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Microorganisms 2020, 8, 478; doi:10.3390/microorganisms8040478 www.mdpi.com/journal/microorganisms
Article
Heliocephala variabilis and Pseudopenidiella
vietnamensis: Two New Hyphomycetous Species in
the Microthyriaceae (Dothideomycetes) from
Vietnam
Isabel
Iturrieta-González, Dania García *, Josep Guarro and Josepa Gené
Unitat de Micologia, Facultat de Medicina i Ciències de la Salut and IISPV, Universitat Rovira i Virgili, Reus,
Tarragona 43201, Spain; isabeliturrieta@gmail.com (I.I.-G.); josep.guarro@urv.cat (J.Gu.);
josepa.gene@urv.cat (J.Ge.)
* Correspondence: dania.garcias@urv.cat; Tel.: + 34-673735073
Received: 29 February 2020; Accepted: 26 March 2020; Published: 27 March 2020
Abstract: In a survey of microfungi from plant debris collected in Vietnam, two new
hyphomycetous species were found, which belong to the genera Heliocephala and Pseudopenidiella
and the family Microthyriaceae (Microthyriales, Dothideomycetes). Maximum Likelihood and Bayesian
Inference sequence analyses of the internal transcribed spacers (ITS) and large subunit (LSU) of the
ribosomal DNA barcodes allowed assessing the phylogenetic relationships of the new species with
other species of the respective genera. Heliocephala variabilis sp. nov. was closely related to
Heliocephala elegans, Heliocephala gracilis, and Heliocephala zimbabweensis, from which it was
morphologically distinguished by its smaller conidiophores and non-rostrate conidia of up to four
septa on the natural substratum. Pseudopenidiella vietnamensis sp. nov. was related to Pseudopenidiella
piceae and Pseudopenidiella podocarpi and differed from the former principally by its lack of
microcondiophores and from P. podocarpi by having larger macroconidiophores and smooth
conidia. Key morphological features to distinguish the accepted species in Heliocephala and
Pseudopenidiella are also provided. In addition, Pseudopenidiella pini was excluded from the genus on
the basis of its morphological features.
Keywords: hyphomycetes; dematiaceous fungi; phylogeny; taxonomy; Vietnam
1. Introduction
Vietnam is one of the twenty most bio-diverse countries in the world [1–3]. Its very diverse
ecological niches, climatic conditions, and high level of plant endemism would suggest that the
country also has great fungal diversity, although this has been not extensively studied so far [4–6].
During a survey of asexual microfungi from plant debris carried out in the Northeast of Vietnam,
two interesting specimens, belonging to the genera Heliocephala and Pseudopenidiella (Microthyriaceae,
Microthyriales, Dothideomycetes) [7] were isolated and deposited in the culture collection at the
Medicine Faculty of the Universitat Rovira i Virgili (Reus, Spain) for subsequent studies.
The genus Heliocephala was introduced by Rao et al. [8] based on Heliocephala proliferans, a
hyphomycetous fungus characterized by the production of solitary macronematous conidiophores,
bearing a terminal compact clusters of monoblastic conidiogenous cells that can arise more or less
radially from short branches (metula-like) and produce obclavate, rostrate, or hooked conidia. Based
on DNA data and morphological features, the genus was emended by Heredia-Abarca et al. [9] to
include the species of Holubovaniella [10], the latter subsequently being considered a synonym of
Heliocephala. Currently, Heliocephala comprises seven species, most of them isolated from plant
Microorganisms 2020, 8, 478 2 of 11
material, with the exception of Heliocephala natarajanii, which was found along with a basidiocarp of
Pisolithus tinctorius [11].
Crous et al. [12] introduced the genus Pseudopenidiella to accommodate Pseudopenidiella piceae
recovered from the needle litter of Picea abies in the Czech Republic. Morphologically, the genus is
characterized by the presence of micro- and macroconidiophores, with aseptate conidia and
ramoconidia arranged in branched acropetal chains. It shows a conidiogenous apparatus similar to
those of the genera Cladosporium, Digitopodium, and Penidiella, but mainly differs from them by the
lack of darkened and coronate-type scars in both conidia and conidiogenous cells and by the absence
of rhizoids associated with the conidiophores. Phylogenetically, three species have been recognized
in the genus, Pseudopenidiella podocarpi being the most recently described on leaves of Podocarpus
latifolus, collected in South Africa [13].
