. M. Gomzhina
gomzhina91@mail.ru
1
All-Russian Institute of Plant Protection, Shosse Podbel’skogo 3,
Saint Petersburg, Russia
Mycological Progress (2020) 19:185–194
https://doi.org/10.1007/s11557-020-01558-8
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mycoherbicides and produce bioactive compounds with her-
bicide characteristics (Guntli et al. 1998; Poluektova et al.
2018).
The taxonomical diversity of phytopathogenic fungi,
which can infect plants of the family Convolvulaceae,
was examined in Russia and other countries. Several
phoma-like species were revealed on these plants and
are listed in Table 1. Three of those species produce sev-
eral compounds with mycoherbicide activity against
C. arvensis:Phoma proboscis (Heiny and Templeton
1991,1995), Phomopsis convolvuli (Ormeno-Nuñez
et al. 1988a,b;Watsonetal.1993; Vogelsang et al.
1998), and Stagonospora convolvuli (Pfirter and Defago
1998; Pfirter et al. 1999;Defagoetal.2001).
Due to extensive analysis of fungal biodiversity on
weeds in Russia and neighboring countries in 1990–2010,
pure cultures of fungi isolated from Convolvulaceae were
collected. This collection is stored in the laboratory of
Mycology and Phytopathology of the All-Russian
Institute of Plant Protection. It includes 70 isolates of
phoma-like fungi obtained from C. arvensis and
C. sepium. All isolates in this collection were identified
based on morphological features. A considerable number
of isolates in this collection were not identified at the spe-
cies level and have unclear definitions, such as Ascochyta
sp., Mycosphaerella sp., and Phoma sp. According to pre-
liminary molecular phylogenetic data (Gomzhina et al., un-
published), the collection consists of at least ten genera of
phoma-like fungi. Among them, eleven isolates were iden-
tified as species of the genus Paraphoma.
The aim of this study was to correctly reidentify Russian
Paraphoma isolates collected from Convolvulaceae and to
taxonomically revise the fungal isolates, based primarily on
a molecular phylogenetic approach and traditional morpho-
logical analysis.
Materials and methods
Isolates
As a result of the extensive studies of fungal biodiversity on
Convolvulaceae weeds carried out in 1990–2010 in different
geographical locations in Russia and territories of neighboring
countries, eleven Paraphoma isolates (Table 2)werecollected
by authors from the leaves of C. arvensis and C. sepium that
exhibited typical leaf spot symptoms. To isolate a pure culture
of fungus from the leaves, fragments of infected material were
surface sterilized with 20 ml of 5% sodium hypochlorite
(NaClO) solution. Firstly washed for 2 min with 0.1% sodium
dodecyl sulfate (SDS), washed with 5% sodium hypochlorite,
and then washed three times with 20 ml of sterile water. After
surface sterilization, the samples were placed on potato-
sucrose agar (PSA) (Samson et al. 2000) containing antibi-
otics (100 μg/ml ampicillin, streptomycin, penicillin,
HyClone, GE Healthcare Life Science, Austria) and 0.4 μl/l
Triton X-100 (PanReac, Spain) to restrict the growth of fungi.
The Petri dishes were incubated at 24 °C in the dark and were
analyzed on days 7–10 of cultivation. Samples of infected
leaves were deposited in the Mycological Herbarium (LEP)
of All-Russian Institute of Plant Protection (VIZR). All
Paraphoma isolates were stored in plastic microtubes on
PSA at + 4 °C in the VIZR pure culture collection.
DNA isolation, PCR, and sequencing
Mycelium was scraped from 20-day-old cultures on oatmeal
agar (OA, Boerema et al. 2004) and macerated with 0.3-mm
glass sand on a Retsch MM400 mixer mill (Retsch, Germany).
Genomic DNA was then extracted according to the standard
CTAB/chloroform method (Doyle and Doyle 1990).
Table 1 Phytopathogenic
phoma-like fungi that can infect
plants of the family
Convolvulaceae
Pathogenic fungus Host References
Phoma sepium Brunaud Calystegia sepium
(L.) R. Br.
