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STUDIES IN MYCOLOGY 50: 415–430. 2004.
Calonectria species and their Cylindrocladium anamorphs: species with
sphaeropedunculate vesicles
Pedro W. Crous1*, Johannes Z. Groenewald1, Jean-Michel Risède2, Philippe Simoneau3 and Nigel L.
Hywel-Jones4
1Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands;
2CIRAD-FLHOR, Station de Neufchâteau, 97130 Capesterre Belle Eau, Guadeloupe, French West Indies; 3UMR PaVé N°77-
Faculté des Sciences, Université d’Angers 2, Bd Lavoisier 49045 Angers cedex, France; 4Biotec Central Research Unit,
National Center for Genetic Engineering and Biotechnology, 113 Paholyothin Rd., Klong 1, Klong Luang, Pathum Thani
12120, Thailand
*Correspondence: Pedro W. Crous, crous@cbs.knaw.nl
Abstract: Species of Cylindrocladium have wide host ranges, and are commonly distributed in soils of tropical and subtropi-
cal regions of the world. In the present study several isolates, which have been baited from soils from various parts of the
world, are compared based on morphology, as well as DNA sequence data from their β-tubulin, histone, elongation factor 1-α
and calmodulin gene regions. As a result of these studies, eight new species with sphaeropedunculate vesicles and 1-septate
conidia are described. An emended key is provided to distinguish these species from others in the Cy. floridanum species
complex.
Taxonomic novelties: Calonectria asiatica Crous & N.L. Hywel-Jones sp. nov. (anamorph Cylindrocladium asiaticum
Crous & N.L. Hywel-Jones sp. nov.), Calonectria colombiensis Crous sp. nov. (anamorph Cylindrocladium colombiense
Crous sp. nov.), Calonectria hongkongensis Crous sp. nov. (anamorph Cylindrocladium hongkongense Crous sp. nov.),
Cylindrocladium chinense Crous sp. nov., Cylindrocladium indonesiae Crous sp. nov., Cylindrocladium malesianum Crous
sp. nov., Cylindrocladium multiphialidicum Crous, P. Simoneau & J.-M. Risède sp. nov., Cylindrocladium sumatrense Crous
sp. nov.
Key words: Ascomycetes, Calonectria, Cylindrocladium, Hypocreales, leaf spots, soil fungi, systematics.
INTRODUCTION
Species of Cylindrocladium Morgan (Cy.) are com-
monly associated with a wide range of disease symp-
toms, including leaf spot, stem rot, canker, blight,
root and pod rot, to name but a few (Crous 2002).
Wherever sexual reproduction is known to occur,
species of Cylindrocladium have Calonectria De Not.
(Ca.) (Nectriaceae, Hypocreales, Ascomycetes)
teleomorphs (Rossman 1979). These have been
reported for 28 of the 41 species currently recognized
(Crous 2002, Crous et al. 2002). In the past, species
were chiefly identified based on the morphology of
their anamorph (Peerally 1991). In recent years a
more integrated approach has been advocated, inte-
grating morphology with DNA sequence data and
sexual compatibility studies. New molecular data
sets, however, have indicated considerable variation
that was easily overlooked when morphology and
sexual compatibility were employed as sole charac-
ters (Schoch et al. 1999, 2001, Kang et al. 2001). As
shown for Cy. gordoniae Leahy, T.S. Shub. & El-
Gholl (Crous et al. 2002), as well as several strains of
Cy. insulare C.L. Schoch & Crous (Schoch et al.
2001), minute morphological differences that could
be perceived as variation within a morphological or
biological species could, in fact, be indicative of
distinct but closely related species. Such species
frequently remain sexually compatible with the other
biological species, but are clearly separable based on
molecular data.
Cylindrocladium species with sphaeropedunculate
to globose vesicles have been reported as pathogens
from a wide range of hosts in most tropical to sub-
tropical countries (Victor et al. 1997, Crous 2002).
To address speciation within this complex, Kang et
al. (2001) used multi-allelic sequence data to deline-
ate species within the Cy. floridanum Sobers & C.P.
Seym. and Cy. spathiphylli Schoult., El-Gholl &
Alfieri species complexes, describing Cy. canadense
J.C. Kang, Crous & C.L. Schoch, Cy. pacificum J.C.
Kang, Crous & C.L. Schoch and Cy. pseudospathi-
phylli J.C. Kang, Crous & C.L. Schoch as new spe-
cies. As noted by Kang et al. (2001), however, some
isolates did not fit in either of these taxa. This was
also the case for some recent collections of Cylindro-
cladium isolates with sphaeropedunculate vesicles
that were obtained from various localities. The aim
of the present study, therefore, was to analyze these
strains by means of morphology, and DNA sequence
415
CROUS ET AL.
416
analysis of their β-tubulin, calmodulin, elongation
factor 1-α and histone gene regions.
MATERIALS AND METHODS
Isolates
Isolates were obtained from debris, or baited from
soil as explained in Crous (2002). They were studied
on divided plates containing 2 % malt extract agar
(MEA) (2 g/L) (Biolab, Midrand, South Africa) in
one half, and carnation leaf agar (CLA) [1 % water
agar (1 g/L) (Biolab) with autoclaved carnation
leaves placed onto the medium] in the other. These
plates were incubated for 7 d at 25 °C under continu-
ous near-UV light, to promote sporulation.
DNA phylogeny
The protocol of Lee & Taylor (1990) was used to
isolate genomic DNA from fungal mycelium grown
on MEA plates. Four loci were amplified, namely,
part of the β-tubulin gene, amplified with primers T1
(O’Donnell & Cigelnik 1997) and Bt-2b (Glass &
Donaldson 1995); part of the histone 3 (H3) gene
with primers H3-1a and H3-1b (Glass & Donaldson
1995); part of the elongation factor 1-α gene with
primers EF1-728F and EF1-986R (Carbone & Kohn
1999), and part of the calmodulin gene with primers
CAL-228F and CAL-737R (Carbone & Kohn 1999).