Based on a polyphasic approach, we propose two novel hyphomycetous fungi from Vietnam,
Heliocephala variabilis and Pseudopenidiella vietnamensis, which are described and illustrated below.
2. Material and Methods
2.1. Samples and Isolates
Samples of plant debris were collected in the Northeast region of Vietnam in 2011 and treated
according to Hernández-Restrepo et al. [14]. They were placed in moist chambers and incubated at
room temperature (22 °C) in darkness, being examined periodically under a stereomicroscope over a
3-month period. The fungi were isolated on potato dextrose agar (PDA; Pronadisa, Madrid, Spain),
and the interesting specimens were preserved at room temperature on PDA slant cultures covered
with mineral oil.
2.2. DNA Extraction, PCR, Sequencing and Phylogenetic Analyses
Isolates were grown on PDA for 14 days at 25 °C in darkness, and DNA was extracted from
mycelium through the modified protocol of Müller et al. [15]. For identification purposes, we
performed the PCR using the primer pairs ITS5/ITS4 and NL1/NL4b to amplify the region spanning
the internal transcribed spacers (ITS) 1 and 2, including the 5.8S gene, and the D1/D2 domain of the
large subunit (LSU) of the nuclear ribosomal (nr)DNA, respectively, following the protocol of Cano
et al. [16]. The PCR products were purified and stored at −20 °C until sequencing at Macrogen
(Madrid, Spain).
ITS and LSU sequences of the unidentified isolates were compared with those of other fungi
deposited at the National Center for Biotechnology Information (NCBI) by the Basic Local Alignment
Search Tool (BLAST). To assess the taxonomic position and phylogenetic relationships of our fungi,
we carried out single analyses of the ITS and LSU sequences obtained here and those available of
Heliocephala and Pseudopenidiella species and other members of the family Microthyriaceae. Since a
similar topology of the phylogenetic trees obtained from the previous analyses was observed, a
combined analysis of the two markers was performed to obtain a more robust phylogeny of the fungi
studied. All the sequences, which were mainly taken from previous studies [7,9,12,13,17–20], were
retrieved from GenBank, including those of Venturia catenospora and Venturia inaequalis used as
outgroup (Table 1). The alignments were made in the MEGA (Molecular Evolutionary Genetics
Analysis) software v.6.0. [21], using the ClustalW algorithm [22] and refined with MUSCLE [23] or
manually if necessary, on the same platform. Phylogenetic reconstructions were made using
Maximum Likelihood (ML) and Bayesian Inference (BI) approaches using the MEGA software v. 6.0.
[21] and MrBayes v. 3.2.6 [24], respectively.
Microorganisms 2020, 8, 478 3 of 11
Microorganisms 2020, 8, 478; doi:10.3390/microorganisms8040478 www.mdpi.com/journal/microorganisms
Table 1. Species included in this study, their substrate, origin, and GenBank accession numbers.
Species Strain Number
1
Substrate Country Genbank Accession No.
2
ITS LSU
Chaetothyriothecium elegans
CPC 21375
T
Leaves of
Castanopsis
sp.
Thailand - KF268420
Heliocephala elegans
MUCL 39003 Fallen leaf of
Andira inermis
Cuba HQ333478 HQ333478
H. gracilis
CBS 369.86
IT
Fallen leaf of
Matayba oppositifolia
Cuba HQ333479 HQ333479
H. natarajanii
MUCL 43745
T
Basideocarp of Pisolithus tinctorius
India
HQ333480
HQ333480
H. zimbabweensis
CBS 691.97
T
Unidentified leaf litter Zimbabwe HQ333481 HQ333481
H. variabilis
sp. nov.
FMR 17592
T
Unidentified dead leaves Vietnam LR536989 LR588212
Microthyrium buxicola
MFLUCC 15-0212
T
Leaves of
Buxus
sp.
Italy - KT306551
MFLUCC 15-0213
Leaves of Buxus sp.
Italy
-
KT306552
M. microscopicum
CBS 115976
T
-
The Netherlands - GU301846
M. propagulensis
IFRD 9037
T
Fallen leaves of
Castanopsis histrix
China - KU948989
Pseudopenidiella piceae
CBS 131453
T
Needle litter of
Picea abies
Czech Republic JX069868 JX069852
P. gallaica
CBS 121796
T
Unidentified dead leaves
Spain
LT984842
LT984843
P. podocarpi
CBS 146067
T
Leaves of
Podocarpus latifolius
South Africa MN562140 MN567647
CPC 37094 Leaves of
P. latifolius
South Africa MN562141 MN567648
P. vietnamensis
sp. nov.
