STUDIES IN MYCOLOGY 50: 149–157. 2004.
Hormonema carpetanum sp. nov., a new lineage of dothideaceous black
yeasts from Spain
Gerald F. Bills*, Javier Collado, Constantino Ruibal, Fernando Peláez and Gonzalo Platas
Centro de Investigación Básica, Merck Sharp and Dohme de España, Josefa Valcárcel 38, Madrid, E-28027, Spain
*Correspondence: G.F. Bills, gerald_bills@merck.com
Abstract: Strains of an unnamed Hormonema species were collected from living and decaying leaves of Juniperus species,
plant litter, and rock surfaces in Spain. Strains were recognized and selected based on their antifungal activity caused by the
triterpene glycoside, enfumafungin, or based on morphological characteristics and ITS1-5.8S-ITS2 rDNA (ITS) sequence
data. Examination of 13 strains from eight different sites demonstrated that they share a common set of morphological features. The strains have identical or nearly identical sequences of rDNA from ITS regions. Phylogenetic analyses of ITS
region and intron-containing actin gene sequences demonstrated that these strains comprised a lineage closely allied to, but
distinct from, Hormonema dematioides, and other dothideaceous ascomycetes with Hormonema anamorphs. Therefore, a
new species, Hormonema carpetanum, is proposed and illustrated. The strains form a pycnidial synanamorph that resembles
the coelomycete genus Sclerophoma in agar culture and on sterilized juniper leaves.
Taxonomic novelty: Hormonema carpetanum Bills, Peláez & Ruibal sp. nov.
Key words: Dothideales, endophyte, enfumafungin, Kabatina, Rhizosphaera, rock-inhabiting fungi, Sclerophoma, Sydowia.
INTRODUCTION
Enfumafungin is a hemiacetal triterpene glycoside
that is produced in fermentations of a Hormonema sp.
associated with living leaves of Juniperus communis
L. (Liesch et al. 1998, Peláez et al. 2000, Schwartz et
al. 2000). Enfumafungin is one, among several, new
fungal triterpenoid glycosides that were discovered
because of their potent in vitro antifungal activity.
The mode of the antifungal action of enfumafungin
and other fungal triterpenoid glycosides was determined to be inhibition of fungal cell wall glucan
synthesis by their specific action on (1,3)- -D-glucan
synthase (Onishi et al. 2000, Peláez et al. 2000).
Three enfumafungin-producing Hormonema Lagerb.
& Melin strains from the province of Madrid were
compared with ascomycetes that have Hormonema
anamorphs, e.g., Sydowia polyspora (Bref. & Tavel)
E. Müll., Kabatina R. Schneid. & Arx species,
known Hormonema species, and other black yeastlike fungi, and were judged to represent an unnamed
Hormonema species (Peláez et al. 2000). However,
the Hormonema strains were not formally described
as a new species.
During recent years, additional strains of the same
Hormonema species were collected from living and
decaying leaves of Juniperus species, plant litter, and
rock surfaces in Spain (Table 1). Some of the strains
were recognized and selected based on their antifungal activity caused by enfumafungin, while others
were recognized based on morphological characteristics and ITS1-5.8S-ITS2 rDNA (ITS) sequence data.
Examination of 13 strains from 8 different sites
demonstrated that they share a set of morphological
features common to the enfumafungin-producing
Hormonema sp. The strains have identical or nearly
identical sequences of rDNA from ITS regions.
Phylogenetic analyses of ITS region and actin gene
sequences demonstrated that these strains comprised
a lineage closely allied to, but distinct from, Hormonema dematioides Lagerb. & Melin, and other
dothideaceous ascomycetes with Hormonema anamorphs. In this report we describe the set of isolates
as a new species, Hormonema carpetanum, observe
that the strains form a pycnidial synanamorph in agar
culture, and present an expanded analysis of their
molecular, morphological, and ecological characteristics.
