Available via license: CC BY 4.0
Content may be subject to copyright.
Page 1/18
Debunking Korunomyces
Bruno W. Ferreira
UFV DFP: Universidade Federal de Vicosa Departamento de Fitopatologia
Janaina L. Alves
UFV DFP: Universidade Federal de Vicosa Departamento de Fitopatologia
Pedro W. Crous
Westerdijk Institute: Westerdijk Fungal Biodiversity Institute
Robert Barreto ( rbarreto@ufv.br )
Departamento de Fitopatologia Centro de Ciências Agrárias https://orcid.org/0000-0001-8920-4760
Research Article
Keywords: Coniella, Multi-gene phylogeny, New taxa, Reappraisal, Taxonomy
DOI: https://doi.org/10.21203/rs.3.rs-859512/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full
License
Page 2/18
Abstract
Korunomyces
is a genus including fungi that produce stipitate, profusely branched, multicellular asexual
reproductive structures (propagules) on leaves and in culture. Three species have been described in the genus:
Korunomyces terminaliae
– the type species,
K. prostratus
and
K. zapatensis
. No molecular studies have ever
been conducted to elucidate the phylogenetic placement of
Korunomyces
. Recently, DNA sequences were
obtained from pure cultures of
K. prostratus
and
K. terminaliae
, enabling an elucidation of their taxonomic
placement. Isolates of
K. prostratus
obtained from diseased tissues of
Miconia calvescens
were observed for
the rst time to form pycnidial conidiomata in culture. A multi-gene phylogeny, including the large subunit of the
nrDNA (nc LSU rDNA), internal transcribed spacer (ITS) region, polymerase II second largest subunit (
RPB2
) and
translation elongation factor 1-α (
TEF1
), placed
K. prostratus
and
K. terminaliae
within
Coniella
(
Schizoparmaceae
). As
Korunomyces
is younger than
Coniella
, it is reduced to synonymy, and a new name and
a new combination are proposed for these two species, namely:
Coniella ferreirense
nom. nov. and
Coniella
prostrata
comb. nov. An emended description of
Coniella
to include the occasional formation of distinct and
elaborate asexual propagules is also provided.
Introduction
The genus
Korunomyces
was proposed by Hodges & Ferreira (1981) for a fungus found producing stipitate,
profusely branched, multicellular asexual reproductive structures (propagules) on infected leaves and in culture.
The type species of the genus was described as
K. terminaliae
, causing a leaf spot disease on
Terminaliae
ivorensis
in Brazil (Hodges & Ferreira, 1981).
Korunomyces zapatensis
was later described from dead leaves of
Nectandra coriaceae
in Cuba (Holubová-Jechová & Castaneda, 1986). The latest addition to the genus was
K.
prostratus
, found causing foliage blight on
Miconia calvescens
in Brazil (Seixas et al., 2007).
Although originally described from
Terminalia ivorensis
in Brazil,
K. terminaliae
was also reported to cause leaf
spots on members of the
Combretaceae
family, such as
Bouchenavia
sp.,
T. catappa, T. ivorensis
and
T.
myriocarpa
(Hodges & Ferreira, 1981; Farr & Rossman, 2021). Hodges and Ferreira (1981) compared the fungus
on
T. ivorensis
with
Cristulariella
,
Papulaspora viridis
(=
Trichoderma matsushimae
) and
Aegerita candida
(=
Bulbillomyces farinosus
), all of which are known to produce non-conidial asexual propagules with more or less
elaborate morphology. The differences in branching, hyphal width and colour, as well as the branching pattern
of the propagules were regarded by Hodges and Ferreira (1981) as sucient to distinguish each of these
agonomycetous fungi from
Korunomyces
. One additional distinctive feature of
Korunomyces
separating it from
Aegerita candida
was the absence of clamp connections, which are present on the propagules and hyphae of
the latter.
Papulaspora
, besides being restricted to a lignicolous (damp wood) habitat is a dematiaceous
hyphomycete whereas
Korunomyces
mycelium and propagules were found to be hyaline.
Cristulariella
depraedans
, similarly to
K. terminaliae
(and also
K protratus
) is a leaf parasite, but when growing in culture it
produces phialoconidia and sclerotia (Redhead 1975). Based on such distinctions, Hodges and Ferreira (1981)
decided to propose the new genus
Korunomyces
to accommodate the fungus on
T. ivorensis
.
Korunomyces zapatensis
was the second species of
Korunomyces
included in the genus. Contrarily to the two
other species, it was not associated with leaf spots, but was originally collected from leaf litter of
Nectandrae
coriaceae
in Cuba.
Korunomyces zapatensis
produces asexual propagules which are morphologically similar to
Page 3/18
those of
K. terminaliae
, but differences in the width of the propagule branches and sizes of the terminal cells
and stalk were recognized as sucient to propose it as a new species (Holubová-Jechová & Castaneda 1986).
The third species to be included in the genus was
Korunomyces prostratus.
It was proposed by Seixas et al.
(2007) based on a fungus found in Brazil associated to leaf spots which coalesce to cause leaf blight on
M.
calvescens
. Despite the signicant similarities with
K. terminaliae
, the propagules of
K. prostratus
– as
indicated by the name – are always prostrate, whereas in
K. terminaliae
these are always formed in an upright
position. It was speculated that the propagules in
K. prostratus
function as infection pads, whereas in
K.
terminaliae
these might serve as fungal analogues of the wind-dispersed pappus-bearing achenes produced by
many plants of the
Asteraceae
.
No additions to the genus were made since 2007. The absence of a sexual morph or other morphological
markers, such as clamp connections and the lack of molecular information for a phylogenetic study left the
taxonomic placement of
Korunomyces
unresolved.
Here we report the results of a study involving the recollection of
K. prostratus
combined with the study of the
ex-type culture of
K. terminaliae
aimed at resolving the taxonomy of this agonomycetous genus.