Saccardo 1895
P. minuta Weh m. C. sepium Alcalde 1952
P. macrocollum Alcalde C. sepium Alcalde 1952
P. c o n v o l vu l i We hm. Convolvulus
glomeratus
Choisy
Weh me yer 1946
P. c a p s u l ar u m Cooke & Harkn. Ipomoea purpurea
(L.) Roth.
Saccardo 1895
P. proboscis Heiny Convolvulus
arvensis L.
Heiny 1990
Phomopsis calystegiae (Cooke) Petr. & Syd. C. sepium Alcalde 1952
Diaporthe convolvuli (Ormeno-Nuñez, Reeleder & A.K.
Watson) R.R. Gomes, C. Glienke & Crous
Convolvulus
arvensis
Ormeno-Nuñez
et al. 1988a,b
Phyllosticta batatas (Thüm.) Cooke Ipomoea batatas
(L.) Lam.
Punithalingam
1982
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The ITS and LSU regions of rDNA were amplified and
sequenced for all 11 isolates. The RNA polymerase II
(RPB2)andβ-tubulin (TUB) genes were sequenced for 10
and 9 isolates, respectively. All obtained sequences were de-
posited into GenBank, and accession numbers are listed in
Tab le 3.
The primers ITS1F (Gardes and Bruns 1993) and ITS4
(White et al. 1990) and the primers LR0R (Rehner and
Samuels 1994) and LR5 (White et al. 1990) were used to
amplify the ITS and LSU regions of the ribosome genes,
respectively. The primer T1/T2 (O’Donnell and Cigelnik
1997; Saleh and Leslie 2004) was used to amplify part of
the TUB gene, and fRPB2-7cR/fRPB2-5f2 (Liu et al. 1999)
was used to amplify part of the RPB2 gene.
The amplification reactions had a total reaction volume of
25 μl, which was composed of dNTPs (200 μМ), each of the
forward and reverse primers (ITS1F/ITS4, LR0R/LR5, T1/
T2, fRPB2-7cR/fRPB2-5f2) (0.5 μМ), Taq DNA-
polymerase (5 U/μl), 10× PCR buffer with Mg
2+
and NH
4
+
ions, and 1–10 ng of total genomic DNA.
Table 2 Information of the
geographical location and dates of
sample collections, from which
the investigated isolates were
obtained
Isolate/culture collection
no.
Location Substrate Date of collection
MF-9.88. Ex-type Russia, Saint Petersburg Convolvulus
arvensis
17 September
2002
MF-9.95. Ex-type Russia, Saint Petersburg C. arvensis 17 September
2002
MF-9.182.1 Ukraine,Tchernigovskaya oblast C. arvensis 01 August 2004
MF-9.222 Kazakhstan, Almatinskaya
oblast
C. arvensis 16 June 2006
MF-9.240 Russia, Vladivostok C. arvensis 07 September
2006
MF-9.294 Russia, Saint Petersburg C. arvensis 02 October 2009
MF-9.296.1 Russia, Saint Petersburg C. arvensis 18 August 2009
MF-9.265 Russia, Saint Petersburg Calystegia sepium 14 September
2007
MF-9.298.1 Russia, Saint Petersburg C. sepium 07 September
2009
MF-9.300.1 Russia, Saint Petersburg C. sepium 05 August 2009
MF-9.301.1 Russia, Saint Petersburg C. sepium Summer 2009
Table 3 Collection details and GenBank accession numbers of isolates
Isolate Isolate/culture
collection no.
