Content uploaded by Miroslav Kolarik
Author content
All content in this area was uploaded by Miroslav Kolarik on Sep 11, 2019
Content may be subject to copyright.
Morphological and molecular characterisation of Geosmithia
putterillii,G. pallida comb. nov. and G. flava sp. nov.,
associated with subcorticolous insects
Miroslav KOLAR
ˇI
´K
1
,
2
, Alena KUBA
´TOVA
´
1
, Sylvie PAZ
ˇOUTOVA
´
2
and Petr S
ˇRU
˚TKA
3
1
Department of Botany,Faculty of Science,Charles University,Bena
´tska
´2,128 0,Praha 2,Czech Republic.
2
Institute of Microbiology CAS,142 20 Praha 4,Czech Republic.
3
Faculty of Forestry,Czech University of Agriculture,Kamy
´cka
´129,165 21 Praha 6,Czech Republic.
E-mail :mkolarik@biomed.cas.cz
Received 8 March 2004; accepted 7 May 2004.
Geosmithia putterillii is an anamorphic fungus with connections to bark beetles. Genetic variability of 89 isolates
traditionally grouped in G.putterillii and G. lavendula isolated from different geographical regions from subcorticolous
insects and from other unspecific substrata was assessed using RAPD, sequencing of the ITS region (ITS1-5.8SrDNA-
ITS2) and morphological characters. RAPD analysis revealed eight distinct groups. One group was represented by
G. lavendula type strain and showed no relations to any other isolate. Five RAPD-types with similar ITS sequences and
phenotype were related to the ex-type strain of Penicillium pallidum (generally given as a synonym of G. putterillii).
Because of unique phylogenetic position and a phenotype markedly different from G. putterillii, the new combination
G. pallida is made here. For another group of isolates formerly identified as G. putterillii the new species G. flava is
described based on a characteristic RAPD-type, a unique ITS sequences and a different phenotype. These newly
recognized species are stable in culture and with worldwide distribution.
INTRODUCTION
Geosmithia typified by G. lavendula represents a poly-
phyletic morphogenus introduced to accommodate
some Penicillium species (Pitt 1979). Currently, there
are ten species accepted in the genus (Pitt 1979, Pitt &
Hocking 1985, Yaguchi, Miyadoh & Udagawa 1993,
Yaguchi, Someya & Udagawa 1994). Raper & Thom
(1949) and Ramirez (1982) regarded P. putterillii and
P. pallidum as two distinct species differing in the ar-
rangement of conidial chains, which are in well-defined
columns in P. putterillii, or divergent, becoming
tangled with age, in P. pallidum. Pitt (1979) examined
several new isolates with intermediate properties and
placed P. pallidum in synonymy with G. putterillii, but
this complex has not yet been investigated using mol-
ecular methods.
Recent rDNA sequence analyses, found Geosmithia
to be polyphyletic with affinities to : (1) hypocrealean
fungi, G. lavendula,G. putterillii ; (2) Penicillium, some
with Talaromyces teleomorphs, notably G. argillacea,
G. cylindrospora,G. eburnea (teleomoph T.eburneus),
G. emersonii (teleomorph T. emersonii), G. swiftii (tele-
omorph T. bacillisporus), and G. viridis ; (3) two poly-
phyletic groups in the Eupenicillium lineage with
Penicillium anamorphs : G. malachitea (teleomorph
Chromocleista malachitea) and G. namyslowskii (Yoshi-
mura & Sugiyama 1997, Ogawa, Ogawa & Sugiyama
2000, Peterson 2000, Iwamoto et al. 2002). The hypo-
crealean Geosmithia members (hereafter also as ‘ Geos-
mithia s. str.’), containing the type species for whole
genus, are closely related to Acremonium alternatum
and cleisthothecial genera like Emericellopsis, and form
a separate clade inside the family Bionectriaceae
(Rossman et al. 1999, Rossman, McKemy & Pardo-
Schultheiss 2001). The polyphyly in Geosmithia is also
supported by differences in the major ubiquinone sys-
tem (Ogawa, Yoshimura & Sugiyama 1997) and in the
constitution of cell wall polysaccharides (Prieto et al.
2002).
The ecology of Geosmithia species is variable. Some
Geosmithia species are thermotolerant or thermophi-
lous species (i.e. G. argillacea,G. emersonii,G. eburnea,
and G. swiftii). Other species are mesophiles isolated
from miscellaneous substrata (soil, plants, wood, foods).
G. putterillii is regarded as an uncommon taxon on
various foodstuffs (Pitt & Hocking 1997). Several iso-
lates have been acquired from wood or subcorticolous
insects. Kirschner (1998, 2001) found that this fungus
is strongly associated with five phloem-feeding bark
Mycol. Res. 108 (9): 1053–1069 (September 2004). fThe British Mycological Society 1053
DOI: 10.1017/S0953756204000796 Printed in the United Kingdom.
Table 1. Hypocrealean Geosmithia isolates used in this study.
Species
a
/Original no.
b
Origin
Collection site/year/collector
c
(from Czech Republic unless
otherwise noted)
GenBank
Accession
no.
d
G. lavendula
NRRL 2146 (=IMI 40570) Laboratory contamination USA, Illinois, 1947
G. flava AJ578484
IMI 224697 (obtained as
G. putterillii)
Ulmus glabra UK, 1978
IMI 158645 (obtained as
G. putterillii)
Hordeum sp., grain UK, 1971
CCF 3333 (=MK 101) Xiphydria sp. (Hymenoptera:Siricidae)
on Castanea sativa
Eastern Bohemia, Pr
ˇelouc
ˇ,
near Senı
´k, 2000, K
AJ578483
MK 603 Leperisinus orni (Coleoptera:
Scolytidae)onFraxinus ornus
France, Poitou Charentes,
Jonzac, 2003, K
MK 570 Hypoborus ficus (Coleoptera:Scolytidae)
on Ficus carica
France, Aquitaine, Ondres, 2003, K
MK 587 Hypoborus ficus on Ficus carica France, Poitou Charentes,
Jonzac, 2003, K
CCF 3356 (=MK 321),
MK 322, MK 325, MK 329
Ernoporus tiliae (Coleoptera:Scolytidae)
on Tilia cordata
North Bohemia, Louny,
Bls
ˇansky´ Chlum hill, 2002, K
MK 353 Ernoporus tiliae on Tilia cordata North Bohemia, Podbor
ˇany, Dubovy´ vrch hill, 2002, K & P
MK 668 Ernoporus tiliae on Tilia cordata South Bohemia, Tr
ˇebon
ˇ, near Stare Jezero lake, 2003, K
CCF 3357 (=MK 300) Leperisinus fraxini (Coleoptera:
Scolytidae)onFraxinus excelsior
North Bohemia, Louny, 2002, K
CCF 3354 (=MK 264) Leperisinus fraxini on Fraxinus excelsior Central Slovakia, Mura
´n
ˇplain,
near Mura
´n
ˇsky´ hrad castle, 2002, K
MK 142 Scolytus intricatus (Coleoptera:
Scolytidae)onQuercus robur
South Bohemia, Bohemian Forest, Povydr
ˇı
´region,
near Hornı
´Hra
´dky, 2001, K
CCF 3349 (=MK 123) S. rugulosus (Coleoptera:
Scolytidae)onPrunus sp.