Materials And Methods
Sample collection processing and observation of fungus morphology
Samples of diseased foliage of
Miconia calvescens
were collected from the type locality (Angra dos Reis, state
of Rio de Janeiro, Brazil). These were screened under a dissecting microscope and parts of the samples bearing
sporulating colonies of the fungi were selected and dried in a plant press. Fungal structures were removed from
the sample surface with a scalpel and mounted in lactophenol and lactofuchsin. Observations were made with
an Olympus BX53 compound microscope adapted with differential contrast lighting and equipped with a digital
camera (Olympus Q-Color 3 ™). Biometric data were obtained from at least 30 observations per representative
fungal structure. A representative specimen was deposited in the fungarium at the Universidade Federal de
Viçosa – state of Minas Gerais, Brazil (Herbarium VIC).
Isolations were performed by aseptic transfer of hyphal tips from the leaf surfaces onto 2 % potato dextrose-
agar (PDA) plates with a sterile scalpel. Culture descriptions were based on the observation of 14-day-old (
K.
prostratus
) colonies formed in plates containing either PDA, vegetable broth-agar (according to Pereira et al,
2002) or potato carrot-agar (PCA) (Crous
et al.
2019), maintained at 25 °C under a 12-h day/night light regime
(light provided by two white and one near-UV lamps placed 35 cm above the plates). The colour terminology
followed Rayner (1970).
DNA isolation
Total genomic DNA was extracted from 7-day-old cultures on PDA by using Wizard® Genomic DNA Purication
Kit (Promega Corporation, WI, USA) following the manufacturer’s instructions and the steps described in Pinho
et al. (2012).
PCR amplication
Page 4/18
The large subunit of the nrDNA (nc LSU rDNA), internal transcribed spacer (ITS), polymerase II second largest
subunit (
RPB2
) and translation elongation factor 1-α (
TEF1
) regions from each fungus included in the study
were sequenced with the primers LSU1Fd (Crous et al. 2009) and LR5 (Vilgalys and Hester 1990) and IT5 + ITS4
(White et al. 1990), EF1Fd + EF2Fd (Groenewald et al. 2013) or EF1-728F + EF1- 986R (Carbone & Kohn 1999) or
EF-2 (O'Donnell et al. 1998) and fRPB2-5F + fRPB2-7cR (Liu et al. 1999), respectively. PCR amplications were
performed in a total volume of 12.5 μL containing 10–20 ng of template DNA, 1× PCR buffer, 0.63 μL DMSO
(99.9 %), 1.5 mM MgCl2, 0.5 μM of each primer, 0.25 mM of each dNTP, 1.0 U BioTaq DNA polymerase (Bioline
GmbH Luckenwalde, Germany). Conditions for PCR amplication consisted of an initial denaturation step of 5
min at 94 ºC followed by 35 cycles of 30 s at 94 ºC, 30 s at 48 ºC and 90 s at 72 ºC for nc LSU rDNA, ITS and 40
cycles of 30 s at 94 ºC, 30s at 52 ºC / 59 ºC and 45 s at 72 ºC for
TEF1
and a nal elongation step of 7 min at
72 ºC. The partial
RPB2
gene was obtained by using a touchdown PCR protocol: start step of 5 min at 94 °C,
followed by 5 cycles of 45 s at 94 °C, 45 s at 60 °C annealing temperature, and 2 min at 72 °C; 5 cycles of 45 s
at 94 °C, 45 s at 58 °C annealing temperature, and 2 min at 72 °C; 30 cycles of 45 s at 94 °C, 45 s at 54 °C
annealing temperature, and 2 min at 72 °C followed by a nal step of 8 min at 72 °C. Amplicons were analysed
on 0.8 % agarose electrophoresis gels stained with GelRed (InstantAgarose) in a 1× TAE buffer and visualized
under UV light to check for amplication size and purity. PCR products were puried and sequenced by
Macrogen Inc. (http://www.macrogen.com).
Phylogenetic analysis
The nucleotide sequences were edited and contigs were generated with software SeqAssem v. 07/2008
(Hepperle 2004). The consensus sequences were compared with others deposited in the GenBank database
using the MegaBLAST program.Sequences obtained from GenBank (www.ncbi.nlm. nih.gov) and the novel
sequences generated during this study were aligned using MEGA v. 6 (Tamura et al. 2013) (Table 1).
Bayesian inference (BI) analyses employing a Markov Chain Monte Carlo method were performed with all
sequences, rst with each locus separately and then with the concatenated sequences. Before launching the BI,
the best nucleotide substitution models were determined for each gene with MrMODELTEST 2.3 (Posada and
Buckley 2004). Once the likelihood scores were calculated, the models were selected according to the Akaike
Information Criterion (AIC). The SYM + I + G model of evolution was used for ITS region, GTR + I +G was used
for LSU and
RPB2
,
and SYM + I + G for
TEF1
. One concatenated tree with the four regions was generated with
Mesquite v. 3.1 (Maddison and Maddison 2011) and estimated on the CIPRES web portal using MrBayes on
XSEDE v. 3.2.6 (Miller et al. 2011).Phylogenetic trees were visualized with the program FigTree v. 1.3.1
(Rambaut 2009).
Additionally, a Maximum likelihood (ML) tree was generated using CIPRES web portal.The trees inferred by
means of ML used the RAxML-HPC v. 8.2.12 (Stamatakis 2014). The bootstrap resampling was congured to
perform 10,000 bootstraps, seeking to assess the stability of the inferred trees and dene the best tree. The
chain stabilities of the phylogenetic tree were assessed by using the bootstrap re-sampling strategy with 1000
bootstrap test replicates.
The resulting tree topologies using the two methods (ML and BI) were then compared and the phylogram was
edited with InkScape 0.91 (www.inkscape.org).
Page 5/18
Sequences of
Melanconiella hyperopta
(CBS 131696) were used as the outgroups in the
Coniella
phylogeny.
