available online at www.studiesinmycology.org
StudieS in Mycology 58: 105–156. 2007.
doi:10.3114/sim.2007.58.05
Biodiversity in the Cladosporium herbarum complex (Davidiellaceae, Capnodiales),
with standardisation of methods for Cladosporium taxonomy and diagnostics
K. Schubert1*, J. Z. Groenewald2, U. Braun3, J. Dijksterhuis2, M. Starink2, C.F. Hill4, P. Zalar5, G.S. de Hoog2 and P.W. Crous2
1
Botanische Staatssammlung München, Menzinger Strasse 67, D-80638 München, Germany; 2CBS Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands;
Martin-Luther-Universität, Institut für Biologie, Geobotanik und Botanischer Garten, Herbarium, Neuwerk 21, D-06099 Halle (Saale), Germany; 4Plant & Environment Laboratory
Biosecurity NZ, Ministry of Agriculture & Forestry, P.O. Box 2095, Auckland 1140, New Zealand; 5Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana,
Slovenia
3
*Correspondence: Konstanze Schubert, konstanze.schubert@gmx.de
Abstract: The Cladosporium herbarum complex comprises ive species for which Davidiella teleomorphs are known. Cladosporium herbarum s. str. (D. tassiana), C.
macrocarpum (D. macrocarpa) and C. bruhnei (D. allicina) are distinguishable by having conidia of different width, and by teleomorph characters. Davidiella variabile is
introduced as teleomorph of C. variabile, a homothallic species occurring on Spinacia, and D. macrospora is known to be the teleomorph of C. iridis on Iris spp. The C. herbarum
complex combines low molecular distance with a high degree of clonal or inbreeding diversity. Entities differ from each other by multilocus sequence data and by phenetic
differences, and thus can be interpreted to represent individual taxa. Isolates of the C. herbarum complex that were formerly associated with opportunistic human infections,
cluster with C. bruhnei. Several species are newly described from hypersaline water, namely C. ramotenellum, C. tenellum, C. subinlatum, and C. herbaroides. Cladosporium
pseudiridis collected from Iris sp. in New Zealand, is also a member of this species complex and shown to be distinct from C. iridis that occurs on this host elsewhere in the
world. A further new species from New Zealand is C. sinuosum on Fuchsia excorticata. Cladosporium antarcticum is newly described from a lichen, Caloplaca regalis, collected
in Antarctica, and C. subtilissimum from grape berries in the U.S.A., while the new combination C. ossifragi, the oldest valid name of the Cladosporium known from Narthecium
in Europe, is proposed. Standard protocols and media are herewith proposed to facilitate future morphological examination of Cladosporium spp. in culture, and neotypes or
epitypes are proposed for all species treated.
Taxonomic novelties: Cladosporium antarcticum K. Schub., Crous & U. Braun, sp. nov., C. herbaroides K. Schub., Zalar, Crous & U. Braun, sp. nov., C. ossifragi (Rostr.) U.
Braun & K. Schub., comb. nov., C. pseudiridis K. Schub., C.F. Hill, Crous & U. Braun, sp. nov., C. ramotenellum K. Schub., Zalar, Crous & U. Braun, sp. nov., C. sinuosum K.
Schub., C.F. Hill, Crous & U. Braun, sp. nov., C. subinlatum K. Schub., Zalar, Crous & U. Braun, sp. nov., C. subtilissimum K. Schub., Dugan, Crous & U. Braun, sp. nov., C.
tenellum K. Schub., Zalar, Crous & U. Braun sp. nov., Davidiella macrocarpa Crous, K. Schub. & U. Braun, sp. nov., D. variabile Crous, K. Schub. & U. Braun, sp. nov.
Key words: Clonality, Davidiella, homothallism, new species, phylogeny, recombination, taxonomy.
InTroducTIon
Cladosporium herbarum (Pers. : Fr.) Link, type species of the genus
Cladosporium Link, is one of the most common environmental fungi
to be isolated worldwide. It abundantly occurs on fading or dead
leaves of herbaceous and woody plants, as secondary invader
on necrotic leaf spots, and has frequently been isolated from
air (Samson et al. 2000), soil (Domsch et al. 1980), foodstuffs,
paints, textiles, humans (de Hoog et al. 2000) and numerous
other substrates. It is also known to occur on old carpophores
of mushrooms and other fungi (Heuchert et al. 2005) and to be
a common endophyte (Riesen & Sieber 1985, Brown et al. 1998,
El-Morsy 2000), especially in temperate regions. Under favourable
climatic conditions C. herbarum also germinates and grows as an
epiphyte on the surface of green, healthy leaves (Schubert 2005).
Persoon (1794) introduced C. herbarum as Dematium
herbarum Pers., which was later reclassiied by Link (1809) as
Acladium herbarum (Pers.) Link. In 1816, Link included C. herbarum
together with three additional species in his newly described genus
Cladosporium. Clements & Shear (1931) proposed C. herbarum
as lectotype species of the latter genus, a decision followed by de
Vries (1952) and Hughes (1958). Several authors provided detailed
treatments of C. herbarum (de Vries 1952, Ellis 1971, Domsch et al.
1980, Prasil & de Hoog 1988), and there are literally thousands of
records of this species in the literature. McKemy & Morgan-Jones
(1991) and Ho et al. (1999) examined C. herbarum in culture and
published detailed descriptions of its features in vitro.
Cladosporium macrocarpum Preuss, a second component
within the herbarum complex, has hitherto been known and treated
as an allied, but morphologically distinct species on the basis of its
Copyright © 2007 CBS Fungal Biodiversity Centre
wider and somewhat larger, frequently 2–3-septate, more regularly
verrucose conidia, shorter conidial chains and more pronounced
prolongations of the conidiophores. Dugan & Roberts (1994) carried
out examinations of morphological and reproductive aspects of both
species, and in so doing demonstrated a morphological continuum
between C. macrocarpum and C. herbarum, concluding that the
name herbarum should have preference. Therefore, Ho et al. (1999)
introduced the new combination C. herbarum var. macrocarpum
(Preuss) M.H.M. Ho & Dugan. Although transitional forms have
been discussed to occur between the two species, several authors
still prefer to retain C. macrocarpum as a separate species.
In an attempt to elucidate the species within the C. herbarum
complex, therefore, a multilocus DNA sequence typing approach
was used, employing ive genes, namely the internal transcribed
spacers of the rDNA genes (ITS), actin, calmodulin, translation
elongation factor 1-α, and histone H3. These data were
supplemented with morphological examinations under standardised
conditions, using light and scanning electron microscopy, as well as
cultural characteristics and growth studies.
MATerIAl And MeThods
Isolates
Isolates included in this study were obtained from the culture
collection of the Centraalbureau voor Schimmelcultures (CBS),
Utrecht, Netherlands, or were freshly isolated from a range of
different substrates. Single-conidial and ascospore isolates were
obtained using the techniques as explained in Crous (1998) for
species of Mycosphaerella Johanson and its anamorphs. Isolates
105
Schubert et al.
were inoculated onto 2 % potato-dextrose agar (PDA), synthetic
nutrient-poor agar (SNA), 2 % malt extract agar (MEA) and oatmeal
agar (OA) (Gams et al. 2007), and incubated under continuous
near-ultraviolet light at 25 °C to promote sporulation. All cultures
obtained in this study are maintained in the culture collection of
the CBS (Table 1). Nomenclatural novelties and descriptions were
deposited in MycoBank (www.MycoBank.org).
DNA isolation, ampliication and sequence analysis
Fungal colonies were established on agar plates, and genomic
DNA was isolated as described in Gams et al. (2007). Partial gene
sequences were determined as described by Crous et al. (2006)
for actin (ACT), calmodulin (CAL), translation elongation factor 1alpha (EF), histone H3 (HIS) and part (ITS) of the nuclear rDNA
operon spanning the 3’ end of the 18S rRNA gene (SSU), the
irst internal transcribed spacer, the 5.8S rRNA gene, the second
internal transcribed spacer and the 5’ end of the 28S rRNA gene
(LSU). The nucleotide sequences were generated using both PCR
primers to ensure good quality sequences over the entire length of
the amplicon. Subsequent sequence alignment and phylogenetic
analysis followed the methods of Crous et al. (2006). Gaps longer
than 10 bases were coded as single events for the phylogenetic
analyses; the remaining gaps were treated as new character
states. Sequence data were deposited in GenBank (Table 1) and
the alignment and tree in TreeBASE (www.treebase.org).
Morphology
As the present study represents the irst in a series dealing
with Cladosporium spp. and their Davidiella Crous & U. Braun
teleomorphs in culture, a speciic, standardised protocol was
established by which all species complexes will be treated in
future.
Morphology of the anamorph: Microscopic observations were made
from colonies cultivated for 7 d under continuous near-ultraviolet
light at 25 °C on SNA. Preparations were mounted in Shear’s
solution (Gams et al. 2007). To study conidial development and
branching patterns, squares of transparent adhesive tape (Titan
Ultra Clear Tape, Conglom Inc., Toronto, Canada) were placed on
conidiophores growing in the zone between the colony margin and
2 cm inwards, and mounted between two drops of Shear’s solution
under a glass coverslip. Different types of conidia are formed by
Cladosporium species for which different terms need to be adopted.
ramoconidia are conidia with usually more than one (mostly 2
or 3) conidial hilum, which typically accumulate at the tip of these
conidia. Conidiogenous cells with more than one conidiogenous
locus are irst formed as apical parts of conidiophores. Such apical
data analysis
The number of entities in the dataset of 79 strains was inferred
with Structure v. 2.2 software (Pritchard et al. 2000, Falush et al.
2003) using an UPGMA tree of data of the ACT gene compared
with CAL, EF and HIS with the exclusion of the nearly invariant ITS
region. For this analysis group indications were derived from a tree
produced with MrAic (Nylander 2004). The length of the burn-in
period was set to 1 000 000, number of MCMC repeats after burn-in
10 000, with admixture ancestry and allele frequencies correlated
models, assuming that all groups diverged from a recent ancestral
population and that allele frequencies are due to drift. Uniform prior
for ALPHA was set to 1.0 (default) and allele frequencies with λ set to
1.0 (default). The numbers of MCMC repetitions after burn-in were
set as 10 000 and 100 000. The number of clusters (K) in Structure
was assumed from 5 to 7. Population differentiation FST (index: θ)
was calculated with 1–6 runs using the same software. The null
hypothesis for this analysis is no population differentiation. When
observed theta (θ) is signiicantly different from those of random
data sets (p < 0.05), population differentiation is considered.
Association of multilocus genotypes was screened with the
multilocus option in bionuMericS v. 4.5. To test for reproductive
mode in each population, the standardised index of association
(ISA; Haubold et al. 1998) was calculated with StArt2 software
(Jolley et al. 2001). The null hypothesis for this analysis is complete
panmixia. The values of ISA were compared between observed and
randomised datasets. The hypothesis would be rejected when p <
0.05. Mean genetic diversity (H) and diversities of individual loci
were calculated with liAn v. 3.5 (Haubold & Hudson 2000). Degrees
of recombination or horizontal gene transfer were also visualised
using SplitStree v. 4.8 software (Huson & Bryant 2006). Split
decomposition was carried out with default settings, i.e., character
transformation using uncorrected (observed, “P”) distances, splits
transformation using “equal angle”, and optimise boxes iteration set
to 2.
106
Fig. 1. Cladosporium conidiophore with ramoconidia, secondary ramoconidia,
intercalary conidia, and small, terminal conidia. Scale bar = 10 µm. K. Schubert
del.
Cladosporium herbarum SpecieS coMplex
Aculeate
Spinulose
Digitate
Muricate
than the small terminal conidia. In older literature true ramoconidia
were often cited as “ramoconidia s. str.”, whereas secondary
ramoconidia have been referred to as “ramoconidia s. lat.”
Morphology of the teleomorph: Teleomorphs were induced by
inoculating plates of 2 % tap water agar onto which autoclaved
stem pieces of Urtica dioica (European stinging nettle) were placed.
Inoculated plates were incubated on the laboratory bench for 7 d,
after that period they were further incubated at 10 °C in the dark for
1–2 mo to stimulate teleomorph development. Wherever possible,
30 measurements (× 1 000 magniication) were made of conidia
and ascospores, with the extremes of spore measurements given
in parentheses. Cultural characteristics: Colonies were cultivated
on PDA, MEA and OA plates for 14 d at 25 °C in the dark, after
which the surface and reverse colours were rated using the charts
of Rayner (1970). Linear growth was determined on MEA, PDA and
OA plates by inoculating three plates per isolate for each medium,
and incubating them for 14 d at 25 ºC, after that period colony
diameters were determined.
Low-temperature scanning electron microscopy
Granulate
Colliculate
Pustulate
Pedicellate
Fig. 2. Terms used to describe conidium wall ornamentation under the cryo-electron
microscope. Adapted from David (1997).
parts of conidiophores are called ramoconidia if they secede at
a septum from the conidiophore (Kirk et al. 2001). The septum at
which the ramoconidium secedes often appears to be somewhat
refractive or darkened. Ramoconidia are characterised by having
a truncate, undifferentiated base (thus they lack a differentiated,
coronate basal hilum formed in the context of conidiogenesis)
and they can be very long, aseptate to sometimes multi-septate.
Although they were formed initially as part of the conidiophore,
they function as propagules. Only few of the species known
until now have the ability to form true ramoconidia. secondary
ramoconidia also have more than one distal conidial hilum but
they always derive from a conidiogenous locus of an earlier formed
cell, which can be either a conidiogenous cell or a ramoconidium.
Secondary ramoconidia are often shorter but somewhat wider than
ramoconidia; they are often septate, and typically have a narrowed
base with a coronate hilum (Fig. 1). Conidia in Cladosporium are
cells with a coronate basal hilum, which is formed in the context of
conidiogenesis and with either a single (when formed as intercalary
units in unbranched parts of chains) or without any distal conidial
hilum (when formed at the tip of conidial chains). For the irst, the
term “intercalary conidium” and for the latter, “small terminal
conidium” is used. Intercalary conidia typically are larger and more
pigmented and have a more differentiated surface ornamentation
www.studiesinmycology.org
Isolates of Cladosporium spp. were grown on SNA with 30 g agar/L
for 3–4 d at room temperature under black light. Relevant parts of
the small colonies with conidiophores and conidia were selected
under a binocular, excised with a surgical blade as small agar (3
× 3 mm) blocks, and transferred to a copper cup for snap-freezing
in nitrogen slush. Agar blocks were glued to the copper surface
with frozen tissue medium (KP-Cryoblock, Klinipath, Duiven,
Netherlands) mixed with 1 part colloidal graphite (Agar Scientiic,
Stansted, U.K.). Samples were examined in a JEOL 5600LV
scanning electron microscope (JEOL, Tokyo, Japan) equipped
with an Oxford CT1500 Cryostation for cryo-electron microscopy
(cryoSEM). Electron micrographs were acquired from uncoated
frozen samples, or after sputter-coating by means of a gold/
palladium target for 3 times during 30 s (Fig. 2). Micrographs of
uncoated samples were taken at an acceleration voltage of 3 kV,
and consisted out of 30 averaged fast scans (SCAN 2 mode), and
at 5 kV in case of the coated samples (PHOTO mode).
resulTs
Phylogeny and differentiation
The manually adjusted alignment contained 80 sequences (including
the outgroup sequence) and the ive loci were represented by a
total of 1 516 characters including alignment gaps which were
used in the analysis. Of the 1 516 characters, 369 were parsimonyinformative, 259 were variable and parsimony-uninformative, and
888 were constant.
Forty equally most parsimonious trees (TL = 1 933 steps; CI
= 0.569; RI = 0.786; RC = 0.447), one of which is shown in Fig.
3, were obtained from the parsimony analysis of the combined
genes. Neighbour-joining analysis using three substitution models
(uncorrected “p”, Kimura 2-parameter and HKY85) on the sequence
data yielded trees with identical topologies. These differed from
the tree presented in Fig. 3 with regard to the placement of C.
macrocarpum strain CPC 12054 which was placed as a sister
branch to the C. bruhnei Linder clade in the distance analyses
(results not shown) because it shares an identical CAL sequence.
All cryptic species consisting of multiple strains are clustering in
well-supported clades with bootstrap support values ranging from
71 % (C. herbarum) to 100 % [e.g. C. ramotenellum K. Schub.,
107
Anamorph
Teleomorph
Accession number1
host
Country
collector
GenBank numbers2
Cladosporium antarcticum
—
CBS 690.92* (ex-type)
Caloplaca regalis
Antarctica
C. Möller
EF679334, EF679405, EF679484, EF679560, EF679636
Cladosporium bruhnei
Davidiella allicina
CBS 134.31 = ATCC 11283
—
Germany
—
EF679335, EF679406, EF679485, EF679561, EF679637
CBS 157.82
Quercus robur
Belgium
—
EF679336, EF679407, EF679486, EF679562, EF679638
CBS 159.54 = ATCC 36948
Man, skin
The Netherlands
—
EF679337, EF679408, EF679487, EF679563, EF679639
CBS 161.55
Man, sputum
The Netherlands
—
EF679338, EF679409, EF679488, EF679564, EF679640
CBS 177.71
Thuja tincture
The Netherlands
—
EF679339, EF679410, EF679489, EF679565, EF679641
CBS 188.54 = ATCC 11290 = IMI 049638 = CPC
3686
CBS 366.80
—
—
—
AY251077, EF679411, EF679490, EF679566, EF679642
Man, skin
The Netherlands
—
EF679340, EF679412, EF679491, EF679567, EF679643
CBS 399.80
Man, skin
The Netherlands
—
AJ244227, EF679413, EF679492, EF679568, EF679644
CBS 521.68
Air
The Netherlands
—
EF679341, EF679414, EF679493, EF679569, EF679645
CBS 572.78
Polyporus radiatus
Russia
V.K. Melnik
DQ289799, EF679415, DQ289866, DQ289831, EF679646
CBS 813.71
Polygonatum odoratum
Czech Republic
—
EF679342, EF679416, EF679494, EF679570, EF679647
(ITs, eF, AcT, cAl, hIs)
Cladosporium cladosporioides
complex
CBS 110024
Industrial water
Germany, Nordrhein-Westfalen —
EF679343, EF679417, EF679495, EF679571, EF679648
CBS 115683 = ATCC 66670 = CPC 5101
CCA-treated Douglas-ire pole
U.S.A., New York
AY361959, EF679418, AY752193, AY752224, AY752255
CBS 121624* = CPC 12211 (neotype)
Hordeum vulgare
Belgium
J.Z. Groenewald EF679350, EF679425, EF679502, EF679578, EF679655
CPC 11386
Tilia cordata
Germany, Sachsen-Anhalt
K. Schubert
EF679344, EF679419, EF679496, EF679572, EF679649
CPC 11840
Ourisia macrophylla
New Zealand
A. Blouin
EF679345, EF679420, EF679497, EF679573, EF679650
CPC 12042 = EXF-389
Hypersaline water from salterns Slovenia
P. Zalar
EF679346, EF679421, EF679498, EF679574, EF679651
CPC 12045 = EXF-594
Hypersaline water from salterns Spain
P. Zalar
EF679347, EF679422, EF679499, EF679575, EF679652
CPC 12046 = EXF-680
Air conditioning system
Slovenia
P. Zalar
EF679348, EF679423, EF679500, EF679576, EF679653
CPC 12139
Hordeum vulgare
The Netherlands
—
EF679349, EF679424, EF679501, EF679577, EF679654
CPC 12212
Hordeum vulgare
Belgium
J.Z. Groenewald EF679351, EF679426, EF679503, EF679579, EF679656
CPC 12921
Eucalyptus sp.
Australia
—
EF679352, EF679427, EF679504, EF679580, EF679657
—
CBS 673.69
Air
The Netherlands
—
EF679353, EF679428, EF679505, EF679581, EF679658
Davidiella sp.
CBS 109082
Silene maritima
United Kingdom
A. Aptroot
EF679354, EF679429, EF679506, EF679582, EF679659
—
CPC 11606
Musa sp.
India
M. Arzanlou
EF679355, EF679430, EF679507, EF679583, EF679660
India
—
CPC 11609
Musa sp.
Cladosporium herbaroides
—
CBS 121626* = CPC 12052 = EXF-1733 (ex-type)
Hypersaline water from salterns Israel
Cladosporium herbarum
Davidiella tassiana CBS 111.82
C.J. Wang
M. Arzanlou
EF679356, EF679431, EF679508, EF679584, EF679661
P. Zalar
EF679357, EF679432, EF679509, EF679585, EF679662
Arctostaphylos uva-ursi
Switzerland
E. Müller
AJ238469, EF679433, EF679510, EF679586, EF679663
CBS 300.49
Biscutella laevigata
Switzerland
J.A. von Arx
EF679358, EF679434, EF679511, EF679587, EF679664
CBS 121621* = CPC 12177 (epitype)
Hordeum vulgare
The Netherlands
—
EF679363, EF679440, EF679516, EF679592, EF679670
CPC 11600
Delphinium barbeyi
U.S.A., Colorado
A. Ramalay
DQ289800, EF679435, DQ289867, DQ289832, EF679665
CPC 11601
Delphinium barbeyi
U.S.A., Colorado
A. Ramalay
EF679359, EF679436, EF679512, EF679588, EF679666
CPC 11602
Delphinium barbeyi
U.S.A., Colorado
A. Ramalay
EF679360, EF679437, EF679513, EF679589, EF679667
CPC 11603
Delphinium barbeyi
U.S.A., Colorado
A. Ramalay
EF679361, EF679438, EF679514, EF679590, EF679668
Schubert et al.
108
Table 1. Isolates subjected to DNA sequence analyses and morphological examinations.
www.studiesinmycology.org
Cladosporium iridis
Davidiella
macrospora
Cladosporium macrocarpum
Davidiella
macrocarpa
Cladosporium ossifragi
—
CPC 11604
Delphinium barbeyi
U.S.A., Colorado
A. Ramalay
EF679362, EF679439, EF679515, EF679591, EF679669
CPC 12178
Hordeum vulgare
The Netherlands
—
EF679364, EF679441, EF679517, EF679593, EF679671
CPC 12179
Hordeum vulgare
The Netherlands
—
EF679365, EF679442, EF679518, EF679594, EF679672
CPC 12180
Hordeum vulgare
The Netherlands
—
EF679366, EF679443, EF679519, EF679595, EF679673
CPC 12181
Hordeum vulgare
The Netherlands
—
EF679367, EF679444, EF679520, EF679596, EF679674
CPC 12183
Hordeum vulgare
The Netherlands
—
EF679368, EF679445, EF679521, EF679597, EF679675
CBS 107.20
Iris sp.
—
—
EF679369, EF679446, EF679522, EF679598, EF679676
CBS 138.40* (epitype)
Iris sp.