However, some of these primer pairs failed to am-
plify with all of the isolates included in this study,
and therefore new primers were designed. For β-
tubulin, we designed a primer (CYLTUB1R: 5’-AGT
TGT CGG GAC GGA AGA G-3’) annealing at a
position 58 nt internal to the first nucleotide position
of primer Bt-2b (nucleotide positions 371–389 of
GenBank sequence AY305703). For histone two
primers were designed, a forward primer (CYLH3F:
5’-AGG TCC ACT GGT GGC AAG-3’; nucleotide
positions 1675–1692 of GenBank sequence
AY062173) annealing at a point 18 nt internal to the
first nucleotide position of primer H3-1a, and a
reverse primer (CYLH3R: 5’-AGC TGG ATG TCC
TTG GAC TG-3’; nucleotide positions 1273–1292 of
GenBank sequence AY062173) annealing at seven nt
internal to the first nucleotide position of primer H3-
1b. A new reverse primer (CylEF-R2: 5’-CAT GTT
CTT GAT GAA (A/G)TC ACG-3’; nucleotide posi-
tions 783–803 of GenBank sequence X96615) that
allows amplification of an extended region of the 3'
end of the elongation factor gene was designed from
sequences available on GenBank. The PCR reaction
mixture used to amplify the different loci consisted of
0.5 units Biotaq polymerase (Bioline, London, U.K.),
1× PCR buffer, 0.5–1.5 mM MgCl2, 0.2 mM of each
dNTP, 5 pmol of each primer, approximately 10 to 30
ng of fungal genomic DNA and was made up to a
total volume of 25 µL with sterile water. Reactions
were performed on a GeneAmp PCR System 9700
(Applied Biosystems, Foster City, CA) with cycling
conditions consisting of denaturation for 5 min at 96
°C, followed by 30 cycles at 96 °C (30 s), 52 °C (30
s), and 72 °C (60 s), with a final 5 min extension step
at 72 °C to complete the reaction. PCR conditions
were the same for all loci, except that the MgCl2
concentration varied. Histone and elongation factor
amplifications used 1.0 mM MgCl2; β-tubulin used
0.5 mM and calmodulin used 1.5 mM. PCR products
were separated by electrophoresis at 80 V for 1 h in a
0.8 % (w/v) agarose gel in 0.5 × TAE running buffer
(0.4 M Tris, 0.05 M NaAc, and 0.01 M EDTA, pH
7.85) and visualised under UV light using a
GeneGenius Gel Documentation and Analysis Sys-
tem (Syngene, Cambridge, U.K.) following ethidium
bromide staining. The amplification products were
purified according to the manufacturer’s instructions
using a commercial kit (GFX PCR DNA and Gel
Band Purification Kit, Amersham Pharmacia Biotech
Europe GmbH, Freiburg, Germany). Sequencing
reactions were carried out using the PCR primers in
ABI PRISM Big Dye Terminator Cycle v. 3.0 Se-
quencing Ready Reaction Kit (Applied Biosystems)
according to the manufacturer’s recommendations.
The reaction was analysed on an ABI Prism 3100
Genetic Analyser (Applied Biosystems).
The sequences generated in this study were added
to other sequences obtained from GenBank
(http://www.ncbi.nlm.nih.gov) and TreeBASE
(http://www.treebase.org) and the alignment was
assembled using Sequence Alignment Editor v.
2.0a11 (Rambaut 2002) with manual adjustments for
improvement made visually where necessary. Se-
quences for Cylindrocladiella peruviana (Bat., J.L.
Bezerra & M.M.P. Herrera) Boesew. and Cylindro-
cladiella lageniformis Crous, M.J. Wingf. & Alfenas
were added to the alignments as outgroups. The
phylogenetic analyses of sequence data were done
using PAUP (Phylogenetic Analysis Using Parsi-
mony) v. 4.0b10 (Swofford 2000). Phylogenetic
analysis of both datasets in PAUP consisted of dis-
tance and parsimony analysis. For distance analysis,
neighbour-joining with the uncorrected (“p”), the
Jukes-Cantor and the Kimura 2-parameter substitu-
tion model were performed. Alignment gaps were
treated as missing data and all characters were unor-
dered and of equal weight. Any ties were broken
randomly when encountered. For parsimony analy-
sis, alignment gaps were treated as a fifth character
state and all characters were unordered and of equal
weight. A heuristic search was performed for each
dataset with 100 random taxon additions and tree
bisection and reconstruction (TBR) as the branch
swapping algorithm. Branches of zero-length were
collapsed and all multiple, equally parsimonious trees
were saved. Measures calculated for parsimony
included tree length, consistency index, retention
index and rescaled consistency index (TL, CI, RI and
CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS
417
RC, respectively). The robustness of the resulting
phylogenetic trees was evaluated by 1000 bootstrap
replications (Hillis & Bull 1993) and the trees were
printed with TreeView v. 1.6.6 (Page 1996). A
partition homogeneity test (Farris et al. 1994) was
conducted in PAUP to evaluate the feasibility of
combining the sequence data sets. Sequences were
deposited in GenBank (Accession numbers
AY725612–AY725775) and the alignments in Tree-
BASE (accession number S1147).
Taxonomy
All morphological examinations were made from
cultures sporulating on CLA. Structures were
mounted in lactic acid, and 30 measurements at ×
1000 magnification were made of each structure.
The 95 % confidence levels were determined, and the
extremes of spore measurements given in parenthe-
ses. Colony reverse colours were noted after 6 d on
MEA at 25 °C in the dark, using the colour charts of
Rayner (1970) for comparison. All cultures studied
are maintained in the culture collection of the Cen-
traalbureau voor Schimmelcultures (CBS), Utrecht,
the Netherlands (Table 1).
RESULTS
DNA phylogeny
The partition homogeneity test showed that the β-
tubulin and histone datasets could not be combined
(P > 0.05); therefore these datasets were analysed
separately. It was possible to combine the smaller
subset of taxa (P = 0.064) sequenced for all four loci
into a single analysis.
For the β-tubulin gene, approximately 480–550
bases were determined for the isolates indicated in
Table 1. The manually adjusted alignment contained
54 taxa (including the two outgroups) and 523 char-
acters including alignment gaps. Of the 523 charac-
ters used in the analysis, 216 were parsimony-
informative, 81 were variable and parsimony-
uninformative, and 226 were constant. Neighbour-
joining analysis using the three substitution models,
as well as parsimony analysis, yielded trees with
similar topology and bootstrap values. Parsimony
analysis of the alignment yielded 64 most parsimoni-
ous trees (TL = 673 steps; CI = 0.738; RI = 0.867;
RC = 0.640), one of which is shown in Fig. 1. The
phylogenetic tree obtained shows a number of well-
supported clades. Although the first cluster does not
have a bootstrap support value, it does contain sev-
eral well-supported clades, namely Cy. floridanum
(100 % bootstrap support), Cy. hongkongense (98 %),
Cy. malesianum (83 %), Cy. indonesiae (92 %), Cy.
chinense (100 %) and Cy. canadense (100 %). The
Cy. floridanum, Cy. hongkongense and Cy. male-
sianum clades cluster together with a bootstrap
support value of 100 %, as do the Cy. indonesiae and
Cy. chinense clades. The second main clustering
(bootstrap support value of 100 %) contains several
taxa and clades, namely Cy. curvisporum CPC 765
and Cy. parasiticum CBS 112217; a Cy. parasiticum
clade containing isolates mainly from Hawaii (Clade
1, 95 % bootstrap support), a second Cy. parasiticum
clade containing isolates from Indonesia and U.S.A.