Location Substrate GenBank accession number
ITS LSU TUB RPB2
Paraphoma melnikiae MF-9.88. Ex-type Russia, Saint Petersburg Convolvulus
arvensis
MG764063 MG764065 MG779456 MG779466
P. melnikiae MF-9.95. Ex-type Russia, Saint Petersburg C. arvensis MG764054 MG764067 –MG779462
P. melnikiae MF-9.182.1 Ukraine, Tchernigovskaya
oblast
C. arvensis MG764058 MG764068 MG779454 MG779463
P. c o n v o l vu l i MF-9.222 Kazakhstan, Almatinskaya
oblast
C. arvensis MG764055 MG764069 ––
P. melnikiae MF-9.240 Russia, Vladivostok C. arvensis MG764061 MG764070 MG779453 MG779464
P. melnikiae MF-9.294 Russia, Saint Petersburg C. arvensis MG764059 MG764072 MG779455 MG779471
P. melnikiae MF-9.296.1 Russia, Saint Petersburg C. arvensis MG764056 MG764073 MG779458 MG779465
Paraphoma cf.
convolvuli
MF-9.265 Russia, Saint Petersburg Calystegia
sepium
MG764062 MG764071 MG779457 MG779467
Paraphoma cf.
convolvuli
MF-9.298.1 Russia, Saint Petersburg C. sepium MG764057 MG764074 MG779459 MG779468
Paraphoma cf.
convolvuli
MF-9.300.1 Russia, Saint Petersburg C. sepium MG764064 MG764066 MG779460 MG779469
Paraphoma cf.
convolvuli
MF-9.301.1 Russia, Saint Petersburg C. sepium MG764060 MG764075 MG779461 MG779470
Mycol Progress (2020) 19:185–194 187
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Table 4 GenBank accession numbers of reference isolates
Isolate Strain/culture
collection no.
GenBank accession number References
ITS LSU TUB RPB2
Neosetophoma samarorum CBS 138.96 KF251160.1 KF251664 KF252655.1 KF252168.1 Quaedvlieg et al. 2013
Neostagonospora caricis CBS 135092 KF251163.1 KF251667 KF252658.1 KF252171.1 Quaedvlieg et al. 2013
Paraphoma
chlamydocopiosa
UMPc01; BRIP 65168 KU999072 –KU999084 –Moslemi et al. 2017
P. c h r y s a nt h e m i c o l a CBS 172.70 KF251165.1 KF251669 KF252660.1 KF252173.1 Quaedvlieg et al. 2013
P. dioscoreae CBS 135100 KF251167.1 KF251671 KF252662.1 KF252175.1 Quaedvlieg et al. 2013
P. f i m e t i CBS 170.70 KF251170.1 KF251674 KF252665.1 KF252178.1 Quaedvlieg et al. 2013
P. pye UMPp04; BRIP 65171 KU999075 –KU999087 –Moslemi et al. 2017
P. radicina CBS 111.79 KF251172.1 KF251676 KF252667.1 KF252180.1 Quaedvlieg et al. 2013
P. rhaphiolepidis CBS 142524 KY979758.1 KY979813.1 KY979924.1 KY979851.1 Crous et al.
P. v i n a c e a UMPV004 KU176887.1 KU176891.1 KU176895.1 –Moslemi et al. 2016,Moslemi
et al. 2017
Parastagonospora nodorum CBS 110109 KF251177.1 KF251681 KF252672.1 KF252185.1 Quaedvlieg et al. 2013
Phaeosphaeriopsis
glaucopunctata
CBS 653.86 KF251199.1 KF251702 KF252693.1 KF252206.1 Quaedvlieg et al. 2013
Phaeosphaeria oryzae CBS 110110 KF251186.1 KF251689 KF252680.1 KF252193.1 Quaedvlieg et al. 2013
Setophoma terrestris CBS 335.29 KF251246.1 KF251749 KF252729.1 KF252251.1 Quaedvlieg et al. 2013
Stagonospora convolvuli 12–039 KC634206.1 –––Not published
S. convolvuli 01–634 HQ677906 –––Not published
Vrystaatia aloeicola CBS 135107 KF251278.1 KF251781 KF252759.1 KF252283.1 Quaedvlieg et al. 2013
Xenoseptoria neosaccardoi CBS 120.43 KF251280.1 KF251783 KF252761.1 KF252285.1 Quaedvlieg et al. 2013
Fig. 1 Maximum-likelihood phylogenetic tree inferred from ITS, representing all Paraphoma species and reference of Stagonospora convolvuli
188 Mycol Progress (2020) 19:185–194
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The PCR conditions were as follows: predenaturation of
DNA at 95 °Сfor 5 min; 35 cycles of denaturation at 92 °С
for 50 s, annealing at 55 °C for 40 s (ITS1F/ITS4 and Т1/Т2)
or at 55 °C for 50 s (LR0R/LR5), and elongation at 72 °Сfor
75 s, followed by a final elongation step for 5 min at 72 °С.