FMR 17593
T
Unidentified dead leaves Vietnam LR536990 LR536991
Stomiopeltis betulae
CBS 114420
Betula sp.
Sweden
GU214701
GU214701
Tumidispora shoreae
MFLUCC 12-0409
T
Dead leaves of
Shorea
sp.
Thailand - KT314073
MFLUCC 14-0574 Dead leaves of
Shorea
sp.
Thailand - KT314074
Venturia catenospora
CBS 447.91
T
Leaf spot of
Salix triandra
Germany EU035427 MH873940
V. inaequalis
CBS 476.61
Fruit of Malus sylvestris
Belgium
EU282478
GU456336
1
CBS: culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CPC: culture collection of Pedro Crous housed at the CBS; IFRD:
International Fungal Research and Development Centre Research Institute of Resource Insects, Kunming; FMR: Facultat de Medicina, Universitat Rovira i Virgili, Reus,
Spain; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Ria, Thailand; MUCL: Mycothèque de L’Université Catholique de Louvain, Louvain-la-Neuve,
Belgium; T and IT: ex-type and ex-isotype strain, respectively.
2
Sequences newly generated in this study are indicated in bold.
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Microorganisms 2020, 8, 478; doi:10.3390/microorganisms8040478 www.mdpi.com/journal/microorganisms
For the ML analysis of the ITS and LSU regions, the best nucleotide substitution model
determined by the same program was the Tamura–Nei model with Gamma distribution and
invariant sites (T93+G+I); ML bootstrap values (BML) ≥ 70 % were considered significant. For the BI
analysis, the best nucleotide substitution model was determined using jModelTest [25]. For the ITS
region, we used the Kimura 2-parameter with Gamma distribution (K80+G) and for the LSU region,
we used General Time Reversible with Gamma distribution and invariant sites (GTR+G+I). The
parameters used were two simultaneous runs of 5,000,000 generations, four Markov chains, sampled
every 1000 generations. The 50% majority-rule consensus tree and posterior probability values (PP)
were calculated after discarding the first 25% of the samples. A PP value ≥ 0.95 was considered
significant.
Sequences of the fungi studied here were deposited in GenBank (Table 1), and the alignment
used was deposited in TreeBASE (submission number S25760).
2.3. Phenotypic Study
Microscopic characterization of the isolates was carried out on potato carrot agar (PCA; potato
20 g, carrot 20 g, agar 13 g, distilled water 1 L) and oatmeal agar (OA; Oatmeal 30 g, agar 13 g, distilled
water 1 L) after 30 days of incubation to get sporulation. Measurements and descriptions of the
structures were taken from the specimens mounted in Shear’s solution or lactic acid (100% v/v).
Photomicrographs were obtained using a Zeiss Axio-Imager M1 light microscope (Zeiss,
Oberkochen, Germany) with a DeltaPix Infinity X digital camera.
Macroscopic characterization of the colonies was done on PDA, OA, and PCA after 30 days of
incubation at 25 °C in darkness. Cardinal temperatures for growth were obtained on PDA incubated
at 5, 15, 20, 25, 30, 35, 37, and 40 °C after 14 days in darkness. The colony colors in descriptions are
based on Kornerup and Wanscher [26].
Nomenclatural novelties and descriptions were deposited in MycoBank [27]. Ex-type cultures
and holotypes, which were dried cultures, were deposited at the Westerdijk Fungal Biodiversity
Institute (CBS, Utrecht, The Netherlands).