(Clade 2, 51 % bootstrap support), Cy. pacificum (87
%), Cy. asiaticum (95 %), Cy. colombiense (67 %)
and Cy. sumatrense (100 %). Basal to the other
clades is a clade (100 %) containing Cy. multiphi-
alidicum and Cy. pseudonaviculatum as sister taxa.
For the histone gene, approximately 430 bases
were determined for the isolates in Table 1. The
manually adjusted alignment contained 55 taxa
(including the two outgroups) and, for each taxon,
489 characters including alignment gaps were ana-
lysed. Among these characters was an insertion of 55
nucleotides in the outgroup taxa, and this was coded
as a single event for analysis purposes, leaving a total
of 434 characters. Of these 175 were parsimony-
informative, 65 were variable and parsimony-
uninformative, and 194 were constant. Neighbour-
joining analysis as described previously yielded trees
with similar topology and bootstrap values, except in
regard to the placement of Cy. multiphialidicum and
Cy. pseudonaviculatum. Using the Kimura 2-
parameter model, these two isolates formed a basal
polytomy, whereas the other two models placed Cy.
pseudonaviculatum as a sister clade (low bootstrap
support value) to the clade containing Cy. florida-
num, Cy. hongkongense and Cy. malesianum, leaving
Cy. multiphialidicum sitting basal to all the other
isolates (data not shown). Parsimony analysis
yielded 24 most parsimonious trees (TL = 631 steps;
CI = 0.648; RI = 0.838; RC = 0.543), one of which is
shown in Fig. 2. The main difference between the
neighbour-joining and parsimony analyses is in the
placement of Cy. multiphialidicum and Cy. pseu-
donaviculatum; parsimony supported the placement
given by the Kimura 2-parameter model (data not
shown). Figure 2 shows a number of well-supported
clades. As in the β-tubulin tree, the first clustering of
clades lacks bootstrap support, but it does show
excellent support for the same clades seen in the β-
tubulin analysis. The Cy. floridanum, Cy. hongkon-
gense and Cy. malesianum clades cluster together
with 98 % bootstrap support, and the Cy. indonesiae
and Cy. chinense clades together have a 100 % sup-
port. The second main cluster (98 % support) con-
tains several entities, firstly a clade (71 %) containing
Cy. curvisporum CPC 765 and Cy. parasiticum CBS
112217, as well as a Cy. parasiticum clade containing
isolates mainly from Hawaii (Clade 1, 88 %) and a
second Cy. parasiticum clade containing isolates
from Indonesia and the U.S.A. (Clade 2, 62 %). The
second main cluster also contains a Cy. pacificum
CROUS ET AL.
clade (92 %), a Cy. colombiense clade (99 %), and a
Cy. sumatrense clade (98 %).
10 changes
Cy. floridanum
100
Cylindrocladiella peruviana AY725653
Cylindrocladiella lageniformis AY725652
AF333401
AF348215
AF348218
AF348219
CBS 114828
CBS 114711
CBS 112710
CBS 112752
CBS 112840
CBS 112834
CBS 112823
CBS 112744
CBS 114827
CBS 111037
AF348224
AF348212
CBS 112217
CBS 112216
CBS 112211
CBS 112218
CBS 112214
CBS 112223
CBS 112210
CBS 111805
AF333411
CBS 112215
CBS 190.50
CBS 113783
CBS 113903
CPC 10450
CBS 112213
CBS 112212
CBS 112219
CBS 112209
Cy. curvisporum AF333395
AF348223
AF348222
CBS 114037
CBS 114038
CBS 112705
AF348220
CBS 112952
CBS 114073
CBS 112711
CBS 112938
AF333413
CBS 112221
CBS 112934
CBS 112829
CBS 112832
CBS 112757
Cy. pseudonaviculatum AF449453
Cy. multiphialidicum CBS 112678
Cy. hongkongense
Cy. malesianum
Cy. indonesiae
Cy. chinense
Cy. canadense
Cy. parasiticum
Cy. pacificum
Cy. asiaticum
Cy. colombiense
Cy. sumatrense
83
98
100
100
73 99
100
92
98
100
100
100
99
100
100
95
51
87
71
95
85
67
100
Clade 1
Clade 2
Fig. 1. One of 64 most parsimonious trees obtained from a
heuristic search with 100 random taxon additions of the β-
tubulin sequence alignment. The scale bar shows 10
changes; bootstrap support values from 1000 replicates are
shown at the nodes. Thickened lines indicate the strict
consensus branches. The tree was rooted to two Cylindro-
cladiella species.
The Cy. asiaticum isolates did not form a clade but are
a basal polytomy in this second main cluster. The two
Cy. canadense isolates form a sister clade (100 %) to
the first and second main clusters. Basal to the other
clades is a clade (100 % support) containing Cy.
multiphialidicum and Cy. pseudonaviculatum as sister
taxa.
For the combined analysis of the four loci, 523,
434, 538 and 493 characters (including alignment gaps
and respectively for β-tubulin, histone, elongation
factor 1-α and calmodulin) were included (Table 1).
The manually adjusted alignment contained 36 taxa,
including the outgroup, and 1988 characters including
alignment gaps were used in the analysis. Of these,
541 were parsimony-informative, 433 were parsi-
mony-uninformative, and 1014 were constant.