Central Bohemia, Prague,
Hostivar
ˇsky´ les forest, 2001, K
MK 58 S. rugulosus on Prunus sp. Central Bohemia, Velky´ Osek,
Libicky´ luh forest, 1999, K & S
MK 97 S. rugulosus on Prunus sp. North Bohemia, C
ˇeske
´str
ˇedohor
ˇı
´Mts.,
Velemı
´n, 2000, K
MK 168 S. rugulosus on P. domestica Central Slovakia, Mura
´n
ˇplain,
near Mura
´n
ˇsky´ hrad castle, 2002, K
MK 135 S. rugulosus,Malus domestica North Bohemia, Louny, Opoc
ˇno, 2001, K
MK 357 S. rugulosus on P. domestica Central Bohemia, Zruc
ˇnad Sa
´zavou,
near Ka
´cov, 2002, K & S
MK 334 S. carpini on Carpinus betulus Central Bohemia, Bohemian Karst,
Svaty´ Jan pod Skalou, 2003, K
MK 368 Taphrorychus bicolor (Coleoptera:Scolytidae),
associated with S. intricatus on Fagus sylvatica
North Bohemia, Louny,
near Hr
ˇivice, 2003, K
MK 347 Rhaphitropis marchicus (Coleoptera:Anthribidae),
associated with S. mali on M. domestica
North Bohemia, Litome
ˇr
ˇice,
near R
ˇedhos
ˇt
’, 2002, K
Geosmithia putterillii s. lat. 1054
G. pallida, RAPD-type I. ‘pallida’
NRRL 2037 (=IMI 40214, CCF 3053) Cotton yarn UK (England), 1927 AJ578486
IMI 054224 (obtained as G. putterillii) Soil Nigeria, 1954,
RK ‘5.Bork 3’ Bark beetle, medulla of
young deciduous tree
Taiwan, Tainan, Hsin-hua, 2002
G. pallida, RAPD-type II. ‘macrospora’
IMI 155478a (obtained as G. putterillii) Trees in apple orchard Cyprus, 1971
IMI 190744 (obtained as G. putterillii)Cucumis melo Peru, 1975
CCF 3340
d
(=MK 115), MK 81 S. intricatus on Q. robur North Bohemia, Louny,
near Br
ˇinkov, 2000, K
AJ578485
(from CCF 3340)
MK 250, MK 183 S. intricatus on Q. dalechampii Central Slovakia, Mura
´n
ˇplain,
near Mura
´n
ˇsky´ hrad castle, 2002, K
MK 91, MK 102 S. rugulosus,Prunus sp. Central Bohemia, Velky´ Osek,
Libicky´ luh forest, 1999, V, K & S
MK 172, MK 203 S. rugulosus on P. domestica Central Slovakia, Mura
´n
ˇplain,
near Mura
´n
ˇsky´ hrad castle, 2002, K
MK 356 S. rugulosus on P. domestica Central Bohemia, Zruc
ˇnad Sa
´zavaou,
Ka
´cov, 2002, K & S
MK 89 S. rugulosus on Prunus sp. North Bohemia, C
ˇeske
´str
ˇedohor
ˇı
´Mts.,
Velky´ vrch hill near Vrs
ˇovice, 2000, K
MK 46 S. rugulosus on Frangula alnus Central Bohemia, Velky´ Osek,
Libicky´ luh forest, 1999, V, K & S
CCF 3336 (=MK 134) S. rugulosus on Malus domestica North Bohemia, Louny,
Opoc
ˇno, 2001, K
MK 350 Rhaphitropis marchicus, asociated
with S. mali on M. domestica
North Bohemia, Litome
ˇr
ˇice,
near R
ˇedhos
ˇt
’, 2002, K
G. pallida, RAPD-type III ‘velutinosa’
CCM 8281 Roots of Q. robur Central Bohemia, Kr
ˇivokla
´t region,
Dr
ˇevı
´c
ˇ, 1998
CCF 309 Soil Czech Republic, 1965
CCF 2809 (=AK 132/93) Dead branch of Q. petraea Central Bohemia, Bohemian Karst, Srbsko – Pla
´ne
ˇ, 1993 AJ578488
MK 18, MK 22 S. intricatus on Q. robur Central Bohemia, Velky´ Osek, Libicky´ luh forest, 1999, V, K & S
AK 121/98, AK 123/98, AK 124/98,
AK 144/98, AK 145/98, AK 197/98,
AK 198/98, AK 199/98, AK 200/98,
AK 201/98
S. intricatus on Q. robur Central Bohemia, Velky´ Osek,
Libicky´ luh forest, 1998
CCF 3344 (=MK 114) S. intricatus on Q. robur North Bohemia, Louny,
near Br
ˇinkov, 2000, K
AK 42/97 S. intricatus on Q. petraea Central Bohemia, Kr
ˇivokla
´t region,
Zbiroh, Kohoutov Nature Reserve, 1997
AK 124/97l, AK 109/97, AK 125/97 S. intricatus on Q. petraea Central Bohemia, Kr
ˇivokla
´t region,
Karlova Ves, Mlyna
´r
ˇu˚ v luh forest, 1997
MK 129 S. intricatus,Quercus sp. Central Bohemia, Prague,
Kunraticky´ les forest, 2001, K
CCF 3353 (=MK 194), MK 190 S. intricatus on Q. dalechampii Central Slovakia, Mura
´n
ˇplain,
near Mura
´n
ˇsky´ hrad castle, 2002, K
MK 56 S. rugulosus on Prunus sp. Central Bohemia, Velky´ Osek, Libicky´ luh forest, 1999, V, K & S
AK 29/98, AK 75/98 S. intricatus on Q. polycarpa Central Bohemia, Velky´ Osek, Libicky´ luh forest, 1998
M. Kolar
ˇı
´k and others 1055
Table 1. (Cont.)
Species
a
/Original no.
b
Origin
Collection site/year/collector
c
(from Czech Republic unless
otherwise noted)
GenBank
Accession
no.
d
MK 382
S. intricatus on Fagus sylvatica North Bohemia, Louny, near Hr
ˇivice, 2003, K
G. pallida, RAPD-type IV ‘nova’
IMI 051240b (obtained as G. putterillii)Persea gratissima associated with
Scolytid beetles
Seychelles, 1952 AJ578489
IMI 191599 (obtained as G. putterillii) Branches of Malus pumila Cyprus, 1975
RK ‘Bork 1’ Phloem-feeding bark beetle,
bark of Mangifera indica L.
Taiwan, Pking-tung,
near Fenggang, 2002
G. pallida, RAPD-type V ‘funiculosa’
IMI 192499 (obtained as G. putterillii)Scolytus beetle UK, 1975
CCF 3341 (=AK 108/97),
AK 51/97, AK 107/97, AK 202/98,
AK 192/98, AK 122/98, AK 195/98,
S. intricatusi on Q. robur Central Bohemia, Velky´ Osek,
Libicky´ luh forest, 1998
AJ578487
(sequence of the strain CCF 3341)
AK 126/97, AK 208/97 S. intricatus on Q. petraea Central Bohemia, Kr
ˇivokla
´t region,
Karlova Ves, Mlyna
´r
ˇu˚ v luh forest, 1997
MK 439 S. multistriatus on Ulmus carpinifolia North Bohemia, Louny, K
MK 512 Ernoporus tiliae on Tilia platyphylos Hungary, Mecsek Mts., Mare-var
near Magya
´rgre
´gy, V, K
MK 548 Pteleobius vittatus (Coleoptera: Scolytidae)
on Ulmus sp.
Hungary, Bakony range, Keris
ˇhill, V, K
G. putterillii
NRRL 2024 (=IMI 40212,
CCF 3052)
Discolored timber of Beilschmiedia tawa New Zealand, 1946
CCF 3342 (=MK 103) S. rugulosus on Prunus sp. North Bohemia, C
ˇeske
´str
ˇedohor
ˇı
´Mts.,
Velemı
´n, 2000, K
CCF 3442 (=MK 596) Liparthrum colchicum (Coleoptera :Scolytidae)
on Laurus nobilis
France, Aquitaine, Ondres 2003, K AJ628350
a
The species identity as revealed by RAPD analysis in the course of this study, ex-type strains are printed bold.
b
AK, personal collection of A. Kuba
´tova
´(maintained at CCF), Prague; CCF, Culture Collection of Fungi, Prague ; CCM, Czech Collection of Microorganisms, Brno, Czech Republic ; IMI, CABI
Bioscience, Egham; MK, personal collection of M. Kolar
ˇı
´k (maintained at CCF); RK, personal collection of R. Kirschner.
c
Noted in isolates obtained in the current study. Abbreviations of collectors : K, M. Kolar
ˇı
´k; K & S, M. Kolar
ˇı
´k & P. S
ˇru˚ tka; K & P, M. Kolar
ˇı
´k & O. Peksa (Department of Botany, Faculty of Science,
Charles University, Prague).
d
Noted in sequences obtained in the current study.
e
Strains isolated from larvae of Agrillus sp. (Coleoptera:Buprestidae), associated with S. intricatus galleries.
Geosmithia putterillii s. lat. 1056
beetle species infesting deciduous trees and conifers.
These bark beetles were associated with very few and
unspecialized fungal species (except Ophiostoma piceae
s. lat.). The same result was obtained during a survey of
fungi associated with the oak bark beetle Scolytus
intricatus (Kuba
´tova
´, Novotny´ & Pra
´s
ˇil 1999, 2002,
Kuba
´tova
´et al. 2004) and with other subcorticolous
insects (Kolar
ˇı
´k 2002), in the Czech Republic.
In order to revise the G. putterillii species complex,
we analysed the morphological, physiological and
DNA characteristics of ex-type cultures, some cultures
used by Pitt (1979) to represent G. putterillii, and a set
of new isolates from phloem and sapwood inhabiting
insect. We believe that G. putterillii sensu Pitt (1979)
represents three genetically isolated cryptic species with
a worldwide distribution.
MATERIALS AND METHODS
Isolates
89 hypocrealean Geosmithia isolates were studied
(Table 1): (1) isolates of M. K., obtained during this
study from 13 insect species (acronym MK) ; (2) isolates
of A.K. from oak bark beetle (Kuba
´tova
´et al. 2004,
acronym AK); (3) isolates of R. Kirschner (J. W.
Goethe-Universita
¨t Frankfurt am Main, acronym RK)
from various bark beetles in Taiwan ; and (4) isolates
from public culture collections (CBS, CCM, CCF, IMI,
NRRL; Table 1). Strain NRRL 2037, derived from the
type of P. pallidum, and 1–2 isolates from each of the
seven RAPD-types, were used for DNA sequencing.
The conidiophore ontogenesis of eurotialean Geos-
mithia members was studied in G. argillacea (CCF
2544, CBS 101.69=IMI 156096), G. namyslowskii
(CBS 353.48=NRRL 1070=IMI 40033), G. viridis
(CBS 252.87=IMI 288716) and G. cylindrospora
(NRRL 2673), and compared with morphology of
Penicillium varians (CCF 2199, CCF 2941).
Cultivation and isolation
Media used were 2% malt agar (MA2), respective 4 %
malt agar (MA4): brewery malt 2xBalling, respective
4xBalling 1 l, agar 15 g, modified after Fassatiova
´
(1986); malt extract agar (MEA ; Pitt 1980) ; Czapek
yeast autolysate agar (CYA ; Pitt 1980) supplemented
with trace elements (0.001 % ZnSO
4
.7H
2
0 and
0.0005% CuSO
4
.5H
2
O); and potato carrot agar
(PCA; Fassatiova
´1986).