Sequences derived from this study were lodged in GenBank (http://www.ncbi.nlm.nih.gov/genbank) (Table 1).
Taxonomic novelties were deposited in MycoBank (www.MycoBank.org).
Results
Phylogeny
Phylogenetic analysis using the ITS, nc LSU rDNA,
RPB2
and
TEF1
regions were based on 51
Coniella
strains,
one isolate of
K. terminaliae
, two isolates of
K. prostratus
and one outgroup sequence (Fig. 1). The combined
alignment was comprised of 3576 characters with gaps (777 for ITS, 1314 for nc LSU rDNA, 768 for
RPB2
and
717 for
TEF1
). The phylogenetic analyses generated by Maximum likelihood (ML), and Bayesian inference (BI)
indicate that
K. terminaliae
and
K. prostratus
grouped within the genus
Coniella
and formed a monotypic well-
supported clade (100%/1.00, ML/BI supports, respectively). Additionally,
K. prostratus
formed a distinct lineage
and was a sister to a strain of
K. terminaliae
.
Taxonomy
Coniella
Höhn., Ber. dt. bot. Ges. 36 (7): 316 (1918), emend.
Synomyms
:
Schizoparme
Shear, Mycologia 15: 120. 1923.
Baeumleria
Petr. & Syd., Beih. Reprium nov. Spec.Regni veg. 42: 268. 1927.
Pilidiella
Petr. & Syd., Beih. Reprium nov. Spec. Regni veg. 42: 462. 1927.
Anthasthoopa
Subram.& K. Ramakr., Proc. Indian Acad. Sci., Sect. B 43: 173. 1956.
Cyclodomella
Mathur
etal.
, Sydowia 13: 144. 1959.
Embolidium
Bat., Brotéria, N.S. 33(3–4): 194. 1964 non Sacc. 1978.
Korunomyces
Hodges & F.A. Ferreira, Mycologia 73: 335, 1981.
Pathogens, saprobes.
Ascomata
brown to black, collapsed collabent, erumpent, becoming supercial, globose,
papillate, with central periphysate ostiole.
Asci
clavate to subcylindrical, with distinct apical ring, oating free at
maturity.
Paraphyses
lacking.
Ascospores
ellipsoid, aseptate, hyaline, at times becoming pale brown at maturity,
smooth, with or without mucoid caps.
Conidiomata
pycnidial, immersed to semi-immersed, unilocular, glabrous,
ostiolate. Ostiole central, circular or oval, often situated in a conical or rostrate neck.
Conidiomata wall
brown to
dark brown or black wall of thin, pale brown
textura angularis
on exterior, and hyaline, thin-walled,
textura
prismatica
in the inner layers except at base, which has a convex, pulvinate tissue of hyaline
textura angularis
giving rise to conidiophores or conidiogenous cells.
Conidiophores
mostly reduced to conidiogenous cells,
occasionally septate and branched at base, invested in mucus.
Conidiogenous cells
discrete, cylindrical,
subcylindrical, obclavate or lageniform, hyaline, smooth-walled, proliferating percurrently, or with visible
periclinal thickening.
Conidia
ellipsoid, globose, napiform, fusiform or naviculate with a truncate base and an
obtuse to apiculate apex, unicellular, thin- or thick-walled, smooth, olivaceous brown to brown, sometimes with a
Page 6/18
longitudinal germ-slit, with or without a mucoid appendage extending from apex to base on one side; basal
hilum with or without short tubular basal appendage.Spermatophores formed in same conidioma, hyaline,
smooth, 1-septate with several apical conidiogenous cells, or reduced to conidiogenous cells.
Spermatogenous
cells
hyaline, smooth, lageniform to subcylindrical, with visible apical periclinal thickening.
Spermatia
hyaline,
smooth, red-shaped with rounded ends.Synasexual morph, when present, agonomicetous composed of
complex, multicellular, repeatedly dichotomous/dendrictily branched, chandelier-like propagules ended in
digitate projections, formed on a simple cylindrical stalk and erect or directly from hyphae and prostrate.
Typus
:
Coniella
fragariae (Oudem.) B. Sutton 1977 (syn.
Coniella
pulchella Höhn. 1918).
Coniella ferreirense
B.W. Ferreira & R.W. Barreto, nom. nov.
MycoBank: MB840982
Etymology: Named after the forest pathologist and mycologist Francisco Alves Ferreira (1950–2018) (Chico
Fungo) who rst collected and described the fungus on
T. ivorensis
, and proposed the genus
Korunomyces
.
≡ Korunomyces terminaliae
Hodges & F.A. Ferreira, Mycologia 73(2): 335 (1981)
Typus
: Brazil: Pará: Belém, on
Miconia calvescens
, 22 Sep 1979, F. A. Ferreira, USDA United States National
Fungus Collections (BPI 71913 – culture ATCC 42410 and CBS 224.80).
Notes: Although the type culture (CBS 224.80) was recovered from culture and found to have remained viable,
only undifferentiated mycelium and sterile pycnidium-like structures were formed. A recollection and novel
examination of material from the type locality would be of interest for a more detailed examination of
C.
ferreirense
in fresh cultures.
Coniella prostrata
(Seixas & R. W. Barreto) B.W. Ferreira & R.W. Barreto, comb. nov. and emend.(Fig. 2).
MycoBank: MB840983
≡ Korunomyces prostratus
Seixas & R.W. Barreto, Mycologia 99 (1): 105 (2007)
Leaf spots
necrotic, initially circular, greyish brown centrally with a brown periphery, becoming irregular with age
with concentric dark brown peripheral rings often resulting in a scale-like pattern, with a yellowish halo,
coalescing and leading to extensive leaf blight; older parts of lesions often cracking and falling out to leave
irregular holes in the leaf lamina. External mycelium
amphigenous, branched, septate, initially hyaline becoming
yellow or orange-brown later. Internal mycelium
indistinct.