10 changes
Cy. floridanum
81
Cylindrocladiella peruviana AY725700
Cylindrocladiella lageniformis AY725699
Cy. multiphialidicum CBS 112678
Cy. pseudonaviculatum CPC 3570
CBS 114552
AF348235
AF348234
AF348231
CBS 114828
CBS 114711
CBS 112710
CBS 112752
CBS 112840
CBS 112834
CBS 112823
CBS 112744
CBS 114827
CBS 111037
Cy. curvisporum CPC 765
CBS 112217
CBS 112216
CBS 113783
CBS 113903
CPC 4873
CBS 112210
CBS 111805
CBS 112211
CBS 112218
CBS 112214
CBS 112223
CBS 112215
CBS 190.50
CPC 10450
CBS 112213
CBS 112212
CBS 112219
CBS 112209
AF348239
AF348238
CBS 114037
CBS 114038
CBS 112705
CBS 112952
AF348236
CBS 114073
CBS 112711
CBS 112938
CBS 112220
CBS 112221
CBS 112757
CBS 112934
CBS 112829
CBS 112832 AF348240
AF348228
Cy. hongkongense
Cy. malesianum
Cy. indonesiae
Cy. chinense
Cy. parasiticum
Cy. pacificum
Cy. asiaticum
Cy. colombiense
Cy. sumatrense
Cy. canadense
Clade 1
Clade 2
59
59
67
100
98
100
100
100
100
100
100
98
98
82
75
56
99
92
86
65
71
88
59
63
62
Fig. 2. One of 24 most parsimonious trees obtained from a
heuristic search with 100 random taxon additions of the
histone sequence alignment. The scale bar shows 10
changes; bootstrap support values from 1000 replicates are
shown at the nodes. The tree was rooted to two Cylindro-
cladiella species.
Neighbour-joining analysis using the three substitu-
tion models yielded trees with similar topology and
bootstrap values, except that the placement of the
clade containing Cy. canadense, Cy. indonesiae and
Cy. chinense differed in the Jukes-Cantor substitution
model. Parsimony analysis yielded twelve most
parsimonious trees (TL = 1942 steps; CI = 0.754; RI =
0.849; RC = 0.640), one of which is shown in Fig. 3.
418
CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS
10 changes
Cy. floridanum
100
Cylindrocladiella peruviana
Cy. canadense UFV76
CBS 114552
ATCC 18882
ATCC 18834
CBS 114828
CBS 114711
CBS 112710
CBS 112752
CBS 112840
CBS 112834
CBS 112823
CBS 112744
CBS 114827
CBS 111037
CBS 113783
CBS 112216
CBS 113903
CBS 112215
CBS 190.50
IMI35429
IMI35428
CPC 682
CBS 112705
CBS 112952
CBS 114073
CBS 112711
CBS 112938
CBS 112220
CBS 112221
CBS 112757
CBS 112934
CBS 112829
CBS 112832
Cy. pseudonaviculatum CPC 3570
Cy. multiphiali dicum CBS 112678
Cy. hongkongense
Cy. pacificum
Cy. indonesiae
Cy. chinense
Cy. parasiticum
Cy. asiaticum
Cy. colombiense
Cy. sumatrense
Cy. malesianum
100
99
100
100
100
100
100
100
95
100
100
100
65
78
100
62
92
79
91
87
53
100
65
100
100
Fig. 3. One of 12 most parsimonious trees obtained from a
heuristic search with 100 random taxon additions of the
combined β-tubulin, histone, elongation factor 1-α and
calmodulin sequence alignment. The scale bar shows 10
changes and bootstrap support values from 1000 replicates
are shown at the nodes. Thickened lines indicate the strict
consensus branches. The tree was rooted to Cylindrocladi-
ella peruviana (AY725653, AY725700, AY725736 and
AY725775).
The main difference between the neighbour-joining
and parsimony analyses was in bootstrap support
values, which were higher in the parsimony analysis
(data not shown). The parsimony tree shows a num-
ber of well-supported clades. As in the β-tubulin and
histone phylograms, the first cluster of clades lacks
bootstrap support, but it does contain the identical
well-supported clades for Cy. floridanum and other
species. Cylindrocladium floridanum, Cy. hongkon-
gense and Cy. malesianum cluster together with 100
% support, as do Cy. indonesiae and Cy. chinense.
The next cluster with 100 % support contains strongly
supported clades for Cy. parasiticum, Cy. pacificum,
Cy. asiaticum, Cy. colombiense and Cy. sumatrense.
Basal to the other clades is a strongly supported clade
containing Cy. multiphialidicum and Cy. pseudonavi-
culatum.
Taxonomy
Calonectria asiatica Crous & Hywel-Jones, sp.
nov. MycoBank MB500102. Figs 4–10.
Anamorph: Cylindrocladium asiaticum Crous &
Hywel-Jones, sp. nov.
Etymology: Named after Asia, from where it was
collected.
Calonectriae kyotensi similis, sed ascosporis brevioribus,
(28–)30–38(–40) × (5–)6(–7) µm (in medio 33 × 6 µm).
Perithecia solitary or in groups of up to 6, orange,
becoming orange-brown with age; in section, apex and
body orange, base red-brown, subglobose to ovoid,
280–400 µm high, 200–350 µm diam, body turning
red, and base dark red-brown (KOH+); perithecial
walls rough, consisting of two thick-walled layers:
outer layer of textura globulosa, 20–70 µm thick, cells
becoming more compressed towards the inner layer of
textura angularis, 10–15 µm thick, cells becoming
thin-walled and hyaline towards the center; outermost
cells 15–35 × 10–25 µm, cells of inner layer 8–15 ×
3–6 µm; perithecial base up to 100 µm thick, consist-
ing of dark red, angular cells, merging with an erum-
pent stroma; cells of the outer wall layer continuous
with the pseudoparenchymatous cells of the erumpent
stroma. Asci 8-spored, clavate, 70–120 × 12–20 µm,
tapering into a long thin stalk. Ascospores aggregated
in the upper third of the ascus, hyaline, guttulate,
fusoid with rounded ends, straight to slightly curved,
1-septate, constricted at the septum, (28–)30–38(–40)
× (5–)6(–7) µm (mean = 33 × 6 µm). Homothallic.
Cylindrocladium asiaticum Crous & Hywel-
Jones, sp. nov. MycoBank MB500103.
Cylindrocladio floridano simile, sed vesiculis latioribus
(12–17 µm diam) et conidiis maioribus (42–)48–55(–65) ×
(4–)5(–5.5) µm, in medio 53 × 5 µm.
Conidiophores consisting of a stipe bearing a penicil-
late arrangement of fertile branches, a stipe extension,
and a terminal vesicle; stipe septate, hyaline, smooth,
60–170 × 7–8 µm; stipe extensions septate, straight to
flexuous, 200–280 µm long, 4–5 µm wide at apical
septum, terminating in a sphaeropedunculate vesicle,
12–17 µm diam; lateral stipe extensions (90° to main
axis) also abundant.