The Netherlands
—
EF679370, EF679447, EF679523, EF679599, EF679677
CBS 175.82
Water
Romania
—
EF679371, EF679448, EF679524, EF679600, EF679678
CBS 223.31 = ATCC 11287
Mycosphaerella tulasnei
—
—
AF222830, EF679449, EF679525, EF679601, EF679679
CBS 299.67
Triticum aestivum
Turkey
—
EF679372, EF679450, EF679526, EF679602, EF679680
CBS 121811* = CPC 12755 (neotype)
Spinacia oleracea
U.S.A.
—
EF679376, EF679454, EF679530, EF679606, EF679684
CPC 11817
Corylus sp.
U.S.A.
—
EF679373, EF679451, EF679527, EF679603, EF679681
CPC 12054 = EXF-2287
Hypersaline water from salterns Slovenia
P. Zalar
EF679374, EF679452, EF679528, EF679604, EF679682
CBS H-19855 = CPC 12752 = CBS 121623
Spinacia oleracea
U.S.A.
—
EF679375, EF679453, EF679529, EF679605, EF679683
CPC 12756
Spinacia oleracea
U.S.A.
—
EF679377, EF679455, EF679531, EF679607, EF679685
CPC 12757
Spinacia oleracea
U.S.A.
—
EF679378, EF679456, EF679532, EF679608, EF679686
CPC 12758
Spinacia oleracea
U.S.A.
—
EF679379, EF679457, EF679533, EF679609, EF679687
CPC 12759
Spinacia oleracea
U.S.A.
—
EF679380, EF679458, EF679534, EF679610, EF679688
CBS 842.91* (epitype)
Narthecium ossifragum
Norway
M. di Menna
EF679381, EF679459, EF679535, EF679611, EF679689
CBS 843.91
Narthecium ossifragum
Norway
M. di Menna
EF679382, EF679460, EF679536, EF679612, EF679690
New Zealand
Cladosporium pseudiridis
—
CBS 116463* = LYN 1065 = ICMP 15579 (ex-type)
Iris sp.
C.F. Hill
EF679383, EF679461, EF679537, EF679613, EF679691
Cladosporium ramotenellum
—
CBS 121628* = CPC 12043 = EXF-454 (ex-type)
Hypersaline water from salterns Slovenia
P. Zalar
EF679384, EF679462, EF679538, EF679614, EF679692
CPC 12047 = EXF-967
Air conditioning system
Slovenia
P. Zalar
EF679385, EF679463, EF679539, EF679615, EF679693
CBS 121629* = CPC 11839 = ICMP 15819 (extype)
CBS 102044
Fuchsia excorticata
New Zealand
A. Blouin
EF679386, EF679464, EF679540, EF679616, EF679694
Hypersaline water from salterns Slovenia
S. Soujak
EF679387, EF679465, EF679541, EF679617, EF679695
CBS 119907* = CPC 12040 = EXF-334 (ex-type)
Hypersaline water from salterns Slovenia
P. Zalar
EF679388, EF679466, EF679542, EF679618, EF679696
Cladosporium sinuosum
—
Cladosporium spinulosum
—
—
CBS 121630* = CPC 12041 = EXF-343 (ex-type)
Hypersaline water from salterns Slovenia
P. Zalar
EF679389, EF679467, EF679543, EF679619, EF679697
Cladosporium sp.
—
CBS 172.52 = ATCC 11320
Carya illinoensis
U.S.A.
—
EF679390, EF679468, EF679544, EF679620, EF679698
CBS 113741
Grape berry
U.S.A.
—
EF679391, EF679469, EF679545, EF679621, EF679699
CBS 113742
Grape berry
U.S.A.
—
EF679392, EF679470, EF679546, EF679622, EF679700
CBS 113744
Grape bud
U.S.A.
—
EF679393, EF679471, EF679547, EF679623, EF679701
CPC 12484
Pinus ponderosa
Argentina
A. Greslebin
EF679394, EF679472, EF679548, EF679624, EF679702
Cladosporium subtilissimum
109
Cladosporium tenellum
—
—
CPC 12485
Pinus ponderosa
Argentina
A. Greslebin
EF679395, EF679473, EF679549, EF679625, EF679703
CBS 113753
Bing cherry fruits
U.S.A.
—
EF679396, EF679474, EF679550, EF679626, EF679704
CBS 113754*
Grape berry
U.S.A.
—
EF679397, EF679475, EF679551, EF679627, EF679705
CPC 12044 = EXF-462
Hypersaline water from salterns Slovenia
P. Zalar
EF679398, EF679476, EF679552, EF679628, EF679706
CBS 121634* = CPC 12053 = EXF-1735 (ex-type)
Hypersaline water from salterns Israel
P. Zalar
EF679401, EF679479, EF679555, EF679631, EF679709
Cladosporium herbarum SpecieS coMplex
Cladosporium subinlatum
110
ACT: partial actin gene, CAL: partial calmodulin gene, EF: partial elongation factor 1-alpha gene, HIS: partial histone H3 gene, ITS: internal transcribed spacer region.
Ex-type cultures.
Zalar, Crous & U. Braun and C. ossifragi (Rostr.) U. Braun & K.
Schub.]. The intraspeciic variation in the C. bruhnei clade is due
to genetic variation present in the sequence data of all loci except
for ITS, those in the C. macrocarpum clade in all loci except for ITS
and ACT, and those in the C. herbarum clade in all loci except for
ITS and CAL (data not shown). However, none of the variation for
these species could be linked to host speciicity or morphological
differences. In general, ITS data did not provide any resolution
within the C. herbarum complex, whereas EF data provided species
clades with very little intraspeciic variation and ACT, CAL and HIS
revealed increasing intraspeciic variation (ACT the least and HIS
the most).
The mean genetic diversity (H) of the entire data set excluding
the nearly invariant ITS region was 0.9307, with little difference
between genes (ACT = 0.9257, CAL = 0.9289, EF = 0.9322, HIS
= 0.9361). The loci showed different numbers of alleles (ACT: 22,
CAL: 16, EF: 21, HIS: 20, ITS: 6). Differentiation of entities when
calculated with Structure software using the admixture/correlated
model showed highest value with K = 6. At this value FST varied
between 0.1362 and 0.3381. Linkage disequilibrium calculated
using the standardised index of association (ISA) for the entire
dataset (observed variance Vo = 0.5602, expected variance Ve =
0.2576) was 0.3914 (P = 0.0001), consistent with a small amount
of recombination that did not destroy the linkage between alleles.
Only few groups appeared to be separated for all alleles; degrees
of gene low are indicated in Fig. 4. SplitStree software produced
unresolved star-shaped structures for all genes, without any sign of
reticulation (Fig. 5).
*
2
EF679404, EF679483, EF679559, EF679635, EF679713
E. Müller
Trisetum distichophyllum
CBS 290.49
Switzerland
AY152552, EF679482, EF679558, EF679634, EF679712
ATCC: American Type Culture Collection, Virginia, U.S.A.; CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS; EXF: Extremophilic Fungi Culture Collection of the Department
of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia; ICMP: International Collection of Micro-organisms from Plants, Landcare Research, Private Bag 92170, Auckland, New Zealand; IMI: International Mycological Institute, CABI-Bioscience,
Egham, Bakeham Lane, U.K.
1
EF679403, EF679481, EF679557, EF679633, EF679711
E. Müller
Switzerland
Allium schoenoprasum
CBS 289.49
Davidiella sp.
—
EF679402, EF679480, EF679556, EF679632, EF679710
—
U.S.A.
Spinacia oleracea
EF679400, EF679478, EF679554, EF679630, EF679708
Davidiella variabile CBS 121636* = CPC 12751 (epitype)
CPC 12753
—
U.S.A.
EF679399, EF679477, EF679553, EF679629, EF679707
Cladosporium variabile
Spinacia oleracea
P. Zalar
D. Glawe
Hypersaline water from salterns Israel
CPC 12051 = EXF-1083
U.S.A.
Phyllactinia sp. on Corylus sp.
CPC 11813
(ITs, eF, AcT, cAl, hIs)
host
Accession number1
Teleomorph
Anamorph
Table 1. (Continued).
Country
collector
GenBank numbers2
Schubert et al.
Cladosporium herbarum SpecieS coMplex
100
74
100
10 changes
Cercospora beticola CPC11557
CPC 11609
CBS 109082
Cladosporium cladosporioides complex
98
CBS 673.69
CPC 11606
Davidiella sp. CBS 290.49
100
CPC 12043
Cladosporium ramotenellum
CPC 12047
100 CBS 842.91 Cladosporium ossifragi
80
CBS 843.91
Davidiella sp. CBS 289.49
CPC 12053
100
91
CPC 11813 Cladosporium tenellum
64
CPC 12051
Cladosporium sp. CBS 113744
CBS 113753
100
63
Cladosporium subtilissimum
CBS 113754
98 CPC 12044
CPC 12139
67 CBS 159.54
54 CBS 188.54
CBS 177.71
CBS 399.80
94
CBS 521.68
54
CBS 115683
62 CBS 366.80
72
81
CBS 134.31
CBS 813.71
66
CBS 110024
Cladosporium bruhnei
CPC 12921
CPC
12046
91
CBS 572.78
CPC 12045
CPC 11840
51
CBS 157.82
CPC 12212
CPC 12211
CPC 12042
CPC 11386
CBS 161.55
Cladosporium sp. CPC 12485
63 CBS 102044
CPC 12040 Cladosporium spinulosum
Cladosporium sp. CPC 12484
Cladosporium sp. CBS 172.52
74
Cladosporium sp. CBS 113741
83
Cladosporium antarcticum CBS 690.92
58
Cladosporium sp. CBS 113742
Cladosporium subinflatum CPC 12041
67
Cladosporium pseudiridis CBS 116463
Cladosporium sinuosum CPC 11839
85
100 CBS 107.20 Cladosporium iridis
CBS 138.40
Cladosporium herbaroides CPC 12052
100 CPC 12751
93
CPC 12753 Cladosporium variabile
CPC 11817
51 9564 CPC 12758
CPC 12759
99
CPC 12054
CBS 175.82
66
Cladosporium macrocarpum
CBS 223.31
81
51 CPC 12757
56 CBS 299.67
CPC 12756
65 67 CPC 12755
CPC 12752
CPC 11603
CBS 300.49
CPC 11600
71 CPC 11604
CPC 11602
94
CPC 11601
Cladosporium herbarum
CBS 111.82
CPC 12177
61
CPC 12183
CPC 12181
100 CPC 12180
CPC 12179
CPC 12178
Fig. 3. One of 40 equally most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the combined sequence alignment (ITS, ACT, CAL, EF,
HIS). The scale bar shows ten changes, and bootstrap support values from 1 000 replicates are shown at the nodes. Thickened lines indicate the strict consensus branches
and strain numbers in bold represent ex-type sequences. The tree was rooted to sequences of Cercospora beticola strain CPC 11557 (GenBank accession numbers AY840527,
AY840458, AY840425, AY840494, AY840392, respectively).
www.studiesinmycology.org
111
Schubert et al.
100
95
90
80
ACT
85
Global (Gapcost:0%)
D. allicina
D. alliacea
D. Variabile D. tassiana
D. variabile
D. macrospora
Davidiella sp.D. macrosporum
D. tassiana
D. macrocarpa
D. macrocarpa
Davidiella sp.
z
z
z
z
¼
2
2
¼
2
2
¼
2
2
2
G
)
~
¼
G
)
G
)
G
)
G
)
G
)
¼
5
¼
~
2
¼
"
¼
"
¼
"
Î
8
Î
8
Î
/
Î
/
Î
/
Î
/
_
~
Î
8
8
¼
8
Î
8
_
_
_
_
_
_
_
~
_
_
z
Î
z
Î
z
Î
z
Î
z
Î
~
Î
~
Î
~
Î
z
Î
z
Î
z
Î
~
_
~
_
~
_
~
_
~
_
~
_
z
_
Â
1
Â
1
Â
1
0
5
0
5
5
6
.CBS 673.69
.
CPC
11606
.
CBS
109082
.
CPC
11609
CBS
110024
.
.
CBS
157.82
.
CBS
161.55
.
CBS
572.78
.
CPC
11386
.
CPC
12042
.
CPC
12046
.
CPC
12211
.
CPC
12212
.
CPC
11840
.
CBS
188.54
.
CBS
366.80
.
CPC
12921
.
CBS
159.54
.
CBS
177.71
.
CBS
399.80
.
CBS
521.68
.
CPC
12139
.
CBS
115683
.
CPC12041
.
CPC
11839
.
CBS
134.31
.
CBS
813.71
.
CPC
12045
.
CBS
113753
.
CBS
113754
.
CPC
12044
.
CPC
12485
.
CBS
690.92
.
CBS
113742
.
CBS
113741
.
CBS
172.52
.
CBS
116463
.
CBS
102044
.
CPC
12040
.
CBS
289.49
.
CBS
842.91
.
CBS
843.91
.
CBS
113744
.
CPC
12484
.
CPC
12751
.
CPC
12753
CBS
107.20
.
.
CBS
138.40
.
CPC
11600
.
CPC
11601
.
CPC
11602
.
CPC
11604
.
CBS
111.82
.
CBS
300.49
.
CPC
11603
.
CBS
175.82
.
CBS
223.31
.
CBS
299.67
.
CPC
11817
.
CPC
12054
.
CPC
12752
.
CPC
12755
.
CPC
12756
.
CPC
12758
.
CPC
12759
.
CPC
12757
.
CPC
12177
.
CPC
12178
.
CPC
12179
.
CPC
12180
.
CPC
12181
.
CPC
12183
.
CPC
12052
.
CPC
11813
.
CPC
12051
.
CPC
12053
.
CPC
12043
.
CPC
12047
.
CBS
290.49
Cladosporium cladosporioides complex
Cladosporium cladosporioides complex
Cladosporium cladosporioides complex
Cladosporium cladosporioides complex
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium subinflatum
Cladosporium sinuosum
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium bruhnei
Cladosporium subtilissimum s.str.
Cladosporium subtilissimum s.str.
Cladosporium subtilissimum s.str.
Cladosporium subtilissimum s.lat.
Cladosporium antarcticum
Cladosporium subtilissimum s.lat.
Cladosporium subtilissimum s.lat.
Cladosporium subtilissimum s.lat.
Cladosporium pseudiridis
Cladosporium spinulosum
Cladosporium spinulosum
Davidiella sp.
Cladosporium ossifragi
Cladosporium ossifragi
Cladosporium subtilissimum s.lat.
Cladosporium subtilissimum s.lat.
Cladosporium variabile
Cladosporium variabile
Cladosporium iridis
Cladosporium iridis
Cladosporium herbarum
Cladosporium herbarum
Cladosporium herbarum
Cladosporium herbarum
Cladosporium herbarum
Cladosporium herbarum
Cladosporium herbarum
Cladosporium macrocarpum
Cladosporium macrocarpum
Cladosporium macrocarpum
Cladosporium macrocarpum
Cladosporium macrocarpum
Cladosporium macrocarpum
Cladosporium macrocarpum
Cladosporium macrocarpum
Cladosporium macrocarpum
Cladosporium macrocarpum
Cladosporium macrocarpum
Cladosporium herbarum
Cladosporium herbarum
Cladosporium herbarum
Cladosporium herbarum
Cladosporium herbarum
Cladosporium herbarum
Cladosporium herbaroides
Cladosporium tenellum
Cladosporium tenellum
Cladosporium tenellum
Cladosporium ramotenellum
Cladosporium ramotenellum
Davidiella sp.
Water
Wood
Human
Fungus
Tree
Hypersaline
Airco
Grass
Grass
Plant
?
Human
Tree
Human
Chemical
Human
Air
Grass
Treated pole
Hypersaline
Plant
?
Plant
Hypersaline
Cherry
Grape
Hypersaline
Wood
Lichen
Grape
Grape
Plant
Plant
Hypersaline
Hypersaline
Plant
Plant
Plant
Grape
Wood
Spinach
Spinach
Plant
Plant
Plant
Plant
Plant
Plant
Plant
Plant
Plant
Water
?
Straw
Branch
Hypersaline
Spinach
Spinach
Spinach
Spinach
Spinach
Spinach
Grass
Grass
Grass
Grass
Grass
Grass
Saltern
Fungus
Hypersaline
Hypersaline
Hypersaline
Airco
Plant
Germany
Belgium
Netherlands
Russia
Germany
Slovenia
Slovenia
Belgium
Belgium
Australia
?
Netherlands
Australia
Netherlands
Netherlands
Netherlands
Netherlands
Netherlands
USA
Slovenia
New Zealand
Germany
Czechia
Spain
USA .
USA
Slovenia
.
Argentina
Antarctica
USA
USA
USA
New Zealand
Slovenia
Slovenia
Switzerland
Norway
Norway
USA
Argentina
USA
USA
Netherlands
Netherlands
USA
USA
USA
USA
Switzerland
Switzerland
USA
Romania
?
Turkey
USA
Slovenia
USA
USA
USA
USA
USA
USA
Netherlands
Netherlands
Netherlands
Netherlands
Netherlands
Netherlands
Israel
USA
Slovenia
Slovenia
Slovenia
Slovenia
Switzerland
Fig. 4. Distance tree of the Cladosporium herbarum complex based on ACT sequence data generated with UPGMA, showing Structure analysis at K = 6 under admixture model
with correlated allele frequencies. Group indications (18) are taken from a tree based on EF sequences with AIC under the HKYG model.
112
Cladosporium herbarum SpecieS coMplex
B
A
C
D
Fig. 5. Split decomposition of the Cladosporium herbarum complex using SplitStree of 16–22 unique alleles obtained from 79 Cladosporium isolates for four loci. The star-like
structures suggest clonal development. A = ACT, B = CAL, C = HIS, D = EF. Scale bars = 0.01 nucleotide substitutions per site.
Taxonomy
Key to the Cladosporium species treated
Morphological features used in the key to distinguish the species treated in this study were determined after 7 d growth at 25 ºC on SNA using
light microscopy, and cultural characteristics after 14 d incubation on PDA.
1. Conidia usually smooth, rarely minutely verruculose .................................................................... C. cladosporioides (species complex)
1. Conidia with different surface ornamentation, minutely to distinctly verruculose, verrucose to echinulate or spiny
................................................................................................................................................................................................................... 2
2. Conidiophores uniform, macronematous; conidia solitary, sometimes formed in short unbranched chains ............................................. 3
2. Conidiophores both macronematous and micronematous; conidia always catenate, usually formed in branched chains
................................................................................................................................................................................................................... 5
www.studiesinmycology.org
113
Schubert et al.
3. Conidiophores due to geniculations often growing zigzag-like, (4–)5–7 µm wide; conidia 9–21 × (5–)6–8 µm, 0–1-septate;
conidiogenous loci and conidial hila 1.2–2(–2.2) µm diam ................................................................................................... C. sinuosum
3. Conidiophores not growing zigzag-like, wider, 6–11 µm; conidia very large and wide, 15–75(–87) × (7–)10–19(–21) µm, often with more
septa; conidiogenous loci and hila wider, (2–)2.5–4 µm diam .................................................................................................................. 4
4. Conidia (18–)30–75(–87) × (7–)10–16(–18) µm, (0–)2–6(–7)-septate, walls thickened, especially in older conidia, up to 1 µm thick
........................................................................................................................................................................................................ C. iridis
4. Conidia shorter and wider, 15–55 × (9–)11–19(–21) µm, 0–3-septate, walls distinctly thickened, up to 2 µm, usually appearing zonate
............................................................................................................................................................................................. C. pseudiridis
5(2) Macronematous conidiophores nodulose or nodose with conidiogenous loci usually conined to swellings .......................................... 6
5. Macronematous conidiophores non-nodulose or only occasionally subnodulose due to geniculate proliferation, but conidiogenous loci not
conined to swellings ............................................................................................................................................................................... 11
6. Macronematous conidiophores 3–6 µm wide, swellings 5–11 µm wide ................................................................................................... 7
6. Macronematous conidiophores somewhat narrower, (1.5–)2.5–5 µm wide, swellings 3–8 µm wide ....................................................... 8
7. Aerial mycelium twisted; conidial septa often distinctly darkened, becoming sinuous with age, apex and base of the conidia often appear
to be distinctly darkened; slower growing in culture (29 mm after 14 d on PDA) ..................................................................... C. variabile
7. Aerial mycelium not twisted; conidial septa as well as apex and base not distinctly darkened, septa not sinuous with age; faster growing
in culture (on average 38 mm after 14 d on PDA) .......................................................................................................... C. macrocarpum
8. Macronematous conidiophores (1.5–)2.5–4.5(–5.5) µm wide, swellings 3–6.5 µm wide; conidia 4–17(–22) µm long, ornamentation
variable, but usually densely echinulate, spines up to 0.8 µm long ................................................................................... C. subinlatum
8. Macronematous conidiophores slightly wider, 3–5 µm, swellings (4–)5–8(–9) µm wide; conidia longer, up to 25(–35) µm, ornamentation
minutely verruculose to verrucose, but not echinulate or spiny ................................................................................................................. 9
9. Conidia formed by macronematous conidiophores 3–33 × (2–)3–6(–7) µm, with age becoming wider, (3.5–)5–9(–11) µm, darker and
more thick-walled ............................................................................................................................................................... C. herbaroides
9. Conidia formed by macronematous conidiophores not becoming wider and darker with age, usually up to 7 µm wide
................................................................................................................................................................................................................. 10
10. Conidiophores usually with small head-like swellings, sometimes also with a second intercalary nodule; small terminal conidia
4–9 × 2.5–3.5 µm, secondary ramoconidia and occasionally formed ramoconidia 10–24(–31) × 3–5(–7) µm ........................ C. bruhnei
10. Conidiophores with a single or often numerous swellings in short succession giving the stalk a knotty/gnarled appearance;
conidia wider, small terminal conidia 4–10 × 3–5(–6) µm, intercalary conidia 6–16 × 4–6 µm, secondary ramoconidia 12–25(–35) ×
(3–)5–7(–9) µm ...................................................................................................................................................................... C. herbarum
11(5) Small terminal and intercalary conidia 4–15 × 3–5 µm, secondary ramoconidia 16–36(–40) × (4–)5–8 µm, 0–3(–4)-septate,
ramoconidia absent ................................................................................................................................................................. C. ossifragi
11. Small terminal conidia, ramoconidia and secondary ramoconidia distinctly narrower, 2–5(–6) µm wide, 0–2(–3)-septate
................................................................................................................................................................................................................. 12
12. Mycelium dimorphic, narrow hyphae 1–3 µm wide, hyaline to subhyaline, thin-walled, hyphae of the second type wider, 3.5–8(–9)
µm, pale to dark greyish olivaceous or olivaceous-brown, thick-walled, sometimes even two-layered, 1(–1.5) µm thick, hyphae
appearing consistently enveloped in polysaccharide-like material or covered by a slime coat; conidiophores usually several times slightly
to distinctly geniculate towards the apex, with numerous conidiogenous loci crowded towards the apex, up to 14 per conidiogenous cell
............................................................................................................................................................................................ C. antarcticum
12. Mycelium not dimorphic, neither enveloped in polysaccharide-like material nor covered by a slime coat; conidiophores usually not
geniculate, occasionally only slightly so .................................................................................................................................................. 13
13. Conidial ornamentation distinctly echinulate, spiny (baculate, digitate or capitate under SEM), spines 0.5–1.3 µm long, loose to moderately
dense, conidial hila usually situated on small peg-like prolongations or denticles ............................................................. C. spinulosum
13. Conidial ornamentation different, minutely verruculose to verruculose, conidial hila not situated on peg-like prolongations
................................................................................................................................................................................................................. 14
14. Small terminal conidia narrowly obovoid, limoniform or fusiform, but neither globose nor subglobose; conidiogenous loci and conidial hila
0.5–2(–2.5) µm diam ........................................................................................................................ C. subtilissimum (species complex)
14. Numerous small globose or subglobose terminal conidia formed, also ovoid or limoniform; conidiogenous loci and conidial hila somewhat
smaller, 0.5–1.5(–2) µm diam ................................................................................................................................................................. 15
114
Cladosporium herbarum SpecieS coMplex
15. Conidiophores usually with numerous conidiogenous loci forming sympodial clusters of pronounced scars at the apex, sometimes up to
10 or even more denticulate loci; conidia 3–20(–28) × 2.5–5(–6) µm, 0–1(–2)-septate, often with several apically crowded hila, up to 7(–9)
................................................................................................................................................................................................. C. tenellum
15. Conidiophores usually only with few conidiogenous loci, mostly 1–3; conidia longer and narrower, 2.5–35 × 2–4(–5) µm, 0–3-septate,
usually with up to three distal conidial hila ...................................................................................................................... C. ramotenellum
Key to the Davidiella species treated
1. Ascospores frequently wider than 7 µm when mounted in Shear’s solution or lactic acid, apical cell obtusely rounded
................................................................................................................................................................................................................... 2
1. Ascospores not wider than 7 µm when mounted in Shear’s solution or lactic acid, apical cell acutely rounded, ascospores (20–)25–27
(–30) × (5.5–)6–7 µm ................................................................................................................................................................. D. allicina
2. Pseudoparaphyses prominent; asci frequently >95 µm; ascospores (22–)23–26(–28) × (6–)6.5–7(–8) µm ..................... D. macrocarpa
2. Pseudoparaphyses mostly absent in older ascomata; asci <95 µm ......................................................................................................... 3
3. Ascospores (22–)26–30(–35) × (7–)7.5–8(–9) µm; asci wider than 18 µm ............................................................................. D. variabile
3. Ascospores (17–)20–23(–25) × (6–)7(–8) µm; asci not wider than 18 µm .............................................................................. D. tassiana
Generic concept of the teleomorph
The introduction of the teleomorph genus Davidiella was mainly
based on phylogenetic studies within the Mycosphaerellaceae
(Braun et al. 2003), where it could be demonstrated that
“Mycosphaerella” species with Cladosporium anamorphs formed
a sister clade to Mycosphaerella (Crous et al. 2000, 2001). Braun
et al. (2003) transferred ive species to Davidiella based on prior
established anamorph-teleomorph connections, though no details
were provided pertaining to morphological differences between
Davidiella and Mycosphaerella. Aptroot (2006) transferred several
additional species to Davidiella, and distinguished them from true
Mycosphaerella species by the presence of distinct, irregular cellular
inclusions (lumina) in their ascospores. Furthermore, Schoch et al.