Propagulophores either absent or dicult to
distinguish from ordinary hyphae, cylindrical, simple, length indeterminate, individual cells 11–27 μm long, 3–4
μm diam at the base, increasing to about 5–8 μm diam immediately below the propagule, hyaline, smooth,
point of rupture indistinct or absent. Propagules
complex,repeatedly branched, chandelier-like, subglobose to
irregular outline at maturity, formed on prostrate hyphae or occasionally on erect propagulophores, multicellular,
composed of primary branches with an initial dichotomous branching pattern, becoming dendritic at maturity,
69–273 × 64–272 μm, branch elements 4–10 μm diam, terminal elements digitate, 4–5 × 7–13 μm, initially
hyaline becoming orange when mature, smooth.. Additional synasexual morph formed in pure culture (on VBA):
Conidiomata pycnidial, globose to slightly depressed globose, 100–260 × 100–370 μm, wall composed of 1–3
Page 7/18
cell-thick layers dark brown
textura angularis
, 7–12 μm diam; dehiscence ostiolate, central. Conidiophores
formed on a dense, basal, cushion-like aggregation of hyaline cells, mostly reduced to conidiogenous cells,
subcylindrical, branched next to base, 7–13 × 3–4 μm, smooth, hyaline, 1–2-septate. Conidiogenous cells
enteroblastic, phialidic with apical periclinal thickening, 7–12 × 2–3 μm, smooth, hyaline, with minute collarette.
Conidia mostly broadly ellipsoidal, often somewhat attened on one side, oblong, subreniform, ovoid to
subovoid, 9–12 × 3–5 μm, apex rounded to subtruncate, hilum sometimes slightly protuberant, aseptate,
hyaline when immature, becoming chestnut-brown at maturity, smooth, guttulate.
In culture: on PDA and PCA, fast-growing (7–7.4 cm diam in 7 days), colonies with cottony-woolly aerial
mycelium, orange centrally and becoming white at the margin, diurnal zonation distinct; dark-orange to umber
or ochreous to orange reverse on PDA; on PCA colonies with attened aerial mycelium surrounded by isolated
areas of sparse aerial mycelium and strongly irregular supercial growth; sporulation (pycnidiospores)
abundant on both media (but appearing only after 14 d).
Material examined: Holotype: Brazil: Rio de Janeiro: Angra dos Reis, Ilha Grande, 04 Jan 2000, VIC 22213.
Paratype: Brazil: Rio de Janeiro: Angra dos Reis, Ilha Grande, road from Vila Abrahão to Dois Rios, 13 Jan 2002,
VIC 22218. Epitype: Brazil: Rio de Janeiro:Angra dos Reis, Praia Brava, on
Miconia calvescens
, 28 Jul 2018, R.
W. Barreto, Herbarium Universidade Federal de Viçosa (VIC 47147 – epitype designated here, MBT 10002681,
ex-epitype culture COAD 2597).
Additional material: Brazil: Rio de Janeiro: Estrada de Guapiaçu, Cachoeiras do Macacú, on
Miconia calvescens
,
8 Jan 2021, R. W. Barreto VIC 47491, culture COAD 3306).
Notes: The ex-type culture was no longer viable. Hence, a new isolate obtained from the same region from
where the type material originated was collected to serve as epitype, as indicated above. An ex-epitype culture
was obtained, deposited in the culture collection and used, together with a supplementary specimen obtained
during the study.
Discussion
In the present study multigene phylogenetic analyses revealed two of the three
Korunomyces
spp. known from
culture,
K. terminaliae
and
K. prostratus
, to form a well-supported clade within the genus
Coniella
, resolving
Korunomyces
as an agonomycetous synasexual morph of
Coniella
(
Schizoparmaceae
). Since
Coniella
has
nomenclatural priority over
Korunomyces
,
Korunomyces
is reduced herein to a synonym for
Coniella
and a new
name and combination are proposed here for
K. terminaliae
and
K. prostratus
:
Coniella ferreirense
and
Coniella
prostrata
. Unfortunately the taxonomic anity of
K. zapatensis
will remain unclear until this fungus is
recollected and epitypied.
Coniella
was introduced by von Höhnel (1918) and typied by
Coniella pulchella
(=
Coniella fragariae
). Many
species of
Coniella
are known as plant pathogens, causing leaf, fruit, stem, and root diseases of a wide range of
hosts, including economically important species, and have received considerable attention in phytopathological
literature (van Niekerk et al. 2004; Alvarez et al. 2016; Chethana et al. 2017). Other species in this genus have a
saprobic lifestyle, occurring in litter, decaying bark and in soil, whereas others occur as endophytes or as
Page 8/18
secondary invaders of plant tissues infected by other organisms or injured by other causes (Alvarez et al. 2016;
Ferreira et al. 1997).
Seixas et al. (2007) when differentiating
C. prostrata
from
C. ferreirense
, speculated that the propagules of
C.
prostrata
would not be functional as dispersion units due to the prostrate condition of the structure and were
likely to function, instead, as infection pads. The authors suggested that the dispersion in
C. prostrata
would
probably depend on some spore stage that had not been observed until then. Several attempts to induce
sporulation of
C. prostrata
were made by the authors, but without success. In this study we observed for the rst
time the formation of sporulating pycnidia in
C. prostrata
in culture, and found it to produce a typical
Coniella
asexual morph. The
Schizoparme
sexual morph is yet to be observed, but it is likely that the pycnidial phase
may be formed on infected leaves of
M. calvescens
at an advanced stage of the necrosis and will represent the
dispersal stage of this fungus, as suggested earlier by Seixas et al. (2007).
In the phylogenetic tree
C. ferreirense
and the two isolates of
C. prostrata
formed distinct lineages within a clade
separated from the other species of
Coniella
.