The RPB2 gene was amplified according to the touchdown
program. All steps were the same as described below, but the
annealing temperature consequently declined from 5 cycles of
60 °Сfor 40 s and 5 cycles of 58 °Сfor 40 s to 30 cycles of
54 °Сfor 40 s.
After PCR, amplicons were purified according to a stan-
dard method with a DNA-binding silica matrix (Boyle and
Lew 1995). Visualization and concentration measurements
of the purified PCR products were implemented by electro-
phoresis in 1% agarose gel stained with ethidium bromide and
MassRuler 100 bp as a marker of concentration.
Fig. 2 Maximum-likelihood phylogenetic tree inferred from ITS and TUB
Fig. 3 Maximum-likelihood phylogenetic tree inferred from ITS, TUB,andRPB2
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Amplicons were sequenced by Sanger’smethod(1977)on
ABI Prism 3500 (Applied Biosystems, Hitachi, Japan), with
the Big Dye Terminator v3.1 Cycle Sequencing Kit (ABI,
Foster City, USA), according to the manufacturer’s
instructions.
Phylogenetic analysis
Sequences were assembled using Vector NTI advance v. 11.0
(Invitrogen, Thermo Fischer Scientific, Waltham, USA) and
aligned with ClustalX 1.8 (Thompson et al. 1997). All known
representative Paraphoma strains and type species of all gen-
era in the family Phaeosphaeriaceae, with Phaeosphaeriopsis
glaucopunctata as an outgroup, were obtained from GenBank
(Table 4) and included in the analysis. The phylogenetic trees
were inferred with RAxML (randomized accelerated maxi-
mum likelihood) software (v. 7.2.8, Stamatakis 2006)bythe
maximum-likelihood (ML) method. Bootstrap support values
with 1000 replications were calculated for tree branches.
Morphological analysis
Pure cultures were incubated on PSA and OA amended with
200 mg/ml streptomycin sulfate for up to 2 weeks under stan-
dard conditions (Boerema et al. 2004). Petri dishes were
placed for 1 week in darkness and then for a week under 12-
h near-ultraviolet light/12-h dark to stimulate sporulation.
Colony diameter was measured after 7 days, and colony mor-
phology was examined after 14 days of incubation. Colony
colors on the surface and underside of the inoculated Petri
dishes were assessed according to the color charts of
Bondartsev (1953). Isolates that did not produce pycnidia on
agar medium were cultivated on autoclaved grain (millet, oat,
and pearl barley) under near-ultraviolet conditions for 14days.
Observations and measurements of 50 replicates of conidia
and conidiomata were conducted with a stereomicroscope
Olympus SZX16 (Olympus, Japan) and microscope
Olympus BX53.
Results
Phylogeny
The adjusted and aligned sequences in the phylogenetic anal-
ysis had the following lengths: ITS region, 483 bp; LSU,
845 bp; TUB,490bp;andRPB2, 736 bp; the number of
polymorphic sites per genome locus was 44 (9.1%), 2
(0.2%), 28 (5.7%), and 32 (4.3%), respectively.
Fig. 4 Leaf spots on Convolvulus arvensis caused by Paraphoma melnikiae sp. nov. from the type material
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Nucleotide sequences of 11 isolates were used for 2-gene
phylogenetic analysis (sequences of ITS and LSU regions).
Sequences of 10 isolates in the 3-gene phylogenetic analysis
(ITS, LSU, and RPB2) and sequences of only nine isolates in
the 4-gene phylogenetic analysis (ITS, LSU, TUB,andRPB2)
were suitable for analysis. Three phylogenetic trees were de-
veloped: an individual tree of the ITS region, a combined tree
for the ITS and TUB genes, and a combined phylogram for all
studied loci.