Various broadleaved trees infested by subcorticolous
insects were sampled during 1999–2003 on eighteen
localities in the Czech Republic, three localities in Slo-
vakia, two localities in Hungary and on two localities in
France (Table 1). Adult subcorticolous insects (i.e. liv-
ing adults before emergence and adults in the larval
feeding stage) or their larvae were excised from bark
and separately washed with 5–10 ml of wash solution
(sterile water and 0.02 % Tween 80) in sonicator (7 min,
44 kHz). Washed insect body and 0.5 ml of the wash
solution or detritus from galleries were inoculated onto
Petri dishes with MA2. The wood specimens were held
in moisture chambers for one month in room tem-
perature and the fungal growth was observed directly in
the insect galleries. Isolated fungi were inoculated into
CYA and MEA for identification.
The fungal cultures were maintained on PCA slants
at 10 xC with periodic transfers. A dried herbarium
specimen of the holotype of the new species is deposited
in the herbarium of the Mycological Department,
National Museum in Prague (PRM). Ex-holotype and
other representative strains were freeze-dried in skim-
med milk and are deposited in the Culture Collection of
Fungi (CCF), Department of Botany, Faculty of Sci-
ence, Charles University, Prague.
Cultural and morphological characteristics
The identification media and morphological charac-
terization of the Geosmithia strains followed the
protocol of Pitt (1980). The isolates described above
were cultured on MEA and CYA. In addition, the
representative strains of each RAPD-type were culti-
vated on Czapek’s solution agar (Raper & Thom 1949).
The cultures were incubated in dark at 25 xand 37 x.
Colour names are given according Kornerup &
Wanscher (1981). Conidiophore and substrate conidia
ontogenesis was observed in plate cultures according
to Cole, Nag Raj & Kendrick (1969), incubated in
daylight for the best development of conidiophore
roughness. Micromorphology was studied on 7 d old
colonies grown on MEA, the conidiophores were taken
from margins and near colony centers, as well as from
areas which differed in their texture. 20 randomly
selected conidia were measured from each strain. The
substrate mycelium from the colony margins was
studied for presence of substrate conidia. Mounts were
prepared in Melzer’s reagent and lactic acid with cotton
blue. Photomicrographs were made on Olympus BX-51
with differential interference contrast or phase contrast.
DNA analysis
Genomic DNA was obtained from mycelium grown on
cellophane discs laid on MEA plates. One to five day
old mycelium was ground in liquid nitrogen and DNA
was extracted using phenol-chloroform procedure as
previously described (Pazˇ outova
´et al. 2000b). RAPD
was performed with primers 8F (5k-GCTCTGAGA-
TTGTTCCGGCT-3k) and 10R (5k-GGCCAGTGT-
GAATATGC-3k), OPA–02 (5k-TGCCGAGCTG-3k),
OPA–20 (5k-GTTGCGATCC-3k). The binary matrix
was analysed using Treecon 1.3b (van de Peer & De
Wachter 1994). A similarity matrix was calculated
using the formula of Nei & Li (1979) and relatedness
of the isolates was evaluated by UPGMA (Sneath &
Sokal 1973). Bootstrap analysis was conducted using
500 iterations. Amplicon containing rDNA region
M. Kolar
ˇı
´k and others 1057
ITS1-5.8S-ITS2 was obtained by PCR reaction with
primers ITS1 and ITS4 (Pazˇ outova
´et al. 2000b). The
mixture (25 ml) contained 50 ng of genomic DNA,
20 pmol of each primer, 0.2 mMdNTPks (dNTP Mas-
termix, Invitek, Berlin), and 1 U of DynaZyme with the
respective buffer (Finnzymes, Oy, Finland). The reac-
tion mixtures were subjected to 32 cycles under the
following temperature regime : 95 xfor 3 min, 55 xfor
30 s, 72 xfor 1 min (1r), 95 xfor 30 s, 55 xfor 30 s, 72 x
for 1 min (30r), and 95 xfor 30 s, 55 xfor 30 s, 72 xfor
10 min (1r). Amplified fragments were purified by the
JetQuick PCR Purification kit (Genomed, Bad Oeyn-
hausen, Germany). The product was sequenced using
the same primers at Microsynth GmbH (Balgach,
Switzerland). All PCR reactions were carried out on
Mastercycler Gradient (Eppendorf, Germany).
Phylogenetic analysis
The sequences were deposited in EMBL Nucleotide
Sequence Database and their GenBank accession
numbers are given in Table 1. Other sequences used in
Figs 11–12 were obtained from GenBank and come
from ex-type cultures of the species indicated. Se-
quences were aligned by Sequence Alignment and
Modelling System (SAM) using the hidden Markov
model (Krogh et al. 1994, Hughey & Krogh 1996) with
the sequence of Acremonium alternatum as an outgroup
(Rossman, McKemy & Pardo-Schultheiss 2001). The
software is available at the server of Computational
Biology group, Computer Science and Engineering,
University of California, Santa Cruz (http://www.cse.
ucsc.edu/research/compbio/HMM-apps/tuneup-dna.
html). The alignment was edited manually using Bio-
Edit version 4.7.1 (Hall 1999). Insertions or deletions
were recoded as missing information except single
indels informative for parsimony. Distance analysis
was performed using PUZZLE 4.0.2 (Strimmer & von
Haeseler 1996). Because of unequal rate of nucleotide
substitution among the positions, the matrix of maxi-
mum likelihood distances was computed using model
of Tamura & Nei (1993) with gamma distance correc-
tions for substitution rate heterogeneity. Parsimony
analyses, obtaining of bootstrap values were done using
seqboot, dnapars and consense from the package
PHYLIP 3.5c (Felsenstein 1993). DNAPARS was run
with the ‘jumble ’ option changing 100rthe species
input order. Bootstrapped (500r) dataset was also run
with ‘jumble ’ 100roption.
RESULTS
Morphology
Although Geosmithia s. str. isolates resemble morpho-
logically genus Penicillium, unique characters were
found. The trend to increase number of diaspores is evi-
dent within Geosmithia s. str. species, and is ensured by
following mechanisms : (1) multiply branched penicilli,
closely packed in a velutinous colony with prodigious
conidiogenesis, the number of phialides is multiplied by
proliferation of all penicillus elements, especially in
older conidiophores; (2) hyphal fragments (arthroco-
nidia) arising in huge number from monilioid con-
idiophores (Fig. 1) and chalmydospore-like conidia on
substrate mycelium, observed in G. flava (Fig. 51 ; (3)
production of substrate conidia (holoblastic synana-
morph) (Figs 8–9) ; and (4) additional conidial pro-
duction on phialides arising on aerial mycelium, or
sessile on the mycelium.
The crucial morphological characters for distin-
guishing Geosmithia s. str. species are colony texture
and colour, and the whole conidiophore pattern.
Although a high conidium size variability occurs in all
Geosmithia s. str. species, the conidium size is useful
taxonomical character.
Size of rami, ramuli and metulae showed high infra-
specific variability and was important from a configur-
ational viewpoint. The whole conidiophore of all
Geosmithia s. str. isolates is roughened, especially in
colonies incubated in daylight. Mounting in Melzer’s
reagent or warming up of the mounts in lactic acid
dissolved the roughness.
Growth rates have limited taxonomical value. Many
isolates varied in growth rate within one RAPD-type,
especially when the rate was measured in freshly iso-
lated strains vs. those after several subculturings. Also
other cultural characteristics like colony texture and
colour were codified within one RAPD-type during the
course of several transfers. Some isolates failed to
Figs 1–2. Geosmithia flava.Fig. 1. Monilioid conidiophore
forming arthroconidia (IMI 158645). Fig. 2. Two con-
idiophores connected by their peg foots (MK 357).
Bar=10 mm.
Geosmithia putterillii s. lat. 1058
growth on CYA to the expected diameters and formed
deep and irregular colony margins. All Geosmithia
s. str. species have specific smell suggesting sour cream
(on MEA) or racy of the soil (on CYA). Members of
Geosmithia s. str. failed to produce a teleomorph either
on natural substrate or after prolonged incubation on
agar plates.
In all Geosmithia s. lat. isolates three types of stipe
ontogenesis were observed. (1) A proportion of stipes is
separated from the fertile hypha by a septum which was
placed immediately in the ramification, or there were
no septa in this branching area. (2) The septa delimiting
stipes from fertile hypha were located randomly, super-
ficially resembling Aspergillus foot cells. These foot
cell-like initials usually have had slightly thicker walls
than adjacent cellular elements as was observed in all
Geosmithia s. lat. isolates and in Penicillium varians
(Figs 3, 5–6). The smooth, vertical part of this element
belonged to the parent hypha, as has been visible
after conidiophore collapse (Fig. 6). (3) Formation of
the element named here peg foot has been observed in
G. flava,G. pallida and G. putterillii isolates. It is a peg
shaped, smooth, and often curved cellular element,
from which mostly a conidiophore arises (Figs 4 and 7).