(2006) placed Davidiella in a separate family (Davidiellaceae) in the
Capnodiales. During the course of the present study, several fresh
specimens of Davidiella spp. were collected or induced in culture,
making it possible to circumscribe the genus as follows:
Davidiella Crous & U. Braun, Mycol. Progr. 2: 8. 2003, emend.
Ascomata pseudothecial, black to red-brown, globose, inconspicuous and immersed beneath stomata to supericial, situated on
a reduced stroma, with 1(–3) short, periphysate ostiolar necks;
periphysoids frequently growing down into cavity; wall consisting of
3–6 layers of textura angularis. Asci fasciculate, short-stalked or not,
bitunicate, subsessile, obovoid to broadly ellipsoid or subcylindrical,
straight to slightly curved, 8-spored. Pseudoparaphyses frequently
present in mature ascomata, hyaline, septate, subcylindrical.
Ascospores bi- to multiseriate, hyaline, obovoid to ellipsoid-fusiform,
with irregular luminar inclusions, mostly thick-walled, straight to
slightly curved; frequently becoming brown and verruculose in
asci; at times covered in mucoid sheath. Cladosporium anamorph
usually produced in culture, but not in all taxa.
Type species: Davidiella tassiana (De Not.) Crous & U. Braun,
Mycol. Progr. 2: 8. 2003.
description of Cladosporium species
Based on morphological examinations (David 1997) and phylogenetic
studies employing DNA sequence data (Crous et al. 2000, 2001,
www.studiesinmycology.org
2007 – this volume, Braun et al. 2003), the generic concept of
the genus Cladosporium has been stabilised. Cladosporium is
conined to Davidiella (Davidiellaceae, Capnodiales) anamorphs
with coronate conidiogenous loci and conidial hila consisting of a
central convex dome and a raised periclinal rim.
Cladosporium antarcticum K. Schub., Crous & U. Braun, sp.
nov. MycoBank MB504573. Figs 6–8.
Etymology: Refers to Antarctica, where the fungus was collected.
Differt a Cladosporio licheniphilo conidiophoris saepe non-ramosis, frequentibus
geniculatis, angustioribus, (2–)3–4.5 µm, conidiis longioribus et angustioribus, 4–30
× 2.5–5 µm, 0–3-septatis, verruculosis vel verrucosis.
Mycelium immersed and supericial, dimorphic, branched, often
with short lateral outgrowths, narrow hyphae 1–3 µm wide,
hyaline to subhyaline, thin-walled, hyphae of the second type
wider, 3.5–8(–9) µm, pluriseptate, often somewhat constricted at
the septa, sometimes swollen, pale to dark greyish olivaceous or
olivaceous-brown, smooth or verruculose, thick-walled, sometimes
even two-layered (two distinct wall layers visible), 1(–1.5) µm thick,
hyphae appearing consistently enveloped in polysaccharide-like
material or covered by a slime coat. Conidiophores micronematous
and macronematous, solitary or in loose groups, arising from
plagiotropous or ascending hyphae, terminally or usually laterally.
Macronematous conidiophores erect to somewhat decumbent,
straight to somewhat lexuous or bent, cylindrical, once or several
times slightly to distinctly geniculate towards the apex due to
sympodial proliferation, unbranched or once branched, up to
120 µm long, 3–4.5 µm wide, sometimes slightly attenuated
towards the apex, pluriseptate, up to eight septa, occasionally
slightly constricted at the septa, pale to medium or even dark
olivaceous-brown or greyish brown, paler towards apices, smooth
to somewhat rough-walled, walls thickened but thinner-walled
towards apices, sometimes slightly swollen at the base, up to 6 µm
wide. Conidiogenous cells integrated, terminal and intercalary, once
or several times slightly to distinctly geniculate, 10–33 µm long,
proliferation sympodial, with several or numerous conidiogenous
loci, at irst terminal, later turning to one side of the stalk and
situated on small lateral shoulders, up to 14 per cell, protuberant,
denticulate, 1–1.5(–2) µm diam, thickened and darkened-refractive.
Micronematous conidiophores as short lateral, peg-like outgrowths
with a single apical scar or somewhat longer, occasionally once
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Schubert et al.
Fig. 6. Cladosporium antarcticum (CBS 690.92). Macro- and micronematous conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
geniculate with several conidiogenous loci at the apex, 2–22 ×
2–3 µm, pale greyish olivaceous, loci denticulate. Ramoconidia
occasionally occurring, cylindrical, up to 30 µm long, 4–5 µm
wide, 0–1-septate, concolorous with the tips of conidiophores, with
a broadly truncate, unthickened and not darkened base, without
dome and rim, 2.5 µm wide. Conidia catenate, in branched chains,
straight, small terminal conidia obovoid, limoniform or narrowly
ellipsoid, 4–14 × 2.5–4 µm [av. ± SD, 8.5 (± 3.3) × 3.5 (± 0.6)],
0(–1)-septate, secondary ramoconidia ellipsoid to cylindrical, often
with several or numerous conidial hila crowded at the distal end, up
to 12, 13–30 × 4–5 µm [av. ± SD, 20.1 (± 5.8) × 4.3 (± 0.5) µm],
0–3-septate, sometimes slightly constricted at the median septum,
pale olivaceous-brown or greyish brown, minutely verruculose to
verrucose (granulate under SEM), walls more or less thickened,
rounded or slightly attenuated towards apex and base, hila
protuberant, denticulate, 0.8–1.5(–2) µm diam, thickened and
darkened-refractive; microcyclic conidiogenesis occurring.
Cultural characteristics: Colonies on PDA attaining 9 mm diam after
14 d at 25 ºC, greenish olivaceous to grey-olivaceous, at the margin
becoming dull green, reverse with a pale olivaceous-grey centre
and a broad olivaceous-black margin, margin narrow, regular,
entire edge, white, feathery, aerial mycelium sparse but colonies
appearing felty, growth lat with somewhat elevated colony centre,
prominent exudates not formed, sporulation dense, covering almost
the whole colony. Colonies on MEA attaining 12 mm diam after 14
d at 25 ºC, olivaceous-grey to iron-grey, iron-grey reverse, velvety
to powdery, aerial mycelium sparse, sporulation profuse. Colonies
116
on OA attaining 4 mm after 14 d at 25 ºC, olivaceous-grey, aerial
mycelium sparse, diffuse, growth lat, without prominent exudates,
sporulating.
Specimen examined: Antarctica, King George, Arctowski, isolated from the lichen
Caloplaca regalis (Teloschistaceae), C. Möller, No. 32/12, 1991, CBS-H 19857,
holotype, isotype HAL 2024 F, culture ex-type CBS 690.92.
Substrate and distribution: On the lichen Caloplaca regalis;
Antarctica.
Notes: This is the second genuine lichenicolous species of the
genus Cladosporium. Cladosporium licheniphilum Heuchert & U.
Braun, occurring on apothecia of Pertusaria alpina in Russia, is
quite distinct from C. antarcticum by having subcylindrical or only
slightly geniculate-sinuous, wider conidiophores, 5–8 µm, with
numerous characteristic terminal branches and much shorter, 0–
1-septate, smooth conidia, 3.5–13 × 3–7 µm (Heuchert & Braun
2006). Cladosporium lichenicola Linds. was invalidly published and
C. arthoniae M.S. Christ. & D. Hawksw. as well as C. lichenum
Keissl. are to be excluded from the genus Cladosporium since
they do not possess the typical cladosporioid scar structure but
inconspicuous, unthickened conidiogenous loci and conidial hila
(Hawksworth 1979, Heuchert et al. 2005). The fungicolous species
C. uredinicola Speg. and the foliicolous species C. alneum Pass.
ex K. Schub. and C. psoraleae M.B. Ellis are morphologically
supericially similar. However, C. uredinicola, a widespread fungus
on rust fungi, downy mildews and powdery mildew fungi, differs
in having somewhat longer and wider, smooth conidia, 3–39 × 2–
6.5(–8) µm, and wider conidiogenous loci and conidial hila, 0.5–3
Cladosporium herbarum SpecieS coMplex
Fig. 7. Cladosporium antarcticum (CBS 690.92). A.
Overview of the growth pattern on SNA. Note the very
large bulbous cells formed at the base of different
conidiophores. Other conidiophores sprout from the
agar surface. B. Overview of conidiophores and conidia.
Note the large distance of the scars on the conidiophore
and the different stages of conidial formation on the
tips of other conidia. The long secondary ramoconidia
are also visible, and sparse aerial hyphae. C. Detail
of B with details of the ornamentation and scars. The
absence of ornamentation at the apical (spore-forming)
end of the secondary ramoconidium is clearly visible. D–
E. Tubular structures on coniophore (D) and secondary
ramoconidium (E). Scale bars: A–B = 10 μm, C–D = 5
µm, E = 2 µm.
Fig. 8. Cladosporium antarcticum (CBS 690.92). A–B. Macronematous conidiophores. C, G. Mycelium enveloped by a polysaccharide-like layer. D, F. Conidia. E. Micronematous
conidiophore. H. Ramoconidium with numerous distal scars. Scale bars = 10 µm.
µm (Heuchert et al. 2005); C. alneum, which causes leaf spots on
Alnus glutinosa, possesses longer and wider conidiophores, 25–
260 × (2–)3–7(–8.5) µm, and somewhat shorter, smooth conidia
(Schubert 2005, Schubert et al. 2006); and C. psoraleae, known
www.studiesinmycology.org
from Myanmar on Psoralea corylifolia, can easily be distinguished
from C. antarcticum by its smooth and wider conidia, 3.5–7 µm,
and wider conidiogenous loci and conidial hila, 1–3 µm diam (Ellis
1972, Schubert 2005).
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Schubert et al.
Fig. 9. Cladosporium bruhnei (CPC 12211). Macro- and micronematous conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
Cladosporium bruhnei Linder, Bull. Natl. Mus. Canada 97: 259.
1947. Figs 9–12.
≡ Hormodendrum hordei Bruhne, in W. Zopf, Beitr. Physiol. Morph. nied.
Org. 4: 1. 1894, non C. hordei Pass., 1887.
≡ Cladosporium herbarum (Pers.: Fr.) Link var. (δ) cerealium Sacc. f.
hordei (Bruhne) Ferraris, Flora Ital. Crypt., Pars I, Fungi, Fasc.13: 882.
1914.
≡ Cladosporium hordei (Bruhne) Pidopl., Gribnaja Flora Grubych Kormov:
268. 1953, nom. illeg., homonym, non C. hordei Pass., 1887.
Teleomorph: Davidiella allicina (Fr. : Fr.) Crous & Aptroot, in
Aptroot, Mycosphaerella and its anamorphs: 2. Conspectus of
Mycosphaerella. CBS Biodiversity Ser. 5: 30. 2006.
Basionym: Sphaeria allicina Fr., Kongl. Vetensk. Acad. Handl. 38:
247. 1817, sactioned by Fr., Syst. Mycol. 2: 437. 1823.
≡ Sphaerella allicina (Fr. : Fr.) Auersw., in Gonn. & Rabenh., Mycol.
Europaea 5–6: 19. 1869.
Ascomata pseudothecial, black, supericial, situated on a small
stroma, globose, up to 250 µm diam; ostioles periphysate, with
apical periphysoids present; wall consisting of 3–6 layers of
reddish brown textura angularis. Asci fasciculate, bitunicate,
subsessile, obovoid to broadly ellipsoid, straight to slightly curved,
8-spored, 65–90 × 16–25 µm; with pseudoparenchymatal cells
of the hamathecium persistent. Ascospores tri- to multiseriate,
overlapping, hyaline, with irregular lumina, thick-walled, straight
to slightly curved, fusoid-ellipsoidal with obtuse basal end, and
acutely rounded apical end, widest near the middle of the apical
cell, medianly 1-septate, not to slightly constricted at the septum,
(20–)25–27(–30) × (5.5–)6–7 µm.
Mycelium supericial, hyphae branched, 1.5–8 µm wide,
pluriseptate, broader hyphae usually slightly constricted at the
septa and somewhat swollen, hyaline to subhyaline, almost smooth
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to somewhat verruculose or irregularly rough-walled, sometimes
appearing to have a slime coat, walls unthickened. Conidiophores
macronematous, sometimes also micronematous, arising as lateral
or terminal branches from plagiotropous or ascending hyphae, erect,
straight to more or less lexuous, sometimes geniculate, nodulose,
usually with small head-like swellings, sometimes also with
intercalary nodules, sometimes swellings protruding and elongated
to one side, unbranched, occasionally branched, (7–)20–330 µm,
sometimes even longer, (2–)3–5 µm wide, swellings (4–)5–8 µm
wide, pluriseptate, not constricted at the septa, septa sometimes
not very conspicuous, subhyaline to pale brown or pale olivaceous,
smooth or somewhat verruculose, walls unthickened or almost so,
more thickened with age. Conidiogenous cells integrated, usually
terminal, cylindrical with a terminal head-like swelling, sometimes
with a second swelling, 15–40 µm long, proliferation sympodial,
with few conidiogenous loci conined to swellings, up to ive per
swelling, loci protuberant, conspicuous, 1–2 µm diam, thickened
and darkened-refractive. Conidia catenate, formed in branched
chains, straight to slightly curved, small terminal conidia subglobose,
ovoid to obovoid or somewhat limoniform, 4–9 × 2.5–3.5 µm [av. ±
SD, 6.5 (± 1.5) × 3.1 (± 0.5) µm], aseptate; secondary ramoconidia
and occasionally formed ramoconidia ellipsoid to subcylindrical
or cylindrical, 10–24(–31) × 3–5(–7) µm [av. ± SD, 16.1 (± 4.1)
× 4.1 (± 0.8) µm], rarely up to 40 µm long, 0–1(–3)-septate, very
rarely 5-septate, subhyaline to pale brown or pale olivaceous,
minutely verruculose to verrucose (mostly granulate with some
muricate projections under SEM), walls unthickened or almost so,
apex rounded or slightly attenuated towards apex and base, hila
protuberant, conspicuous, 1–2 µm wide, up to 1 µm high, thickened
and darkened-refractive; microcyclic conidiogenesis occurring.
Cladosporium herbarum SpecieS coMplex
Belgium, isolated from Quercus robur (Fagaceae), CBS 157.82; Kampenhout,
isolated from Hordeum vulgare (Poaceae), 26 June 2005, J.Z. Groenewald, CBS-H
19856, neotype designated here of C. bruhnei, isoneotype HAL 2023 F, cultures
ex-type CBS 121624 = CPC 12211, CPC 12212. Czech Republic, Lisen, isolated
from Polygonatum odoratum (Liliaceae), CBS 813.71, albino mutant of CBS 812.71.
Germany, CBS 134.31 = ATCC 11283 = IMI 049632; Nordrhein-Westfalen, Mühlheim
an der Ruhr, isolated from industrial water, IWW 727, CBS 110024; Sachsen-Anhalt,
Halle (Saale), Robert-Franz-Ring, isolated from leaves of Tilia cordata (Tiliaceae),
2004, K. Schubert, CPC 11386. netherlands, isolated from air, CBS 521.68; isolated
from Hordeum vulgare, 1 Jan. 2005, P.W. Crous, CPC 12139; isolated from man,
skin, CBS 159.54 = ATCC 36948; Amsterdam, isolated from Thuja tincture, CBS
177.71; Geleen, St. Barbara Ziekenhuis, isolated from man, skin, CBS 366.80, CBS
399.80; isolated from man, sputum, Aug. 1955, CBS 161.55. new Zealand, Otago,
Lake Harris, isolated from Ourisia macrophylla (Scrophulariaceae), 30 Jan. 2005,
A. Blouin, Hill 1135, CPC 11840. Russia, Moscow region, isolated from Polyporus
radiatus (Polyporaceae), Oct. 1978, CBS 572.78 = VKM F-405. slovenia, Ljubljana,
isolated from an air conditioning system, 2004, M. Butala, EXF-680 = CPC 12046;
Sečovlje, isolated from hypersaline water from salterns (reserve pond), 2005, P.
Zalar, EXF-389 = CPC 12042. spain, Ebro Delta, isolated from hypersaline water
from salterns (crystallisation pond), 2004, P. Zalar, EXF-594 = CPC 12045. sweden,
Skåne, on tip blight of living leaves of Allium sp. (Alliaceae), Fr. no. F-09810, UPSFRIES, holotype of Davidiella allicina. u.s.A., New York, Geneva, isolated from
CCA-treated Douglas-ir pole, CBS 115683 = ATCC 66670 = CPC 5101.
Substrate and distribution: Living and decaying plant material, man,
air, hypersaline and industrial water; widespread.
Literature: Saccardo (1899: 1076), Linder (1947: 289).
Fig. 10. Cladosporium bruhnei (CPC 12211). A. Conidiophore with characteristic
long secondary ramoconidium and complex conidiophore. B. Detail of hila on
secondary ramoconidia. C. Details of prominent ornamentation on conidia. Scale
bars: A = 10 µm, B = 2 µm, C = 5 µm.
Cultural characteristics: Colonies on PDA reaching 22–32 mm diam
after 14 d at 25 ºC, olivaceous-grey to iron-grey, sometimes whitish,
smoke-grey to pale olivaceous due to abundant aerial mycelium
covering almost the whole colony, with age collapsing becoming
olivaceous-grey, occasionally zonate, velvety to loccose, margin
narrow, entire edge, white, glabrous to somewhat feathery, aerial
mycelium sparse to abundant, white, luffy, growth regular, lat to
low convex, sometimes forming few exudates in the colony centre,
sporulating. Colonies on MEA reaching 21–32 mm diam after 14
d at 25 ºC, grey-olivaceous, olivaceous-grey to dull green or irongrey, sometimes whitish to pale smoke-grey due to abundant aerial
mycelium, olivaceous-grey to iron-grey reverse, velvety, margin
narrow, entire edge to slightly undulate, white, radially furrowed,
glabrous to slightly feathery, aerial mycelium sparse to abundant,
mainly in the centre, white, luffy, growth convex to raised, radially
furrowed, distinctly wrinkled in the colony centre, without prominent
exudates, sporulating. Colonies on OA reaching 20–32 mm diam
after 14 d at 25 ºC, smoke-grey, grey-olivaceous to olivaceous-grey,
greenish black or iron-grey reverse, margin narrow, entire edge,
colourless to white, glabrous, aerial mycelium sparse to abundant,
dark smoke-grey, diffuse, high, later collapsed, felty, growth lat,
without prominent exudates, sporulation profuse.
Specimens examined: Sine loco et dato, CBS 188.54 = ATCC 11290 = IMI
049638. Australia, N.S.W., Barrington Tops National Park, isolated from leaves
of Eucalyptus stellulata (Myrtaceae), 3 Jan. 2006, B. Summerell, CPC 12921.
www.studiesinmycology.org
Fig. 11. Davidiella allicina (F-09810, UPS-FRIES, holotype). Ascus and ascospores.
Scale bar = 10 µm. P.W. Crous del.
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Fig. 12. Cladosporium bruhnei (CPC 12211) and its teleomorph Davidiella allicina. A–B. Macronematous conidiophores. C. Conidial chains. D. Micronematous conidiophore. E.
Ascomata of the teleomorph formed on the host. F–G. Asci. Scale bars: A–B, D, F = 10 µm, E = 200 µm.