Other species of
Coniella
have been described from
Terminalia
spp., namely:
C. crousii
on
T. chebula
from India
(Alvarez et al. 2016),
C. macrospora
on
T. ivorensis
from the Ivory Coast (Alvarez et al. 2016),
C. pseudogranati
on
T. stuhlmannii
from Zambia (Alvarez et al. 2016; Chethana et al. 2017),
C. fragariae
on
T. chebula
and
T.
paniculata
from India (Rajeshkumar et al. 2011),
C. terminaliae
on
T. tomentosa
from India (Rajeshkumar et al.
2011; Alvarez et al. 2016) and
C. terminaliicola
on
T. superba
from Ecuador (Alvarez et al. 2016).
Coniella
crousii
,
C. macrospora
,
C. pseudogranati
, and
C. fragariae
are phylogenetically distant from
C. prostrata
and
C.
ferreirense
. There are no sequences available on GenBank for
C. terminaliae
and
C. terminaliicola
. Pycnidial
formation was not described for
C. terminaliicola
and
C. ivorensis
, making morphological comparison
impossible.
Coniella terminaliae
has globose to subglobose spores, 2–8 × 2–3.5 µm, whereas
C. prostrata
has
ellipsoidal conidia, 9–12 × 3–5 µm. Although the size, shape and colour of the conidia are overlapping
characteristics in some species, the formation of propagules and propagulophores are unique for
C. ferreirense
and
C. prostrata
.
Seixas et al. (2007) and Hodges & Ferreira (1981) performed pathogenicity tests with
C. ferreirense
and
C.
prostrata
. The results of these inoculations showed that
C. prostrata
was capable of causing necrosis on leaves
of
M. calvescens
,
T. ivorensis
and
E. grandis
, but not on
T. catappa
, whereas
C. ferreirense
was able to infect
three species of
Terminalia
, but not
Eucalyptus grandis
(Hodges and Ferreira 1981). Despite the partial overlap
of the host range, there are differences between the two species. Based on the DNA phylogeny and morphology
presented here and host differences reported by Seixas et al. (2007) and Hodges & Ferreira (1981), the two
species are considered as distinct.
Unfortunately, a phylogenetic study of
K. zapatensis
was not possible, because the species is only represented
by herbarium material available only in Cuba and there are no ex-type cultures or DNA sequences available for
this species (Holubová-Jechová & Castaneda, 1986). However, the morphological similarity with the other
species previously belonging to
Korunomyces
, indicates that this is yet another agonomycetous synasexual
morph of
Coniella
. For now, this particular species should be treated as
incertae sedis
.
Declarations
Page 9/18
Acknowledgements
This publication forms part of a thesis submitted by B. W. Ferreira to the Departamento de Fitopatologia,
Universidade Federal de Viçosa as a requirement for a DSc. We would like to dedicate this paper to the late Dr.
Francisco A. Ferreira – “Chico Fungo” – who collected and described
Korunomyces
and made many other
important contributions to the elds of mycology and forest pathology.Electron microscopy studies were
performed at the Núcelo de Microscopia e Microanálise da Universidade Federal de Viçosa (NMM-UFV).
Funding
This study received nancial support from the Brazilian Conselho Nacional de Desenvolvimento Cientíco e
Tecnológico (CNPq) andCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Author information
Aliations
Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, 36570-900, Brazil
Bruno W. Ferreira,Janaina L. Alves, Robert W. Barreto
Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands
Pedro W. Crous
Corresponding author
Correspondence to Robert W. Barreto (rbarreto@ufv.br).
Contributions
BWF conducted the isolation of strains, DNA extractions, PCR amplications, phylogenetic analyses and wrote
the manuscript. JLA and PWC prepared the morphological characterization and participated in writing of the
manuscript. RWB is the research leader. He corrected the text and guided throughout the development of the
study.
Ethics declarations
Conict of interest
The authors declare that they have no conict of interest.
Data availability
The datasets generated and analysed during the current study are available either in GenBank at NCBI (National
Center for Biotechnology Information), as indicated in the text, or available from the corresponding author.
References
Page 10/18
1. Alvarez LV, Groenewald JZ, Crous PW (2016) Revising the
Schizoparmaceae
:
Coniella
and its synonyms
Pilidiella
and
Schizoparme
. Stud Mycol 85:1–34. https://doi.org/10.1016/j.simyco.2016.09.001
2. Ahmad S (1967) Contributions to the fungi of west Pakistan. VI Biologia 13:38
3. Ahmad S (1968) Contribution to the fungi of west Pakistan. VII Biologia 14:4
4. Carbone I, Kohn LM (1999) A method for designing sets for speciation studies in lamentous ascomycetes.
Mycologia 91:553–556. https://doi.org/10.2307/3761358
5. Chethana KWT, Zhou Y, Zhang W, Liu M, Xing QK, Hyde KD, Yan JY, Li XH (2017)
Coniella vitis
sp. nov. is the
common pathogen of white rot in Chinese vineyards. Plant Dis 101:2123–2136.
https://doi.org/10.1094/PDIS-12-16-1741-RE
. Crous PW, Schoch CL, Hyde KD, Wood AR, Gueidan C, Hoog GS, Groenewald JZ (2009) Phylogenetic
lineages in the Capnodiales. Stud Mycol 64:17–47. https://doi.org/10.3114/sim.2009.64.02
7. Crous PW, Shivas RG, Quaedvlieg W, van der Bank M et al (2014) Fungal Planet description sheets: 214–
280. Persoonia 32:184–306. http://dx.doi.org/10.3767/003158514X682395
. Crous PW, Schumacher RK, Wingeld MJ, Lombard L, Giraldo A, Christensen M, Gardiennet A, Nakashima
C, Pereira OL, Smith AJ, Groenewald JZ (2015b) Fungal systematics and evolution: FUSE 1. Sydowia