MATERIALS AND METHODS
Isolates
Living cultures of H. carpetanum are maintained in
the Merck Research Laboratories Microbial Resources Culture Collection in Rahway, New Jersey,
U.S.A. (MRL), the Centro de Investigación Básica,
Merck Sharp and Dohme in Madrid (CIBE), and
unless indicated otherwise (Table 1). Reference
strains of Hormonema, Kabatina and Sydowia species were obtained from the Centraalbureau voor
Schimmelcultures (CBS), Utrecht, The Netherlands.
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Table 1. Details pertaining to isolates of Hormonema carpetanum studied.
Isolate
F131395
F138213
F0599041
F0594611
MF61671, ATCC
74360, IMI 392072
F154715
F154786
TRN24
TRN252, CBS
115712
TRN31, IMI 392073
TRN40, CBS 115713
TRN201
TRN278
1
Sequence accession No.
(ITS, actin)
AY616203, AY616225
Geographic origin
Host or substratum
Circo de Gredos, Ávila, Spain
AY616200, AY616226
AY616210, AY616224
AY616209, AY616223
AF182375, AY616222
Sierra de Rubión, Covarrubias, Burgos, Spain
Navalquejigo, Madrid, Spain
Navalquejigo, Madrid, Spain
Navalquejigo, Madrid, Spain
Living leaves, Juniperus communis
subsp. nana
Living leaves, Juniperus oxycedrus
Living leaves, Juniperus communis
Living leaves, Juniperus communis
Living leaves, Juniperus communis
AY616207, AY616227
AY616208, AY616228
AY616202, AY616220
AY616205, AY616217
Trevélez, Granada, Spain
Ossa de Montiel, Albacete, Spain
Cancho Gordo, La Cabrera, Madrid, Spain
Cancho Gordo, La Cabrera, Madrid, Spain
Leaf litter, Cistus albidus
Leaf litter, Juniperus sabina
Granite
Granite
AY616206, AY616218
AY616201, AY616221
AY616204, AY616219
AY616199, AY616216
Cancho Gordo, La Cabrera, Madrid, Spain
Cancho Gordo, La Cabrera, Madrid, Spain
La Tejera, Checa, Guadalajara, Spain
Atazar, Madrid, Spain
Granite
Granite
Sandstone
Slate
Strains previously characterised in Peláez et al. (2000). 2Holotype and ex-type culture.
Morphology
All isolates were cultured in at least three different
media to study their macro- and microscopic characters. The set of media used for characterization was
based on their previous efficiency in the induction of
sexual or asexual states, and included: potatodextrose agar (PDA, Difco), oatmeal agar (OA,
Difco), and Czapek-yeast extract agar (CYA) (Pitt &
Hocking 1997). For some isolates, additional observations were made on cornmeal agar (Sigma-Aldrich)
and potato-carrot agar (Gams et al. 1998). Colony
diameter, texture, pigmentation, margin appearance,
exudates, and colours in the descriptions were recorded after 2 wk at 22 ºC, unless noted otherwise.
Colour designations, e.g., 4F6–2, are from Kornerup
and Wanscher (1978) and those in capital letters are
from Ridgway (1912). Pycnidial conidiomata were
induced in a subset strains by inoculating flasks of
Sabouraud’s maltose broth containing autoclaved
fresh leaves of Juniperus oxycedrus L. with four 5mm agar discs of mycelia and conidia. Flasks were
incubated 5 d at 22 ºC at 220 rpm on a rotary shaker.
Mycelia-covered leaves were removed from liquid
cultures with forceps and incubated on water agar.
Additional observations on pycnidial conidiomata
were made from 3- to 6-wk-old cultures on PDA and
OA. Procedures for isolating strains from rock surfaces were described in Ruibal et al. (2005).
Microscopic features were evaluated by 1) observing structures mounted in 5 % KOH; 2) growing
fungal colonies on cover glasses immersed in dilute
malt extract agar (0.5 % malt extract Difco, 1.5 %
agar) and supported on 2 % water agar. Colonies on
cover glasses were fixed in 95 % ethanol, mounted in
5 % KOH, and photographed.