Notes: Cladosporium bruhnei proved to be an additional component
of the herbarum complex. The species resembles C. herbarum s.
str. as already stated by Linder (1947), but possesses consistently
narrower conidia, usually 2.5–5 µm wide, and the conidiophores
often form only a single apical swelling. The species was described
by Bruhne (l.c.) as Hormodendrum hordei from Germany but type
material could not be located. Linder (1947) examined No. 1481a-5
(Canada, N. Quebec, Sugluk, on Elymus arenarius var. villosus,
31 Jul. 1936, E. Meyer), presumably in the National Museum, and
stated that this specimen agreed well with the description and
illustration given by Bruhne (l.c.). Although the species occurs
on numerous substrates and is widely distributed, it has not yet
been recognised as a distinct species since it has probably been
interpreted as a narrow variant of C. herbarum.
Based on morphology and DNA sequence data, the CBS strain
CBS 177.71 chosen by Prasil & de Hoog (1988) as representative
living strain of C. herbarum, rather clusters together with isolates
of C. bruhnei. The strain CBS 813.71 is an albino mutant of the
latter species as it does not appear to contain colour pigment.
Furthermore, all isolates from humans treated until now as C.
herbarum proved to be conspeciic with the narrow-spored C.
bruhnei.
Although Davidiella tassiana (ascospores 17–25 × 6–8.5 µm,
RO) was treated as synonymous to D. allicina (ascospores 20–27
× 6–7 µm, UPS) in Aptroot (2006), they differ in apical ascospore
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taper, with ascospores of D. allicina being acutely rounded, while
those of D. tassiana are obtusely rounded. The same ascospore
taper was also observed in the teleomorph of C. bruhnei, and thus
the name D. allicina is herewith linked to C. bruhnei, which is distinct
from C. herbarum, having D. tassiana as teleomorph.
Cladosporium herbaroides K. Schub., Zalar, Crous & U. Braun,
sp. nov. MycoBank MB504574. Figs 13–15.
Etymology: Refers to its morphological similarity to Cladosporium
herbarum.
Differt a Cladosporio herbaro conidiis polymorphis, 3–33 × (2–)3–6(–7) µm,
postremo latioribus, (3.5–)5–9(–11) µm, fuscis et crassitunicatis; et a Cladosporio
macrocarpo conidiophoris leniter angustioribus, 3–5 µm latis, nodulis angustioribus,
5–8 µm latis.
Mycelium branched, (1–)2–8 µm wide, septate, often with small
swellings and constrictions, subhyaline to pale brown or pale
olivaceous-brown, smooth or almost so to somewhat verruculose,
walls unthickened or almost so. Conidiophores macronematous
and micronematous, arising lateral from plagiotropous hyphae or
terminally from ascending hyphae. Macronematous conidiophores
erect, straight to slightly lexuous, often geniculate, nodulose, with
unilateral or multilateral swellings, often numerous swellings in
short succession giving them a gnarled appearance, often forming
somewhat protruding or prolonged lateral swellings or a branch-
Cladosporium herbarum SpecieS coMplex
Fig. 13. Cladosporium herbaroides (CPC 12052). Macro- and micronematous conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
like prolongation below the terminal swelling (due to sympodial
proliferation), unbranched or sometimes branched, 30–230 µm
long or even longer, 3–5 µm wide, swellings 5–8 µm wide, septate,
not constricted at septa, pale to medium olivaceous-brown,
smooth or almost so, walls slightly thickened. Conidiogenous cells
integrated, terminal or intercalary, cylindrical, usually nodulose to
nodose forming distinct swellings, sometimes geniculate, 15–55
µm long, with numerous conidiogenous loci usually conined to
swellings or situated on small lateral shoulders, sometimes on the
top of short peg-like prolongations or denticles, loci protuberant,
1–2 µm diam, thickened and darkened-refractive. Micronematous
conidiophores much shorter, narrower, paler, neither nodulose nor
geniculate, arising laterally from plagiotropous hyphae, often only
as short lateral denticles or branchlets of hyphae, erect, straight,
conical to cylindrical, unbranched, 3–65 × 2–3 µm, mostly aseptate,
sometimes up to ive septa, subhyaline, smooth, walls unthickened.
Conidiogenous cells integrated, terminal or conidiophores reduced
to conidiogenous cells, conidiogenous loci solitary or sometimes
as sympodial clusters of pronounced denticles, protuberant, 1–1.5
µm diam, thickened and somewhat darkened-refractive. Conidia
polymorphous, two main morphological types recognisable, formed
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by the two different types of conidiophores, conidia formed by
macronematous conidiophores catenate, in branched chains,
straight to slightly curved, subglobose, obovoid, limoniform, ellipsoid
to cylindrical, 3–33 × (2–)3–6(–7) µm [av. ± SD, 14.5 (± 7.9) ×
5.2 (± 1.2) µm], 0–2(–3)-septate, sometimes slightly constricted at
septa, septa median or somewhat in the lower half, pale to medium
olivaceous-brown, verruculose to verrucose (granulate under
SEM), walls slightly thickened, with up to three rarely four distal
scars, with age becoming medium or even dark brown (chocolate
brown), wider and more thick-walled, 5.5–33 × (3.5–)5–9(–11) µm
[av. ± SD, 14.4 (± 6.9) × 7.2 (± 1.9) µm], walls up to 1 µm thick,
hila protuberant, 0.8–2(–2.5) µm diam, thickened and darkenedrefractive; microcyclic conidiogenesis occurring. Conidia formed by
micronematous conidiophores paler and narrower, mostly formed
in unbranched chains, sometimes in branched chains with up to
three distal hila, straight to slightly curved, limoniform, narrowly
fusiform, almost iliform to subcylindrical, 10–26(–35) × 2–3.5
µm [av. ± SD, 15.6 (± 6.2) × 2.9 (± 0.5) µm], 0–1(–3)-septate,
subhyaline to pale brown, almost smooth to minutely verruculose,
walls unthickened, hila protuberant, 1–1.5 µm diam, thickened and
somewhat darkened-refractive.
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Schubert et al.
Fig. 14. Cladosporium herbaroides (CPC 12052). A–B, D. Macronematous conidiophores. C. Conidial chain. E. Micronematous conidiophore. F. Microcyclic conidiogenesis. G.
Conidia formed by micronematous conidiophores. Scale bars = 10 µm.
Cultural characteristics: Colonies on PDA attaining 23 mm diam
after 14 d at 25 ºC, grey-olivaceous to olivaceous, olivaceous-grey
reverse, velvety, margin regular, entire edge, narrow, feathery, aerial
mycelium abundantly formed, loose, with age covering large parts
of the colony, woolly, growth lat with somewhat elevated colony
centre, folded, regular, deep into the agar, with few prominent
exudates, sporulation profuse. Colonies on MEA attaining 24 mm
diam after 14 d at 25 ºC, grey- to greenish olivaceous, olivaceousgrey or iron-grey reverse, velvety to powdery, margin narrow,
colourless, entire edge, somewhat feathery, aerial mycelium pale
olivaceous-grey, sparse, growth convex, radially furrowed, folded
in the colony centre, without prominent exudates, sporulating.
Colonies on OA attaining 23 mm diam after 14 d at 25 ºC, greyolivaceous, margin more or less regular, entire edge, colourless,
somewhat feathery, aerial mycelium whitish to smoke grey, at irst
sparse, later more abundantly formed, growth lat, without exudates,
sporulation profuse.
Specimen examined: Israel, from hypersaline water of Eilat salterns, 2004, coll.
N. Gunde-Cimerman, isol. M. Ota, CBS-H 19858, holotype, isotype HAL 2025 F,
culture ex-type CBS 121626 = EXF-1733 = CPC 12052.
Substrate and distribution: Hypersaline water; Israel.
Notes: Cladosporium herbaroides is morphologically similar to C.
herbarum but differs in having somewhat longer conidia becoming
wider, darker and even more thick-walled with age [at irst conidia
3–33 × (2–)3–6(–7) µm, with age (3.5–)5–9(–11) µm wide]. Besides
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that, the species often produces a second conidial type formed on
micronematous conidiophores, giving rise to unbranched conidial
chains which are almost iliform, limoniform, narrowly fusiform to
subcylindrical, much narrower and paler than the ones formed by
macronematous conidiophores, 10–26(–35) × 2–3.5 µm. In C.
herbarum, conidia formed by micronematous conidiophores do not
occur as frequently as in C. herbaroides, and differ in being often
clavate and somewhat wider, up to 4(–5) µm wide. Cladosporium
macrocarpum is easily distinguishable by having somewhat wider
conidiophores (3–)4–6 µm, with distinctly wider swellings, 5–10 µm
wide, and the conidia are usually (3–)5–9(–10) µm wide.
Cladosporium herbarum (Pers. : Fr.) Link, Ges. Naturf. Freunde
Berlin Mag. Neuesten Entdeck. Gesammten Naturk. 7: 37. 1816:
Fr., Syst. mycol. 3(2): 370. 1832. Figs 16–19.
Basionym: Dematium herbarum Pers., Ann. Bot. (Usteri) 11: 32.
1794: Fr., Syst. mycol. 3(2): 370. 1832.
= Dematium epiphyllum var. (β) chionanthi Pers., Mycol. eur. 1: 16. 1822, syn.
nov.
For additional synonyms see Dugan et al. (2004), Schubert
(2005).
Teleomorph: Davidiella tassiana (De Not.) Crous & U. Braun,
Mycol. Progr. 2: 8. 2003.
Basionym: Sphaerella tassiana De Not., Sferiacei Italici 1: 87.
1863.
≡ Mycosphaerella tassiana (De Not.) Johanson, Öfvers. Förh. Kongl.
Svenska Vetensk.-Akad. 41: 167. 1884.
Cladosporium herbarum SpecieS coMplex
Fig. 15. Cladosporium herbaroides (CPC 12052). A. Overview of the growth characteristics of this fungus. Broad hyphae run over the surface of the agar, and possibly give rise
to conidiophore branches. The conidiophores of this fungus can be rather long, resembling aerial hyphae. Clusters of conidia are clearly visible in this micrograph. B. The very
wide surface hyphae can anastomose. C. Conidiophore with secondary ramoconidia and conidia. Note the variation in scar size. D. A very elaborate, complex conidiophore with
different scars of variable size, one being more than 2 μm wide! E. Details of secondary ramoconidia and hila. Note the rather strong ornamentation in which smaller “particles”
are between larger ones. F. Three conidia in a row. Note the scar formation in the chain and the reduction of the size of the cells throughout the spore-chain. The inset shows
the resemblance of the scars on a conidiophore and on a secondary ramoconidium. Scale bars: A = 50 µm, B–C, F (inset) = 10 µm, D–E = 5 µm, F = 2 µm.
Ascomata pseudothecial, black, globose, erumpent to supericial,
up to 200 µm diam, with 1(–3) short, periphysate ostiolar necks;
wall consisting of 3–6 layers of medium red-brown textura
angularis. Asci fasciculate, bitunicate, subsessile, obovoid to
broadly ellipsoid, straight to slightly curved, 8-spored, 65–85 × 13–
17 µm. Pseudoparaphyses absent in host material, but remnants
observed when studied in culture, hyaline, septate, subcylindrical,
anastomosing, 3–4 µm wide. Ascospores tri- to multiseriate,
overlapping, hyaline, with irregular luminar inclusions, thickwalled, straight to slightly curved, fusoid-ellipsoidal with obtuse
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ends, widest near middle of apical cell, medianly 1-septate, not to
slightly constricted at the septum, tapering towards both ends, but
more prominently towards the lower end, (17–)20–23(–25) × (6–)
7(–8) µm; becoming brown and verruculose in asci. Ascospores
germinating after 24 h on MEA from both ends, with spore body
becoming prominently constricted at the septum, but not distorting,
up to 7 µm wide, hyaline to pale brown and appearing somewhat
verruculose, enclosed in a mucoid sheath, with germ tubes being
irregular, somewhat nodular.
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Schubert et al.
almost smooth to minutely verruculose or irregularly rough-walled,
sometimes forming clavate conidia, up to 33 µm long, 0–2-septate.
Conidiogenous cells integrated, terminal or conidiophores reduced
to conidiogenous cells, narrowly cylindrical or iliform, with a single
or two loci. Conidia catenate, in unbranched or loosely branched
chains with branching mostly occurring in the lower part of the chain,
straight to slightly curved, small terminal conidia without distal hilum
obovoid, 4–10 × 3–5(–6) µm [av. ± SD, 7.8 (± 1.9) × 4.7 (± 0.9) µm],
aseptate, intercalary conidia with a single or sometimes up to three
distal hila limoniform, ellipsoid to subcylindrical, 6–16 × 4–6 µm
[av. ± SD, 12.4 (± 1.6) × 5.3 (± 0.6) µm], 0–1-septate, secondary
ramoconidia with up to four distal hila, ellipsoid to cylindrical-oblong,
12–25(–35) × (3–)5–7(–9) µm [av. ± SD, 18.8 (± 4.5) × 6.2 (± 0.9)
µm], 0–1(–2)-septate, rarely with up to three septa, sometimes
distinctly constricted at the septum, septum median or somewhat
in the upper or lower half, pale greyish brown or brown to medium
brown or greyish brown, minutely verruculose to verrucose, walls
slightly to distinctly thickened, guttulate to somewhat granular,
usually only slightly attenuated towards apex and base, apex
obtuse or slightly truncate, towards the base sometimes distinctly
attenuated with hila situated on short stalk-like prolongations, hila
slightly to distinctly protuberant, truncate to slightly convex, (0.8–)
1–2.5(–3) µm wide, 0.5–1 µm high, somewhat thickened and
darkened-refractive; microcyclic conidiogenesis occurring, conidia
forming micro- and macronematous secondary conidiophores.
Fig. 16. Davidiella tassiana (RO, holotype). Ascus and ascospores. Scale bar = 10
µm. P.W. Crous del.
Mycelium supericial, loosely branched, (0.5–)1–5 µm wide, septate,
sometimes constricted at septa, hyaline, subhyaline to pale brown,
smooth or almost so to verruculose or irregularly rough-walled,
sometimes appearing irregular in outline due to small swellings
and constrictions, walls unthickened to somewhat thickened, cell
lumen appearing to be granular. Conidiophores both macro- and
micronematous, arising laterally from plagiotropous hyphae or
terminally from ascending hyphae. Macronematous conidiophores
erect, straight to lexuous, somewhat geniculate-sinuous, nodulose
to nodose with unilateral or multilateral swellings, with a single to
numerous swellings in short succession giving the stalk a knotty/
gnarled appearance, unbranched or occasionally branched, up to
three times, sometimes with a lateral branch-like proliferation below
or at the apex, 10–320 × 3.5–5 µm, swellings 5–8(–9) µm wide,
pluriseptate, septa sometimes constricted when formed after a
node, pale to medium brown, older ones almost dark brown, paler
towards the apex, smooth or minutely verruculose, walls thickened,
sometimes even two-layered. Conidiogenous cells integrated,
terminal or intercalary, nodulose to nodose, with a single or up
to ive swellings per cell, 10–24 µm long, proliferation sympodial,
with several conidiogenous loci conined to swellings, mostly
situated on small lateral shoulders, more or less protuberant,
broadly truncate to slightly convex, 1.5–2.5 µm diam, thickened
and somewhat darkened-refractive. Micronematous conidiophores
hardly distinguishable from hyphae, sometimes only as short lateral
outgrowth with a single apical scar, short, conical to almost iliform
or narrowly cylindrical, non-nodulose, not geniculate, unbranched,
5–120 × 1.5–3(–4) µm, pluriseptate, not constricted at septa,
cells usually very short, 5–15 µm long, subhyaline to pale brown,
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Cultural characteristics: Colonies on PDA reaching 19–37 mm diam
after 14 d at 25 ºC, grey-olivaceous to olivaceous-grey, whitish
to smoke-grey or pale olivaceous-grey due to abundant aerial
mycelium, velvety, reverse olivaceous-grey or iron-grey, margin
almost colourless, regular, entire edge, glabrous to feathery,
aerial mycelium abundant mainly in the colony centre, dense,
felty, woolly, sometimes becoming somewhat reddish brown,
fawn coloured, growth regular, lat to low convex with an elevated
colony centre, sometimes forming few large prominent exudates,
sporulation profuse. Colonies on MEA reaching 17–37 mm diam
after 14 d at 25 ºC, smoke-grey to pale olivaceous-grey towards
margin, olivaceous-grey to iron-grey reverse, velvety, margin white,
entire edge to slightly undulate, aerial mycelium abundant, dense,
luffy to felty, growth low convex or raised, radially furrowed, folded
and wrinkled in the colony centre, without prominent exudates but
sporulating. Colonies on OA reaching 12–28 mm diam after 14
d at 25 ºC, olivaceous-grey to iron-grey, due to abundant aerial
mycelium pale olivaceous-grey, olivaceous-grey reverse, margin
narrow, more or less undulate, white, aerial mycelium white, loose to
dense, high, luffy to felty, covering large parts of the colony, growth
lat to low convex, without prominent exudates, sporulating.
Specimens examined: Sine loco, sine dato, L 910.225-733, lectotype of C.
herbarum, selected by Prasil & de Hoog, 1988. Sine loco, on leaves of Chionanthus
sp. (Oleaceae), L 910.255-872 = L-0115833, holotype of Dematium epiphyllum
var. (β) chionanthi. netherlands, Wageningen, isolated from Hordeum vulgare
(Poaceae), 2005, P.W. Crous, CBS-H 19853, epitype designated here of C.
herbarum and D. tassiana, isoepitype HAL 2022 F, ex-type cultures, CPC 12177 =
CBS 121621, CPC 12178–12179, 12181, 12183. Italy, on upper and lower surface
of dead leaves of Carex nigra [“fusca”] (Cyperaceae), Tassi no. 862, RO, holotype
of Davidiella tassiana. u.s.A., Colorado, San Juan Co., above Little Molas Lake,
isolated from stems of Delphinium barbeyi (Ranunculaceae), 12 Sep. 2004, A.
Ramaley, CBS-H 19868 (teleomorph), single ascospore isolates, CBS 121622 =
CPC 11600, CPC 11601–11604.
Substrate and distribution: On fading and decaying plant material,
on living leaves (phylloplane fungus), as secondary invader, as an
endophyte, isolated from air, soil, foodstuffs, paints, textiles and
numerous other materials; cosmopolitan.
Cladosporium herbarum SpecieS coMplex
Fig. 17. Cladosporium herbarum (CPC 11600). Macro- and micronematous conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
Literature: de Vries (1952: 71), Hughes (1958: 750), Ellis (1971:
313), Domsch et al. (1980: 204), Sivanesan (1984: 225), Ellis &
Ellis (1985: 290, 468, 1988: 168), Prasil & de Hoog (1988), Wang
& Zabel (1990: 202), McKemy & Morgan-Jones (1991), Dugan &
Roberts (1994), David (1997: 59), Ho et al. (1999: 129), de Hoog et
al. (2000: 587), Samson et al. (2000: 110), Samson et al. (2001).
Notes: De Vries (1952) incorrectly selected a specimen of Link’s
herbarium at herb. B as lectotype. Prasil & de Hoog (1988) discussed
this typiication and designated one of Persoon’s original specimens
as lectotype in which C. herbarum could be recognised. The latter
material, which is in poor condition, could be re-examined within
the course of these investigations and showed conidia agreeing
with the current species concept of C. herbarum being (6–)9.5–
14.5(–21) × (5–)6–7(–8) µm. Since the identity of the strain CBS
177.71 chosen by Prasil & de Hoog (1988) as representative living
strain of C. herbarum could not be corroborated, an epitype with
a living ex-epitype culture is designated. The holotype specimen
of D. tassiana (RO) is morphologically similar to that observed on
the epitype of C. herbarum, having ascospores which are (17–)21–
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23(–25) × (6–)7–8(–8.5) µm, turning brown and verruculose in asci
with age. However, no hamathecial remnants were observed in
ascomata in vivo.
The connection to the teleomorph D. tassiana could be
conirmed, which is in agreement with the indings of von Arx (1950)
and Barr (1958). Ascospore isolates formed the typical C. herbarum
anamorph in culture, and these anamorph cultures developed
some immature fruiting bodies within the agar. When inoculated
onto water agar plates with nettle stems, numerous ascomata with
viable ascospores were formed in culture.
Cladosporium iridis (Fautrey & Roum.) G.A. de Vries, Contr.
Knowl. Genus Cladosporium: 49. 1952. Figs 20–21.
Basionym: Scolicotrichum iridis Fautrey & Roum., Rev. Mycol.
(Toulouse) 13: 82. 1891.
≡ Heterosporium iridis (Fautrey & Roum.) J.E. Jacques, Contr. Inst. Bot.
Univ. Montréal 39: 18. 1941.
For additional synonyms see Dugan et al. (2004).
Teleomorph: Davidiella macrospora (Kleb.) Crous & U. Braun,
Mycol. Progr. 2: 10. 2003.
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Fig. 18. Cladosporium herbarum (CPC 11600) and its teleomorph Davidiella tassiana (from the host and CPC 12181). A–B. Macronematous conidiophores. C. Micronematous
conidiophore. D. Microcyclic conidiogenesis. E. Conidial chain. F. Ascomata on the leaf. G. Ascomata formed in culture on nettle stems. H–I. Asci on the host. J–K. Ascospores
in culture. L. Asci in culture. Scale bars: A, E, H, J–L = 10 µm, F–G, I = 200 µm.
Basionym: Didymellina macrospora Kleb., Ber. Deutsch. Bot. Ges.
42: 60, 1924. 1925.
≡ Mycosphaerella macrospora (Kleb.) Jørst., Meld. Stat. Plantepatol. Inst.
1: 20. 1945.
Mycelium branched, 2–8 µm wide, septate, not constricted at the
septa, hyaline to pale brown, smooth, walls slightly thickened,
sometimes guttulate. Conidiophores very long, usually terminally
arising from ascending hyphae, erect to subdecumbent, slightly
to distinctly lexuous, geniculate-sinuous, usually several times,
subnodulose due to geniculate, sympodial proliferation forming
swollen lateral shoulders, unbranched, rarely branched, up to
720 µm long, 6–11 µm wide, swellings 8–11(–14) µm wide,
pluriseptate, often very regularly septate, not constricted at
the septa, pale to medium olivaceous-brown, somewhat paler
towards the apex, smooth to minutely verruculose, walls only
slightly thickened. Conidiogenous cells integrated, terminal as
well as intercalary, cylindrical-oblong, 15–55 µm long, proliferation
percurrent to sympodial, usually with a single geniculation forming
laterally swollen shoulders often below a septum, conidiogenous
loci conined to swellings, usually one locus per swelling, rarely
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two, protuberant, (2–)2.5–4 µm diam, somewhat thickened
and darkened-refractive. Conidia solitary, sometimes in short,
unbranched chains, straight to curved, young conidia pyriform to
subcylindrical, connection between conidiophore and conidium
being rather broad, subhyaline to pale olivaceous-brown, walls
slightly thickened, then enlarging and becoming more thick-walled,
cylindrical-oblong, soleiform with age, both ends rounded, usually
with a slightly to distinctly bulbous base, visible from a very early
stage, but broadest part often towards the apex not at the base, (18–)
30–75(–87) × (7–)10–16(–18) µm [av. ± SD, 53.3 (± 17.8) × 12.6 (±
2.2) µm], (0–)2–6(–7)-septate, usually not constricted at the septa,
rarely slightly constricted, septa often becoming sinuous with age,
pale to medium olivaceous-brown, sometimes darker, verrucose to
echinulate, walls thickened, especially in older conidia, up to 1 µm
thick, hila protuberant, often stalk-like or conically prolonged, up
to 2 µm long, (2–)2.5–3.5(–4) µm diam, with age becoming more
sessile, sometimes just visible as a thickened plate just below the
outer wall layer, especially in distal scars of branched conidia,
periclinal rim often distinctly visible, hila somewhat thickened and
darkened-refractive; microcyclic conidiogenesis not observed.