67:81–118
9. Crous PW, Summerell BA, Shivas RG, Burgess TI et al (2012) Fungal Planet description sheets: 107–127.
Persoonia 28:138–182. http://dx.doi.org/10.3767/003158512X652633
10. Crous PW, Wingeld MJ, Guarro J, Hernández-Restrepo M et al (2015a) Fungal Planet description sheets:
320–370. Persoonia 34:167–266. http://dx.doi.org/10.3767/003158515X688433
11. Crous PW, Verkley GJM, Groenewald JZ et al (2019) Fungal biodiversity, Westerdijk Laboratory Manual
Series 1. Westerdijk Fungal Biodiversity Institute, The Netherlands
12. Dianese JC, Medeiros RB, Santos LTP, Sutton BC (1993)
Coniella costae
sp. nov. on leaves of
Myrcia
tomentosa
from Brazilian cerrado. Mycol Res 97:1234–1236. https://doi.org/10.1016/S0953-
7562(09)81291-4
13. Farr DF, Rossman AY (2021) Fungal Databases, U.S. National Fungus Collections, ARS, USDA.
https://nt.arsgrin.gov/fungaldatabases/fungushost/fungushost.cfm. Accessed 20 Jan 2021
14. Ferreira FA, Alfenas AC, Coelho L (1997) Portas-de-entrada para
Coniella fragariae
em folhas de eucalipto.
Revista Árvore 21:307–311
15. Firdousi SA, Sharma CD, Vyas KM (1994) A new species of
Coniella
from India. Acta Bot Indica 22:134–
135
1. Groenewald JZ, Nakashima C, Nishikawa J et al (2013) Species concepts in
Cercospora
: spotting the
weeds among the roses. Stud Mycol 75:115–170. https://doi.org/10.3114/sim0012
17. Hepperle D (2004) SeqAssem©. Win32–Version. A sequence analysis tool contig assembler and trace data
visualization tool for molecular sequences. Available at: http://www.sequentix.de
1. Hodges CS, Ferreira FA (1981)
Korunomyces
, a new genus of fungi imperfecti from Brazil. Mycologia
73:334–342. https://doi.org/10.2307/3759653
19. Holubová-Jechová V, Castañeda Ruiz RF (1986) Studies on Hyphomycetes from Cuba III. New and
interesting dematiaceous taxa from leaf litter. Ceská Mykol 40:74–85
Page 11/18
20. Jayawardena RS, Hyde KD, Jeewon R, Ghobad-Nejhad M, Wanasinghe DN, Liu N et al (2019) One stop shop
II: taxonomic update with molecular phylogeny for important phytopathogenic genera: 26–50 (2019).
Fungal Divers 94:41–129. https://doi.org/10.1007/s13225-019-00418-5
21. Liu YJ, Whelen S, Hall BD (1999) Phylogenetic relationships among ascomycetes: evidence from an RNA
polymerase II subunit. Mol Biol Evol 16:1799–1808.
https://doi.org/10.1093/oxfordjournals.molbev.a026092
22. Maddison WP, Maddison DR (2011) Mesquite: a modular system for evolutionary analysis. Version 3.51
http://www.mesquiteproject.org. Accessed 3 Jan 2021
23. Marin-Felix Y, Groenewald JZ, Cai L, Chen Q, Marincowitz S et al (2017) Genera of phytopathogenic fungi:
GOPHY. Stud Mycol 86:99–216. https://doi.org/10.1016/j.simyco.2017.04.002
24. Miller MA, Pfeiffer W, Schwartz T (2011) The CIPRES science gateway: a community resource for
phylogenetic analyses. In Proceedings of the 2011 TeraGrid Conference: extreme digital discovery, pp1–8.
https://doi.org/10.1145/2016741.2016785
25. Miranda BEC, Barreto RW, Crous PW, Groenewald JZ (2012)
Pilidiella tibouchinae
sp. nov. associated with
foliage blight of
Tibouchina granulosa
(quaresmeira) in Brazil. IMA Fungus 3:1–7.
https://doi.org/10.5598/imafungus.2012.03.01.01
2. Nag Raj TR (1993) Coelomycetous anamorphs with appendage-bearing conidia. Mycologue Publications,
Waterloo
27. O'Donnell K, Cigelnik E, Nirenberg HI (1998) Molecular systematics and phylogeography of the
Gibberella
fujikuroi
species complex. Mycologia 90:465–493. https://doi.org/10.2307/3761407
2. Pereira JM, Barreto RW, Ellison CA, Maa LA (2003)
Corynespora cassiicola
f. sp.
lantanae
: a potential
biocontrol agent for
Lantana camara
from Brazil. Biol Control 26:21–31. https://doi.org/10.1016/S1049-
9644(02)00112-3
29. Petrak F (1955) Neue Mikromyzeten der australischen Flora. Sydowia 9:559–570
30. Pinho DB, Firmino AL, Ferreira-Junior WG, Pereira OL (2012) An ecient protocol for DNA extraction from
Meliolales
and the description of
Meliola centellae
sp. nov. Mycotaxon 122:333–345.
http://dx.doi.org/10.5248/122.333
31. Posada D, Buckley TR (2004) Model selection and model averaging in phylogenetics: advantages of Akaike
information criterion and Bayesian approaches over likelihood ratio tests. Syst Biol 53:793–808.
https://doi.org/10.1080/10635150490522304
32. Rajeshkumar KC, Hepat RP, Gaikwad SB, Singh SK (2011)
Pilidiella crousii
sp. nov. from the northern
Western Ghats, India. Mycotaxon 115:155–162. https://doi.org/10.5248/115.155
33. Rambaut A (2009) FigTree 1.2.2. Available at: http://tree.bio.ed.ac.uk/software/gtree/. Accessed on Dez
15 2020
34. Rayner RW (1970) A mycological colour chart. CMI and British Mycological Society, Kew
35. Redhead SA (1975) The genus
Cristulariella
. Can J Bot 53:700–707
3. Samuels GJ, Barr ME, Lowen R (1993) Revision of
Schizoparme
(
Diaporthales, Melanconidaceae
).