DNA extraction, amplification, DNA analyses
DNA extraction was performed by the methods
described in Peláez et al. (1996). The first amplification of both internal transcribed spacers (ITS1 and
ITS2) and the 5.8S gene of these isolates was per-
150
formed using primers 18S-3 (5’-GAT GCC CTT
AGA TGT TCT GGG G-3’) and ITS4A (Larena et
al. 1999). To further test relationships at the interand intraspecific level, an intron-containing portion
of the actin gene was amplified using primers ACT512F and ACT-783R (Carbone & Kohn 1999).
Polymerase chain reactions were performed
following standard procedures (5 min at 93 ºC followed by 40 cycles of 30 s at 93 ºC, 30 s at 53 ºC and
2 min at 72 ºC) with Taq DNA polymerase (QbioGene) following the procedures recommended by
the manufacturer. Amplification products (0.1
g/mL) were sequenced using the BigDye Terminators version 1.1 (Applied Biosystems, Foster City,
CA) following the procedures recommended by the
manufacturer. For all the amplification products, each
strand was sequenced with the same primers used for
the initial amplification. Separation of the reaction
products by electrophoresis was performed in an ABI
PRISM 3700 DNA Analyzer (Applied Biosystems,
Foster City, CA). Partial sequences were assembled
manually, and a consensus sequence was generated.
Sequence matching with public or proprietary
databases was performed with BLAST2N and FastA
(GCG WISCONSIN PACKAGE Version 10.3UNIX, Accelrys Inc). A selection of the best matching sequences were aligned manually using GENEDOC (Nicholas et al. 1997). Phylogenetic relationships were determined from the aligned sequences
using PAUP version 4 (Swofford 2000). Nucleotide
substitutions were treated as unordered, unweighted
characters. Maximum parsimony trees were inferred
using the heuristic search options with stepwise
addition and the tree bisection reconnection algorithm. Data were resampled with 1000 bootstrap
replicates by using the heuristic search option of
PAUP. The percentage of bootstrap replicates that
yielded greater than 50 % for each group was used as
a measure of statistical confidence. Consensus trees
were calculated using 50 % majority rule. Sequence
alignments and phylogenetic trees were deposited in
A NEW HORMONEMA FROM SPAIN
TreeBase as accession numbers S1171, M2027 and
M2028.
In addition to sequences of Hormonema carpetanum listed in Table 1, new sequences of the ITS1,
ITS2 internal transcribed spacers and the 5.8S rDNA
gene obtained in this study are: Hormonema prunorum (Dennis & Buhagiar) Herm.-Nijh. CBS 933.72
(AY616213), Kabatina juniperi R. Schneid. & Arx
CBS 239.66 (AY616211), CBS 466.66 (AY616212).
New sequences of actin gene fragment from H.
carpetanum are listed in Table 1. In addition, the
fragment was sequenced from Sydowia polyspora
CBS 128.66 (AY616214) and Kabatina juniperi CBS
239.66 (AY616215).
The following sequences, largely derived from
previous studies on dothideaceous fungi of the
Aureobasidium and Hormonema complexes (de Hoog
et al. 1999, Yurlova et al. 1999, Hambleton et al.
2003), were obtained from GenBank in order to
supplement analyses of newly obtained sequences;
Hormonema dematioides AJ278925, AJ278925,
AJ278926, AJ278927, AJ278928, AJ278929,
AJ278939, AF013228, AF462439, AY160202, H.
macrosporum Voronin AJ244247, Rhizosphaera
kalkhoffii Bubák AF01321, Scleroconidioma sphagnicola Tsuneda, Currah & Thormann AY220610,
Sclerophoma pythiophila (Corda) Höhn. AF462438,
Sydowia polyspora AJ244262.
RESULTS
Phylogenetic analysis
Sequencing of the rDNA of the 13 isolates of H.
carpetanum revealed that the ITS1-5.8S-ITS2 gene
fragment was 510 nucleotides long. The percentage
homology among isolates of the H. carpetanum clade
(Fig. 1) ranged between 99 to 100 %. The lengths of
the actin gene fragments from H. carpetanum varied
from 237 to 232 nucleotides among the 13 isolates.