Cladosporium herbarum SpecieS coMplex
Fig. 19. Cladosporium herbarum (CPC 11600). A. Overview of hyphal growth and conidiophore formation of a colony on SNA. Conidiophores are often formed on very wide
(approx. 10 μm), septate hyphae that often grow near the agar surface. B. A more detailed view on colony organisation reveals the ornamented conidia. Note the septum near the
conidiophore (arrow). C. Detail of spore ornamentation and hila on a secondary ramoconidium (arrow). Ornamentation is visible during early stages of spore formation (arrow).
D. Structure of the conidiophore, illustrating the complex morphology of the spore-forming apparatus. In addition, secondary ramoconidia, conidia, and a hilum on the conidium
are visible. E. Complex structure of the spore-forming apparatus. F. Details of secondary ramoconidia with complex scar-pattern on the right cell. G. Details of a secondary
ramoconidium giving rise to conidia. Note the lack of ornamentation at the location of spore formation. Scale bars: A = 50 µm, B, F = 10 µm, C–E, G = 5 µm.
Cultural characteristics: Colonies on PDA reaching 19–23 mm
diam after 14 d at 25 ºC, pale greenish olivaceous, smoke-grey
to olivaceous-grey due to abundant aerial mycelium, greenish
olivaceous to olivaceous reverse, margin broad, regular, entire
edge to slightly undulate, feathery, aerial mycelium abundantly
formed, felty, luffy, covering large parts of the colony, mainly in
the central parts, high, growth low convex with a somewhat raised
colony centre. Colonies on MEA reaching 9–23 mm diam after 14 d
at 25 ºC, pale olivaceous-grey to olivaceous-grey, olivaceous-grey
reverse, felty, margin slightly undulate, white, somewhat raised,
aerial mycelium abundant, loose, diffuse, high, growth low convex,
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radially furrowed, slightly folded. Colonies on OA reaching 10–19
mm diam after 14 d at 25 ºC, olivaceous, margin broad, undulate,
white, aerial mycelium white, very high, loose, diffuse, hairy, growth
lat, due to the mycelium low convex, without prominent exudates
and sporulating on all media.
Specimens examined: Isolated from Iris sp. (Iridaceae), CBS 107.20. France,
Cote d’Or, Jardin de Noidan, on leaves of Iris germanica, Jul. 1880, F. Fautrey,
Roumeguère, Fungi Sel. Gall. Exs. No. 5689, PC, lectotype of C. iridis, selected
by David, 1997; K, isolectotype. netherlands, Boterenbrood, isolated from leaves
of Iris sp., Aug. 1940, CBS-H 19859, epitype designated here of C. iridis, culture
ex-epitype CBS 138.40.
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Schubert et al.
Fig. 20. Cladosporium iridis (CBS 138.40). Conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
Fig. 21. Cladosporium iridis (teleomorph Davidiella macrospora) (CBS 138.40). A–C. Conidiophores with conidia. D. Conidium. Scale bar = 10 µm.
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Cladosporium herbarum SpecieS coMplex
Fig. 22. Cladosporium macrocarpum (CBS 299.67). Macro- and micronematous conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
Substrates and distribution: Leaf spot and blotch of Iris spp.
including I. crocea, I. lorentina, I. foetidissima, I. germanica, I.
gueldenstaedtiana, I. kamaonensis, I. pallida, I. plicata (= I. swertii
Hort.), I. pseudacorus, I. pumila, I. spuria ssp. halophila, and other
species, also on Belacamanda chinensis (= Gemmingia chinensis),
Hemerocallis fulva, Gladiolus gandavensis; Africa (Algeria,
Morocco, South Africa, Zambia, Zimbabwe), Asia (Armenia,
Azerbaijan, China, Georgia, India, Iran, Israel, Japan, Kazakhstan,
Kirgizstan, Korea, Russia, Turkey, Turkmenistan, Uzbekistan),
Australasia (Australia, New Zealand), Europe (Austria, Belgium,
Belorussia, Cyprus, Czech Republic, Denmark, Estonia, France,
Germany, Great Britain, Greece, Italy, Latvia, Lithuania, Malta,
Moldavia, Montenegro, Netherlands, Norway, Poland, Romania,
Russia, Serbia, Spain, Sweden, Ukraine), North America (Canada,
U.S.A.), Central & South America (Argentina, Chile, Jamaica,
Panama, Uruguay).
Literature: Ellis (1971: 312), Ellis & Waller (1974), Sivanesan (1984:
222), McKemy & Morgan-Jones (1990), David (1997: 43), Shin et
al. (1999).
Notes: The description of the morphological parameters in culture
is based on the isolate sporulating on PDA, since sporulation on
SNA was not observed. The conidiophores and conidia in vivo are
usually wider than in culture [conidiophores (6–)9–15(–17) µm
wide, conidia (11–)15–23(–28) µm].
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Cladosporium macrocarpum Preuss, in Sturm, Deutsch. Fl.
3(26): 27. 1848. Figs 22–25.
≡ Cladosporium herbarum var. macrocarpum (Preuss) M.H.M. Ho &
Dugan, in Ho et al., Mycotaxon 72: 131. 1999.
= Dematium herbarum var. (β) brassicae Pers., Syn. meth. fung. 2: 699. 1801,
syn. nov.
= Dematium graminum Pers., Mycol. eur. 1: 16. 1822, syn. nov.
= Dematium vulgare var. (δ) typharum Pers., Mycol. eur. 1: 14. 1822, syn.
nov.
= Dematium vulgare var. (β) foliorum Pers., Mycol. eur. 1: 14. 1822, syn. nov.
For additional synonyms see Dugan et al. (2004), Schubert
(2005).
Teleomorph: Davidiella macrocarpa Crous, K. Schub. & U. Braun,
sp. nov. MycoBank MB504582.
Davidiellae tassianae similis, sed pseudoparaphysibus prominentibus et ascosporis
maioribus, (22–)23–26(–28) × (6–)6.5–7(–8) µm.
Ascomata supericial on a small stroma, black, up to 200 µm
diam, globose, separate, but developing with 1–3 necks with age;
ostioles consisting of pale brown to subhyaline cells, periphysate,
with periphysoids growing into the cavity; wall consisting of 3–6
layers of medium brown textura angularis. Pseudoparaphyses
present, hyaline, subcylindrical, septate, anastomosing, 3–4
µm diam; hamathecial cells persistent in cavity. Asci fasciculate,
bitunicate, subsessile, broadly ellipsoid with a long tapered stalk,
straight to curved, 8-spored, 70–110 × 15–20 µm. Ascospores
tri- to multiseriate, overlapping, hyaline, guttulate, irregular lumina
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Schubert et al.
Fig. 23. Cladosporium macrocarpum (CBS 299.67) and its teleomorph Davidiella macrocarpa (CPC 12755). A–C. Macronematous conidiophores and conidia. D–G.
Micronematous conidiophores. H. Microcyclic conidiogenesis. I. Ascomata formed on nettle stems in culture. J. Periphyses. K, M–N. Asci. L. Ostiole. Scale bars: A, D–H, J–N
= 10 µm, I = 200 µm.
rarely observed, thick-walled, straight to slightly curved, fusoidellipsoidal with obtuse ends, widest in the middle of the apical
cell, medianly 1-septate, not to slightly constricted at the septum,
tapering towards both ends, but more prominently towards lower
end, (22–)23–26(–28) × (6–)6.5–7(–8) µm; mucoid sheath rarely
observed, mostly absent.
Mycelium unbranched or loosely branched, 1–4.5(–5) µm wide,
septate, sometimes slightly constricted at septa, hyaline to pale
brown, smooth to minutely verruculose, walls unthickened or slightly
thickened. Conidiophores micronematous and macronematous,
130
solitary, arising terminally from plagiotropous hyphae or terminally
from ascending hyphae. Macronematous conidiophores erect,
straight to somewhat lexuous, cylindrical-oblong, nodulose to
nodose, with a single apical or usually several swellings either
somewhat distinct from each other or often in short succession giving
conidiophores a knotty appearance, swellings sometimes laterally
elongated or formed at the top of a branch-like outgrowth below
the apical swelling, sometimes distinctly geniculate, unbranched,
sometimes branched, 12–260 × (3–)4–6 µm, swellings 5–10 µm
wide, pluriseptate, sometimes slightly constricted at septa, pale to
medium brown or olivaceous-brown, somewhat paler at apices,
Cladosporium herbarum SpecieS coMplex
Fig. 24. Cladosporium macrocarpum (CBS 299.67). A. Survey of a conidiophore that forms several secondary ramoconidia and conidia. Several aerial hyphae are also visible
in this picture. B. Conidiophore with broadly ellipsoid secondary ramoconidia and obovoid conidia. Note the different scars on the conidiophore at the lower left. C. Ellipsoid
or obovoid conidia with notable areas of scar formation. The ornamentation is relatively widely distributed over the body of the cell and similar to C. variabile. D. Detail of
a conidiophore (see B) with scars. Note the relatively shallow rings of the scars. E. Details of conidia and a secondary ramoconidium. F. Conidiophore with a secondary
ramoconidium and conidia. Note the hila on several spores and the lack of ornamentation at the site where spores are formed. Scale bars: A–C, = 10 µm, D, F = 5 µm, E = 2
µm.
smooth to minutely verruculose or verruculose, walls somewhat
thickened, sometimes even two-layered. Conidiogenous cells
integrated, terminal or intercalary, cylindrical, nodulose with lateral
shoulders or nodose with swellings round about the stalk, with
conidiogenous loci conined to swellings, 12–37 µm long, with up to
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12 loci per cell, usually with up to six, loci conspicuous, protuberant,
(1–)1.5–2 µm diam, somewhat thickened and darkened-refractive.
Micronematous conidiophores almost indistinguishable from
hyphae, straight, narrowly iliform, non-nodulose or with a single or
few swellings, mostly with small head-like swollen apices, usually
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Schubert et al.
or branched chains, subglobose, obovoid to limoniform, ellipsoid
or fusiform, 2.5–16 × 1.5–5 µm, 0(–1)-septate, few longer conidia
subcylindrical to clavate, up to 37(–43) µm long, 0–2(–3)-septate,
occasionally with up to four septa, sometimes slightly constricted
at the septa, subhyaline to pale brown, almost smooth to minutely
verruculose, walls unthickened, hila 0.8–1.2 µm diam, thickened
and darkened-refractive.
Cultural characteristics: Colonies on PDA reaching 30–43 mm
in diam after 14 d at 25 ºC, dark dull green to olivaceous-grey,
olivaceous-grey, dark olivaceous- to iron-grey reverse, pulvinate,
velvety, sometimes somewhat zonate, paler zones towards the
margin, margin regular, entire edge, almost colourless to white,
glabrous to feathery, aerial mycelium sparse to more abundant
in the colony centre or covering large areas of the colony, hairy,
luffy or felty, whitish to smoke-grey, sometimes becoming reddish,
livid red to vinaceous, growth lat, regular, sometimes forming
few prominent exudates, exudates sometimes slightly reddish,
sporulation profuse with two kinds of conidiophores, low and high.
Colonies on MEA reaching 31–50 mm in diam after 14 d at 25
ºC, grey-olivaceous to olivaceous-grey or iron-grey, sometimes
pale olivaceous-grey to whitish due to abundant aerial mycelium,
olivaceous-grey or iron-grey reverse, velvety or powdery, margin
narrow, entire edge, colourless to white, glabrous, aerial mycelium
sparse to abundant, hairy or felty, growth regular, lat to low convex,
radially furrowed, without prominent exudates, sporulation profuse.
Colonies on OA reaching 29–40 mm in diam after 14 d at 25 ºC,
grey-olivaceous, olivaceous-grey to dark smoke-grey, olivaceousblack or iron grey reverse, margin entire edge, narrow, colourless
or white, glabrous, aerial mycelium sparse, mainly in the colony
centre, felty, white to smoke-grey or grey-olivaceous, felty, growth
lat, regular, without exudates, sporulating.
Fig. 25. Davidiella macrocarpa (CPC 12755). Ascus and ascospores. Scale bar =
10 µm. P.W. Crous del.
only few micrometer long, 1.5–3 µm wide, aseptate or with only
few septa, subhyaline, smooth or almost so, walls unthickened,
with a single or only few conidiogenous loci, narrow, 0.8–1.2 µm
diam, thickened and somewhat darkened-refractive. Conidia
catenate, in branched chains, small terminal conidia subglobose,
obovoid, oval, limoniform, 4–11 × (3–)4–6 µm [av. ± SD, 7.6 (±
1.9) × 5.0 (± 0.8) µm], aseptate, intercalary conidia broadly ovoidellipsoid, 10–17 × (4.5–)5–9 µm [av. ± SD, 12.7 (± 2.1) × 6.8 (±
0.8) µm], 0–1-septate; secondary ramoconidia broadly ellipsoid
to subcylindrical, 14–25(–30) × (5–)6–9(–10) µm [av. ± SD, 19.4
(± 3.5) × 7.6 (± 1.0) µm], 0–2(–3)-septate, sometimes slightly
constricted at the septa, septa somewhat sinuous with age, pale
brown to medium olivaceous-brown or brown, sometimes even
dark brown, verruculose to echinulate (muricate under SEM),
walls thickened, up to 1 µm thick, mostly broadly rounded at
apex and base, sometimes attenuated, sometimes guttulate
by oil drops, with up to three apical hila, mostly 1–2, hila sessile
(apparently somewhat immersed) to somewhat protuberant, 1–
2(–2.5) µm diam, thickened and darkened-refractive; microcyclic
conidiogenesis occurring with conidia forming secondary microand macronematous conidiophores, conidia often germinating with
long hyphae. Conidia formed by micronematous conidiophores
usually smaller, narrower and paler, catenate, in short unbranched
132
Specimens examined: Sine loco et dato, L 910.255-723 = L-0115836, lectotype
designated here of Dematium graminum. Sine loco, on dead stems of Brassica sp.
(Brassicaceae), No. 601, L 910.255-716 = L-0115849, holotype of D. herbarum var.
(β) brassicae. Sine loco, on leaves of Iris (Iridaceae), Quercus (Fagaceae), Brassica
etc., L 910.255-736 = L-0115871, holotype of D. vulgare var. (β) foliorum, isotype L
910.255-718 = L-0115872. Sine loco et dato, L 910.255-698 = L-0115852, lectotype
designated here for D. vulgare var. (δ) typharum. Isolated from “Mycosphaerella
tulasnei”, CBS 223.32 = ATCC 11287 = IMI 049635. romania, isolated from water,
CBS 175.82. slovenia, Sečovlje, isolated from hypersaline water from salterns
(precrystalisation pond), 2004, P. Zalar, EXF-2287 = CPC 12054. Turkey, Ankara,
Tekeli, isolated from Triticum aestivum (Poaceae), isol. S. Tahsin, ident. A.C. Stolk,
CBS 299.67. u.s.A., Seattle, University of Washington Campus, 47.6263530, 122.3331440, isolated from cleistothecia of Phyllactinia guttata (Erysiphaceae)
on leaves of Corylus sp. (Corylaceae), 16 Sep. 2004, D. Glawe, CPC 11817;
Washington, isolated from Spinacia oleracea (Chenopodiaceae), 1 Jan. 2003,
L. DuToit, CBS-H 19855, neotype designated here for C. macrocarpum, and
holotype of D. macrocarpa, isoneotype HAL 2020 F, isotype HAL 2021 F, culture
ex-type CPC 12752, 12756–12759, CPC 12755 = CBS 121623.
Substrate and distribution: Decaying plant material, human,
hypersaline water, water; widespread.
Literature: de Vries (1952: 76), Ellis (1971: 315), Domsch et al.
(1980: 208), Ellis & Ellis (1985: 290, 468), Matsushima (1985: 5),
McKemy & Morgan-Jones (1991), Dugan & Roberts (1994), David
(1997: 71), Samson et al. (2000: 112).
Notes: In the absence of Preuss’s type material (not preserved)
de Vries (1952) “lectotypiied” C. macrocarpum by a specimen in
Saccardo’s herbarum (Herb. Myc. P.A. Saccardo no. 419, PAD).
This material, subsequently distributed in Mycotheca Italica no.
1396, should correctly be regarded as neotype (David 1997). A
single collection of Saccardo’s Mycotheca Italica no. 1396 from
herb. HBG, which can be considered as isoneotype material,
Cladosporium herbarum SpecieS coMplex
Fig. 26. Cladosporium ossifragi (CBS 842.91). Conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
was re-examined and proved to rather agree with the species
concept of C. herbarum s. str. The conidia were formed in simple,
rarely branched chains, 6–26 × (4–)5.5–8(–9) µm, 0–3-septate,
almost smooth or minutely to densely verruculose or verrucose
(Schubert 2005). However, since de Vries’ “lectotypiication” was
incorrect according to the code (ICBN, Art. 9.2, 9.17), a neotype is
designated.
The delimitation of C. macrocarpum as a morphologically
distinct species from C. herbarum has been controversially
discussed by several authors (McKemy & Morgan-Jones 1991,
Dugan & Robert 1994, Ho et al. 1999). Based on molecular as well
morphological studies, it can be shown that C. macrocarpum is a
well-deined species distinguishable from C. herbarum s. str. by
forming conidiophores with wider nodes, 5–10 µm, wider and more
frequently septate conidia [small terminal conidia 4–11 × (3–)4–6
µm versus 4–10 × 3–5(–6) µm in C. herbarum, intercalary conidia
10–17 × (4.5–)5–9 µm versus 6–16 × 4–6 µm in C. herbarum,
secondary ramoconidia 14–25(–30) × (5–)6–9(–10) µm versus 12–
25(–35) × (3–)5–7(–9) µm in C. herbarum] and by being connected
to Davidiella macrocarpa. On natural substrates the conidiophores
are usually somewhat wider than in culture, 4–8(–10) µm wide, and
also the conidia can be somewhat wider, sometimes up to 13(–15) µm.
www.studiesinmycology.org
Cladosporium graminum, described by Persoon (1822), as
well as C. brunneum and C. gracile, introduced by Corda (1837),
are older synonyms of C. macrocarpum and, according to the
code, would have priority. However, since C. macrocarpum is a
well established, currently used name with numerous records in
literature, a proposal to conserve the name against these older
names is in preparation for formal publication in Taxon.
A characteristic difference between ascomata of C.
macrocarpum in comparison to those of C. herbarum, are the
smaller, globose pseudothecia, asci with longer stalks, prominence
of pseudoparaphyses, and rather inconspicuous luminar ascospore
inclusions.
Cladosporium ossifragi (Rostr.) U. Braun & K. Schub., comb.
nov. MycoBank MB504575. Figs 26–28.
Basionym: Napicladium ossifragi Rostr., Bot. Fǽröes 1: 316.
1901.
≡ Heterosporium ossifragi (Rostr.) Lind, Dan. fung.: 531. 1913.
= Heterosporium magnusianum Jaap, Schriften Naturwiss. Vereins SchleswigHolstein 12: 346. 1902.
≡ Cladosporium magnusianum (Jaap) M.B. Ellis in Ellis, More
Dematiaceous Hyphomycetes: 337. 1976.
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Schubert et al.
Fig. 27. Cladosporium ossifragi (CBS 842.91). A. Macronematous conidiophore. B. Micronematous conidiophore. C–D. Conidia. E. Conidia and microcyclic conidiogenesis.
Scale bars = 10 µm.
Fig. 28. Cladosporium ossifragi (CBS 842.91). A. Survey on different secondary ramoconidia and conidia. B. Details of conidia and hila. Note the very pronounced ornamentation
and the absence of ornamentation near the site of spore formation. C. Detail of the end of a secondary ramoconidium with pronounced hila. D. Formation of a new conidium.
Note the broad scar behind it (> 1 μm). E. Formation of a new conidium from a smooth-walled stalk. F. Hila on a secondary ramoconidium. This micrograph is from the sample
before coating with gold-palladium and shows similar features as the sample after sputter coating. Scale bars: A = 10 µm, B–D, F = 2 µm, E = 5 µm.
134
Cladosporium herbarum SpecieS coMplex
Mycelium abundantly formed, twisted, often somewhat aggregated,
forming ropes, branched, 1–5 µm wide, septate, often irregularly
swollen and constricted, hyaline or subhyaline to pale brown,
smooth, walls unthickened or only slightly thickened. Conidiophores
macronematous and micronematous, arising from plagiotropous
hyphae, terminally or laterally, erect to subdecumbent, more
or less straight to lexuous, cylindrical, sometimes geniculate,
subnodulose with loci often situated on small lateral shoulders,
unbranched, sometimes branched, often very long, up to 350 µm
long, 3–4.5(–5) µm wide, pluriseptate, shorter ones aseptate, not
constricted at septa, pale to pale medium brown, paler towards
apices, sometimes subhyaline, smooth to minutely verruculose,
especially towards apices, walls somewhat thickened, up to 0.5 µm,
sometimes appearing two-layered. Conidiogenous cells integrated,
terminal as well as intercalary, cylindrical, sometimes geniculate,
subnodulose, 5–31 µm long, proliferation sympodial, with few
loci (1–3) per cell, loci usually conined to small lateral shoulders,
protuberant, conspicuous, short cylindrical, 1–2 µm wide, up to
1 µm high, somewhat thickened, darkened-refractive. Conidia
catenate, in short, unbranched or branched chains, straight, small
terminal and intercalary conidia subglobose, obovoid to ellipsoid,
4–15 × 3–5 µm [av. ± SD, 9.3 (± 3.7) × 4.0 (± 0.7) µm], 0–1septate, not constricted at the septa, pale brown, hila 0.8–1 µm
diam, secondary ramoconidia cylindrical, sometimes ellipsoid or
subfusiform, 16–36(–40) × (4–)5–8 µm [av. ± SD, 26.6 (± 7.4) ×
6.0 (± 1.2) µm], (0–)1–3(–4)-septate [in vivo wider, (6–)7–9(–11)
µm, and with up to ive, rarely seven septa], not constricted at
the septa, septa sometimes slightly sinuous, pale brown to pale
medium brown, densely verruculose, verrucose to echinulate
(densely muricate under SEM), walls unthickened to somewhat
thickened, rounded or somewhat attenuated at apex and base,
hila protuberant, conspicuous, sometimes situated on short, small
prolongations, 1–2.5 µm diam, somewhat thickened and darkenedrefractive; microcyclic conidiogenesis occasionally occurring.