419
CROUS ET AL.
Figs 4–7. Calonectria asiatica and its anamorph Cylindro-
cladium asiaticum (CBS 114073). 4. Vertical section
through a perithecium. 5. Section through lateral perithe-
cial wall. 6. Ascospores. 7. Conidiophore. Scale bars: 4 =
45 µm, 5, 6 = 10 µm, 7 = 15 µm.
Conidiogenous apparatus 40–90 µm long, 40–80
µm wide; primary branches aseptate or 1-septate, 20–
30 × 4–7 µm; secondary branches aseptate, 15–20 ×
4–5 µm, tertiary branches aseptate, 10–15 × 3–5 µm,
additional branches –5, aseptate, 10–15 × 3–4 µm,
each terminal branch producing 2–6 phialides; phi-
alides doliiform to reniform, hyaline, aseptate, 10–13
× 3–4 µm; apex with minute periclinal thickening and
inconspicuous collarette. Conidia cylindrical,
rounded at both ends, straight, (42–)48–55(–65) ×
(4–)5(–5.5) µm (mean = 53 × 5 µm), 1-septate, lack-
ing a visible abscission scar, held in parallel cylindri-
cal clusters by colorless slime. Megaconidia and
microconidia unknown.
Holotype: Thailand, Prathet Thai, on leaf litter, 2001, N.L.
Hywel-Jones (herb. CBS 9889, holotype of Calonectria
asiatica and Cylindrocladium asiaticum, cultures ex-type
CBS 114073 = CPC 3900 = SFE 726).
Cultural characteristics: Colonies with feathery,
irregular margins, abundant white to cream-coloured
aerial mycelium, surface rust-coloured (7’i); reverse
with cream-coloured to white outer margin, and rust
(7’i) inner region, becoming chestnut (7’m) towards
the centre. Colonies reaching 42–64 mm diam after 7
d on MEA in the dark at 25 °C.
Figs 8–10. Calonectria asiatica and its anamorph Cylindro-
cladium asiaticum (CBS 114073). 8. Conidiophore and
sphaeropedunculate vesicles. 9. Ascospores. 10. Conidia.
Scale bar = 10 µm.
Substrate: Debris, soil.
Distribution: Indonesia, Thailand.
Notes: Cylindrocladium asiaticum is morphologically
similar to Cy. floridanum [vesicles 6–12 µm diam,
conidia (35–)45–50(–55) × 3–4(–5) µm, mean = 40 ×
3.5 µm], but can be distinguished by having wider
vesicles (12–17 µm diam) and larger conidia (42–)48–
55(–65) × (4–)5(–5.5) µm, mean = 53 × 5 µm.
Additional cultures examined: Thailand, Prathet Thai, on
leaf litter, 2001, N.L. Hywel-Jones, CBS 112711 = CPC
3898 = SFE 744). Indonesia, Northern Sumatra, soil
collected under canopies of Eucalyptus trees, 2001, M.J.
Wingfield, CBS 112938 = CPC 4513.
Cylindrocladium chinense Crous, sp. nov.
MycoBank MB500104. Figs 11, 12.
Etymology: Named after the country from which it
was collected, China.
Cylindrocladio floridano simile, sed conidiis longioribus (in
medio 45 × 4 µm) et paucis ramis (–3) conidiophororum
differens.
Teleomorph unknown. Conidiophores consisting of a
stipe bearing a penicillate arrangement of fertile
branches, a stipe extension, and a terminal vesicle;
420
CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS
stipe septate, hyaline, smooth, 40–150 × 6–7 µm; stipe
extensions septate, straight to flexuous, 120–150 µm
long, 2.5–3.5 µm wide at the apical septum, terminat-
ing in a sphaeropedunculate vesicle, 6–9 µm diam;
lateral stipe extensions (90° to main axis) common.
Conidiogenous apparatus 40–60 µm long and wide;
primary branches aseptate or 1-septate, 20–30 × 5–6
µm; secondary branches aseptate, 15–30 × 4–6 µm,
tertiary branches aseptate, 10–20 × 4–5 µm, each
terminal branch producing 2–4 phialides; phialides
elongate doliiform to reniform, hyaline, aseptate, 10–
20 × 3–4 µm; apex with minute periclinal thickening
and inconspicuous collarette. Conidia cylindrical,
rounded at both ends, straight, (38–)41–48(–56) ×
(3.5–)4(–4.5) µm (mean = 45 × 4 µm), 1-septate,
lacking a visible abscission scar, held in parallel
cylindrical clusters by colorless slime. Megaconidia
and microconidia unknown.
Holotype: China, soil, 2000, E.C.Y. Liew (holotype herb.
CBS 9887; culture ex-type CBS 114827 = CPC 4101).
Cultural characteristics: Colonies fast growing with
abundant aerial mycelium, consisting of strands and
tufts of white to cream-coloured hyphae; surface
sienna (13i); reverse with cream-coloured to white
outer region, sienna (13i) inner region, and rust-
coloured (7’i) area near the centre. Colonies reaching
51–72 mm diam after 7 d on MEA in the dark at 25
°C.
Substrate: Soil.
Distribution: China.
Figs 11, 12. Cylindrocladium chinense (CBS 114827). 11.
Conidia. 12. Conidiophore and vesicles. Scale bar = 10 µm.
Notes: This species, which is part of the Cylindrocla-
dium floridanum complex, is only known from China,
inclusive of Hong Kong. Morphologically, Cy.
chinense can be distinguished based on a combination
of characters, namely having conidia of intermediate
length (mean = 45 × 4 µm), up to three conidiophore
branches, and, commonly, lateral stipe extensions.
Additional cultures examined: Hong Kong, soil, Jun.
1995, M.J. Wingfield, CBS 111037 = CPC 1154;
China, soil, 2000, E.C.Y. Liew, CBS 112744 = CPC
4104.
Calonectria colombiensis Crous, sp. nov.
MycoBank MB500105. Figs 13–22.
Anamorph: Cylindrocladium colombiense Crous, sp.
nov.
Etymology: Named after Colombia, from where it was
collected.
Calonectriae kyotensi simile, sed ascosporis brevioribus,
(28–)30–35(–40) × (4–)5(–6) µm (in medio 33 × 5 µm)
differens.