Percentages of homology of the actin gene fragments
ranged from 92 to 100 % among the isolates, with
isolate TRN278 having the highest sequence divergence (Fig. 2). To estimate intergeneric distances, the
actin gene was also sequenced in Sydowia polyspora
CBS 128.64 and Kabatina juniperi CBS 239.66.
Attempts at amplification and sequencing of the actin
gene fragment of Kabatina juniperi CBS 466.66 and
Hormonema prunorum CBS 933.72 yielded chromatograms with poorly defined nucleotide sequences,
suggesting the presence of more than one actin sequences within the same isolate (data not shown).
Morphology, blast search results, and alignments
of the ITS1-5.8S-ITS2 rDNA clearly associated H.
carpetanum with species classified among the Dothideales, especially a complex of fungi that have in
common Hormonema conidial states (Peláez et al.
2000). Recognition and differentiation of H. carpeta-
num as a distinct and monophyletic lineage was well
supported by parsimony analysis of both genes
fragments (Figs 1, 2). In both single-gene genealogies, the isolates formed a discrete group supported in
90 % and 100 % of the bootstrap-resampled ITS15.8S-ITS2 and actin datasets, respectively (Figs 1, 2).
Small infraspecific differences were evident among
isolates of H. carpetanum, and infraspecific grouping
varied depending on the gene fragment analysed.
Fig. 1. Relationships of Hormonema carpetanum isolates
and selected reference strains inferred by maximum
parsimony consensus of aligned sequences of the ITS15.8-ITS2 rDNA. Statistical support (1000 bootstrap) values
of > 50 % indicated at branch points. Tree parameters:
total characters = 528, constant characters = 454, variable
characters parsimony-uninformative = 23, variable characters parsimony-informative = 51, tree length = 103, consistency index (CI) = 0.777 and retention index (RI) = 0.932.
Outgroup taxon: R. kalkhoffii.
Neither gene fragment grouped isolates of H. carpetanum according to their origins from living plants,
plant litter, or rock surfaces (Figs 1, 2).
Isolates referable to Sydowia polyspora and its H.
dematioides and Sclerophoma pythiophila synanamorphs also formed a distinct and well supported
lineage parallel to that of H. carpetanum (Fig. 1). The
infraspecific variation among the ITS1-5.8S-ITS2
dataset of H. carpetanum was similar to the differences observed among the same dataset from isolates
referable to Sydowia polyspora and its synanamorphs
(Fig. 1). The homology among isolates of the S.
polyspora–H. dematioides clade ranged between 97
to 99.5 %. Pairwise comparisons of sequence homologies between strains of the H. carpetanum and
the S. polyspora–H. dematioides clade ranged between 92 to 94 %, and most of the nucleotide differences between strains of the two clades were concentrated in the ITS2 region.
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BILLS ET AL.
Fig. 2. Relationship of Hormonema carpetanum isolates
and selected reference strains inferred by maximum
parsimony consensus of aligned sequences of an introncontaining fragment of the actin gene. Statistical support
(1000 bootstrap) values of > 50 % indicated at branch
points. Tree parameters: total characters = 248, constant
characters = 152, variable characters parsimonyuninformative = 78, variable characters parsimonyinformative = 25, tree length = 142, consistency index (CI)
= 0.915 and retention index (RI) = 0.750. Outgroup taxon:
S. polyspora.
The relatively low bootstrap values within the S.
polyspora–H. dematioides and H. carpetanum species clusters suggested that further delineation of
isolates based on the ITS1-5.8S-ITS2 dataset alone is
unreliable. However, some infraspecific grouping of
H. carpetanum strains was apparent based on actin
gene sequences (Fig. 2).
Taxonomic Part
Hormonema carpetanum Bills, Peláez & Ruibal, sp. nov. MycoBank MB500039. Figs 3–21.
= Hormonema sp., Peláez et al., Syst. & Appl.
Microbiol. 23: 333. 2000.
Synanamorph: Sclerophoma sp.
Etymology: Carpetanus (Latin), in reference to the
ancient Roman name for the Central Cordillera of
Spain.