Cultural characteristics: Colonies on PDA reaching 53 mm diam
after 14 d at 25 ºC, greenish olivaceous, grey-olivaceous to
olivaceous-grey or iron-grey, appearing somewhat zonate, dull
green to olivaceous-black reverse, margin colourless, regular,
entire edge, aerial mycelium abundantly formed, covering at irst
the colony centre later most of the surface, dense, high, growth lat
with elevated colony centre, somewhat folded. Colonies on MEA
reaching 54 mm diam after 14 d at 25 ºC, pale olivaceous-grey
to olivaceous-grey in the centre, iron-grey reverse, velvety, margin
colourless to white, entire edge, radially furrowed, aerial mycelium
abundantly formed, luffy to felty, growth lat with somewhat raised,
folded colony centre. Colonies on OA attaining 52 mm diam after
14 d at 25 ºC, olivaceous-grey to iron-grey, iron-grey to greenish
black reverse, margin white, entire edge, aerial mycelium diffuse,
loose, growth lat, prominent exudates absent, sporulation profuse
on all media.
Specimens examined: denmark, Undallslund, on leaves of Narthecium ossifragum
(Melanthiaceae), 13 Sep. 1885, E. Rostrup, CP, neotype designated here of C.
ossifragi; Tǿnder, Rǿmǿ near Twismark, 19 Aug. 1911, H. Sydow, Sydow, Mycoth.
Germ. 1047, M. Germany, Hamburg, Eppendorfer Moor, on leaves of Narthecium
ossifragum, 12 Sep. 1897, O. Jaap, HBG, lectotype selected here of C.
magnusianum; 4 Sep. 1903, O. Jaap, Jaap, Fungi Sel. Exs. 49, M; Wernerwald near
Cuxhaven, Aug. 1927, A. Ludwig, Petrak, Mycoth. Gen. 146, M. norway, Bjerkreim
County, isolated from leaves of Narthecium ossifragum, M. di Menna, CBS-H 19860,
epitype designated here of C. ossifragi, culture ex-epitype CBS 842.91 = ATCC
200946; Møre og Romsdal County, isolated from leaves of Narthecium ossifragum,
M. di Menna, CBS 843.91.
www.studiesinmycology.org
Substrate and distribution: Causing leaf spots on Narthecium
ossifragum; Europe (Austria, Denmark, Germany, Great Britain,
Ireland, Norway).
Literature: Ellis & Ellis (1985: 390), David (1995a; 1997: 85–86,
88), Ho et al. (1999: 132).
Notes: Type material of Napicladium ossifragi is not preserved in
Rostrup’s herbarium (on Narthecium ossifragum, Faeroe Islands,
Viderö, Viderejde and Österö, Svinaa, sine dato, leg. Ostenfeld &
Harz). However, other authentic collections seen and examined
by Rostrup are deposited at CP. Lind (1913) re-examined these
samples, synonymised N. ossifragi with H. magnusianum and
correctly introduced the combination H. ossifragi. Nevertheless,
the correct oldest name for this fungus has been ignored by
most authors. David (1997), who clearly stated that N. ossifragi is
the earliest name for this species, preferred to use the name C.
magnusianum because the typiication of Rostrup’s name was still
uncertain. Despite the lacking type material, there is no doubt about
the correct identity of N. ossifragi since authentic material of this
species, examined by and deposited in Rostrup’s herbarium (CP),
is preserved. Therefore, there is no reason to reject the oldest valid
name for this species. The original collection of C. magnusianum
cited by Jaap (1902) (on leaves of Narthecium ossifragum, Denmark,
Tǿnder, Rǿmǿ, peatbog by Twismark, Jul.–Aug. 1901, Jaap), but
not designated as type, is not preserved (David 1997). It is neither
deposited at B, HBG nor S. However, in the protologue Jaap (1902)
also referred to material of this species found near Hamburg, which
is, hence, syntype material available for lectotypiication.
Cladosporium pseudiridis K. Schub., C.F. Hill, Crous & U. Braun,
sp. nov. MycoBank MB504576. Figs 29–30.
Etymology: Epithet derived from its similar morphology to
Cladosporium iridis.
Differt a Cladosporio iridis conidiis 0–3-septatis, brevioribus et latioribus, 15–55 ×
(9–)11–19(–21) µm.
Mycelium sparingly branched, 2–7 µm wide, septate, not constricted
at the septa, subhyaline to pale brown, smooth or almost so, walls
somewhat thickened, guttulate or protoplasm appearing granular,
sometimes enveloped by a slime coat. Conidiophores arising
mostly terminally from ascending hyphae, sometimes also laterally
from plagiotropous hyphae, erect, more or less straight, broadly
cylindrical-oblong, once or several times slightly to distinctly
geniculate-sinuous, forming more or less pronounced lateral
shoulders, nodulose, unbranched, 100–320(–500) × 7–11 µm,
swellings 10–14 µm wide, becoming narrower and paler towards the
apex, septate, not constricted at the septa, septa mainly basal, apical
cell often very long, pale to medium olivaceous-brown, subhyaline
at the apex, smooth or almost so, sometimes minutely verruculose,
walls usually distinctly thickened, sometimes even two-layered, up
to 1(–2) µm thick, protoplasm granular, often clearly contrasting
from the outer wall. Conidiogenous cells integrated, terminal and
intercalary, cylindrical-oblong, slightly to distinctly geniculatesinuous, nodulose with conidiogenous loci conined to swellings
or lateral shoulders, 30–110 µm long, proliferation percurrent to
sympodial, with a single or three, sometimes up to ive geniculations
per cell, usually only a single locus per swelling, protuberant, very
prominent, short cylindrical, peg-like, clearly composed of a dome
and surrounding rim, dome often higher than the periclinal rim,
broad, somewhat paler than rim, conically narrowed, (2–)2.5–4
µm wide, up to 2 µm high, thickened and darkened-refractive.
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Schubert et al.
Fig. 29. Cladosporium pseudiridis (CBS 116463). Conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
Conidia solitary, sometimes in short unbranched chains of two or
three, straight to slightly curved, young conidia small, 0–1-septate,
broadly ovoid to pyriform, 15–26 × (9–)11–16(–18) µm [av. ± SD,
19.2 (± 4.3) × 14.2 (± 3) µm], irst septum somewhat in the upper
half, the upper cell is much smaller but gradually extending as the
conidium matures, mature conidia 1–3-septate, broadly pyriform,
cylindrical-oblong or soleiform, usually with a distinctly bulbous
base, 30–55 × 12–19(–21) µm [av. ± SD, 41.5 (± 6.8) × 17.1 (± 2.1)
µm], broadest part of conidia usually at the bulbous base, mostly
attenuated towards the basal septum, septa becoming sinuous with
age, pale to medium olivaceous-brown or brown, usually echinulate,
sometimes coarsely verrucose, walls distinctly thickened, up to 2
136
µm thick, often appearing layered with a large lumen in the centre
of the cell, broadly rounded to lattened at apex and base, hila often
very prominent, often peg-like elongated, up to 3 µm long, with age
becoming less prominent, visible as a thickened lat plate just below
the outer echinulate wall layer, slightly raised towards the middle,
2–3.5 µm diam, thickened and darkened-refractive; microcyclic
conidiogenesis not observed.
Cultural characteristics: Colonies on PDA attaining 6 mm diam
after 14 d at 25 ºC, whitish, smoke-grey to pale olivaceous-grey
due to abundant aerial mycelium, olivaceous-black reverse, margin
narrow, white, more or less crenate, aerial mycelium zonate, luffy,
covering most of the colony, mainly in the colony centre, growth
Cladosporium herbarum SpecieS coMplex
Fig. 30. Cladosporium pseudiridis (CBS 116463). A–C. Conidiophores and conidia. D. Part of a conidiogenous cell showing a protuberant cladosporioid conidiogenous locus.
E–F. Conidia. Scale bars = 10 µm.
convex to raised, deep into the agar, with age few large prominent
exudates formed, sparingly sporulating. Colonies on MEA attaining
7 mm diam after 14 d at 25 ºC, olivaceous-grey, pale olivaceousgrey to pale rosy-buff due to abundant aerial mycelium covering
almost the whole colony, iron-grey reverse, margin colourless or
white, broad, regular, more or less glabrous, aerial mycelium luffy,
dense, high, growth convex to umbonate, sometimes with elevated
colony centre, prominent exudates lacking, sporulation sparse.
Colonies on OA attaining 8 mm diam after 14 d at 25 ºC, white, pale
buff to pale olivaceous-grey in the centre, margin grey-olivaceous,
olivaceous- to iron-grey reverse, margin entire edge or somewhat
undulate, somewhat feathery, growth raised with a somewhat
depressed centre forming an elevated outer rim, without prominent
exudates, sporulation more abundant.
Specimen examined: new Zealand, Auckland, Mt. Albert, Carrington Road, Unitec
Campus, isolated from large leaf lesions on Iris sp. (Iridaceae), 15 Aug. 2004, C.F.
Hill, CBS-H 19861, holotype, culture ex-type CBS 116463 = LYN 1065 = ICMP
15579.
Substrate and distribution: On living leaves of Iris sp.; New
Zealand.
Notes: Cladosporium pseudiridis closely resembles C. iridis, a
common and widespread species causing leaf spots on numerous
Iris spp. and a few additional hosts of the host family Iridaceae,
but the latter species is easily distinguishable by having longer
and narrower, more frequently septate conidia, (18–)30–75(–87) ×
(7–)10–16(–18) µm, (0–)2–6(–7)-septate.
www.studiesinmycology.org
It is unlikely that C. pseudiridis is of New Zealand origin since
the genus Iris is not indigenous to New Zealand. All Iris species
that are found in this country have been introduced, mainly for
horticultural purposes. The species is, therefore, probably more
common than indicated above. However, within the course of the
recent monographic studies in the genus Cladosporium numerous
herbarium specimens, mainly of European origin, have been
examined and proved to be correctly identiied agreeing with the
species concept of C. iridis. Additional collections and cultures are
necessary to determine its distribution.
Cladosporium ramotenellum K. Schub., Zalar, Crous & U. Braun,
sp. nov. MycoBank MB504577. Figs 31–33.
Etymology: Refers to the morphological similarity with Cladosporium
tenellum.
Differt a Cladosporio cladosporioide conidiophoris et conidiis leniter angustioribus,
2–4(–5) µm latis, conidiis 0–2(–3)-septatis, semper verruculosis; et a Cladosporio
tenello locis conidiogenis non numerosis et non aggregatos ad apicem, conidiis
longioribus et angustioribus, 2.5–35 × 2–4(–5) µm, 0–3-septatis.
Mycelium unbranched or only sparingly branched, 1.5–4 µm wide,
septate, without swellings and constrictions, hyaline or subhyaline,
smooth, sometimes irregularly rough-walled, walls unthickened.
Conidiophores solitary, macronematous and micronematous,
arising as lateral branches of plagiotropous hyphae or terminally
from ascending hyphae, erect, straight or slightly lexuous,
cylindrical, neither geniculate nor nodulose, without head-like
swollen apices or intercalary swellings, unbranched, sometimes
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Schubert et al.
Fig. 31. Cladosporium ramotenellum (CPC 12043). Conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
branched, branches often only as short lateral prolongations,
mainly formed below a septum, 14–110 × 2–4 µm, septate, not
constricted at the septa, subhyaline to pale olivaceous or brown,
smooth to minutely verruculose, walls unthickened, sometimes
guttulate. Conidiogenous cells integrated, terminal, sometimes also
intercalary, cylindrical, not geniculate, non-nodulose, 10–28(–50)
µm long, proliferation sympodial, with few conidiogenous loci,
mostly 1–3, loci sometimes situated on small lateral prolongations,
protuberant, 0.5–1.5(–2) µm diam, thickened and somewhat
darkened-refractive. Ramoconidia formed, cylindrical-oblong, up
to 47 µm long, 2–4 µm wide, 0–1-septate, rarely up to 4-septate,
subhyaline to very pale olivaceous, smooth or almost so, with a
broadly truncate base, without any dome and raised rim, 2–3 µm
wide, not thickened but somewhat refractive. Conidia numerous,
polymorphous, catenate, in branched chains, straight, sometimes
slightly curved, small terminal conidia numerous, globose,
subglobose or ovoid, obovoid or limoniform, 2.5–7 × 2–4(–4.5) µm
[av. ± SD, 5.1 (± 1.3) × 3.1 (± 0.6) µm], aseptate, without distal
hilum or with a single apical scar, intercalary conidia ellipsoid to
138
subcylindrical, 8–15 × 3–4(–4.5) µm [av. ± SD, 11.5 (± 2.4) × 3.6
(± 0.5) µm], 0–1-septate; secondary ramoconidia subcylindrical
to cylindrical-oblong, 17–35 × 3–4(–5) µm [av. ± SD, 22.5 (±
5.6) × 3.7 (± 0.5) µm], 0–3-septate, not constricted at the septa,
subhyaline to very pale olivaceous, minutely verruculose (granulate
under SEM), walls unthickened or almost so, apex broadly rounded
or slightly attenuated towards apex and base, sometimes guttulate,
hila protuberant, conspicuous, 0.8–1.5(–2) µm diam, somewhat
thickened and darkened-refractive; microcyclic conidiogenesis
occurring.
Cultural characteristics: Colonies on PDA reaching 46–49 mm diam
after 14 d at 25 ºC, olivaceous to grey-olivaceous due to abundant
sporulation, appearing zonate in forming concentric zones, margin
entire edge to slightly undulate, white, glabrous, aerial mycelium
absent or sparse, growth lat with a somewhat folded and wrinkled
colony centre, without prominent exudates, sporulation profuse.
Colonies on MEA reaching 48–49 mm diam after 14 d at 25 ºC,
grey-olivaceous to olivaceous-grey, velvety, olivaceous-grey to
Cladosporium herbarum SpecieS coMplex
Fig. 32. Cladosporium ramotenellum (CPC 12043). A, C. Macronematous conidiophore. B. Conidial chain. D. Micronematous conidiophore. E. Ramoconidia and conidia. Scale
bars = 10 µm.
Fig. 33. Cladosporium ramotenellum (CPC 12043). A. Survey of colony development showing a large bulbous “foot cell” that gives rise to conidiophores, which can be branched.
B. Details of conidiophores showing secondary ramoconidia and conidia. The inset shows scar formation on a conidiophore. C. Conidiophore and several conidia. D. Details
of ornamentation on conidia. Note the wide, but relatively low ornamentation units. E. A micrograph illustrating the organisation within a conidiophore. Scale bars A–D = 5 µm,
E = 10 µm.
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139
Schubert et al.
Fig. 34. Cladosporium sinuosum (CPC 11839). Conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
iron-grey reverse, margin entire edge to undulate, radially furrowed,
colourless, glabrous to feathery, aerial mycelium sparse, diffuse,
growth lat with slightly elevated colony centre, distinctly wrinkled,
prominent exudates not formed, abundantly sporulating. Colonies
on OA attaining 40 mm diam after 14 d at 25 ºC, grey-olivaceous,
margin entire edge, colourless or white, glabrous, aerial mycelium
absent or sparse, growth lat, without exudates, sporulation
profuse.
Specimens examined: slovenia, Ljubljana, isolated from an air conditioning system
(bathroom), 2004, M. Butala, CBS 121627 = CPC 12047 = EXF-967; Sečovlje,
isolated from hypersaline water from reverse ponds, salterns, 2005, P. Zalar, CBS-H
19862, holotype, isotype HAL 2026 F, culture ex-type CBS 121628 = CPC 12043
= EXF-454.
Substrate and distribution: Hypersaline water, air; Slovenia.
Notes: Cladosporium ramotenellum, which appears to be a
saprobe in air and hypersaline water, morphologically resembles
C. cladosporioides and C. tenellum K. Schub., Zalar, Crous &
U. Braun, but is quite distinct from C. cladosporioides by having
somewhat narrower conidiophores and conidia, 2–4(–5) µm
140
wide, and 0–3-septate, always minutely verruculose conidia.
Cladosporium tenellum, a newly introduced species (see below)
isolated from hypersaline water and plant material, possesses
conidiophores with numerous conidiogenous loci, usually crowded
towards the apex forming sympodial clusters of pronounced scars,
and shorter and somewhat wider, 0–1(–2)-septate conidia, 3–20(–
28) × (2.5–)3–5(–6) µm. Besides these morphological differences,
C. ramotenellum is faster growing in culture than C. tenellum.
Cladosporium arthrinioides Thüm. & Beltr. and C. hypophyllum
Fuckel are also close to C. ramotenellum, but C. arthrinioides,
known from Italy on leaves of Bougainvillea spectabilis, deviates in
having shorter and wider, 0–1(–2)-septate, mostly smooth conidia
(2–18 × 2–6.5 µm) which become larger and more frequently
septate with age (up to 32 µm long and with up to four septa);
and C. hypophyllum occurring in Europe on leaves of Ulmus
minor differs in having often mildly to distinctly geniculate-sinuous,
sometimes subnodulose conidiophores and shorter and somewhat
wider, 0–1(–3)-septate conidia, 4–17(–19) × 2–5 µm, becoming
distinctly swollen, darker, longer and wider with age, 5–7 µm, with
the septa often being constricted (Schubert 2005).
Cladosporium herbarum SpecieS coMplex
Fig. 35. Cladosporium sinuosum (CPC 11839). A–D. Conidiophores. E–F. Conidia. Scale bars = 10 µm.
Cladosporium sinuosum K. Schub., C.F. Hill, Crous & U. Braun,
sp. nov. MycoBank MB504578. Figs 34–35.
Etymology: Refers to the usually distinctly sinuous conidiophores.
Differt a Cladosporio herbaro conidiophoris distincte sinuosis, conidiis solitariis vel
breve catenatis, catenis non ramosis, echinulatis.
Mycelium sparingly branched, 1–7 µm wide, septate, not
constricted at the septa, subhyaline to pale brown, smooth to
minutely verruculose, walls unthickened or slightly thickened,
sometimes with small swellings. Conidiophores arising laterally
from plagiotropous hyphae or terminally from ascending hyphae,
erect, more or less straight to lexuous, often once or several times
slightly to distinctly geniculate-sinuous, sometimes even zigzag-like,
nodulose with small to large lateral shoulders, shoulders somewhat
distant from each other or in close succession giving them a knotty/
gnarled appearance, unbranched or once branched, 25–260 × 5–7
µm, shoulders up to 10 µm wide, pluriseptate, septa sometimes
in short succession, not constricted at the septa, pale brown to
medium brown, smooth to minutely verruculose, walls thickened,
often distinctly two-layered, up to 1 µm thick. Conidiogenous
cells integrated, terminal or intercalary, often slightly to distinctly
geniculate-sinuous, nodulose with small to large laterally swollen
shoulders, 8–30 µm long, proliferation sympodial, with a single
or up to three conidiogenous loci, usually conined to lateral
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shoulders, protuberant, often denticle-like or on the top of short
cylindrical stalk-like prolongations, 1.2–2(–2.2) µm diam, mainly
2 µm, somewhat thickened and darkened-refractive, dome often
slightly higher than the surrounding rim. Conidia solitary or in short
unbranched chains with up to three conidia, straight, obovoid, oval,
broadly ellipsoid to subcylindrical or sometimes clavate (broader at
the apex), 9–21 × (5–)6–8 µm [av. ± SD, 14.5 (± 2.5) × 6.6 (± 0.7)
µm], 0–1-septate, not constricted at the septa, septum more or less
median, pale greyish brown, densely echinulate, spines up to 1 µm
long, walls thickened, apex mostly broadly rounded or sometimes
attenuated, towards the base mostly distinctly attenuated forming a
peg-like prolongation, up to 2 µm long, hila protuberant, 1.2–2 µm
diam, mainly 2 µm, somewhat thickened and darkened-refractive;
microcyclic conidiogenesis not observed.
Cultural characteristics: Colonies on PDA attaining 20 mm
diam after 14 d at 25 ºC, pale olivaceous-grey due to abundant
aerial mycelium, olivaceous-grey towards margins, iron-grey
to olivaceous-black reverse, margin regular, entire edge, aerial
mycelium abundant, cottony, dense, high, growth regular, low
convex, radially furrowed in the centre, growing deep into the agar,
with age numerous small to large prominent exudates, sporulation
sparse. Colonies on MEA attaining 16 mm diam after 14 d at 25 ºC,
white to pale smoke-grey, fawn reverse, velvety, margin undulate,
glabrous, aerial mycelium abundant, dense, high, luffy, growth
raised with elevated colony centre, laterally furrowed, without
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Schubert et al.
Fig. 36. Cladosporium spinulosum (CPC 12040). A. Overview on agar surface with conidiophores arising from the surface. The spore clusters on the conidiophore are very
compact. Note several simple, tubular conidiophore ends. The inset shows details of a conidium showing two pronounced hila and a unique, very distinct ornamentation on the
cell wall. B. Conidiophore with globose or subsphaerical secondary ramoconidia and conidia. Note the newly forming cells and hila. C. Two conidiophores. D. Details of spores
and spore formation. E. The end of a conidiophore and two scars. Scale bars: A = 20 µm, A (inset) = 1 µm, B, D–E = 5 µm, C = 10 µm.
prominent exudates. Colonies on OA attaining 18 mm diam after 14
d at 25 ºC, olivaceous, white to pale olivaceous-grey in the centre
due to abundant aerial mycelium, olivaceous-grey reverse, margin
white, entire edge, glabrous, aerial mycelium loose to dense, high,
luffy to felty, growth lat to low convex, regular, without prominent
exudates, sporulating.
Specimen examined: new Zealand, Te Anau, isolated from leaves of Fuchsia
excorticata (Onagraceae), 31 Jan. 2005, A. Blouin, Hill 1134A, CBS-H 19863,
holotype, culture ex-type CBS 121629 = CPC 11839 = ICMP 15819.
Substrate and distribution: On living leaves of Fuchsia excorticata;
New Zealand.
Notes: This new species is well characterised by its slightly to
distinctly geniculate-sinuous, often zigzag-like conidiophores and
its conidia formed solitary or rarely in short unbranched chains and
is therefore morphologically not comparable with any of the species
described until now. Most Cladosporium species with conidia usually
formed solitary or in short unbranched chains have previously
been treated as species of the genus Heterosporium Klotzsch ex
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Cooke, now considered to be synonymous with Cladosporium. All
of them, including the newly introduced C. arthropodii K. Schub.