All currently known species of Paraphoma and all our
isolates formed a common monophyletic group. Within
this group, all our isolates clustered as three distinct
clades in all phylogenetic trees with a maximum value
of bootstrap support (100%). The composition of these
clades was identical in all trees, and these clades did not
cluster with any Paraphoma species (Figs. 1,2,3). The
first clade consisted of four isolates (MF-9.301, MF-
9.298.1, MF-9.265, MF-9.300.1). The second clade in-
cluded six isolates (MF-9.296.1, MF-9.88, MF-9.294.1,
MF-9.240, MF-9.95, MF-9.182.1) (Figs. 2,3). The third
clade included only the isolate MF-9.222 (Fig. 1).
In the tree of the ITS region (Fig. 1), the isolate MF-
9.222 clustered within the same clade with the two refer-
ence strains of S. convolvuli (12–039; 01–634), which are
represented in GenBank only by these two sequences.
Thus, according to the obtained molecular phylogenetic
data, isolate MF-9.222 was identified as S. convolvuli.
However, due to its position in the Paraphoma cluster,
the new taxonomical combination Paraphoma convolvuli
has been proposed for S. convolvuli.
The other isolates were divided into two subclades in
all phylogenetic trees. The topologies of the combined
phylogenetic trees were more detailed, including the
Fig. 5 Immersed pycnidia of Paraphoma melnikiae sp. nov. on leaves of Convolvulus arvensis from the type material
Fig. 6 Pycnidia of Paraphoma melnikiae sp. nov. on leaves of
Convolvulus arvensis from the type material
Fig. 7 Conidiogenous cells of Paraphoma melnikiae sp. nov. from the
type material
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topology of clade 2, which was more explicit and allowed
the division of isolates MF-9.182, MF-9.299, and MF-
9.240 into two subclades (Figs. 2,3). This clade did not
include any type or representative isolate. It is monophy-
letic and, in all trees, has a maximum value of bootstrap
support. Therefore, the isolates of this clade are consid-
ered a new species, Paraphoma melnikiae.
Morphology and Taxonomy
Paraphoma convolvuli (Dearn. & House) Gomzhina M. M.
&GasichE.L.comb.nov.
MycoBank MB823867
Basionym Stagonospora convolvuli Dearn. & House
Paraphoma melnikiae Gomzhina M. M. & Gasich E. L.,
sp. nov.
MycoBank MB823800
Type specimen LEP 131845. The type specimen represents
dried leaves of C. arvensis with leaf spots collected in Saint
Petersburg on February 17, 2002.
Etymology: Named after Dr. V. A. Melnik (1937–2017), an
outstanding Russian mycologist and taxonomist who dedicat-
ed his work to different fungi, including phoma-like species.
This fungus causes leaf spots on C. arvensis.Spotsare
incorrectly rounded with concentric zones and some
pycnidia (Fig. 4). Pycnidia are diffuse, semisubmerged,
rounded, dark brown, and 100–250 μm(Figs.5,6).
Conidiophores are reduced to phialidic conidiogenous
cells formed from the inner cells of the pycnidial wall,
hyaline, discrete, flask-shaped, and 7.29–10.25 × 3.6–
4.24 μm(Fig.7). Conidia are cylindrical with rounded tips,
straight or slightly curved, with 0–2 transverse septa, 9–
22.5 × 1.5–3.8 μm(Fig.8). On OA (Fig. 9), the colony
diameter is 25–34 mm after 7 days and 42–54 mm after
14 days. Felty-velvet or flocculose felty-velvet aerial
mycelia are not abundant, pale-olivaceous. The colors of
the colonies on the upper and lower parts are from pale to
dark brown, sometimes with dark brown, reddish, and fal-
low sectors. The color of the colonies could also be from
pale to dark vinaceous shades, sometimes with pale-
olivaceous sectors. Margins are regular and slightly
curved. Pycnidia are sparse, scattered, immersed and
Fig. 8 Conidia of Paraphoma melnikiae sp. nov. from the type material
Fig. 9 Morphology of Paraphoma melnikiae sp. nov. colonies on OA (ex-type isolate MF–9.88)
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semi-immersed, rare in aerial mycelium, dark brown,
rounded, hairy, 40–420 μm, with 1–3 ostioles. Conidia
are hyaline, multiguttulate, cylindrical with rounded tips,
straight or slightly curved, with 0–2 transverse septa 7–16
(10.4 ± 0.5) × 1.5–2.5 (2.0 ± 0.1) μm.