Species H. dematioides similis, sed conidiis maioribus et
serie ex nucleicis acidis differt. Coloniae radiatim rugulosae in agaro avenae farinae vel tuberum solani dextrosato;
constantes praecipue ex hyphis submersis radialiter exten-
152
sis; mycelio aerio absente vel exiguo, pycnidiis formatis in
ca. 3–4 hebdomadis in vel sub agari superficie, plerumque
cum massa humecta conidiorum e conidiogenesi myceliali
accumulatorum in guttis vel filis radialibus olivaceo-nigris
ad atris. Conidiophora absentia in mycelio. Cellulae
conidiogenae holoblasticae, incorporataae, intercalares,
mycelio similes. Conidia ad 7–14(–16) µm longa, ad 3–6
µm lata, hyalina ad pallide olivaceo-grisea. Conidiomata
pycnidialia formata in vitro, similia generi Sclerophoma,
ad 350 µm diam, globosa vel subglobosa, unilocularia,
solitaria ad gregaria, olivaceo-brunnea ad atra, glabra vel
setosa, initiata e massa stromatica cellularum
isodiametricarum, strato tenui hypharum obtecta. Cellulae
conidiogenae phialidicae, isodiametricae, 8–15 um diam,
unum vel plura conidia ex uno vel pluribus aegre distinctis
ostiolis proferentes vel per conversionem ipsorum conidia
praebentes. Conidia pycnidialia ellipsoidea ad late
ellipsoidea, aliquando curvata vel constricta in parte
media, glabra, attenuata ad basim, hyalina usque ad
atrogriseo-brunnea, 8–14(–17) 4–6 µm.
Inventa in foliis Juniperi speciei superficialiter disinfectis,
plantarum residuis et superficie saxorum in Hispania
centrali:
Holotype exsiccatum specimen herb. CBS H-13135, (CBS
H-13136 isotype). Et cultura viva TRN 25 = CBS 115712.
Mycelium consisting of torulous, wide, often thickwalled, hyaline to dark olive to olive black, shortcylindrical to subglobose cells, often with hyphal
cells longitudinally septate, in age sometimes developing groups of muriform cells, with individual cells
up to 22 µ m diam, generally sparsely branched, but
sometimes becoming more branched with age. Conidiophores absent on mycelia. Conidiogenous cells
holoblastic, integrated, intercalary, usually not differentiated from main axes of vegetative mycelium,
occasionally arising from an undifferentiated lateral
cell or filament, sometimes with a faint pore or
slightly protruding annellate scar evident at the
conidiogenous locus, often abundant along the entire
radius of young extending hyphae, less so on older,
contorted and melanized hyphae. Conidia up to 7–
14(–16) µm long, up to 3–6 µ m wide, aseptate, or
rarely 1-septate in old cultures, usually ellipsoidal
and tapered towards the base, but quite variable,
occasionally pyriform or subglobose, smooth, often
with one or more budding scars, hyaline to pale
olivaceous-grey, often budding to produce 1 or 2
secondary conidia, accumulating in yeast-like masses
along radial axes of vegetative hyphae.
A NEW HORMONEMA FROM SPAIN
determinate, short cylindrical to isodiametric, 8–15
µm diam, thin- to thick-walled, hyaline to pale,
giving rise to one or more conidia, through one or
more poorly defined openings or by conversion of the
cell’s internal contents directly into to a conidium,
collapsing after conidia are released. Pycnidial conidia ellipsoid to broadly ellipsoid, sometime curved,
indented on one side or constricted at the centre,
smooth, usually tapered toward base, occasionally
with eccentric basal scar, hyaline to dark greyish
brown, 8–14(–17) × 4–6 µm.
Figs 3–6. Hormonema carpetanum in agar culture after
two wks. 3. TRN40, on potato-dextrose agar. 4. TRN40,
on oatmeal agar. 5. F059461, on Czapek yeast extract agar.
6. F059461, on oatmeal agar.
Figs 7–9. Mycelial conidiogenesis and conidia of Hormonema carpetanum. 7, 8. F131395, slide culture on dilute
malt agar. 9. TRN 31, slide culture on dilute malt agar.
Scale bars = 10 µm.