& C.F. Hill from New Zealand, which also belongs to this species
complex (Braun et al. 2006), possess very large and wide, often
pluriseptate conidia quite distinct from those of C. sinuosum (David
1997). Cladosporium alopecuri (Ellis & Everh.) U. Braun, known
from the U.S.A. on Alopecurus geniculatus is also quite different by
having larger and wider conidia, 20–40 × 7–13(–15) µm, and wider
conidiogenous loci and conidial hila, 3.5–5 µm diam (Braun 2000).
Cladosporium herbarum is supericially similar but the
conidiophores of the latter species are sometimes only slightly
geniculate-sinuous but never zigzag-like and the verruculose to
verrucose conidia are frequently formed in unbranched or branched
chains.
Cladosporium spinulosum Zalar, de Hoog & Gunde-Cimerman,
Studies in Mycology 58: 180. 2007 – this volume. Fig. 36.
Note: This new species is described and illustrated in Zalar et al.
(2007 – this volume).
Cladosporium herbarum SpecieS coMplex
Fig. 37. Cladosporium subinlatum (CPC 12041). Macro- and micronematous conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
Cladosporium subinlatum K. Schub., Zalar, Crous & U. Braun,
sp. nov. MycoBank MB504579. Figs 37–39.
Etymology: Refers to its nodulose conidiophores.
Differt a Cladosporio bruhnei conidiophoris cum nodulis angustioribus, 3–6.5 µm
latis, conidiis brevioribus, 4–17(–22) µm longis, spinulosis, cum spinulis ad 0.8 µm
longis; et a Cladosporio spinuloso conidiophoris nodulosis, conidiis spinulosis, cum
spinulis brevioribus, ad 0.8 longis, locis conidiogenis et hilis latioribus, (0.5–)1–2
µm latis.
Mycelium unbranched or occasionally branched, 1.5–3 µm wide,
later more frequently branched and wider, up to 7 µm wide,
septate, not constricted at the septa, hyaline or subhyaline, almost
smooth to somewhat verruculose or irregularly rough-walled, walls
unthickened. Conidiophores mainly macronematous, sometimes
also micronematous, arising terminally from ascending hyphae
or laterally from plagiotropous hyphae, erect or subdecumbent,
straight or lexuous, sometimes bent, cylindrical, nodulose, usually
with small head-like swellings, sometimes swellings also on a
lower level or intercalary, occasionally geniculate, unbranched,
occasionally branched, (5–)10–270 × (1.5–)2.5–4.5(–5.5) µm,
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swellings 3–6.5 µm wide, aseptate or with few septa, not constricted
at the septa, pale brown, pale olivaceous-brown or somewhat
reddish brown, smooth, usually verruculose or irregularly roughwalled and paler, subhyaline towards the base, walls thickened,
sometimes appearing even two-layered, up to 1 µm thick.
Conidiogenous cells integrated, usually terminal or conidiophores
reduced to conidiogenous cells, cylindrical, nodulose, usually with
small head-like swellings with loci conined to swellings, sometimes
geniculate, 5–42 µm long, proliferation sympodial, with several loci,
up to four situated at nodules or on lateral swellings, protuberant,
conspicuous, denticulate, (0.8–)1–2 µm diam, thickened and
darkened-refractive. Conidia catenate, in branched chains, more
or less straight, numerous globose and subglobose conidia, ovoid,
obovoid, broadly ellipsoid to cylindrical, 4–17(–22) × (2.5–)3.5–
5.5(–7) µm [av. ± SD, 11.7 (± 4.6) × 4.5 (± 0.8) µm], 0–1(–2)septate, not constricted at septa, pale brown or pale olivaceousbrown, ornamentation variable, mainly densely verruculose to
echinulate (loosely muricate under SEM), spines up to 0.8 µm
high, sometimes irregularly verrucose with few scattered tubercles
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Schubert et al.
Fig. 38. Cladosporium subinlatum (CPC 12041). A–C. Macronematous conidiophores. D–E. Conidia. Scale bar = 10 µm.
Fig. 39. Cladosporium subinlatum
(CPC 12041). A–G. Images of an 11d-old culture on SNA. A. Overview of
colony with clusters of conidia and
aerial hyphae. Many of the hyphae
have a collapsed appearance. B.
Detail of colony with conidiophores,
conidia and aerial hyphae that
are partly collapsed. C. Detail of a
conidiophore end and a secondary
ramoconidium. Note the scars at the
end of the conidiophore. D. Details
of conidia and ornamentation.
The ornamentation consists out of
markedly deined units, which have
a relatively large distance from each
other. Note the hilum on the right
conidium. E. Conidiophore with
large scars and conidia. F. Different
blastoconidia with very early stages
of new spore formation in the middle
of the picture. G. Pattern of spore
development. Scale bars: A = 20
µm, B, E–G = 5 µm, C = 10 µm, D
= 2 µm.
144
Cladosporium herbarum SpecieS coMplex
or irregularly echinulate, walls unthickened or slightly thickened,
apex rounded or slightly attenuated towards apex and base, hila
conspicuous, protuberant, denticulate, 0.5–2 µm diam, thickened
and darkened-refractive; microcyclic conidiogenesis observed.
Specimen examined: slovenia, Sečovlje, isolated from hypersaline water from
crystallization ponds, salterns, 2005, S. Sonjak, CBS-H 19864, holotype, isotype
HAL 2027 F, culture ex-type CBS 121630 = CPC 12041 = EXF-343.
Cultural characteristics: Colonies on PDA attaining 29 mm diam
after 14 d at 25 ºC, olivaceous-black to olivaceous-grey towards
margin, margin regular, entire edge, narrow, colourless to white,
glabrous to feathery, aerial mycelium formed, luffy, mainly near
margins, growth lat, somewhat folded in the colony centre, deep
into the agar, few prominent exudates formed with age, sporulation
profuse. Colonies on MEA attaining 25 mm diam after 14 d at 25
ºC, olivaceous-grey to olivaceous due to abundant sporulation
in the colony centre, pale greenish grey towards margin, irongrey reverse, velvety to powdery, margin crenate, narrow, white,
glabrous, radially furrowed, aerial mycelium diffuse, growth convex
with papillate surface, wrinkled colony centre, without prominent
exudates, sporulation profuse. Colonies on OA attaining 26 mm
diam after 14 d at 25 ºC, olivaceous, iron-grey to greenish black
reverse, growth lat, deep into the agar, with a single exudate,
abundantly sporulating.
Notes: Cladosporium subinlatum, an additional saprobic species
isolated from hypersaline water, was at irst identiied as C.
spinulosum, but proved to be both morphologically as well as
phylogenetically distinct from the latter species in having somewhat
wider [(1.5–)2.5–4.5(–5.5) µm], nodulose macronematous
conidiophores with conidiogenous loci conined to swellings, wider
conidiogenous loci and hila, (0.8–)1–2 µm, and spiny conidia
with shorter spines than in C. spinulosum (up to 0.8 µm versus
0.5–1.3 µm long) (Zalar et al. 2007). With its narrow, nodulose
macronematous conidiophores and catenate conidia, C. bruhnei
is morphologically also similar but differs by having conidiophores
with wider swellings, (4–)5–8 µm, and longer conidia 4–24(–31)
µm, rarely up to 40 µm long which are minutely verruculose to
verrucose but not spiny.
Substrate and distribution: Hypersaline water; Slovenia.
Fig. 40. Cladosporium subtilissimum (CBS 113754). Macro- and micronematous conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
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Schubert et al.
Fig. 41. Cladosporium subtilissimum (CBS 113754). A–C. Macronematous conidiophores. D. Conidial chain. E. Micronematous conidiophore. F–G. Conidia. Scale bars = 10
µm.
Cladosporium subtilissimum K. Schub., Dugan, Crous & U.
Braun, sp. nov. MycoBank MB504580. Figs 40–42.
Etymology: Refers to its narrow conidiophores and conidia.
Differt a Cladosporio cladosporioide conidiophoris et conidiis semper asperulatis ad
verruculosis, conidiis 0–1(–2)-septatis.
Mycelium unbranched or sparingly branched, 1–5 µm wide, septate,
without swellings and constrictions, hyaline to subhyaline or pale
brown, smooth to minutely verruculose, walls unthickened or almost
so, protoplasm somewhat guttulate or granular. Conidiophores
macronematous and micronematous, arising laterally from
plagiotropous hyphae or terminally from ascending hyphae, erect,
straight to slightly lexuous, iliform to cylindrical-oblong, nonnodulose, sometimes geniculate towards the apex, unbranched or
once branched, branches short to somewhat longer, usually formed
below a septum, sometimes only short, denticle-like or conical,
25–140 × 2–4 µm, 0–4-septate, not constricted at the septa,
subhyaline to pale brown, almost smooth, minutely verruculose
to verruculose, sometimes irregularly rough-walled in the lower
part, walls unthickened or slightly thickened, protoplasm guttulate
or somewhat granular. Conidiogenous cells integrated, terminal
or pleurogenous, sometimes also intercalary, iliform to narrowly
cylindrical, non-nodulose, sometimes geniculate, 14–57 µm long,
with usually sympodial clusters of pronounced conidiogenous loci
at the apex or on a lower level, denticle-like or situated on short
lateral prolongations, up to ive loci, intercalary conidiogenous
cells usually with a short denticle-like lateral outgrowth below a
septum, protuberant, denticulate, somewhat truncate, 1.2–2 µm
diam, thickened and darkened-refractive. Ramoconidia sometimes
occurring, conidiogenous cells seceding at one of the upper septa
146
of the conidiophore and behaving like conidia, iliform or cylindrical,
20–40(–55) µm long, 1.5–4 µm wide, 0–1-septate, concolorous
with conidiophores, not attenuated towards apex and base, base
broadly truncate, non-cladosporioid, without any dome and raised
rim, 2–3.5 µm wide, neither thickened nor darkened, sometimes
slightly refractive. Conidia catenate, in branched chains, up to 12
or even more in a chain, straight, small terminal conidia numerous,
subglobose, narrowly obovoid, limoniform or fusiform, 4–9 × 2–3.5
µm [av. ± SD, 6.4 (± 1.5) × 2.8 (± 0.4) µm], with up to three distal
scars, aseptate, hila (0.5–)0.8–1 µm diam, intercalary conidia
narrowly ellipsoid, fusiform to subcylindrical, 9–18 × 3–4(–6) µm
[av. ± SD, 13.0 (± 2.5) × 3.8 (± 0.3) µm], 0(–1)-septate, hila 1–1.2(–
1.8) µm diam, with up to four distal scars, secondary ramoconidia
ellipsoid, fusiform or subcylindrical, (13–)17–32(–37) × 3–5(–6) µm
[av. ± SD, 21.4 (± 4.4) × 4.1 (± 0.5) µm], 0–1(–2)-septate, septum
median or somewhat in the lower half, usually not constricted at
the septa, with up to six distal hila crowded at the apex, hila (1.2–)
1.5–2(–2.5) µm diam, apex often somewhat laterally enlarged
or prolonged with hila crowded there, very pale or pale brown or
olivaceous-brown, minutely verruculose to verruculose (granulate
under SEM), walls unthickened or only slightly thickened, often
slightly attenuated towards apex and base, protoplasm often
guttulate or granular, hila protuberant, denticulate, (0.5–)0.8–2(–
2.2) µm diam, thickened and darkened-refractive; microcyclic
conidiogenesis occasionally observed.
Cultural characteristics: Colonies on PDA attaining 24 mm diam
after 14 d at 25 °C, grey-olivaceous to olivaceous, olivaceousgrey, iron-grey or olivaceous-black reverse, velvety, margin
regular, entire edge, white or pale greenish olivaceous, glabrous
to feathery, aerial mycelium sparse, only few areas with abundant
Cladosporium herbarum SpecieS coMplex
Fig. 42. Cladosporium subtilissimum (CBS 113754). A. Overview on the organisation of spore formation. The micrograph shows a large basal secondary ramoconidium which
has chains of secondary ramoconidia, intercalary and small terminal conidia. The conidia are formed in rows of often three cells. Note the size difference in the different cells.
B. Conidiophore showing very pronounced scars that almost appear as branches. C. Detail of (A), illustrating the scar formation between the cells. D. Conidia during different
stages of formation. E. Details of pronounced hila, and prominent ornamentation on secondary ramoconidia with the central dome-formed area. F. Different conidia and hila.
Scale bars: A = 10 µm, B–D, F = 5 µm, E = 2 µm.
mycelium, diffuse, growth regular, lat or with a raised and wrinkled
colony centre, radially furrowed, effuse, usually without prominent
exudates, with age several exudates formed, sporulation profuse,
colonies consisting of two kinds of conidiophores, short and a few
longer ones. Colonies on MEA reaching 25 mm diam after 14 d
at 25 °C, greenish olivaceous to grey-olivaceous in the centre,
olivaceous-grey to iron-grey reverse, velvety, margin entire edge,
crenate or umbonate, narrow, pale greenish olivaceous, sometimes
radially furrowed, aerial mycelium absent or sparse, growth low
convex with distinctly wrinkled colony centre, without prominent
exudates, abundantly sporulating. Colonies on OA attaining 25 mm
diam after 14 d at 25 °C, dark grey-olivaceous to olivaceous due to
profuse sporulation, iron-grey reverse, sometimes releasing some
olivaceous-buff pigments into the agar, velvety, margin regular,
entire edge or crenate, narrow, colourless or white, glabrous or
feathery, aerial mycelium sparse, growth lat with slightly raised
colony centre, prominent exudates lacking, sporulation profuse.
www.studiesinmycology.org
Specimens examined: slovenia, Sečovlje, isolated from hypersaline water from
salterns (reserve pond), 2005, P. Zalar, CPC 12044 = EXF-462. u.s.A., isolated
from bing cherry fruits, F. Dugan, CBS 113753; isolated from a grape berry, F.
Dugan, wf 99-2-9 sci 1, CBS-H 19865, holotype, isotype HAL 2028 F, culture extype CBS 113754.
Excluded strains within the subtilissimum complex: Argentina,
isolated from Pinus ponderosa (Pinaceae), 2005, A. Greslebin,
CPC 12484, CPC 12485. u.s.A., isolated from grape berry, F.
Dugan, CBS 113741, CBS 113742; isolated from grape bud, F.
Dugan, CBS 113744.
Substrate and distribution: Plant material and hypersaline water;
Slovenia, U.S.A.
Notes: Cladosporium cladosporioides is morphologically comparable with the new species but deviates in having usually smooth
conidiophores and conidia, with the conidia being mainly aseptate.
C. subtilissimum is represented by three isolates of different origins
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Schubert et al.
Fig. 43. Cladosporium tenellum (CPC 12053). Macro- and micronematous conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
Fig. 44. Cladosporium tenellum (CPC 12053). A–C, E. Macronematous conidiophore. D. Micronematous conidiophore. F. Ramoconidium and conidia. Scale bars = 10 µm.
148
Cladosporium herbarum SpecieS coMplex
Differt a Cladosporio cladosporioide conidiophoris et conidiis semper asperulatis,
locis conidiogenis apicalibus, numerosis, hilis quoque numerosis, conidiophoris
angustioribus, (1–)1.5–3.5(–4) µm latis; et a Cladosporio subtilissimo loci
conidiogenis et hilis apicalibus, numerosis, angustioribus, saepe 1–1.5 µm latis,
conidiis minutis numerosis, saepe globosis.
Fig. 45. Cladosporium tenellum (CPC 12053). A. A bird’s eye view of a colony of
C. tenellum with its very characteristic bundles of aerial hyphae. Numerous conidia
are visible, formed on simple conidiophores. B. Hyphae that run on the agar surface
give rise to conidiophores and numerous conidia, that are relatively rounded. C.
Conidiophore ends are rather simple and have large scars. D. Hila on a secondary
ramoconidium with non-ornamented area. E. Detail of the prominent ornamentation
on a secondary ramoconidium. Scale bars: A = 20 µm, B = 10 µm, C, E = 2 µm,
D = 5 µm.
and substrates. Besides these strains, several additional isolates
listed under excluded strains are morphologically indistinguishable
from C. subtilissimum in culture, but genetically different, clustering
in various subclades. They are indicated as Cladosporium sp. in
the tree (Fig. 3).
Cladosporium tenellum K. Schub., Zalar, Crous & U. Braun, sp.
nov. MycoBank MB504581. Figs 43–45.
Etymology: Refers to its narrow conidiophores and conidia.
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Mycelium sparingly branched, 1–3 µm wide, septate, septa often
not very conspicuous, not constricted at the septa, sometimes
slightly swollen, subhyaline, smooth, walls unthickened.
Conidiophores macronematous and micronematous, solitary,
arising terminally or laterally from plagiotropous or ascending
hyphae, erect or subdecumbent, almost straight to more or less
lexuous, cylindrical, sometimes geniculate towards the apex, but
not nodulose, sometimes with short lateral prolongations at the
apex, unbranched to once or twice branched (angle usually 30–45°
degree, sometimes up to 90°), branches usually below a septum,
6–200 × (1–)2–4(–5) µm, septate, septa not very conspicuous,
not constricted at the septa, subhyaline to pale brown, almost
smooth to usually asperulate, walls unthickened or almost so.
Conidiogenous cells integrated, terminal or intercalary, sometimes
conidiophores reduced to conidiogenous cells, cylindrical,
sometimes geniculate, non-nodulose, 6–40 µm long, proliferation
sympodial, with several conidiogenous loci often crowded at the
apex and sometimes also at a lower level, situated on small lateral
shoulders, unilateral swellings or prolongations, with up to 6(–10)
denticulate loci, forming sympodial clusters of pronounced scars,
intercalar conidiogenous cells with short or somewhat long lateral
outgrowths, short denticle-like or long branches with several scars
at the apex, usually below a septum, loci protuberant, 1–1.5(–2) µm
diam, thickened and darkened-refractive. Ramoconidia sometimes
occurring, cylindrical, up to 32 µm long, 2.5–4 µm wide, with a
broadly truncate, unthickened base, about 2 µm wide. Conidia
catenate, formed in branched chains, straight, small terminal
conidia globose, subglobose, ovoid, oval, 3–6 × 2.5–3.5 µm [av.
± SD, 4.5 (± 1.3) × 2.8 (± 0.4) µm], aseptate, asperulate, with 0–2
distal hila, intercalary conidia and secondary ramoconidia ellipsoidovoid, ellipsoid to subcylindrical, 3.5–20(–28) × (2.5–)3–5(–6) µm
[av. ± SD, 12.4 (± 5.4) × 4.1 (± 0.7) µm], 0–1-septate, rarely with
up to three septa, sometimes slightly constricted at the septa,
subhyaline, pale brown to medium olivaceous-brown, asperulate
or verruculose (muricate, granulate or colliculate under SEM),
walls unthickened or slightly thickened, apex rounded or slightly
to distinctly attenuated towards apex and base, often forming
several apical hila, up to 7(–9), crowded, situated on small lateral
outgrowths giving them a somewhat irregular appearance, hila
protuberant, 0.5–1.5 µm diam, thickened and darkened-refractive;
microcyclic conidiogenesis sometimes occurring.
Cultural characteristics: Colonies on PDA reaching 27–34 mm diam
after 14 d at 25 ºC, smoke-grey, grey-olivaceous to olivaceous-grey,
olivaceous-grey to iron-grey reverse, velvety to powdery, margin
regular, entire edge, narrow, colourless to white, aerial mycelium
absent or sparingly formed, felty, whitish, growth regular, lat,
radially furrowed, with folded and elevated colony centre, deep into
the agar, with age forming few to numerous prominent exudates,
sporulation profuse, few high conidiophores formed. Colonies on
MEA reaching 25–44 mm diam after 14 d at 25 ºC, olivaceous-grey
to olivaceous- or iron-grey due to abundant sporulation in the colony
centre, velvety, margin regular, entire edge, narrow, colourless,
white to pale olivaceous-grey, aerial mycelium loose, diffuse,
growth convex with papillate surface, radially furrowed, wrinkled,
without prominent exudates, sporulating. Colonies on OA reaching
23–32 mm diam after 14 d at 25 ºC, grey-olivaceous, olivaceous149
Schubert et al.
Fig. 46. Cladosporium variabile (CPC 12751). Macro- and micronematous conidiophores and conidia. Scale bar = 10 µm. K. Schubert del.
grey to olivaceous due to abundant sporulation in the colony centre,
olivaceous- or iron-grey reverse, velvety, margin regular, entire
edge, narrow, colourless or white, aerial mycelium sparse, diffuse,
loccose, growth lat to low convex, radially furrowed, wrinkled,
without prominent exudates, sporulation profuse.
Specimens examined: Israel, Eilat, isolated from hypersaline water from salterns,
2004, N. Gunde-Cimerman, CBS 121633 = CPC 12051 = EXF-1083; Ein Bokek,
isolated from hypersaline water of the Dead Sea, 2004, M. Ota, CBS-H 19866,
holotype, isotype HAL 2029 F, culture ex-type CBS 121634 = CPC 12053 = EXF1735. u.s.A., Seattle, University of Washington campus, isolated from Phyllactinia
sp. (Erysiphaceae) on leaves of Corylus sp. (Corylaceae), 16 Sep. 2004, D. Glawe,
CPC 11813.
Substrates and distribution: Hypersaline water and plant material;
Israel, U.S.A.
Notes: Cladosporium subtilissimum and C. cladosporioides are
morphologically comparable with the new species C. tenellum, but
C. cladosporioides deviates in having usually smooth conidiophores
150
and conidia with only few conidiogenous loci and conidial hila crowded
at the apex and somewhat wider conidiophores, 3–5(–6) µm; and
in C. subtilissimum the small terminal conidia are not globose but
rather narrowly obovoid to limoniform, the conidiogenous loci and
conidial hila are somewhat wider, (0.5–)0.8–2(–2.2) µm, and at the
apices of conidiophores and conidia only few scars are formed.
Cladosporium ramotenellum, which morphologically also
resembles C. tenellum, possesses longer and narrower, 0–3-septate
conidia, 2.5–35 × 2–4(–5) µm, but forms only few conidiogenous
loci and conidial hila at the apices of conidiophores and conidia.
Cladosporium variabile (Cooke) G.A. de Vries, Contr. Knowl.
Genus Cladosporium: 85. 1952. Figs 46–48.
Basionym: Heterosporium variabile Cooke, Grevillea 5(35): 123.
1877.
≡ Helminthosporium variabile Cooke, Fungi Brit. Exs. Ser. 1, No. 360.
1870, nom. inval.
= Cladosporium subnodosum Cooke, Grevillea 17(83): 67. 1889.
Cladosporium herbarum SpecieS coMplex
Fig. 47. Cladosporium variabile and its teleomorph Davidiella variabile (CPC 12751). A–C. Macronematous conidiophores. D, F. Micronematous conidiophores. E, G–H.