3. Results
BLAST analyses with LSU and ITS sequences confirmed the morphological identification of FMR
17592 and FMR 17593 at the genus level but revealed a relatively low percentage of identity with
respect to other species, suggesting they were novel species of Heliocephala and Pseudopenidiella,
respectively. The similarity of LSU sequences between FMR 17592 and other Heliocephala species (i.e.,
Heliocephala gracilis, Heliocephala zimbabweensis, Heliocephala elegans, and H. natarajanii) ranged from
94.04% to 96.73%. In the case of FMR 17593, similarity was 97.68% to P. podocarpi, 96.24% to P. piceae,
and 95.80% to Pseudopenidiella gallaica. ITS sequences showed lower percentages of identity, with a
maximum of 95.10% between FMR 17592 and the Heliocephala species mentioned above and of
≤89.25% between FMR 17593 and the species of Pseudopenidiella analyzed. A combined analysis of the
two loci (ITS and LSU) revealed the status of these fungi with respect to the other species of
Heliocephala and Pseudopenidiella and allied genera of the family Microthyriaceae (Figure 1). The total
alignment included 20 sequences and comprised 1501 bp, from which 604 bp were variable, 864 bp
conserved, and 460 bp phylogenetically informative. FMR 17592 and FMR 17593 were included in
the full-supported clades representatives of the genera above mentioned and were genetically and
morphologically differentiated from their closest phylogenetic relatives.
Microorganisms 2020, 8, 478 5 of 11
Figure 1. Maximum Likelihood (ML) tree constructed with the internal transcribed spacers (ITS) and
large subunit (LSU) sequences of 18 strains representative of the family Microthyriaceae
(Microthyriales). The phylogenetic tree was rooted with V. catenospora and V. inaequalis (Venturiaceae,
Venturiales). Bootstrap support values for ML greater than 70% and Bayesian posterior probabilities
greater than 0.95 are given near nodes. The names of the newly described species are in bold. Branch
lengths are proportional to distance;
T
Ex-type strain;
IT
Ex-isotype strain.
Key morphological features that distinguish the accepted species of Heliocephala and
Pseudopenidiella, including the new taxa described below, are summarized in Tables 2 and 3.
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Table 2. Key morphological features distinguishing the accepted Heliocephala species.
Species Conidiophore
Size *
Conidia References
Size * Septum No. Ornamentation
Rostrum
H. elegans
250–700 × 7–11 8–25 × 3–4 1–3 Smooth Present, straight [10]
H. gracilis
80–350 × 7–10
4–12.5 × 2–5 0–1 Smooth Absent [10]
H. natarajanii up to 109 × 1.5–3.5 (8.5–)17–34(–103) × (1.5–) 2.5–4.5(–6.5) 2(–3) Basal cell
verruculose
Present, straight, curved
or uncinate [11]
H. proliferans up to 210 × 3.5–4 (10–)15–50(–200) × 3–4 2 Basal cell
verruculose
Present, straight or
curved [8]
H. triseptata
21–40 × 7–19 15–27 × 3.5–4.5 3 Smooth Present, straight [9]
H. variabilis up to 153 × 4–6 4–26 × 3–6 (0–)1–3(–4)
Smooth to
verruculose Absent
Present
study
H. vietnamensis
210–340 × 6–8 14–17 × 2.8–3.8 3 Smooth Absent [28]
H. zimbabweensis
180–240 × 3–4 23–125 × 3.5–5.3 2 Smooth Present, straight [29]
* in μm.
Table 3. Key morphological features distinguishing the accepted Pseudopenidiella species.
Species Macroconidiophore
size * Microconidiophore Ramoconidia Size *
Conidia
References
Size *
Ornamentation
P. gallaica
up to 120 × 2–3
Present
7.5–11 × 2–3
6–12 × 1–3
Smooth to verruculose
[7]
P. piceae
up to 150 x 3–4 Present 8–12 × 2–3 (6–)7–9(–10) × (2.5–)3
Finely verruculose
[12]
P. podocarpi 10–110 × 3–4 Absent (9–)12–13 × (2.5–)3–
3.5
(9–)11–12(–15) × 2.5(–
3) Verruculose [13]
P. vietnamensis up to 236 × 3–5 Absent 7–13 × 3–4 5–10 × 2–3 Smooth
Present
study
* in μm.
Microorganisms 2020, 8, 478 7 of 11
Microorganisms 2020, 8, 478; doi:10.3390/microorganisms8040478 www.mdpi.com/journal/microorganisms
Taxonomy
Heliocephala variabilis Iturrieta-González, Gené, Dania García, sp. nov.—MycoBank MB 833179
(Figure 2).
Figure 2. H. variabilis (ex-type FMR 17592). (A–C). Colonies on potato dextrose agar (PDA), potato
carrot agar (PCA), and oatmeal agar (OA), respectively, after 30 days at 25 °C. (D–J). Conidiophores
and conidia. Scale bars: (D,E) = 20 μm; (F–J) = 10 μm.
Etymology: Name refers to the variation in the conidial morphology.