Peg foot originates from a modified vegetative hypha,
arising laterally from a horizontal parent hypha. In
comparison with a horizontal foot cell-like initial, peg
foot was more or less vertical. From the peg foot, the
roughened stipe grew out laterally, and the peg foot
continued its growth terminally. This terminal part
usually collapses and therefore the swollen cellular
element (peg foot) is often asymmetric, with a scar left
by the collapsed terminal hypha (Fig. 4). Some of these
branches, which do not collapse, can also form another
stipe immediately or after short growth (Fig. 2). In
older colonies, after collapse of vegetative hyphae, the
peg foot remained as a part of the entire conidiophore
(Fig. 61).
Besides enteroblastic conidia arising in columns from
phialides, a high production of another conidial type
(substrate conidia) was observed in G. flava,G. lavendula
and G. putterillii. Substrate conidia were cylindrical,
ellipsoidal or clavate, 3–10r2.5–3 mm, often with
truncated base, 1–10 in one cluster, holoblastic. They
were produced within agar medium (Fig. 8) and rarely
from aerial conidiogenous cells (Fig. 9), solitary sessile
on the mycelium or on conidiogenous cells, which were
not separated from a parent hypha by a septum.
Phylogenetic analysis
Preparing of the genomic DNA and RAPD analysis
The best reproducibility of PCR reactions was obtained
using DNA from 1 d old mycelium (0.1–0.5 g). Es-
pecially in Geosmithia pallida samples, it eliminated a
contamination of DNA by an inhibiting brown pig-
ment. A total 300 of reproducible fragments were ampli-
fied with four primers from 89 Geosmithia isolates.
Associations among the fungal isolates as revealed
by UPGMA cluster analysis of the genetic distances
is given in Fig. 10. Eight highly supported groups
(RAPD-types) were found with each of the four pri-
mers (bootstrap values 100 %), although the extent of
polymorphism differed (data not shown). Within each
group, the vast majority of RAPD loci evaluated were
monomorphic bands. Although each RAPD-type con-
tained a large proportion of unique haplotypes, these
differed by only a few polymorphic loci.
RAPD-type I encompassing the ex-type isolate of
Penicillium pallidum NRRL 2037 showed no significant
similarity pattern to the ex-neotype strain of G. putter-
illii. RAPD-type I showed statistically supported re-
lations to RAPD-type II. RAPD-type II and other
brown coloured isolates that were distributed into the
four RAPD’s types (III, IV, V), formed together a
cluster, which was not statistically supported in general,
but with some primers, the groups appeared related. G.
putterillii featured its own unique pattern shared with
isolate CCF 3342 and CCF 3442 (RAPD-type VI). Some
isolates formerly determined as G. putterillii showed no
relations to the neotype of this species, as well as to any
other RAPD-type, and grouped together to a tentative
cryptic species named G. flava (RAPD-type VII). The
other clearly defined and unique RAPD-type consisted
of the type of G. lavendula (RADP-type VIII).
ITS region analysis
Phylogram relating representatives of RAPD-types
to the other described species of Geosmithia was
Figs 3–7. Conidiophore initials (CCF 3357). Fig. 3. Foot cell-
like initial delimited from parent hypha by a septum. Fig. 6.
Aseptate foot cell-like initial and collapsing conidiophore
(arrow). Fig. 5. Asymmetric foot cell-like initial with collaps-
ing branch of the parent hypha. Figs 4, 7. Conidiophores
arising from the vertical initials called peg foot. Figs 8–9.
Geosmithia putterillii (CCF 3342). Fig. 8. Substrate conidia
arising from substrate mycelium. Fig. 9. Substrate conidia
(arrow) arising from aerial mycelium nearby penicillate con-
idiophore. Bar=10 mm.
M. Kolar
ˇı
´k and others 1059
constructed based on rDNA sequences. The alignment
(including gaps) had 560 positions, from which 432
were constant. Only three differences were found be-
tween G. flava IMI 224697 and CCF 3333 and four
between G. putterillii NRRL 2024 and CCF 3442.
Phylogenetic relationships among Geosmithia s. str.
were inferred from the quartet-puzzling tree with
maximum likelihood branch lengths (Fig. 11) and from
maximum parsimony analysis (Fig. 12) of the aligned
ITS sequences. Topology of both trees was nearly
identical for all branches with over 50 % bootstrap
support. The first strongly supported clade comprised
putative populations of G. pallida with low average
genetic distance (0.00375–0.02220). Isolates of G. flava
formed their own distinct cluster related to G. lavendu-
la. Genetic distance between G. putterillii and G. la-
vendula (0.05530) was similar or smaller than those
between G. putterillii and any of the new species (G.
flava: 0.06529–0.06469 ; G. pallida : 0.04305–0.05149).
TAXONOMY
Geosmithia pallida (G. Sm.) Kolar
ˇı
´k, Kuba
´tova
´&
Pazˇ outova
´,comb. nov. (Figs 13–18, 25–39)
Penicillium pallidum G. Sm., Trans. Br. mycol. Soc.18 :
88 (1933).
Geosmithia putterillii (Thom) Pitt, Can. J. Bot.57 : 2022
(1979) pro parte.
Conidiophores on MEA, born mostly on the substrate
mycelium or on funicules, stipes determinate, erect,
30–400r2–5 mm, smooth to verrucose, septate, arising
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
AK145/98
AK144/98
AK197/98
CCF309
AK30/98
AK123/98
AK42/97
CCF 2809
AK75/98
AK124/97
AK200/98
AK199/98
MK18
MK129
CCM8281
MK190
MK56
MK22
MK382
CCF3353
AK121/98
AK29/98
AK198/98
CCF3344
AK109/97
AK124/98
AK125/97
IMI051240b
IMI191599
Bork1
AK126/97
AK122/98
IMI192499
AK51/97
AK202/98
AK107/97
AK195/98
AK192/98
CCF3341
MK439
AK208/97
MK512
MK548
IMI054224
NRRL2037
Bork3
IMI155478a
IMI190744
MK350
MK172
MK203
MK91
MK102
CCF3340
MK46
MK356
MK89
MK81
MK250
MK183
CCF3336
NRRL2146
NRRL2024
CCF3342
CCF3442
MK587
MK570
IMI158645
CCF3333
IMI224697
MK329
MK334
CCF3356
MK322
MK347
MK142
MK135
MK668
MK97
MK58
CCF3354
CCF3357
MK368
MK603
MK325
CCF3349
MK168
MK357
MK353
59
53
58
50
58
98
60
63
100
50
70
58
100
50
100
77
100 93
93
93
93
93
93
93
93
97
100
100
G.pallida
G.lavendula
G.putterillii
G. flava
63
53
100
82
100
67
50
60
53
100
III
IV
V
I
II
VI
VII
VIII
Fig. 10. Unweighted pair group method with arithmetic
averaging (UPGMA) phenogram based on RAPD data from
isolates belonging to Geosmithia s. str. Clades with less than
50% bootstrap support were collapsed to polytomies. Bold
dots indicate taxa included in rDNA trees (Figs 11–12). The
stability of these branches was evaluated by bootstrap
analysis with 100 replications.
Fig. 11. Quartet puzzling tree with maximum likelihood
branch lengths. Clades with bootstrap support under 50 %
were collapsed to polytomies. Acremonium alternatum was
used as an outgroup.
Geosmithia putterillii s. lat. 1060
from peg foot or cellular initials suggesting foot cells of
Aspergillus;penicilli biverticillate, terverticillate, ir-
regularly quaterverticillate, pentaverticillate or even
more branched (with six branch points between stipes
and conidium), often asymmetric ; rami of different
size, often 15–17r3–3.5 mm or longer (to 80 mm) and
asymmetric in some isolates ; metulae 8–12 (–18)r
2.5–4 mm, 2–4 per branch, usually with verrucose walls.
Conidiogenous cells phialides, 8–11r2–2.5 mm, 3–6 per
metula, typically cylindrical without distinct neck, walls
verruculose to verrucose ; conidia smooth, cylindrical
to ellipsoidal, 3–4 (–4.5)r1.5–2 mm, very variable in
size, conidial chains not persistent, at first roughly
parallel, becoming tangled with age.
MEA,25 x,14 d: Colonies 40–50 mm diam, plane or
radially furrowed, surface texture velutinous (with the
tendency to develop white mycelial overgrowth in cen-
tral areas or floccose sectors with sparse sporulation in
some strains), or strongly funiculose, when young (after
4–7 d) consisting of a white, very tough, submerged
mycelial felt, which later gives rise to funiculose tufts or
ropes with conidiophores ; margins narrow, entire,
submerged (up to 7 mm broad) ; mycelium white ; con-
idiogenesis heavy in velutinous isolates or sometimes
sparse in those more funiculose, coloured yellowish
brown (5E 8–5, 5 D 6–5), light brown (6D 8–5), rarely
pale (2A2) or white ; exudate clear, uncoloured or
brown (6E8), olive brown (4E5) or absent; soluble
pigment absent; reverse dull yellow (3B3), light brown
or olive brown, olive green (2F6), sometimes with
shades of rose or orange. Colonies of G. pallida on
MEA often develop limited floccose texture and less
sporulating sectors or overgrowths (mycelial type).
Subcultures made from these sectors have only few
conidiophores, which are smaller and less branched
than those of typical conidial type ; and conidia are
colourless. Diminished sporulation of mycelial type
handicaps these mutants in the nature and so they are
known only from the laboratory.
MEA,37 x,14 d: No growth.