Conidia. I. Twisted aerial mycelium. J. Ascomata formed on nettle stem in culture. K. Surface view of ascomal wall of textura epidermoidea. L–M. Asci. N–P. Ascospores. Q.
Ascus with a sheath. Scale bars A, D, G–J, K–N = 10 µm, J = 250 µm.
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Schubert et al.
Fig. 48. Cladosporium variabile (CPC 12753). A. Survey of hyphae that grow on the agar surface. Some of the fungal cells have a swollen appearance and could develop into
a “foot cell” that gives rise to a conidiophore. B. A number of aerial hyphae obstruct the swollen, large structures on the agar surface, which give rise to conidiophores. Some
of them appear ornamented. C. A series of conidia formed on a conidiophore (bottom of the micrograph). D. Detail of the ornamented conidia. The ornamentations are isolated
and dispersed. Note also the ornamentation-free scar zone and the hilum of the left cell. E. Two conidia behind an aerial hypha. F. Two conidiophores forming secondary
ramoconidia. Note the bulbous shape of the spore-forming apparatus. This micrograph is from an uncoated sample. Scale bars: A–C, F = 10 µm, D = 2 µm, E = 5 µm.
Teleomorph: Davidiella variabile Crous, K. Schub. & U. Braun, sp.
nov. MycoBank MB504583.
Davidiellae tassianae similis, sed ascosporis maioribus, (22–)26–30(–35) × (7–)7.5–
8(–9) µm, et ascis latioribus, plus quam 18 µm.
Ascomata pseudothecial, black, supericial, situated on a small
stroma, globose, up to 250 µm diam, with 1–3 ostiolate necks;
ostioles periphysate, with apical periphysoids present; wall
consisting of 3–6 layers of dark brown textura angularis, textura
epidermoidea in surface view. Asci fasciculate, bitunicate,
subsessile, obovoid to broadly ellipsoid, straight to slightly curved,
8-spored, 70–95 × 18–28 µm; with pseudoparenchymatal cells
of the hamathecium persistent. Ascospores tri- to multiseriate,
overlapping, hyaline, with irregular lumina, thick-walled, straight
to slightly curved, fusoid-ellipsoidal with obtuse ends, widest near
the middle of the apical cell, medianly 1-septate, not to slightly
constricted at the septum, at times developing a second septum
in each cell, several ascospores with persistent, irregular mucoid
sheath, (22–)26–30(–35) × (7–)7.5–8(–9) µm.
152
Mycelium immersed and supericial, irregularly branched, aerial
mycelium twisted and spirally coiled, 1–3 µm wide, septate,
sometimes with swellings or small lateral outgrowths, hyaline
to subhyaline, smooth, walls unthickened, hyphae which give
rise to conidiophores somewhat wider, 3–4.5 µm, subhyaline to
pale brown, almost smooth to minutely verruculose, sometimes
enveloped by a polysaccharide-like cover. Conidiophores usually
macronematous, but also micronematous, arising terminally
from ascending hyphae or laterally from plagiotropous hyphae.
Macronematous conidiophores erect, more or less straight to
lexuous, often distinctly geniculate-sinuous forming lateral
shoulders or unilateral swellings, sometimes zigzag-like or
somewhat coralloid, nodulose, swellings at irst terminal, then
becoming lateral due to sympodial proliferation, often as distinct
lateral shoulders, unbranched, sometimes once branched, 6–
180 × (2.5–)3–6 µm, swellings (3–)6–11 µm wide, septate, not
constricted at the septa, pale to medium olivaceous-brown or
brown, usually verruculose, walls somewhat thickened, about 1 µm
thick, sometimes appearing to be two-layered. Conidiogenous cells
integrated, terminal and intercalary, cylindrical, nodulose to nodose,
Cladosporium herbarum SpecieS coMplex
with a single or two swellings per cell, swellings apart from each
other or formed in short succession, loci conined to swellings, up to
six per node, protuberant, 1–2 µm diam, thickened and darkenedrefractive. Micronematous conidiophores erect, straight to slightly
lexuous, unbranched, usually without swellings, iliform to narrowly
cylindrical, sometimes only as short lateral outgrowths of hyphae,
often almost indistinguishable from hyphae, up to 50 µm long,
1.5–2.5(–3) µm wide, longer ones pluriseptate, septa appear to be
somewhat more darkened, with very short cells, 4–12 µm long,
subhyaline to pale brown, smooth, walls unthickened or almost so.
Conidiogenous cells integrated, usually terminal, rarely intercalary,
cylindrical, non-nodulose, with a single, two or few conidiogenous
loci at the distal end, protuberant, up to 2 µm diam, thickened
and darkened-refractive. Conidia catenate, in branched chains,
straight, subglobose, obovoid, oval, broadly ellipsoid to cylindrical,
sometimes clavate, 4–26(–30) × (3.5–)5–9(–10) µm [av. ± SD,
16.8 (± 6.9) × 6.5 (± 1.4) µm], 0–3-septate, usually not constricted
at the septa, septa becoming sinuous with age, often appearing to
be darkened, pale to medium or even dark brown or olivaceousbrown, verruculose to densely verrucose or echinulate (granulate
under SEM), walls slightly to distinctly thickened in larger conidia,
apex and base broadly rounded, sometimes broadly truncate or
somewhat attenuated, apex and base often appear to be darkened
or at least refractive, hila protuberant to somewhat sessile (within
the outer wall ornamentation), (0.8–)1–2 µm diam, thickened and
darkened-refractive; microcyclic conidiogenesis occurring.
Cultural characteristics: Colonies on PDA attaining 29 mm diam
after 14 d at 25 ºC, olivaceous to olivaceous-grey or iron-grey, irongrey or olivaceous-grey reverse, velvety to powdery, margin regular,
entire edge to imbriate, almost colourless, aerial mycelium whitish
turning olivaceous-grey, sometimes reddish, greyish rose, woollyfelty, growth lat with elevated colony centre, somewhat folded or
radially furrowed, with age forming several very small but prominent
exudates, sporulation profuse. Colonies on MEA attaining 27 mm
diam after 14 d at 25 ºC, olivaceous-grey to iron-grey, white to pale
olivaceous-grey in the centre due to abundant aerial mycelium,
velvety, margin very narrow, colourless, more or less entire edge,
radially furrowed, aerial mycelium luffy to loccose, dense, growth
low convex with wrinkled and folded centre, without exudates,
sporulation profuse. Colonies on OA attaining 25 mm diam after
14 d at 25 ºC, iron-grey or olivaceous, margin regular, entire edge,
narrow, white, glabrous, aerial mycelium whitish, at irst mainly in
the colony centre, high, dense, loccose, growth lat, abundantly
sporulating, no exudates.
Specimens examined: Great Britain, Wales, Montgomeryshire, Welshpool, Forden
Vicarage, on Spinacia oleracea (Chenopodiaceae), J.E. Vize, Cooke, Fungi Brit. Exs.
Ser. I, No. 360, K, holotype. u.s.A., Washington, isolated from Spinacia oleracea, 1
Jan. 2003, L. DuToit, CBS-H 19867, epitype designated here of C. variabile and D.
variabile, cultures ex-epitype CBS 121635 = CPC 12753, CPC 12751.
Substrate and distribution: Leaf-spotting fungus on Spinacia
oleracea; Asia (China, India, Iraq, Pakistan), Europe (Austria,
Belgium, Cyprus, Denmark, France, Germany, Great Britain,
Hungary, Italy, Montenegro, Netherlands, Norway, Romania, Spain,
Turkey), North America (U.S.A.).
Literature: de Vries (1952: 85–88), Ellis (1971: 315), Ellis & Ellis
(1985: 429), David (1995b; 1997: 94, 96–98), Ho et al. (1999:
144).
Notes: In vivo the conidia are usually longer, somewhat wider and
more frequently septate, (6.5–)10–45(–55) × (4.5–)6–14(–17) µm,
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0–4(–5)-septate (Schubert 2005). In culture the dimensions tend to
be smaller, which was already mentioned by de Vries (1952).
This leaf-spotting fungus supericially resembles C.
macrocarpum, but besides its pathogenicity to Spinacia, C.
variabile differs from the latter species in having distinctly larger
and more frequently septate conidia on the natural host, forming
twisted and spirally coiled aerial mycelium in culture and in having
lower growth rates in culture (29 mm after 14 d on PDA versus
38 mm on average in C. macrocarpum). Furthermore, the conidial
septa of C. variabile are often distinctly darkened, become sinuous
with age and the apex and base of the conidia often appear to
be distinctly darkened. A Davidiella teleomorph has not previously
been reported for this species.
The cladosporioides complex
This species complex will be treated in an additional paper in
this series, dealing with the epitypiication of this common and
widespread species, and with numerous isolates identiied and
deposited as C. cladosporioides.
dIscussIon
In the present study, a multilocus genealogy supported by light
and SEM microscopy, and cultural characteristics was used to
redeine species borders within Cladosporium, especially within
the C. herbarum complex. Most of the diagnostic features used
for species delimitation on host material (Heuchert et al. 2005,
Schubert 2005), proved to be applicable in culture. However,
morphological features were often more pronounced in vivo than
in vitro. For instance, conidiophore arrangement is not applicable
to cultures, conidiophore and conidium widths were often
narrower in culture than on the natural host, and macro- as well
as microconidiophores were often observed in culture, but not on
host material. All species belonging to the C. herbarum complex
are characterised by possessing conidia which are ornamentated,
the ornamentation ranging from minutely verruculose to verrucose,
echinulate or spiny whereas in the C. sphaerospermum complex
species with both smooth-walled as well as ornamented conidia
are included (Zalar et al. 2007). The surface ornamentation varies
based on the length of surface protuberances and in the density
of ornamentation. Furthermore, the conidia are mainly catenate,
formed in unbranched or branched chains. However, species
previously referred to the genus Heterosporium, which usually
produce solitary conidia or unbranched chains of two or three
conidia at the most on the natural host, also belong to this species
complex (e.g., C. iridis). In vitro these chains can become longer
and may even be branched. The conidiophores formed in culture
are mostly macro- but may also be micronematous, sometimes
forming different types of conidia that vary in shape and size from
each other. Most of the species possess nodulose conidiophores
with the conidiogenesis conined to the usually lateral swellings.
However, this phenetic trend is not consistently expressed in all
of the species belonging to the C. herbarum complex. The various
Cladosporium species within the C. herbarum complex were
observed to have subtle differences in their phenotype which were
visible via cryo-electron microscopy (cryoSEM), and are discussed
below.
Fungal colonies: CryoSEM provides the opportunity to study the
organisation of the fungal colony at relatively low magniications.
Cladosporium tenellum proved to be the only fungus able to form
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Schubert et al.
aerial hyphal strands under the conditions studied. Cladosporium
variabile formed abundant aerial hyphae, but in C. spinulosum
these were sparse, and only conidiophores were observed on the
agar surface. Three-day-old colonies of C. subinlatum formed
numerous, long aerial hyphae, and no conidiophores could be
discerned under the binocular. After 11 d the aerial hyphae seemed
to have disappeared, giving rise to conidiophores. Cladosporium
antarcticum, C. variabile and C. ramotenellum showed very large,
swollen (> 10 μm) cells which gave rise to conidiophores. With C.
variabile possible earlier stages of these cells were visible (Fig. 48),
which gave rise to conidiophores. More than one conidiophore could
be formed on such a structure (C. variabile and C. ramotenellum).
Cladosporium herbarum has very wide hyphae on the agar surface,
which gave rise to conidiophores as lateral branches. These wide
hyphae were observed to anastomose, which may provide a irm
interconnected supporting mycelium for these conidiophores. In
C. herbaroides these wide hyphae could also be discerned, but
conidiophore formation was less obvious. Similarily, C. tenellum
has wide, parallel hyphae that gave rise to conidiophores.
These observations reveal fungal structures in Cladosporium
that have not previously been reported on, and that raise intriguing
biological questions. For instance, why are hyphal strands observed
in some species (C. tenellum), and not in others, and what happens
to the aerial hyphae during incubation in some species such as C.
subinlatum? Furthermore, these preliminary results suggest that
CryoSEM provide additional features that can be used to distinguish
the different species in the C. herbarum complex.
Fine details of morphological stuctures: CryoSEM provides the
opportunity to study ine details of the conidiophore, (ramo)conidia
and scars. Samples can be studied at magniication up to
× 8 000, revealing details at a reinement far above what is
possible under the light microscope (LM) (Fig. 2). However, the LM
micrographs provide information about the different compartments
of ramoconidia, as well as the thickness and pigmentation of the
cell wall of different structures. With other words, the different
techniques are complementary, and both reveal fungal details that
build up the picture that deines a fungal species.
Conidiophores can vary with respect to their width and the
length. Cladosporium ramotenellum, C. antarcticum and C. variabile
have tapered conidiophores formed on large globoid “foot cells”.
The conidiophore itself can be branched. Cladosporium spinulosum
has conidiophores that rise from the agar surface, but can have
a common point of origin. These conidiophores are not tapered,
but parallel and slender. The conidiophores of C. bruhnei and C.
herbaroides are rather long, and can appear as aerial hyphae.
An important feature of the conidiophore is the location were the
conidia are formed. Conidiophore ends can be simple and tubular,
or rounded to more complex, several times geniculate, with several
scars. Conidiophore ends become more elaborate over time.
Cladosporium spinulosum and C. tenellum have nearly tubular
conidiophore ends, with often very closely aggregated scars. The
conidiophore ends of C. subinlatum are also near tubular with a
hint of bulbousness. Cladosporium subtilissimum is similar, but with
somewhat more elevated scars that look denticulate. Cladosporium
variabile has nodulose, somewhat swollen apices with often sessile,
almost inconspicuous scars. In the case of C. macrocarpum, these
structures are also nodulose to nodose and somewhat bent, with
only slightly protuberant loci. Cladosporium ramotenellum has
tubular conidiophore ends with pronounced scars. Cladosporium
antarcticum has very characteristic, tapered ends, and widely
dispersed (5 μm) scars. More complex conidiophore ends are
more irregular in shape, and have scars dispersed over a longer
154
distance, such as observed in C. bruhnei, C. herbaroides, and C.
herbarum.
Secondary ramoconidia are usually the irst conidia formed on
a conidiophore. They are often multicellular, and have one basal
cladosporioid hilum, and more at the apex. Few Cladosporium
species additionally form true ramoconidia representing apical
parts of the conidiophore which secede at a septum resulting in
an undifferentiated non-coronate base and function as conidia.
Ramiication of conidial chains is realised through these conidia.
They can occur in up to three stages, which results in elaborated
spore structures. The basal secondary ramoconidium is invariably
the largest, and cell size decreases through a series of additional
secondary ramoconidia, intercalary conidia, and small, terminal
conidia. The elongation of secondary ramoconidia varies among
the different species. Cladosporium macrocarpum has broadly
ellipsoid to cylindrical secondary ramoconidia usually with broadly
rounded ends, like C. variabile, while C. spinulosum has secondary
ramoconidia that can often hardly be discerned from the conidia
that are formed at later stages. The conidia of the other species
roughly fall between these species. The most notable structures on
these conidia are their ornamentation, scar pattern and morphology.
Cladosporium spinulosum forms numerous globose to subsphaerical
spores with digitate, non-tapered surface ornamentation, which
is unique for all the species discussed here. In his study on
Cladosporium wall ornamentation, David (1997) recognised three
classes of echinulate surfaces (aculeate, spinulose, digitate), and
ive classes of verrucose surfaces (muricate, granulate, colliculate,
pustulate and pedicellate) (Fig. 2). The ornamentation particles vary
in shape, width, height and density. The most strongly ornamented
conidia of the species examined by SEM are formed by C. ossifragi,
with the ornamentation both large (up to 0.5 μm wide) and high,
and can be regarded as densely muricately ornamented. Strong
ornamentation is also seen in C. herbaroides, which is mostly
granulate. Cladosporium tenellum (with muricate, granulate and
colliculate tendencies) and C. bruhnei (mostly granulate with some
muricate projections) have relatively large ornamentation structures
with slightly more space between the units than the other two
species. Cladosporium antarcticum, C. ramotenellum, C. variabile
and C. subtilissimum exhibit rather large granulate ornamentations
that have a more irregular and variable shape. Cladosporium
subinlatum shows the widest dispersed structures of the series,
being muricate. In contrast, C. macrocarpum has a very neat and
regular pattern of muricate ornamentation. The area of formation
of new spores on conidia is invariably not ornamented, and hila all
have the typical Cladosporium morphology with a central dome and
a ring-like structure around it.
Branching patterns: Spores usually show a “line of weakness”
between them where the coronate scars form. It seems that scars at
both sides of the line of weakness have the central dome structure,
which appears to play a major role in the effective mechanism
Cladosporium employs for spore dispersal, with the dome actively
pushing the conidia apart. This mechanism is also illustrated in
David (1997, ig. 2E). Indeed, conidia of Cladosporium are very
easily dislodged; even snap freezing or the electrical forces inside
the SEM often result in dislodgement of the spores in a powdery
“wave”. It is no surprise, therefore, that Cladosporium conidia are
to be found in most air samples. In Cladosporium, conidia are
mostly formed in chains, with the size invariably decreasing from
the base to the apex of the row. Upon formation each conidium is
separated from the conidiophore, or previously formed conidium,
and hence from its nutrients. The basal ramoconidium or secondary
ramoconidia have the nutrients and metabolic power to produce
Cladosporium herbarum SpecieS coMplex
a number of additional secondary ramoconidia that in turn could
produce a chain of intercalary conidia, and inally, some small,
single-celled, terminal conidia. Further research is still necessary
to determine if speciic branching patterns can be linked to different
species.
A surprising inding from the present study is the huge diversity
in species and genotypes that exist in nature, be it in the indoor
environment, on fruit surfaces, or in extreme ecological niches such
as salterns, etc. It is clear that detailed studies would be required
to ind and characterise other species of Cladosporium and obtain
a better understanding of their host ranges and ecology. A further
surprise lay in the fact that several of these species are capable
of sexual reproduction, and readily form Davidiella teleomorphs in
culture. The Davidiella states induced here were all from homothallic
species. Further attention now needs to be given to elucidating
teleomorphs from other species which, as in Mycosphaerella
(Groenewald et al. 2006, Ware et al. 2007) could be heterothallic,
and experiencing clandestine sex.
Despite the occurrence of many different genotypes in
variable genes, the degree of diversity in the entire data set was
low. For the majority of the species ITS was almost invariant, with
only six genotypes in the entire dataset. This suggests a very
recent evolution. The standardised index of association (ISA) was
high (0.3914), indicating an overabundance of clonality and / or
inbreeding, the latter possibly matching with observed homothallism
of Davidiella teleomorphs. Clonality was visualised with SplitStree
software, where star-shaped representations without any sign of
reticulation were obtained for all genes, though at different branch
lengths (Fig. 5). With Structure software an optimal subdivision
was achieved at six putative groups. Some of them were distinctly
separated, yielding a theta (θ) around 0.14, but in most cases there
was considerable overlap in representation of motifs, with θ at
signiicantly higher values. Results are dificult to interpret due to
the small size of the data set compared to the number of predicted
groups, and due to unknown but probably large sampling effects.
With optimal subdivision of the 79 strains at a hypothesised value
of K = 6 (Fig. 4), still a large degree of inter-group similarity was
noted, as was the case at any other level of K. This was particularly
obvious when data from the most variable genes (EF and ACT) are
superimposed (Fig. 4). The ACT groups are further subdivided by EF
data, but in many cases the same EF motif (indicated with arrows)
was encountered in different (multilocus) species, for example in C.
antarcticum, C. spinulosum, Davidiella sp., and the various clusters
comprising Cladosporium strains which are phenotypically almost
indistinguishable but genetically distinct from C. subtilissimum. A
similar situation was found with the distribution of EF genotypes
(indicated with doughnuts) in C. herbarum and C. macrocarpum.
Nevertheless, the data set showed signiicant structuring, partly
correlating with geography, e.g. the EF-determined cluster of C.
bruhnei that contained isolates from different sources in The
Netherlands. Differences may be over-accentuated by known
sampling effects, particularly in C. herbarum and C. macrocarpum,
where single-spore isolates from a single collection are included.
Taken together the data suggest a recent, preponderantly clonal
evolution, combined with limited natural selection at a low level of
evolutionary pressure. As a result, many genotypes produced by
hot spots in the genes analysed have survived, leading to nearly
random variation in the data set. Many combinations of motifs that
possibly could emerge have maintained in the course of time due
to the absence of recombination. This indicates that the observed
structure is that of populations within a single species, and
consequently a distinction of clonal “species” could be redundant.
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This conclusion is underlined by the fact that a single source in
a single location can be colonised by various genotypes, such
as grapes in the U.S.A. containing three different, closely related
genotypes. However, the phenomenon of co-inhabitation by
different Mycosphaerella species on the same lesion of Eucalyptus
has been described before (Crous 1998, Crous et al. 2004) and it
is therefore not surprising that different genotypes occurring close
together are also observed for the related genus Cladosporium.
There is no obvious ecological difference between genotypes, and
hence isolates seem to have equal itness.
However, in general we noticed a remarkable concordance
of genetic and phenetic characters. The morphological study was
done prior to sequencing, and nearly all morphotypes clustered
in separate molecular entities. There are some exceptions, such
as with C. antarcticum with striking morphology that was almost
identical on the molecular level to Cladosporium spp. that resemble
C. subtilissimum and would normally have been interpreted to
be a mutant. Conversely, nearly all genetically distinguishable
groups proved to be morphologically different, with the exception
of members of the C. subtilissimum s. lat. complex (indicated as
Cladosporium sp. in Fig. 3 and Table 1). The possibility remains
that the found genetic parameters correlate with phenetic markers
other than morphology, such as virulence, toxins or antifungal
susceptibilities. For this reason we introduce the established
entities here as formal species. They can be diagnosed by ACT
sequencing or by phenetic characters provided in the key. For
simple routine purposes, however, they can be seen and treated
as the “C. herbarum complex”, based on their close phylogenetic
relationships.
AcKnoWledGeMenTs
Several colleagues from different countries provided material and valuable cultures
without which this work would not have been possible. In this regard, we thank
particularly Alina G. Greslebin (CIEFAP, Esquel, Chubut, Argentina), Frank Dugan
and Lindsey DuToit (Washington State Univ., U.S.A.), and Annette Ramaley
(Colorado, U.S.A.). Konstanze Schubert was inancially supported by a Synthesys
grant (No. 2559) which is gratefully acknowledged. We thank Marjan Vermaas for
preparing the photographic plates, Rianne van Dijken for the growth studies, and
Arien van Iperen for maintaining the cultures studied. Bert Gerrits van den Ende is
acknowledged for assisting with the molecular data analyses.
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