Mycelium consisting of branched, septate, subhyaline to pale brown, smooth to verrucose
hyphae 1–2 μm wide. Conidiophores macronematous, rarely semi-macronematous, mononematous,
erect, subcylindrical, with up to seven septa, brown, pale brown towards the apex, smooth-walled,
up to 153 μm long (up to 148 μm long on the natural substratum), 4–6 μm wide, commonly 2–3 closely
packed primary branched, from which 1–2 secondary metula-like branches are commonly present.
Conidiogenous cells terminal, monoblastic, discrete, ampuliform, smooth-walled, pale brown, 3–13
× 2–3 μm. Conidia solitary, broadly ellipsoidal, subcylindrical or obclavate, smooth-walled to slightly
verruculose, pale brown, (0–)1–3-septate (up to 4-septate on the natural substratum): 0–1-septate, 4–
11 × 3–6 μm; 2–3-septate 15–26 × 3–5 μm; 4-septate 24–26 × 3.8–4.3 μm. Sexual morph not observed.
Culture characteristics after 30 days at 25 °C—Colonies on PDA reaching 17–18 mm of diameter,
olive color (3F7) with some areas olive-brown (4E4), velvety, convex, aerial mycelium scarce, margin
slightly irregular; reverse dark brown (7F8) to black. On PCA, reaching 18 mm of diameter, yellowish
brown (5E4), black at the periphery, velvety, flat, aerial mycelium scarce, margin slightly irregular;
Microorganisms 2020, 8, 478 8 of 11
reverse dark brown (7F8) to black. On OA, reaching 23 mm of diameter, yellowish brown (5E4), black
at the periphery, umbonate, slightly floccose at the center, velvety towards the periphery, margin
entire and slightly fimbriate; reverse dark brown (7F8) to black.
Cardinal temperatures for growth: Minimum 15 °C, optimum 25 °C, maximum 30 °C.
Specimen examined: Vietnam, north-east region, on unidentified dead leaf, Aug. 2011, Josep
Guarro (holotype CBS H-24291, culture ex-type CBS 146334 = FMR 17592).
Diagnosis: H. variabilis differs from four closely related species, i.e., H. elegans, H. gracilis, H.
zimbabweensis, and H. natarajanii, by the size of its macronematous conidiophores and the septation
of conidia (Table 2).
Pseudopenidiella vietnamensis Iturrieta-González, Dania García, Gené, sp. nov.—MycoBank MB
833180 (Figure 3).
Figure 3. P. vietnamensis (ex-type FMR 17593). (A–C) Colonies on PDA, PCA, and OA, respectively,
after 30 days at 25 °C. (D–I,L) Conidiophores and conidia. (D,E) Conidiophores under
stereomicroscope. (J–K) Detail of conidiophore basal cells. Scale bars: (F,G) = 20 μm; (H–L) = 10 μm.
Etymology: Name refers to Vietnam, the country where the fungus was collected.
Mycelium consisting of branched, septate, pale brown, smooth-walled hyphae 1–3 μm wide.
Conidiophores macronematous, mononematous, unbranched, erect to slightly flexuous,
subcylindrical, with up to 10-septate, pale brown to brown, smooth-walled, verruculose towards the
apex, swollen and often lobate basal cell, up to 236 μm long, 3–5 μm wide; microconidiophores not
observed. Conidiogenous cells terminal, polyblastic, with up to three inconspicuous conidiogenous
loci, verruculose, pale brown, 12–18 × 2–4 μm. Ramoconidia cylindrical, aseptate, pale brown,
Microorganisms 2020, 8, 478 9 of 11
verruculose, 7–13 × 3–4 μm, forming conidia in acropetal branched chains. Conidia cylindrical,
aseptate, pale brown, smooth-walled to verruculose, 5–10 × 2–3 μm. Sexual morph not observed.
Culture characteristics after 30 days at 25 °C: Colonies on PDA reaching 23 mm of diameter, grey
(7F1) with some regions greyish brown (5D3), black at the edge, velvety, slightly convex, aerial
mycelium scarce, margin entire; reverse dark brown (7F8) to black. On PCA, reaching 17 mm of
diameter, brownish grey (5F2), velvety, slightly convex, aerial mycelium scarce, margin undulate;
reverse dark brown (7F8) to black. On OA, reaching 15 mm of diameter, brownish grey (5F2), black
at the edge, finely granular, flat, aerial mycelium scarce, margin entire; reverse dark brown (7F6) to
black.