Figs 13–18. Calonectria colombiensis and its anamorph
Cylindrocladium colombiense (CBS 112220). 13. Vertical
section through a perithecium. 14. Cells around ostiolar
region of perithecium. 15. Ascospores. 16. Conidiophore.
17. Vesicles. 18. Conidia. Scale bars: 13 = 45 µm, 14–18
= 10 µm.
Perithecia solitary or in groups of up to 6, orange,
becoming orange-brown with age; in section, apex and
body orange, base red-brown, subglobose to ovoid,
200–350 µm high, 200–300 µm diam, body turning
red, and base dark red-brown (KOH+); perithecial
walls rough, consisting of two thick-walled layers:
outside layer of textura globulosa, 20–60 µm thick,
421
CROUS ET AL.
cells becoming more compressed towards inner layer
of textura angularis, 10–15 µm thick, cells becoming
thin-walled and hyaline towards the center, outermost
cells 15–35 × 10–25 µm, cells of inner layer 8–17 ×
3–6 µm; perithecial base up to 160 µm thick, consist-
ing of dark red, angular cells, merging with an erum-
pent stroma; cells of the outer wall layer continuous
with the pseudoparenchymatous cells of the erumpent
stroma. Asci 8-spored, clavate, 90–150 × 11–23 µm,
tapering into a long thin stalk. Ascospores aggregated
in the upper third of the ascus, hyaline, guttulate,
fusoid with rounded ends, straight to slightly curved,
1-septate, becoming constricted at the septum,
(28–)30–35(–40) × (4–)5(–6) µm (mean = 33 × 5 µm).
Cultures were homothallic.
Figs 19–22. Calonectria colombiensis and its anamorph
Cylindrocladium colombiense (CBS 112220). 19. Ascus.
20. Conidiophore and vesicles. 21. Ascospores. 22. Co-
nidia. Scale bar = 10 µm.
Cylindrocladium colombiense Crous, sp. nov.
MycoBank MB500106.
Cylindrocladio parasitico simile, sed conidiis minoribus,
(33–)48–58(–60) × (4–)4.5(–5) µm, in medio 53 × 4.5 µm
differens.
Conidiophores consisting of a stipe bearing a penicil-
late arrangement of fertile branches, a stipe extension,
and a terminal vesicle; stipe septate, hyaline, smooth,
50–70 × 6–7 µm; stipe extensions septate, straight to
flexuous, 130–200 µm long, 3–4 µm wide at the
apical septum, terminating in a sphaeropedunculate
vesicle, 7–12 µm diam; lateral stipe extensions (90° to
main axis) also abundant. Conidiogenous apparatus
40–60 µm long, 25–60 µm wide; primary branches
aseptate or 1-septate, 16–20 × 4–6 µm; secondary
branches aseptate, 10–20 × 3–5 µm, tertiary branches
aseptate, 10–17 × 3–5 µm, additional branches –5,
aseptate, 8–15 × 3–4 µm, each terminal branch pro-
ducing 2–6 phialides; phialides elongate doliiform to
reniform, hyaline, aseptate, 10–18 × 3–5 µm; apex
with minute periclinal thickening and inconspicuous
collarette. Conidia cylindrical, rounded at both ends,
straight, (33–)48–58(–60) × (4–)4.5(–5) µm (mean =
53 × 4.5 µm), 1(–3)-septate, lacking a visible abscis-
sion scar, held in parallel cylindrical clusters by color-
less slime. Megaconidia and microconidia unknown.
Holotype: Colombia, La Selva, soil under Eucalyptus
grandis trees, M.J. Wingfield (herb. CBS 9890, holotype of
Calonectria colombiensis and Cylindrocladium colombi-
ense, cultures ex-type CBS 112220 = CPC 723, CBS
112221 = CPC 724, 725).
Cultural characteristics: Colonies with feathery,
irregular margins, sparse white to sienna (13i) aerial
mycelium, surface rust-coloured (7’i); reverse rust
(7’i). Colonies reaching 26–39 mm diam after 7 d on
MEA in the dark at 25 °C.
Substrate: Soil.
Distribution: Colombia.
Notes: Isolates of Cy. colombiense were initially
treated under Cy. parasiticum by Crous (2002). This
was based on observations that Cy. colombiense
produces 3-septate conidia, and that the conidia are
generally larger, (33–)48–58(–60) × (4–)4.5(–5) µm,
mean = 53 × 4.5 µm, than those of Cy. floridanum,
(35–)45–50(–55) × 3–4(–5) µm, mean = 40 × 3.5 µm,
showing considerable overlap with those of Cy. para-
siticum, (45–)70–82(–90) × (4–)5–6.5(–7) µm, mean =
62 × 6 µm. Although Cy. parasiticum has been re-
ported in the literature to occur on eucalypts from
various tropical countries (Crous 2002), cultures from
this host are presently available to confirm this.
Calonectria hongkongensis Crous, sp. nov.
MycoBank MB500107. Figs 23–29.
Anamorph: Cylindrocladium hongkongense Crous, sp.
nov.
Etymology: Named after Hong Kong, from where it
was collected.
Calonectriae kyotensi simile, sed ascosporis brevioribus,
latioribus, (25–)28–35(–40) × (4–)5–6(–7) µm (in medio 31
× 6 µm) differens.
Perithecia solitary or in groups of up to 3, orange,
becoming red-brown with age; in section, apex and
422
CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS
body orange, base red-brown, subglobose to ovoid,
350–550 µm high, 300–450 µm diam, body turning
red, and base dark red-brown (KOH+); perithecial
Figs 23–25. Calonectria hongkongensis and its anamorph
Cylindrocladium hongkongense (CBS 114828). 23. Co-
nidiophore and vesicles. 24. Ascospores. 25. Conidia. Scale
bar = 10 µm.
Figs 26–32. Calonectria hongkongensis (CBS 114828) and
Cylindrocladium malesianum (CBS 112752). 26–29. Ca.
hongkongensis. 26, 27. Asci. 28. Ascospores. 29.
Conidiophore. 30–32. Cy. malesianum. 30. Conidiophore.