CYA,25 x,14 d: Colonies 40–65 mm diam, plane or
radially furrowed, zonate, with velutinous and funicu-
lose zones, growth of some isolates (like AK 200/98) is
restricted (35 mm), margins are then deep, lobate and
colony raised or crateriform centrally, up to 50 mm
height ; conidiogenesis depends on isolate, usually heavy
in velutinous areas, coloured similarly to the colonies
on MEA; exudate uncoloured, dull yellow, rust (6E8),
olive brown (4E5) or absent ; soluble pigment absent;
other characteristics similar as on MEA.
Infraspecific variability :G. pallida is a clearly recog-
nisable species, both at the morphological and the
molecular levels, but exhibits a high degree of varia-
bility at the infraspecific level. G. pallida consists of five
clearly defined RAPD types characterized also by their
ITS-rDNA sequences, which are world-wide distrib-
uted but they lack major ecological specificity. These
RAPD-types appear to be morphologically uniform,
but isolates of intermediate morphocharacters also
occur. Morphologically, it is possible to distinguish
each population using the size of penicillus, stipes and
conidia, its complexity and the character of colony
texture on MEA. On CYA, less significant colony fea-
tures were observed, therefore MEA and MA4 were
used as the main diagnostic media for all Geosmithia
s. str. species.
RAPD-type I ‘pallida ’. Stipes (30–) 80–120 (–250)r
3–4 mm; penicillus branched 1–3 (–4)r,20–50r20–
50 mm, 1–4rlower than stipes; conidia (3) 3.5–4
(–4.5)r(1.5–) 2 mm ; texture colony on MEA funiculose
or velvety with funicules in the colony centre, sporu-
lation on MEA pale (2A2) or white.
RAPD-type II ‘macrospora ’. Conidia en masse yel-
lowish brown, light brown ; otherwise as the previous
RAPD-type I. Conidia of both types are larger than
those of other RAPD-types.
RAPD-type III ‘velutinosa ’. Stipes (100–) 240–280
(–500)r3.5–5 mm, rarely smaller ; penicillus well bran-
ched 3–5 (–6)r, 80–200r50–80 mm, 3–4rlower than
stipes, rami longer (to 80 mm) and asymmetric ; conidia
3–3.5 (–5)r(1–) 1.5–2; texture colony on MEA veluti-
nous (sometimes with small flocculi of aerial hyphae),
conidia en masse yellowish brown, light brown. Iso-
late AK 109/97 has typical colony pattern for this
group, but its lower stipe size, penicillus complexity,
and large conidia show affinities to RAPD-type V
‘funiculosa ’. Similarly, strain CCF 309 shows affinities
to RAPD-type II ‘ macrospora ’ in lower penicillus
complexity.
RAPD-type IV ‘nova ’ : Velvety with funiculose
zones; penicillus in IMI 191599 typically biverticillate
to terverticillate with stipes up to 150 mm height ;
otherwise similar to RAPD-type III.
Fig. 12. Strict consensus cladogram resulting from maximum
parsimony. Clades with bootstrap support under 50% were
collapsed to polytomies. Acremonium alternatum was used as
an outgroup.
M. Kolar
ˇı
´k and others 1061
Figs 13–24. Colonies on MEA and CYA at 25 xC, after 14 d in the dark. Figs. 13–18. Geosmithia pallida Fig. 13. CCF 309
(MEA). Fig. 14. CCF 3340 (MEA). Fig. 15. IMI 192499 (MEA). Fig. 16. AK 145/98 (CYA). Fig. 17. CCF 3340 (CYA).
Fig. 18. IMI 190744 (CYA). Figs 19–24. Geosmithia flava Fig. 19. MK 142 (MEA). Fig. 20. MK 353 (MEA). Fig. 21. CCF
3333 (MEA). Fig. 22. MK 142 (CYA). Fig. 23. IMI 224697 (CYA). Fig. 24. CCF 3333 (CYA).
Geosmithia putterillii s. lat. 1062
RAPD-type V ‘funiculosa ’. Stipes (10–) 30–120
(–200)r3.5–5 mm; penicillus branched 2–3 (–5)r30–
130r50–80 mm, 1–1.5rlower than stipes ; conidia (3)
3.5–4 (–4.5)r1.5–2 (–2.5), conidia en masse yellowish
brown, light brown ; texture colony on MEA funiculose
(mainly in the centre of a colony).
Distinctive features:G. pallida is characterized by
brown shades of sporulation on MEA (except RAPD-
type I) and CYA, submerged growth of the young col-
ony and its wet appearance, the presence of very tough
close-textured basal felt on both media, by the occur-
rence of smaller conidia (shorter than 4.0 mm) and by
the arrangement of conidial chains which are less per-
sistent than in other Geosmithia s. str. species and
become tangled with age. It is easily distinguishable
from other Geosmithia species. Colonies brown in ob-
verse, smaller conidia and conidial chains arrangement
set G. pallida apart from G. putterillii and G. lavendula.
G. pallida resembles some isolates of G. flava (MK 58)
in yellowish brown colour of sporulation and tough
basal felt, but it can be distinguished from G. flava by
the colour of sporulation on CYA, by conidium size
and shape and by arrangement of conidial chains.
G. pallida superficially resembles the phylogenetically
distant species G. argillacea in the production of small
cylindrical conidia coloured similarly en masse. Never-
theless, G. argillacea is thermophilic, it has acerose
phialides, often clearly clavate conidia and lacks peg
foot. In the past, G. pallida was rarely found, but
as illustrated by the origin of the isolates examined,
it is widely distributed. G. pallida appears to have
affinities with the wide range of subcorticolous insects,
like bark beetles and sawyer beetle Phoracantha semi-
punctata.
Geosmithia flava Kolar
ˇı
´k, Kuba
´tova
´& Pazˇ outova
´,
sp. nov. (Figs 19–24, 40–49)
Geosmithia putterillii (Thom) Pitt, Can. J. Bot.57 : 2022
(1979) pro parte.
Etym.: flava (Latin), refers to the yellowish colour of
sporulation on MEA and CYA.
Pars aversa coloniae culturarum CYA et MEA velutina vel
propinquua floccosa vel funiculosa; conidiogenesis abunda
vel moderata, flava; penicilli verruculosi, biverticillati usque
pentaverticillati; stipites 40–100r3–4.5 mm ; conidia cylin-
drica, ellipsoidea vel doliiformia 3.5–4r2–2.5 mm; catenae
usque 500 mm longae. Species similis Geosmithia putterillii sed
genetice isolata.
Typus:Czech Republic :Bohemia :Pr
ˇelouc
ˇ: forest near Se-
nı
´k, alt. 260 m, ex dead adult of Xiphydria sp. (Hymenoptera :
Siricidae) in sapwood of Castanea sativa, 30 Sept. 2000, M.
Kolar
ˇı´k(PRM 900980 – holotypus ; CCF 3333 (=MK 101)
ex-type culture; PRM 90098, PRM 900982, PRM 900983,
PRM 900984, PRM 900985 – isotypi. GenBank sequence
ex-type AJ578483.
Conidiophores on MEA arising from the subsurface,
surface or aerial mycelium, stipes determinate, erect,
40–100r3–4.5 mm, or indeterminate, verrucose, sep-
tate, arising from initials suggesting foot cells of As-
pergillus or smooth peg foot cell ; penicilli terverticillate
to quaterverticillate, but biverticillate or pentaverticil-
late types present in small number in some isolates,
often symmetric and compact (e.g. with phialides and
metulae of equal size) ; rami 15–20 mmr3–3.5 mm ; ra-
muli in verticils of 2–3, 10–15r3.0–3.5 mm, metulae in
well defined verticils of 3–5, 6–12r2.5–3.5 mm, usually
with verrucose walls ; conidiogenous cells phialides,
9–12r2–3 mm, 3–8 per metula, typically cylindrical
without distinct neck, walls verruculose to verrucose ;
conidia ellipsoidal or narrowly doliiform, (3–) 3.5–4
(–5)r(1.5–) 2–2.5 mm ; sometimes with a collarium,
conidial chains up to 500 mm in length, in well defined,
persistent and parallel columns. Atypical whole mon-
ilioid conidiophores occur in all isolates, on MEA
sometimes forming sporodochia like globose granules
(to 4 mm broad), second type of atypical conidiophores
with normal stipes but the penicilli are multiply bran-
ched, monilioid and do not produce conidia from
phialides, but only terminally delimitated fragments of
monilioid hyphae (arthroconidia) ; substrate conidia in
some isolates, 3.0–8.0r2.5–3.0 mm ; chlamydospore-
like conidia oval, thick walled, 5.0–15.0r5.0–8.0 mm,
1–2 celled, arising only on substrate mycelium.
MEA,25x,14 d: Colonies 40–65 mm diam, plane,
low or slightly raised centrally, surface texture veluti-
nous with crust of conidial chains (in age sometimes
adherent in masses), but often with floccose or funicu-
lose areas or wholly strongly funiculose ; margins
narrow or lobate, submerged (up to 2 mm broad);
Figs 25–32. Geosmithia pallida. Light micrograph of penicillii
and conidia on MEA, 7 d in the dark at 25 xC, mounted in
lactic acid with cotton blue (Fig. 25) (others). Fig. 25. CCF
2809, penicilli. Fig. 26. NRRL 2037, penicilli. Fig. 27. CCF
3341, penicilli. Fig. 28. CCF 3340, conidia. Fig. 29. CCF 2809,
conidia. Fig. 30. CCF 3341, conidia. Fig. 31. AK 200/98,
conidia. Fig. 32. IMI 0541240b, conidia. Bar=10 mm.