Cardinal temperatures for growth: Minimum 15 °C, optimum 25 °C, maximum 30 °C.
Specimen examined: Vietnam, north-east region, on unidentified dead leaf, Aug. 2011, Josep
Guarro (holotype CBS H-24292, culture ex-type CBS 146219 = FMR 17593).
Diagnosis: P. vietnamensis differs from P. piceae and P. gallaica in the lack of microconidiophores,
and from P. podocarpi in the size of their macronematous conidiophores (Table 3).
4. Discussion
Sequence analysis of the ITS and LSU barcodes were enough to resolve the taxonomy of the
fungi under study and attribute the species to the monophyletic genera Heliocephala and
Pseudopenidiella. However, phylogenetic relationships to other genera in the family Microthyriaceae
remained obscure with the present taxon sampling, due to the lack of statistical support in the main
clades obtained in the analysis (Figure 1). Despite DNA data not being available for all species of the
mentioned genera, the novel species, H. variabilis and P. vietnamensis, showed morphological traits
that clearly allowed their distinction from the other species of the respective genera (Tables 2 and 3).
Heliocephala variabilis was phylogenetically close to H. elegans, H. gracilis, and H. zimbabweensis.
The first two species, which were previously included in the genus Holubovaniella [10], could be
differentiated from H. variabilis by having much more robust conidiophores (up to 700 × 11 μm in H.
elegans; up to 350 × 10 μm in H. gracilis) that usually proliferate, showing several clusters of short
branches and intercalary conidiogenous cells. In addition, on the natural substratum, our species
showed conidia with up to four septa, while those of H. elegans and H. gracilis are 1–3- and 0–1-septate,
respectively [10]. Heliocephala zimbabweensis resembles H. proliferans, and both differ from H. variabilis
by having longer conidiophores (up to 210 μm in H. proliferans; up to 240 μm in H. zimbabweensis) and
two-septate conidia with a very long and filiform rostrum, subsequently showing much longer
conidia (10–200 μm in H. proliferans; 23–125 μm in H. zimbabweensis) than the species proposed here.
Another feature exclusive to H. proliferans and H. zimbabweensis is the presence of a secondary cluster
of conidiogenous cells at the apex of the conidial rostrum [8,29]. The great morphological similarity
of these two species suggest they could be conspecific, but the lack of DNA data from the type is a
handicap to elucidating the taxonomy of these fungi. Other two Heliocephala species with no
molecular data are Heliocephala triseptata and Heliocephala vietnamensis [9,28], but the protologue of
both taxa only describes conidia with three septa, and the conidiophores of the former are the smallest
ones in the genus (21–40 μm long), while those of H. vietnamensis are longer (up to 340 μm) than those
observed in H. variabilis (up to 153 μm long).
Although the three species of Pseudopenidiella are phylogenetically well differentiated,
morphologically they are very similar, and even their conidiogenous apparatus resembles that of
other cladosporium-like fungi that belong to the order Capnodiales, such as Penidiella or Apenidiella
and other related genera [30,31]. Pseudopenidiella species can be only distinguished by subtle
differences in their macroconidiophores and by the presence or absence of microconidiophores, as
summarized in Table 3. It is worth mentioning that, based exclusively on morphological data, a fourth
species named Pseudopenidiella pini was introduced in the genus by Kirk [32]. This was based on
Polyscytalum pini, which was described from several specimens collected on decaying needles of Pinus
sylvestry, mainly in the United Kingdom. However, none of these specimens is currently available for
molecular comparison. Some of the features described and illustrated in its protologue, such as the
presence of denticulate conidiogenous cells and one-septate (ramo-) conidia [33], do not fit with the
Microorganisms 2020, 8, 478 10 of 11
generic concept of Pseudopenidiella [12]; therefore, we prefer to exclude P. pini from the genus until
further studies with additional new collections of the species can confirm its position.
Author Contributions: I.I.-G. conceived the ideas, organized and analyzed the data, and joined in the writing;
D.G. and J.Ge. conceived the ideas, analyzed the data, and led the writing; J.Gu. collected the samples and led
the writing. All authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by the Spanish Ministerio de Economía y Competitividad, grant CGL2017-
88094-P.
Conflicts of Interest: The authors declare no conflict of interest.
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