Pycnidial conidiomata in agar or on sterilized leaves
up to 350 µm diam, globose to subglobose, unilocular, solitary to gregarious, rarely confluent in age,
shiny, dark olive-brown to black, smooth, or with
scanty 30–75 µ m long, basally up to 7 µ m wide
setae; conidiomata initiating as stromatic masses of
broadly cylindrical to isodiametric cells, enveloped
by a thin layer of hyphal filaments, dehiscing by
irregular erosion of upper layers, accumulating dark
brown masses of moist conidia when mature. As
basal stromatic cells of conidiomata accumulate and
expand, upper layers of stromatic cells convert into
conidiogenous cells. Conidiogenous cells phialidic,
Cultural characteristics: Colonies on PDA differed
in radial extension depending on the isolate, ranging
from 21–37 mm diam, with margin submerged,
fimbriate, usually radially rugulose or slightly furrowed, often slightly depressed toward the centre,
consisting predominantly of submerged, radially
extending hyphae, shiny to moist, during the first few
weeks, aerial mycelium absent to scant, but abundant
aerial filamentous mycelium may emerge with prolonged incubation (>1 mo), in all strains scattered to
gregarious pycnidia formed in about 3–4 wks at or
below the agar surface, usually with masses of moist
conidia from mycelial conidiogenesis accumulating
in moist drops or in radial strands, pale olive-brown
to dark olive, Yellowish Olive, Dark Olive, Dark
Greenish Olive, 3F6–2, 4F6–2, or dark olive-grey,
Dark Greyish Olive, Olivaceous-Black (1), 30F5–2,
eventually becoming black. Colonies on OM ranged
from 24–32 mm diam; depending on the isolate,
submerged to appressed, radially furrowed, silky to
shiny, moist, dark olive, olivaceous-black to black, in
most strains scattered pycnidia formed at or below
the agar surface in about 3–4 wks. Colonies on CYA
17–21 mm, raised, rugulose, with radially extending
yeast-like hyphae, often with hyaline to buff watery
zones or sectors mixed with dark olivaceous-black
zones. Growth was poor on media with dilute starch
as the primary carbon source, e.g., cornmeal agar or
potato-carrot agar. No growth was observed at 37 °C.
Habitat: Isolated from surface-sterilized leaves of
Juniperus species, plant litter, and rock surfaces.
Known distribution: Spain, from Burgos southward to
Granada and from Ávila eastward to Guadalajara.
Specimens examined: Living cultures listed in Table 1.
Spain, Madrid, La Cabrera, Cancho Gordo, isolated from
surface of granite formation, Aug. 2001, dried holotype
culture with mycelial conidia and pycnidia conidiomata
and ex-holotype culture TRN25 = CBS 115712.
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BILLS ET AL.
Figs 10–14. Pycnidia of Hormonema carpetanum. 10. ATCC 74360, pycnidia on autoclaved leaves of Juniperus oxycedrus.
Scale bar = 500 µm. 11. TRN31, pycnidia on autoclaved leaves of J. oxycedrus. Scale bar = 500 µm. 12. TRN201, pycnidia
from edge of oatmeal agar plate. Scale bar = 500 µm. 13. TRN201, pycnidia from edge of oatmeal agar plate. 14. Scale bar =
250 µm. TRN24, pycnidial initial from potato dextrose agar. Scale bar = 50 µm.
DISCUSSION
The genus Hormonema, typified by H. dematioides,
has been applied to melanized filamentous fungi that
produce slimy, yeast-like conidia that are formed
basipetally in a non-synchronous manner from one or
few loci on cells of undifferentiated vegetative hyphae. Percurrent conidiogenous loci in Hormonema
has served to distinguish the genus from similar fungi
classified in Aureobasidium that produce conidia
synchronously from the conidiogenous loci
(Hermanides-Nijhof 1977, de Hoog & Yurlova
1994). The differences in modes of conidiogenesis
and cluster analysis of ITS1 and ITS2 sequences
clearly separated fungi with Aureobasidium anamorphs from those with Hormonema anamorphs (de
Hoog et al. 1999, Yurlova et al. 1999). Furthermore,
other studies based on ITS data have consistently
154
segregated strains of the S. polyspora–H. dematioides
complex into well-supported clades (Yurlova et al.