M. Kolar
ˇı
´k and others 1063
mycelium white or yellowish white (2A2) ; substrate
mycelium sparse or dense, sometimes form tough basal
felt; conidiogenesis very heavy to heavy, light yellow
(2–4 A 4–5); exudate absent ; soluble pigment absent or
light yellow (1–4 A 4–5); reverse sulphur yellow (1A5),
light yellow, dull yellow or brownish orange (6C8)
in age.
MEA,37x,14 d: No growth.
CYA,25x,14 d: Colonies 35–40 mm diam in freshly
isolated strains, but to 65 mm diam after several sub-
cultures, plane or radially furrowed, raised centrally ;
surface texture mostly velutinous with a crust of
conidia, or granular to nearly floccose with small funi-
cules at the colony margin (often in freshly isolated
strains), or strongly funiculose ; mycelium white or
rusty (sometimes forming rusty ropes in colony centre),
Figs 33–39. Geosmithia pallida.Figs 33–34. RAPD-type II (CCF 3340), penicilli and conidia. Fig. 35. RAPD-type I (NRRL
2037), penicilli and conidia. Figs 36–37. RAPD-type V (CCF 3341), penicilli and conidia. Figs 38–39. Penicilli and conidia
of RAPD-type III (CCF 2809). Bars: Figs 34–36, 38=10 mm ; Figs 33, 37, 39=50 mm.
Geosmithia putterillii s. lat. 1064
conidiogenesis very heavy to heavy, typically deep yel-
low (4A8) or yolk yellow (4B8), rarely salmon (6A4) ;
exudate clear, uncoloured or rusty ; soluble pigment
amber yellow (4B6) to light brown (6D8) or absent ;
reverse dull yellow, yellow-ochre (5C7), light brown or
darker centrally.
Teleomorph: Unknown.
Habitat: In galleries and in surrounding (phloem and
sapwood) of bark beetles feedings, or other insects
(Xiphydria), rarely from various plant substrates.
Known association see Table 1.
Distribution : Known from the Czech Republic,
Slovakia, Hungary, France and from UK.
Infraspecific variability : Our isolates are largely
micromorphologicaly uniform. Colony texture and
diameter on both media vary from velutinous to vel-
utinous with floccose and funiculose areas, depending
on isolate or as a consequence of many transfers. The
salmon colony colour and completely floccose texture
on CYA occurred only in the freshly isolated type
strain. After several subculturings, the colony was
velvety and yellowish, with light salmon shades only.
Both isolates from the UK (IMI 224697, IMI 158645)
are typical in the high frequency of well-branched
monilioid penicilli. Production of chlamydospore-like
conidia on substrate mycelium is known only from
some isolates, and seems to be fairly uncommon in this
species.
Distinctive features :G. flava is typical by yellow
shades of sporulation on MEA and CYA, a presence
of rusty coloured aerial mycelium on CYA, slightly
doliiform conidia (in most of the isolates) and by a
production of monilioid conidiophores. G. flava
resembles G. putterillii by ¡yellow sporulation on
MEA, penicillus type and production of substrate
conidia, whereas G. putterillii differs by longer and
thinner conidia (4–4.5r1.5–2 mm), and often by ab-
sence of yellow shades in conidia produced on CYA.
Geosmithia putterillii (Thom) Pitt, Can. J. Bot.57 : 2022
(1979). (Figs 50–63)
Penicillium putterillii Thom, Penicillia : 368 (1930).
Conidiophores on MEA arising from the subsurface,
surface or aerial mycelium, stipes determinate, erect,
15–100r3–5 mm, arising from peg foot or from initials
suggesting foot cells of Aspergillus ;penicilli mono-
verticillate (those arising from aerial mycelium), bi-
verticillate or terverticillate, often asymmetric ; rami
(first branch) often 10–20r3–4 mm; metulae in verticils
of 2–3, 7–15r2.0–4.0 mm, usually with verrucose
walls; conidiogenous cells phialides 7–13r2mm, 3–7 per
metula, typically cylindrical without distinct neck (ab-
ruptly tapering to an apical pore), walls verruculose to
verrucose; conidia cylindrical to ellipsoidal, (3.5–) 4–4.5
(–5.5)r(1–) 1.5 (–2) mm, conidial chains up to 400 mm
in length, in well defined, persistent and parallel col-
umns; substrate conidia present, 3–8r2.5–3 mm.
Figs 40–44. Geosmithia flava. Light micrograph of penicillii
and conidia on MEA, 7 d in the dark at 25 xC, mounted in
lactic acid with cotton blue (Fig. 41) or in Melzer’s reagent
(others). Figs 40, 41. CCF 3333, penicilli. Fig. 42. IMI 224697,
conidia. Fig. 43. CCF 3333, conidia. Fig. 44. MK 353, co-
nidia, showing collarettes (arrows). Bar=10 mm.
Figs 45–49. Geosmithia flava (CCF 3333). Fig. 45. Conidia.
Fig. 46. Penicilli. Fig. 47. chlamydospore-like conidia on
substrate mycelium. Fig. 48. Conidial chains. Fig. 49. Peg
foot, with a collapsing branch of parent hypha (arrow). Bars :
Figs 45–46=10 mm, Figs 47–49=50 mm.
M. Kolar
ˇı
´k and others 1065
MEA,25 x,14 d: Colonies 35–50 mm diam, plane or
radially furrowed, typically raised centrally, with the
cerebriform pattern in centre ; surface texture velutinous
with crust of conidial chains, with slightly funiculous
zones; margins narrow or lobate, submerged (up to
2 mm broad); mycelium white ; substrate mycelium
sparse and not forming tough basal felt ; conidiogenesis
very heavy, white to pale yellow (2A3), yolk yellow
(4A3), sometimes pale orange (5A3) ; exudate absent or
uncoloured, dull; soluble pigment absent or amber yel-
low (4B6); reverse light yellow to yellow-ochre (5C7).
MEA,37 x,14 d: No growth.
CYA,25 x,14 d: Colonies 50–65 mm, radially fur-
rowed, raised centrally ; surface texture mostly
velutinous with crust of conidia, zonate, central area
wrinkled, sometimes flocosse or funiculose, marginal
area with small funicules, margins narrow, 3 mm broad
submerged area; mycelium white, conidiogenesis very
heavy, white or orange–white (5A2) to yellowish white
(2A2); exudate absent ; soluble pigment absent; reverse
light yellow to yellow–ochre.
Infraspecific variability : Only three genetically nearly
identical strains of this species are known. Strains CCF
3342 and CCF 3442 show some difference in colony
colour (yellowish sporulation) and the conidiophores
are generally large than those of NRRL 2024.
Distinctive features :G. putterillii is characterised by
cylindrical, long and narrow conidia. G. putterillii is
morphologically similar to G. flava. These two species
have a similar colony texture, branched conidiophores,
and parallel conidial columns. Conidia of both species
en masse are shades of yellow on MEA. Both species
produce substrate conidia. The morphological delimi-
tation of these two species is mentioned above. G. put-
terillii appears to be rather uncommon.
DISCUSSION
The foot cell of Aspergillus species is a part of the single
celled stipe, although it may be in some cases delimited
by a septum (Minter et al. 1985). The latter case is
Figs 50–53. Geosmithia putterillii. Colonies on MEA and CYA at 25 xC, after 14 d in the dark. Fig. 50. NRRL 2024 (MEA).
Fig. 51. CCF 3342 (MEA). Fig. 52. CCF 3342 (CYA). Fig. 53. NRRL 2024 (CYA). Figs 54–58. Light micrograph of
penicilli and conidia on MEA, 7 d in the dark at 25 xC. Fig. 54. CCF 3342, terverticillate penicillus. Fig. 55. NRRL 2037,
biverticillate penicilli. Fig. 56. CCF 3342, conidia. Figs 57, 58. NRRL 2024, conidia. Bar=10 mm.
Geosmithia putterillii s. lat. 1066
morphologically identical with initials observed in
Penicillium varians and in all species of Geosmithia s.
lat. However, the term ‘foot cell-like initials ’ is used
here instead of the term foot cell, which we prefer
for Aspergillus descriptions. Footcell- like initials of
Stachybotrys and Gibellula, anamorphs belonging to
Hypocreales, have been observed (Kuba
´tova
´1994,
Tzean, Hsieh & Wu 1997), but their nature is not yet
known and a further examination of their ontogenesis
is needed. A variant of the foot cell-like initial is rep-
resented here by a newly established character called
the ‘peg foot ’, which is a useful taxonomical character
typical of Geosmithia s. str. It is never a part of the
horizontal parent hypha, and it was found only in
G. putterillii,G. pallida, and G. flava.
The sexual state of Geosmithia is not known, but
based on the phylogenetical affinities of G. putterillii
(Rossman et al. 1999), a cleistothecial teleomorph
could be expected. Formation of colony sectors with
the mycelial or conidial type described as showing a
‘dual phenomenon ’ by Hansen (1938), was seen in
Geosmithia and has been observed in many fungal
genera, including Fusarium (Nelson et al. 1983), Peni-
cillium (Raper & Thom 1949, Ramirez 1982) or G.
argillacea (Stolk 1969). It is probably caused by
mutation, but the molecular basis of this phenomenon
remains unclear (Kistler & Miao 1992).