Mycotaxon 46:459–483
Page 12/18
37. Seixas CD, Barreto RW, Killgore E (2007) Fungal pathogens of
Miconia calvescens
(
Melastomataceae
) from
Brazil, with reference to classical biological control. Mycologia 99:99–111.
https://doi.org/10.1080/15572536.2007.11832605
3. Sharma ND, Agarwal GP (1977) Fungi causing plant diseases at Jabalpur Madhya Pradesh India. Part 15.
Some sphaeropsidales. Sydowia 30:290–296. https://doi.org/10.1007/BF02132445
39. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large
phylogenies. Bioinformatics 30:1312–1313. https://doi.org/10.1093/bi-oinformatics/btu033
40. Sutton BC (1980) The
Coelomycetes
: fungi imperfecti with pycnidia, acervuli, and stromata.
Commonwealth Mycological Institute, Kew
41. Sydow H, Sydow P (1913) Novae fungorum species—IX. Ann Mycol 11:62
42. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA 6: molecular evolutionary genetics
analysis version 6.0. Mol Biol Evol 30:2725–2729. https://doi.org/10.1093/molbev/mst197
43. Van Niekerk JM, Groenewald JZ, Verkley GJM, Fourie PH, Wingeld MJ, Crous PW (2004) Systematic
reappraisal of
Coniella
and
Pilidiella
, with specic reference to species occurring on
Eucalyptus
and
Vitis
in
South Africa. Mycol Res 108:283–303. https://doi.org/10.1017/S0953756204009268
44. Vilgalys R, Hester M (1990) Rapid genetic identication and mapping of enzymatically amplied ribosomal
DNA from several Cryptococcus species. J Bacteriol 172:4238–4246.
https://doi.org/10.1128/jb.172.8.4238-4246.1990
45. Von Höhnel F (1918) Dritte vorlauge Mitteilung mycologischer Ergebnisse. Ber Dtsch Bot Ges 36:Nr. 201–
304 309–317.
4. Vu D, Groenewald M, De Vries M, Gehrmann T, Stielow B, Eberhardt U, … Verkley GJM (2019) Large-scale
generation and analysis of lamentous fungal DNA barcodes boosts coverage for kingdom fungi and
reveals thresholds for fungal species and higher taxon delimitation. Stud Mycol 92:135–154.
https://doi.org/10.1016/j.simyco.2018.05.001
47. White TJ, Bruns T, Lee S, Taylor J (1990) Amplication and direct sequencing of fungal ribosomal RNA
genes for phylogenetics. In: Innis GelfandM, Shinsky D, White T (eds) PCR protocols: a guide to methods
and applications. Academic, New York, pp315–322
Tables
Table1. Taxa and collections used for multi-gene phylogenetic analyses in this study.
Page 13/18
Species name Strain
accession
number1
GenBank accession
number2
References
ITS LSU
RPB2 TEF1
Coniella africana
CBS
114133T =
CPC 405
AY339344 AY339293 KX833421 KX833600 Van Niekerk
et al. (2004);
Alvarez et al.
(2016)
C. crousii
HQ264189 – – – Rajeshkumar
et al. (2011)
C. diplodiella
CBS
111858ET =
CPC 3708
AY339323 KX833335 KX833423 KX833603 Van Niekerk
et al. (2004);
Alvarez et al.
(2016)
CBS
111857 =
CPC 3735
AY339325 AY339285 KX833422 KX833602 Alvarez et al.
(2016)
CBS
112333 =
CPC 3775
AY339329 KX833336 KX833424 KX833604 Alvarez et al.
(2016)
C. diplodiopsis
CBS
590.84T =
CPC 3940
AY339334 AY339288 – – Alvarez et al.
(2016)
CBS 10923
= CPC 3933 AY339332 AY339287 KX833440 KX833624 Van Niekerk
et al. (2004);
Alvarez et al.
(2016)
CBS
112637 =
CPC 4228
KX833530 KX833355 KX833441 KX833625 Alvarez et al.
(2016)
CBS
112702 =
CPC 3866
KX833531 KX833356 KX833442 KX833626 Alvarez et al.
(2016)
C. duckerae
VPRI 13689
= CBS
142045T
KY924929 – – – Marin-Felix et
al. (2017)
C. erumpens
CBS
52378T
KX833535 KX833361 KX833446 KX833630 Alvarez et al.
(2016)
C. eucalyptigena
CBS
139893T
KR476725 – – – Crous et al.
(2015a, b)
C. eucalyptorum
CBS
112640T =
CPC3904
AY339338 AY339290 KX833452 KX833637 Van Niekerk
et al. (2004);
Alvarez et al.
(2016)
CBS
110674 = KX833536 KX833362 KX833447 KX833631 Alvarez et al.
(2016)
Page 14/18
CPC 610
CBS
111023 =
CPC 3843
KX833537 KX833363 KX833448 KX833632 Alvarez et al.
(2016)
C. ferreirense
CBS
224.80T
MH861257 MH873026 – – Vu et al.
(2019)
C. fragariae
CBS
17249NT =
CPC 3930
AY339317 AY339282 KX833472 KX833663 Van Niekerk
et al. (2004);
Alvarez et al.
(2016)
CBS 45468 KX833571 KX833393 KX833477 KX833670 Alvarez et al.
(2016)
C. fusiformis
CBS
141596T =
CPC 19722
KX833576 KX833397 KX833481 KX833674 Alvarez et al.
(2016)
CBS
114850 KX833574 KX833395 KX833479 KX833672 Alvarez et al.
(2016)
CBS
114851 KX833575 KX833396 KX833480 KX833673 Alvarez et al.
(2016)
C. granati
CBS
132860 KX833577 KX833400 KX833484 KX833677 Alvarez et al.
(2016)
CBS
130974 =
CPC 19625
JN815312 KX833398 KX833482 KX833675 Alvarez et al.