1999, Hambleton et al. 2003). Peláez et al. (2000)
used a combination of mycelial features, conidiogenesis, and ITS1-5.8S-ITS2 sequences to place the
then unnamed H. carpetanum among dothideacous
fungi with Hormonema anamorphs. Our reanalysis of
an expanded data set, which includes more isolates
and more new sequences of related fungi from public
databases, reconfirms the conclusion that the new
species is a close, but distinct relative of H. dematioides, Kabatina species, Rhizosphaera species, and
the recently described Scleroconidioma sphagnicola
(Hambleton et al. 2003).
The pattern of synanamorphy of H. carpetanum is
consistent with patterns of closely related fungi, viz.
pycnidial conidiomata forming on plant surfaces, or
sometimes in culture, and a mycelial conidial state
(Hormonema) associated with vegetative growth.
A NEW HORMONEMA FROM SPAIN
Figs 15–21. Pycnidial conidiogenous cells and conidia of Hormonema carpetanum. 15. F131395, young stromatic cells and
conidiogenous cell (arrow). 16. F131395, young stromatic cells and conidiogenous cell (arrow). 17. TRN201, stromatic cells
and conidiogenous cell (arrow), note collapsed stromatic cells. 18. TRN201, stromatic cells and conidiogenous cells (arrows),
note collapsed stromatic cells. 19. TRN201, stromatic cells and conidiogenous cells (arrows). 20. F131395, conidiogenous cell
(arrow). 21. TRN31, pycnidial conidia produced on autoclaved leaves of J. oxycedrus. Scale bars = 10 µm.
In the preliminary description of H. carpetanum
(Liesch et al. 1998, Peláez et al. 2000), pycnidial
conidiomata were not mentioned probably because
strains were not incubated for extended intervals on a
variety of media. It is also possible that conidial
masses from pycnidial initials were overlooked and
confused with accumulated masses of dark conidia
from vegetative hyphae. Sequence similarities to other
species with pycnidial synanamorphs led us to suspect
that H. carpetanum should produce a pycnidial state,
at least on host tissue. We were able to artificially
induce pycnidia on autoclaved leaves of J. oxycedrus
(Figs 10, 11). Once pycnidia were recognized on plant
tissue, masses of stromatic cells at and below the agar
surface were recognized as pycnidial initials (Fig. 14),
and sporulating pycnidia were easily distinguished in
3–4 wk-old cultures on PDA and OA (Figs 12, 13).
The development of both pycnidial conidiomata and
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BILLS ET AL.
the Hormonema state of Sydowia polyspora have been
observed in vitro (Sutton & Waterston 1970). In a
monographic revision of the genus Hormonema, the
possibility of in vitro production of acervuli and
pycnidia by Hormonema species was acknowledged
(Hermanides-Nijhof 1977), however, in vitro production of pycnidia by strains of H. dematioides was not
described as a component of its in vitro life cycle.
Pycnidial development and morphology of H.
carpetanum differs from that described in species of
Rhizosphaera, Kabatina, Scleroconidioma, and
Sclerophoma. Rhizosphaera species have discreet,
phialidic conidiogenous cells, which are produced in
intercalary or terminally on chains of cells arising
from the pycnidial wall (Gourbière & Morelet 1979,
Gourbière & Morelet 1980, Sutton 1980, Butin &
Kehr 2000). In Kabatina, erect phialidic conidiogenous cells form on the upper layer of a stroma-like
acervulus (Sutton 1980). In Scleroconidioma, the
outer surface layer of stromatic conidiomata are
converted into conidiogenous cells and produce conidia through percurrently proliferating cells or phialides (Tsuneda et al. 2000, Tsuneda et al. 2001). The
conidiomata of H. carpetanum seem most similar to
those of Sclerophoma pythiophila in which the cells of
the stromatic tissue, consisting of a thick-walled
textura angularis, function as conidiogenous cells.