Production of substrate conidia increases the number
of diaspores and facilitates dispersal by insects. The
presence of more than two independent modes of
conidiogenesis (pleomorphism) is common in deuter-
omycetes, e.g. Botryotrichum piluliferum forms both
solitary holoblastic conidia and basipetal chains of
enteroblastic propagules. We suspect that substrate
conidia in Geosmithia are analogues of those observed
in Acremonium kiliense and A. strictum (Gams 1971)
and in Pochonia suchlasporia var. suchlasporia (Zare,
Gams & Evans 2001). Substrate conidia originate
from the conidiogenous cells that resemble the adelo-
phialides of Acremonium kiliense (Gams 1971), but
conidiogenesis in Geosmithia is holoblastic.
The name G. pallida is based on the ex-type culture
NRRL 2037 used by Smith (1933), Raper & Thom
(1949) and Ramirez (1982) which represents Penicillium
pallidum. Their descriptions of ex-type strain are quite
similar to our observations. NRRL 2037 differs only
slightly in its colony morphology (i.e. the colour on
MEA, and the presence of a soluble pigment) from that
described by Ramirez. However, his description was
based on two strains, one of which (CBS 248.32) has
not been at our disposal. In our reinvestigation of the
ex-type strain of G. pallida, we found smaller conidia
(3–4r1.5–2 mm), as observed by both authors men-
tioned above. Pitt (1979) did not note the smaller
conidial size and the brown colour of the sporulation
observed in the G. pallida strains used in his study, in
his G. putterillii concept. Pitt considered P. pallidum a
synonym of G. putterillii, but using his key G. pallida
strains fail to be identified as G. putterillii.
Our results show that in G. pallida brown coloured
colonies (especially on CYA) and small conidia are
typical, whereas G. putterillii is a lighter species with
white to light yellow colonies and large conidia. The
integration of molecular and morphological characters
shows that G. pallida is a distinct and readily recognis-
able species, different from G. putterillii. Moreover, it
supports a morphological species concept congruent
with the biological species concept. G. pallida includes
five RAPD-types, but it is not yet clear whether they
represent reproductively isolated lineages. The presence
of isolates with phenotypes intermediate between some
of the RAPD-types indicates that recombination may
occur. Therefore, we have not chosen to propose for-
mal names for these groups (e.g. as varieties), despite of
their distinctive individuality.
The ex-type culture of P. putterillii, isolated from
fruit storage in South Africa, was soon lost (Thom
1930). Pitt (1979) neotypified P. putterillii (and so G.
putterillii) on NRRL 2024, the isolate on which the
description by Raper & Thom (1949) was based. Our
observations mostly correspond with the description of
Raper & Thom (1949). The large conidia typical for
this group were also observed by Thom (1930), Raper
& Thom (4–5r2–3 mm) and by Ramirez (4–6.5r1.5–
2.5 mm). Here, we present a more homogenous concept
of G. putterillii than that of Pitt (1979). Pitt’s descrip-
tion is based on ten strains, seven of which were treated
in our study and belong to G. pallida (NRRL 2037, IMI
190744, IMI 155478a, IMI 191599, IMI 051240b),
Figs 59–63. Geosmithia putterillii (CCF 3342). Fig. 59.
Conidial chains. Fig. 60. Penicillus arising from aerial my-
celium. Fig. 61. Penicilli arising from substrate mycelium.
Fig. 62. Substrate conidia. Fig. 63. Conidia. Bars : Figs
61–63=10 mm ; Figs 59–60=50 mm.
M. Kolar
ˇı
´k and others 1067
G. putterillii (NRRL 2024) and G. flava (IMI 158645).
Substrate conidia were found in all G. putterillii isolates,
and were probably overlooked by previous authors.
The G. flava phenotype resembles that of G. putter-
illii, but DNA analysis shows that they are fairly un-
related. We do not pretend to have clearly solved the
morphological delimitation between G. flava and G.
putterillii. Some isolates could not have been classified
unequivocally according to their morphology, but their
RAPD patterns and rDNA-ITS sequences identified
them as G. flava. Therefore, we decided to introduce G.
flava as a new species, despite the difficult morpho-
logical distinction from G. putterillii.
The distribution of isolates within RAPD-types
shows, that there is no geographical or habitat speci-
ficity. Recently published PCR-fingerprinting-based
phylogeny of other fungi associated with insects dem-
onstrated a correlation between the identified phylo-
genetic groups and their geographical origin (Obornı
´k
et al. 1999) or host insect range (Maurer et al. 1997). On
the other hand, many insect-associated fungi, such as
Beauveria bassiana (Gaitan et al. 2002) or Paecilomyces
do not exhibit any such correlation (Cantone & Van-
denberg 1998, Chew et al. 1998). We suspect that the
model of genetic variation of Geosmithia species is
similar to those of Paecilomyces spp. (Obornı
´ket al.
2000). The high mobility of conidia, encountered in
both genera, supports a cosmopolitan distribution,
absence of geographically conserved populations and
wide substratum range. The high stability of RAPD
characters among the strains of wide geographic sep-
aration indicates the stability of newly differentiated
species and their effective clonal isolation, which occurs
in Geosmithia spp. despite their sympatry. This could
be due to heterokaryon incompatibility as has been
reported for other fungi such as B. bassiana (Paccola-
Meirelles & Azevedo 1991) and Trichoderma spp.
(Turner et al. 1997).
We have unpublished results that show that there are
many other Geosmithia s. str. species (including G.
lavendula), associated with bark beetles, and that this
niche is still relatively unexplored. There are few studies
dealing with the diversity of non-ophiostomatoid or
non-entomopathogenetic fungi, and even fewer have
addressed those from subcorticolous insects without
phytopathogenic importance. We believe that this study
will serve as a basis for further work on Geosmithia, and
other fungi associated with subcorticolous insects.
ACKNOWLEDGEMENTS
This project was supported by Czech Grant Agency grant 522/02/
1206, 206/03/H137 and by the Institutional Research Concept No.
AV0Z5020903. We thank Martha Christensen and Dorothy B.
Tuthill (University of Wyoming), Ronald Kirschner (J. W. Goethe-
Universita
¨t Frankfurt am Main) and Zofia Kozakiewicz (CABI
Bioscience) for providing Geosmithia strains; and to Zdene
`k Pouzar
(National Museum, Prague) for the critical reading of the manuscript
and for providing the Latin diagnoses; and to Milos
ˇKnizˇ ek for the
bark beetle determinations.
REFERENCES
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z.,
Miller, W. & Lipman, D. J. (1997) Gapped BLAST and PSI-
BLAST: a new generation of protein database search programs.
Nucleic Acids Research 25: 3389–3402.
Cantone, F. A. & Vandenberg, J. D. (1998) Intraspecific diversity in
Paecilomyces fumosoroseus.Mycological Research 102: 209–215.
Chew, J. S. K., Strongman, D. B. & Mackay, R. M. (1998) Compari-
sons of twenty isolates of the entomopathogen Paecilomyces far-
inosus by analysis of the RAPD markers. Mycological Research
102: 1254–1258.
Cole, G. T., Nag Raj, T. R. & Kendrick, W. B. (1969) A simple
technique for time-lapse photomicrography of microfungi in plate
culture. Mycologia 61: 726–730.
Fassatiova
´, O. (1986) Moulds and Filamentous Fungi in Technical
Microbiology. Elsevier, Amsterdam.
Felsenstein, J. (1993) PHYLIP phylogeny inference package. Version
3.5c. J. Felsenstein, Department of Genetics, University of
Washington, Seattle.
Gaitan, A., Valderrama, A. M., Saldarriaga, G., Velez, P. & Bustillio,
A. (2002) Genetic variability of Beauveria bassiana associated with
the Coffee Berry Borer Hypothenemus hampei and other insect.
Mycological Research 106: 1307–1314.
Gams, W. (1971) Cephalosporium-artige Schimmelpilze (Hyphomy-
cetes). Gustav Fischer Verlag, Stuttgart.
Hall, T. A. (1999) BioEdit: a user-friendly biological sequence align-
ment editor and analysis program for Windows 95/98/NT. Nucleic
Acids Symposium Series 41: 95–98.
Hansen, H. N. (1938) The dual phenomenon in imperfect fungi.
Mycologia 30: 442–455.
Hughey, R. & Krogh, A. (1996) Hidden Markov models for sequence
analysis: extension and analysis of the basic method. Computer
Applications in the Biosciences 12: 95–107.
Iwamoto, S., Tokumasu, S., Suyama, Y. & Kakishima, M. (2002)
Molecular phylogeny of four selected species of the strictly ana-
morphic genus Thysanophora using nuclear ribosomal DNA se-
quences. Mycoscience 43: 169–180.
Kirschner, R. (1998) Diversita
¨t mit Borkenka
¨fern assoziierter fila-
mento
¨ser Mikropilze. PhD thesis, Eberhard-Karls Universita
¨t,
Tu
¨bingen.
Kirschner, R. (2001) Diversity of filamentous fungi in bark beetle
galleries in Central Europe. In Trichomycetes and Other Fungal
Groups (J. K. Misra & B. W. Horn, eds) : 175–196. Science Pub-
lishers, Enfield, NH.