31. Vesicle. 32. Conidia. Scale bars: 26, 27, 29–31 = 15
µm, 28, 32 = 10 µm
walls rough, consisting of two thick-walled layers:
outside layer of textura globulosa, 20–40 µm thick,
cells becoming more compressed towards inner layer
of textura angularis, 10–15 µm thick, cells becoming
thin-walled and hyaline towards the center, outermost
cells 15–30 × 10–20 µm, cells of inner layer 8–15 ×
3–6 µm; perithecial base up to 150 µm thick, consist-
ing of dark red, angular cells, merging with an erum-
pent stroma, cells of the outer wall layer continuous
with the pseudoparenchymatous cells of the erumpent
stroma. Asci 8-spored, clavate, 80–140 × 14–20 µm,
tapering into a long thin stalk. Ascospores aggregated
in the upper third of the ascus, hyaline, guttulate,
fusoid with rounded ends, straight to curved, 1-
septate, becoming constricted at the septum, (25–)28–
35(–40) × (4–)5–6(–7) µm (mean = 31 × 6 µm).
Homothallic.
Cylindrocladium hongkongense Crous, sp. nov.
MycoBank MB500108.
Cylindrocladio floridano simile, sed ramis
conidiophororum numerosis (–8) et penicillo ad 100 µm
alto et lato differens.
Conidiophores consisting of a stipe bearing a penicil-
late arrangement of fertile branches, a stipe extension,
and a terminal vesicle; stipe septate, hyaline, smooth,
40–60 × 5–6 µm; stipe extensions septate, straight to
flexuous, 100–200 µm long, 3–4 µm wide at apical
septum, terminating in a sphaeropedunculate vesicle,
8–14 µm diam; lateral stipe extensions (90° to main
axis) also abundant. Conidiogenous apparatus 70–
100 µm long, 70–120 µm wide; primary branches
aseptate or 1-septate, 17–25 × 4–7 µm; secondary
branches aseptate, 15–20 × 3–5 µm, tertiary branches
aseptate, 10–15 × 3–5 µm, additional branches –8,
aseptate, 8–15 × 3–4 µm, each terminal branch pro-
ducing 2–6 phialides; phialides elongate doliiform to
reniform, hyaline, aseptate, 9–15 × 3–5 µm; apex with
minute periclinal thickening and inconspicuous col-
larette. Conidia cylindrical, rounded at both ends,
straight, (38–)45–48(–53) × 4(–4.5) µm (mean = 46.5
× 4 µm), 1-septate, lacking a visible abscission scar,
held in parallel cylindrical clusters by colorless slime.
Megaconidia and microconidia unknown.
Holotype: Hong Kong, soil, M.J. Wingfield (herb. CBS
9886, holotype of Calonectria hongkongensis and Cylin-
drocladium hongkongense, culture ex-type CBS 114828 =
CPC 4670).
Cultural characteristics: Colonies irregular with
feathery margins and abundant white to sienna (13i)
aerial mycelium, surface rust-coloured (7’i) to pale
white; reverse sienna (13i) to rust-coloured (7’i).
Colonies reaching 15–30 mm diam after 7 d on MEA
in the dark at 25 °C.
Substrate: Soil.
Distribution: China.
423
CROUS ET AL.
Notes: Cylindrocladium honkongense is distinguished
from Cy. floridanum by having numerous conidio-
phore branches (–8), and a conidiogenous apparatus
that is up to 100 µm wide and long, as well as conidia
that have a longer average length (46.5 × 4 µm).
Additional culture examined: China, soil, 8 Nov. 1993, M.J.
Wingfield, CPC 686 = CBS 114711.
Cylindrocladium indonesiae Crous, sp. nov.
MycoBank MB500109. Figs 33, 34.
Figs 33, 34. Cylindrocladium indonesiae (CBS 112823).
33. Conidiophore and vesicles. 34. Conidia. Scale bar = 10
µm.
Etymology: Only known from Indonesia.
Cylindrocladio floridano simile, sed ramis
conidiophororum numerosis (–5) et conidiis longioribus (in
medio 50.5 × 4 µm) differens.
Teleomorph unknown. Conidiophores consisting of a
stipe bearing a penicillate arrangement of fertile
branches, a stipe extension, and a terminal vesicle;
stipe septate, hyaline, but pale brown below, smooth,
40–80 × 5–6 µm; stipe extensions septate, straight to
flexuous, 110–160 µm long, 2.5–3 µm wide at the
apical septum, terminating in a sphaeropedunculate
vesicle, 7–9 µm diam; lateral stipe extensions absent.
Conidiogenous apparatus 40–60 µm long, 60–80 µm
wide; primary branches aseptate or 1-septate, 18–25 ×
4–5 µm; secondary branches aseptate, 10–20 × 4–5
µm, tertiary and additional branches, –5, aseptate, 10–
15 × 4–5 µm, each terminal branch producing 2–6
phialides; phialides elongate doliiform to reniform,
hyaline, aseptate, 8–15 × 3–4 µm; apex with minute
periclinal thickening and inconspicuous collarette.
Conidia cylindrical, rounded at both ends, straight,
(40–)45–55(–60) × (3–)4 µm (mean = 50.5 × 4 µm),
1-septate, lacking a visible abscission scar, held in
parallel cylindrical clusters by colorless slime.
Megaconidia and microconidia unknown.
Holotype: Indonesia, Warambunga, soil, 9 Mar. 1996, M.J.
Wingfield (holotype herb. CBS 9891, culture ex-type CBS
112823 = CPC 4508).
Cultural characteristics: Colonies fast-growing with
feathery margins and moderate to abundant white
aerial mycelium; surface umber (13’i); reverse with
white outer margin, and umber (13’i) inner region,
becoming rust-coloured (7’i) towards the centre.
Colonies reaching 56–80 mm diam after 7 d on MEA
in the dark at 25 °C.
Substrate: Soil.
Distribution: Indonesia.
Notes: Cylindrocladium indonesiae can be distin-
guished from other species in the Cy. floridanum
complex by having numerous conidiophore branches,
by having conidia of medium length (mean = 50.5 × 4
µm), and by lacking lateral stipe extensions.
Additional culture examined: Indonesia, Warambunga,
soil, 9 Mar. 1996, M.J. Wingfield, CBS 112840 = CPC
4554, CBS 112834 = CPC 4547.
Figs 35, 36. Cylindrocladium malesianum (CBS 112752).
35. Conidiophore and vesicles. 36. Conidia. Scale bar = 10
µm.
424
CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS
Cylindrocladium malesianum Crous, sp. nov.
MycoBank MB500110. Figs 30–32, 35, 36.
Etymology: Named after Malaysia, the region from
which it was collected.
425
ia unknown.
nd
hialides.
× 4.5 µm) et appendicibus crassitunicatis
ifferens.
rless
slime. Megaconidia and microconidia unknown.