Descriptions of S. pythiophila describe the cells of the
stromatic tissue which function as phialides. As
conidiogenesis cells produce conidia, they accumulate
in the upper regions of the conidiomata and are released by an irregular erosion of the conidiomatal wall
or membrane (Sutton & Waterston 1970, Sutton 1980,
Butin & Peredo 1986). In H. carpetanum, the stromatic cells of the pycnidia (Figs 15, 16) produce
conidia blastically or internally (Figs 15–20). As the
stromatic cells mature and collapse (Figs 17–20),
conidia are released via erosion of the pycnidial
membrane and accumulate as a moist mass (Figs 10,
12, 13). However, pycnidial conidial dimensions in H.
carpetanum mostly are in the range of 8–14 × 4–6 µ m,
and therefore larger than those reported for S. pythiophila which have been recorded as 4–8 × 2–3 µm.
Pycnidia of S. pythiophila are described as either
multilocular or unilocular, while thus far in vitro only
unilocular conidiomata have been observed in H.
carpetanum. Furthermore, mycelial conidial dimensions in H. carpetanum often range up to 14 µ m,
while conidia of H. dematioides have been reported to
be up to 12 µ m (Hermanides-Nijhof 1977, de Hoog et
al. 2000). We conclude that the pycnidial conidiomata
of H. carpetanum could be accommodated in
Sclerophoma, but we prefer not to formally name this
state of the life cycle because it will likely be accompanied by the prevailing Hormonema state.
Tsuneda et al. (2004) described a new genus and
species, Endoconidioma populi Tsuneda, Hambleton
& Currah to accommodate a pycnidial fungus with a
156
closed peridium and pycnidial locule filled with
conidiogenous cells that form conidia endogenously.
A Hormonema-like synamorph is produced by the
mycelia in culture. ITS sequence data indicated that
the E. populi is a very close relative of H. carpetanum.
Comparison of the morphological descriptions E.
populi and H. carpetanum indicated the two are distinct and different fungi; Tsuneda et al. (2004) hypothesized, based on similarities in ITS sequences,
that the yet undescribed Hormonema strain ATCC
74360 was congeneric with Endoconidioma.
Most fungi with Hormonema anamorphs described
to date are associated with living plants (HermanidesNijhof 1977, Ramaley 1992, Middlehoven & de Hoog
1997, Tsuneda et al. 2000). Most notable among these
fungi are a complex of Rhizosphaera and Kabatina
species associated with needle diebacks in conifers
(Gourbière & Morelet 1980, Sutton 1980, Martínez &
Ramírez 1983, Butin & Kehr 2000), and Sydowia
polyspora and its synanamorphs (Sutton & Waterson
1970, Sutton 1980, Butin & Peredo 1986). However,
examination of the list of isolates of H. carpetanum
(Table 1) suggests on one hand, a pattern of ecological
specialization with regard to foliage of Juniperus
species, yet on the other, the ability to at least survive
on, and perhaps colonize, dead plant litter, and rock
surfaces. Presumably the ability to survive in stone
surfaces is related to heavily melanized vegetative
cells or cell aggregates. Such cells could survive the
high radiation and desiccation of these exposed environments, act as vegetative propagules, and perhaps
propagate and disperse by budding yeast cells during
favourable conditions. Trimmatostroma abietis (Butin
et al. 1996), a fungus that grows epiphytically on
living conifer needles and forms stromatic conidiomata on senescent needles of Abies and Pinus species,
exhibits a similar pattern of distribution. The fungus
has also been isolated from man-made and natural
stone surfaces as well as being observed in certain
traumatic human mycoses. The pattern of distribution
is also reminiscent of that of H. dematioides, where
the mycelial conidial state is frequently observed as an
epiphyte and endophyte of various conifer species,
isolated from dead conifer wood, and occasionally
recovered from human and animal infections
(Hermanides-Nijhof 1977, de Hoog et al. 2000).
ACKNOWLEDGEMENTS
We thank José Manuel Ruiz Vila for his scholarly assistance in preparation of the Latin diagnosis. Portions of this
work were derived from the doctoral dissertation of C.
Ruibal to be submitted to the Faculty of Science, Universidad Autónoma de Madrid.
A NEW HORMONEMA FROM SPAIN
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