(2016)
CBS
130975 =
CPC 19626
JN815313 KX833399 KX833483 KX833676 Alvarez et al.
(2016)
C. hibisci
CBS
109757ET
KX833589 – – KX833689 Marin-Felix et
al. (2017)
C. javanica
CBS
45568T
KX833583 KX833403 KX833489 KX833683 Alvarez et al.
(2016)
C. koreana
CBS 14397 KX833584 AF408378 KX833490 KX833684 Alvarez et al.
(2016)
C. lanneae
CBS
141597T =
CPC 22200
KX833585 KX833404 KX833491 KX833685 Alvarez et al.
(2016)
C. limoniformis
CBS
111021T =
PPRI 3870
KX833586 KX833405 KX833492 KX833686 Alvarez et al.
(2016)
C. lustricola
DAOMC
251731T
MF631778 MF631799 MF651900 MF651899 Jayawardena
et al. (2019)
C. macrospora
CBS
52473T =
KX833587 AY339292 KX833493 KX833687 Alvarez et al.
(2016)
Page 15/18
CPC 3935
C. malaysiana
CBS
141598T =
CPC 16659
KX833588 KX833406 KX833494 KX833688 Alvarez et al.
(2016)
C. musaiaensis
CBS
109757 =
AR 3534
KX833589 AF408337 – KX833689 Alvarez et al.
(2016)
C. nicotianae
CBS
87572T=
PD 72/793
KX833590 KX833407 KX833495 KX833690 Alvarez et al.
(2016)
C. nigra
CBS
16560T =
IMI 181519
AY339319 KX833408 KX833496 KX833691 Van Niekerk
et al. (2004);
Alvarez et al.
(2016)
C. obovata
CBS
111025 =
CPC4196
AY339313 KX833409 KX833497 KX833692 Van Niekerk
et al. (2004);
Alvarez et al.
(2016)
C.
paracastaneicola
CBS
141292T =
CPC 20146
KX833591 KX833410 KX833498 KX833693 Alvarez et al.
(2016)
CPC 25498 KX833592 KX833411 – KX833694 Alvarez et al.
(2016)
C. peruensis
CBS
110394T =
RMF 7401
KJ710463 KJ710441 KX833499 KX833695 Crous et al.
(2015a, b)
C. prostrata
COAD 3306 MZ727003 MZ726999 MZ772857 MZ772859 This study
COAD2597TMZ727004 MZ727000 MZ772858 MZ772860 This study
C. pseudogranati
CBS
137980T
KJ869132 – – – Crous et al.
(2014)
C.
pseudostraminea
CBS
112624T =
IMI 233050
KX833593 KX833412 KX833500 KX833696 Alvarez et al.
(2016)
C. quercicola
CBS
90469NT
KX833595 KX833414 KX833502 KX833698 Alvarez et al.
(2016)
CBS 283.76 KX833594 KX833413 KX833501 KX833697 Alvarez et al.
(2016)
CPC 12133 KX833596 – KX833503 KX833699 Alvarez et al.
(2016)
C. solicola
CBS
76671T
KX833597 KX833416 KX833505 KX833701 Alvarez et al.
(2016)
CBS
114007 = AY339320 KX833415 KX833504 KX833700 Alvarez et al.
(2016)
Page 16/18
IMI 253210
CPC 17308 KX833598 KX833417 – KX833702 Alvarez et al.
(2016)
C. straminea
CBS 14922
= CPC 3932 AY339348 AY339296 KX833506 KX833704 Van Niekerk
et al. (2004);
Alvarez et al.
(2016)
C. tibouchinae
CBS
131595T =
CPC 18512
JQ281774 KX833418 KX833507 JQ281778 Miranda et
al. (2012);
Alvarez et
al.(2016)
CBS
131594T =
CPC 18511
JQ281774 KX833418 KX833507 JQ281778 Alvarez et al.
(2016)
C. vitis
MFLUCC
16–1399T
KX890008 KX890083 – KX890058 Jayawardena
et al. (2019)
C. wangiensis
CBS
132530T =
CPC 19397
JX069873 JX069857 KX833509 KX833705 Crous et al.
(2012);
Alvarez et al.
(2016)
Melanconiella
hyperopta
CBS
131696 JQ926281 JQ926281 KX833510 KX833706 Miranda et
al. (2012);
Alvarez et
al.(2016)
1 ET: ex-epitype culture; NT: ex-neotype culture; T: ex-type culture.
2 ITS: internal transcribed spacers and intervening 5.8S nrDNA; LSU: 28S nrDNA;
RPB2
: DNA-directed RNA
polymerase II second largest subunit;
TEF1
: translation elongation factor 1-alpha.
Figures
Page 17/18
Figure 1
Maximum Likelihood (ML) tree based on combined nc LSU rDNA, ITS, RPB2 and TEF1 showing the relationship
of Coniella ferreirense and C. prostrata with other closely related species within Coniella. Bootstrap support
values or Bayesian posterior probabilities higher than 70 % or 0.90 are indicated above or below thickened
branches (– indicates lack of support). Isolates from this study are indicated by bold text.
Page 18/18
Figure 2
Coniella prostrata comb. nov. (VIC 47147). A Miconia calvescens individual bearing typical leaf blight
symptoms resulting from attack by C. prostrata at type locality. B–C Young propagules and propagulophores. D
Mature conidiomata on PDA. E Ostiolate mature conidiomata. F Conidiogenous cells. G Conidia H Colony on
PDA after 14 days (incubation at 25 °C in 12 h light/dark cycle). I Colony on PCA after 14 days (incubation at 25
°C in 12 h light/dark cycle). Scale bars: a, b, c, e and f = 20 μm; g = 10 μm.