Kistler, J. A. & Miao, V. P. W. (1992) New modes of genetic change in
filamentous fungi. Annual Reviews of Phytopathology 30: 131–152.
Kolar
ˇı
´k, M. (2002) Ekologie a taxonomie hypokrea
´lnı´ch za
´stupcu
˚rodu
Geosmithia. Dipl. Thesis, Department of Botany, Charles Univer-
sity, Prague.
Kornerup, A. & Wanscher, J. H. (1981) Taschenlexikon der Farben.
Muster–Schmidt Verlag, Go
¨ttingen.
Krogh, A., Brown, M., Mian, I. S., Sjolander, K. & Haussler, D. (1994)
Hidden Markov models in computational biology: Applications to
protein modeling. Journal of Molecular Biology 235: 1501–1531.
Kuba
´tova
´, A. (1994) New records of micromycetes from Czech and
Slovak Republic. III. Acremonium furcatum,Gonatobotryum para-
siticum,Stachybotrys bisbyi, and Wardomyces inflatus.Czech My-
cology 47: 151–158.
Kuba
´tova
´, A., Novotny´ , D. & Pra
´s
ˇil, K. (1999) Be
ˇlokaz dubovy´ jako
pr
ˇenas
ˇec
ˇmikroskopicky´ ch hub. In Houby a Les (L. Jankovsky´ ,R.
Krejc
ˇı
´r
ˇ& V. Antonı
´n, eds): 235–236. Mendel University of Agri-
culture and Forestry, Brno.
Kuba
´tova
´, A., Novotny´ , D. & Pra
´s
ˇil, K. (2002) Microscopic fungi
associated with oak bark beetle (Scolytus intricatus) in the Czech
Republic. IMC7 Book of Abstracts: 300. International Mycological
Congress, Oslo.
Kuba
´tova
´, A., Kolar
ˇı
´k, M., Pra
´s
ˇil, K. & Novotny´ , D. (2004) Bark
beetles and their galleries: well-known niche for little known fungi,
case of Geosmithia.Czech Mycology 55: in press.
Geosmithia putterillii s. lat. 1068
Maurer, P., Couteaudier, Y., Girard, P. A., Bridge, P. D. & Riba, G.
(1997) Genetic diversity of Beauveria bassiana and relatedness to
host insect range. Mycological Research 101 : 159–164.
Minter, D. W., Hawksworth, D. L., Onions, A. H. S. & Kozakiewicz,
Z. (1985) Descriptive terminology of the conidiogenous structures
in Aspergillus and Penicillium.InAdvances in Penicillium and
Aspergillus Systematics (R. A. Samson & J. I. Pitt, eds): 73–86.
Plenum Press, New York.
Nelson, P. E., Toussoun, T. A. & Marasas, W. F. O. (1983) Fusarium
species: an illustrated manual for identification. Pennsylvania State
University Press, University Park.
Nei, M. & Li, W.-H. (1979) Mathematical model for studying genetic
variation in terms of restriction endonucleases. Proceedings of the
National Academy of Sciences, USA 76: 5269–5273.
Obornı
´k, M., Stouthamer, R., Meekes, E. & Schilthuittzen, M.
(1999) Molecular characterization and phylogeny of the en-
tomopathogenic fungus Aschersonia spp. Plant Protection Science
35: 1–9.
Obornı
´k, M., Klı
´c
ˇ,M.&Z
ˇizˇ ka, L. (2000) Genetic variability and
phylogeny inferred from random amplified polymorphic DNA
data reflect life strategy of entomopatogenic fungi. Canadian
Journal of Botany 78 : 1150–1155.
Ogawa, H., Yoshimura, A. & Sugiyama, J. (1997) Polyphyletic
origins of species of the anamorphic genus Geosmithia and
the relationships of the cleistothecial genera: Evidence from
18S, 5S and 28S rDNA sequence analyses. Mycologia 89 :
756–771.
Ogawa, H. & Sugiyama, J. (2000) Evolutionary relationships of the
cleistothecial genera with Penicillium,Geosmithia,Merimbla and
Sarophorum anamorphs as inferred from 18S rDNA sequence
divergence. In Integration of Modern Taxonomic Methods for
Penicillium and Aspergillus classification (R. A. Samson & J. I. Pitt,
eds): 149–162. Harwood Publishers, Reading.
Paccola-Meirelles, L. D. & Azevedo, J. L. (1991) Parasexuality in
Beauveria bassiana.Journal of Invertebrate Pathology 57:
172–176.
Pazˇ outova
´, S., Bandyopadhyay, R., Frederickson, D. E. & Mantle,
P. G. (2000a) Relations among sorghum ergot isolates from the
Americas, Africa, India, and Australia. Plant Disease 84 : 437–442.
Pazˇ outova
´, S., Ols
ˇovska
´, J., Linka, M., Kolı
´nska
´, R. & Flieger, M.
(2000b) Chemoraces and habitat specialization of Claviceps pur-
purea populations. Applied and Environmental Microbiology 66:
5419–5425.
Peterson, S. W. (2000) Phylogenetic analysis of Penicillium species
based on ITS and LSU–rDNA nucleotide sequences. In Integration
of Modern Taxonomic Methods for Penicillium and Aspergillus
classification (R. A. Samson & J. I. Pitt, eds): 163–178. Harwood
Academic Publishers, Reading.
Pitt, J. I. (1979) Geosmithia gen. nov. for Penicillium lavendulum and
related species. Canadian Journal of Botany 57: 2021–2030.
Pitt, J. I. (1980) [‘ 1979 ’] The genus Penicillium and its teleomorphic
states Eupenicillium and Talaromyces. Academic Press, London.
Pitt, J. I. & Hocking, A. D. (1985) Interfaces among genera related to
Aspergillus and Penicillium.Mycologia 77: 810–824.
Pitt, J. I. & Hocking, A. D. (1997) Fungi and Food Spoilage, 2nd edn.
Blackie Academic and Professional, London.
Prieto, A., Ahrazem, O., Gomez-Miranda, B., Bernabe, B., Bernabe,
M. & Leal, J. A. (2002) Cell wall polysaccharides F1SS disclose
the relatedness of the genus Geosmithia with Eupenicillium and
Talaromyces.Canadian Journal of Botany 80: 410–415.
Ramirez, C. (1982) Manual and Atlas of the Penicillia. Elsevier
Biomedical Press, New York.
Raper, K. & Thom, B. (1949) A Manual of the Penicillia. Williams &
Wilkins, Baltimore.
Rossman, A. Y., Samuels, G. J., Rogerson, C. T. & Lowen, R. (1999)
Genera of Bionectriaceae,Hypocreaceae, and Nectriaceae (Hypo-
creales,Ascomycetes). Studies in Mycology 42: 1–238.
Rossman, A. Y., McKemy, J. M. & Pardo-Schultheiss, R. A. (2001)
Molecular studies of the Bionectriaceae using large subunit rDNA
sequences. Mycologia 93: 100–110.
Smith, G. (1933) Some new species of Penicillium.Transactions of the
British Mycological Society 18: 88–91.
Sneath, P. H. A. & Sokal, R. R. (1973) Numerical Taxonomy.W.H.
Freeman, San Francisco.
Stolk, A. C. (1969) Penicillium argillaceum sp. nov., a thermotolerant
Penicillium. Transactions of the British Mycological Society 53:
307–311.
Strimmer, K. & von Haeseler, A. (1996) Quartet puzzling : a quartet
maximum likelihood method for reconstructing tree topologies.
Molecular Biology and Evolution 13: 964–969.
Tamura, K. & Nei, M. (1993) Estimation of the number of nucleotide
substitutions in the control region of mitochondrial DNA in hu-
mans and chimpanzees. Molecular Biology and Evolution 10:
512–526.
Thom, Ch. (1930) The Penicillia. Williams & Wilkins, Baltimore.
Turner, D., Kovacs, W., Kuhls, K., Lieckfeldt, E., Peter, B., Arisan-
Atac, I., Strauss, J., Samuels, G. J., Bo
¨rner, T. & Kubicek, C. P.
(1997) Biogeography and phenotypic variation in Trichoderma
sect. Longibrachiatum and associated Hypocrea species. Mycologi-
cal Research 101: 449–459.
Tzean, S. S., Hsieh, L. S. & Wu W. J. (1997) Atlas of Entomo-
pathogenic Fungi from Taiwan. Council of Agriculture Executive,
Yuan, Taipei.
van de Peer, Y. & de Wachter, R. (1994) TREECON for Windows : a
software package for the construction and drawing of evolutionary
trees for the Microsoft Windows environment. Computer Appli-
cations in the Biosciences 10 : 569–570.
Yaguchi, T., Miyadoh, S. & Udagawa, S.-I. (1993) Chromocleista,a
new cleistothecial genus with a Geosmithia anamorph. Transactions
of the Mycological Society of Japan 34: 101–108.
Yaguchi, T., Someya, A. & Udagawa, S.-I. (1994) Two new species of
Talaromyces from Taiwan and Japan. Mycoscience 35: 249–255.
Zare, R., Gams, W. & Evans, H. C. (2001) A revision of Verticillium
section Prostrata. V. The genus Pochonia, with notes on Rotifer-
ophthora.Nova Hedwigia 73: 51–86.
Corresponding Editor: D. L. Hawksworth
M. Kolar
ˇı
´k and others 1069