Chlamydospores were absent. Perithecia were not
observed.
Note: Morphologically, the conidia of P. melnikiae differ
from conidia of the closely related species P. convolvuli in
shape and size. The conidia of P. convolvuli are longer (15–
18 μm)andhavemoretransversesepta(2–3) (Saccardo
1931).
Discussion
Based on the morphological characteristics, all isolates were
primarily identified as S. convolvuli by the authors. However,
the conidia of the studied isolates were broader and less elon-
gated than the conidia of Stagonospora species. Our isolates
did not possess typical morphological features of pycnidia and
conidia to identify them as members of the genus Paraphoma.
The pycnidia of the studied isolates were not setose, and the
conidia were longer than typical Paraphoma conidia. It is
known that such morphological characteristics are highly var-
iable and do not represent phylogenetic relationships among
fungi in this group.
Unlike the traditional morphological approach, molecular-
phylogenetic methods allow the identification of all isolates as
members of the genus Paraphoma. Based on molecular data,
a new combination, P. convolvuli, was proposed for
S. convolvuli. Isolate MF-9.222 should be identified as
P. convolvuli, whereas isolates from clade 1 should be identi-
fied as Paraphoma cf. convolvuli. All Paraphoma isolates
from clade 1 shared similar morphological features with
P. convolvuli isolate MF-9.222 but differed from it by a single
deletion in the ITS sequence and one insertion in the LSU
sequence. Clade 1 was monophyletic and well supported; all
isolates were obtained from C. sepium, not from C. arvensis,
as was isolate MF-9.222 and the reference P. convolvuli.
Apparently, these isolates are new species of the genus
Paraphoma, but this requires subsequent validation.
Isolates of the second phylogenetic clade were treated as a
new species of Paraphoma,P. melnikiae. This new taxon was
proposed according to the polyphasic approach to species rec-
ognition (Consolidated Species Concepts) and based on phy-
logenetic, morphological, and biological characteristics.
To construct phylogenetic hypotheses for closely related
phoma-like species, the most informative loci are ITS, TUB,
and RPB2. The sequencing of these loci was implemented in
this study and resulted in robust, well-supported phylogenetic
clades in the phylograms. Thus, to resolve phylogenetic rela-
tionships among Paraphoma species, this set of loci is also
taxonomically informative. Despite this being used in non-
taxonomic studies, the molecular identification of phoma-
like fungi is often based only on sequences of the ITS region.
Thus, data on sequences of phylogenetic informative loci of
particular phoma-like species in GenBank are often presented
one-sidedly and scantly. The implementation of phylogenetic
studies in such cases becomes difficult. Although it was pre-
viously suggested not to identify phoma-like fungi only by
morphological features and to take into account molecular
traits, now it is not recommended to identify these fungi only
by sequencing the ITS loci, as the most popular region for
phylogenetic studies, but to include sequences of other infor-
mative regions of DNA in the phylogenetic analysis.
AmajorityofParaphoma species are widely distributed,
occurring as soil-borne fungi causing diseases of aboveground
parts of plants. Analysis of the pure culture collection of
phoma-like fungi derived from Convolvulaceae showed that
species of the genus Paraphoma were detected in Russia,
Kazakhstan, and Ukraine. P. convolvuli was found on
C. arvensis only in Kazakhstan, whereas closely related iso-
lates of Paraphoma cf. convolvuli (probably a new species)
were detected only in one location in Russia, in Saint
Petersburg, on C. sepium. The new species P. melnikiae was
found on C. arvensis in two locations in Russia (Saint
Petersburg and Vladivostok) and in Ukraine.
Funding information This study was financially supported by the
Russian Science Foundation, project 19-76-30005.
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