Cylindrocladio floridano simile, sed ramis
conidiophororum numerosis (–6) et conidiis longioribus (in
medio 47.5 × 4 µm) differens.
Teleomorph unknown. Conidiophores consisting of a
stipe bearing a penicillate arrangement of fertile
branches, a stipe extension, and a terminal vesicle;
stipe septate, hyaline, smooth, 70–200 × 5–7 µm; stipe
extensions septate, straight to flexuous, 120–200 µm
long, 3–4 µm wide at the apical septum, terminating in
a sphaeropedunculate to globose vesicle, 8–15 µm
diam; lateral stipe extensions (90° to main axis) also
present. Conidiogenous apparatus 50–60 µm long,
30–80 µm wide; primary branches aseptate or 1-
septate, 20–30 × 5–6 µm; secondary branches
aseptate, 10–30 × 5–6 µm, tertiary and additional
branches, –6, aseptate, 10–15 × 4–6 µm, each terminal
branch producing 2–6 phialides; phialides elongate
doliiform to reniform, hyaline, aseptate, 7–15 × 2.5–4
µm; apex with minute periclinal thickening and
inconspicuous collarette. Conidia cylindrical,
rounded at both ends, straight, (34–)45–52(–55) × (3–
)4 µm (mean = 47.5 × 4 µm), 1-septate, lacking a
visible abscission scar, held in parallel cylindrical
clusters by colorless slime. Megaconidia and
microconid
Holotype: Indonesia, Northern Sumatra, soil, 9 Mar.
1996, M.J. Wingfield (holotype herb. CBS 9885,
culture ex-type CBS 112752 = CPC 4223).
Cultural characteristics: Colonies with feathery,
irregular white margins, surface with moderate sienna
(13i) aerial mycelium and inner region; reverse with
thin sienna (13i) outer region, and chestnut (7’m)
inner region. Colonies reaching 36–47 mm diam after
7 d on MEA in the dark at 25 °C.
Substrate: Soil.
Distribution: Indonesia, Thailand.
Notes: Cylindrocladium malesianum can be
distinguished from other species in the Cy. floridanum
complex by having numerous conidiophore branches,
conidia of medium length (mean = 47.5 × 4 µm), and
numerous lateral stipe extensions.
Additional culture examined: Thailand, Prathet, on leaf
litter, 2001, N.L. Hywel-Jones, CBS 112710 = CPC 3899.
Cylindrocladium multiphialidicum Crous, P.
Simoneau & J.-M. Risède, sp. nov. MycoBank
MB500111. Figs 37–41.
Etymology: Named after its characteristic
conidiophores that form numerous branches a
p
Cylindrocladio floridano simile, sed ramis
conidiophororum numerosis (–8) et conidiis longioribus (in
medio 53
d
Teleomorph unknown. Conidiophores consisting of a
stipe bearing a penicillate arrangement of fertile
branches, a stipe extension, and a terminal vesicle;
stipe septate, hyaline to medium brown, smooth, 80–
150 × 7–10 µm; stipe extensions septate, straight to
flexuous, hyaline to pale brown, thick-walled, 170–
300 µm long, 4–5 µm wide at apical septum,
terminating in a clavate to sphaeropedunculate vesicle,
8–16 µm diam. Conidiogenous apparatus 70–150 µm
long and wide; primary branches aseptate or 1-septate,
20–40 × 5–6 µm; secondary branches aseptate or 1-
septate, 15–25 × 5–6 µm, tertiary branches aseptate,
10–20 × 5–6 µm, additional branches –8, aseptate, 10–
15 × 4–5 µm, each terminal branch producing 2–6
phialides; phialides elongate doliiform to reniform,
hyaline, aseptate, 8–15 × 3–4 µm; apex with minute
periclinal thickening and inconspicuous collarette.
Conidia cylindrical, rounded at both ends, guttulate,
straight, (45–)48–55(–65) × (4–)4.5(–5) µm (mean =
53 × 4.5 µm), 1-septate, lacking a visible abscission
scar, held in parallel cylindrical clusters by colo
CROUS ET AL.
Figs 37, 38. Cylindrocladium multiphialidicum (CBS
112678). 37. Conidiophore and vesicles. 38. Conidia. Scale
bar = 10 µm.
Holotype: Cameroon, on soil surrounding roots of Musa
sp., Mar. 1998, Abadie, (holotype herb. CBS 9887, culture
ex-type CBS 112678 = Cam 13).
426
ycelium.
t (Risède & Simoneau 2004).
phoro.
Cultural characteristics: Colonies fast growing with
irregular margins, moderate to abundant white aerial
mycelium; surface sienna (13i); reverse with sienna
(13i) outer margin, and chestnut (9’m) inner region.
Colonies reaching 70–80 mm diam after 7 d on MEA
in the dark at 25 °C. Microsclerotia (perithecial
initials?) aggregating in clusters on agar surface,
bright red, turning red-brown to brown with age,
eventually becoming covered in m
Substrate: Soil.
Distribution: Cameroon.
Notes: Cylindrocladium multiphialidicum resembles
other taxa in the Cy. floridanum complex. Its conidial
dimensions (mean 53 × 4.5 µm) are larger than those
of Cy. floridanum (mean 40 × 3.5 µm), and more
closely match those of Cy. pacificum (mean 55 × 4.5
µm). Conidial lengths of Cy. pacificum tend to have a
broader range (38–)45–65(–75) µm than those of Cy.
multiphialidicum (45–)48–55(–65) µm. Furthermore,
stipes of Cy. multiphialidicum are thick-walled, which
is not the case in Cy. pacificum. The most
characteristic feature distinguishing Cy.
multiphialidicum from other taxa in the Cy.
floridanum complex is the numerous branches (–8)
and phialides, that are formed on the conidiophores.
Cylindrocladium multiphialidicum has thus far only
been isolated once from Musa roots, which may be
due to the fact that it has been shown to not be highly
virulent to this hos
Cylindrocladium sumatrense Crous, sp. nov.
MycoBank MB500112. Figs 42–46.
Etymology: Named after the location from which it
was collected, Sumatra, Indonesia.
Cylindricladio pacifico simile sed ramis conidiophororum
paucis (–3) et nonnullis appendicibus in quoque
conidio
Teleomorph unknown. Conidiophores consisting of a
stipe bearing a penicillate arrangement of fertile
branches, a stipe extension, and a terminal vesicle;
stipe septate, hyaline, smooth, 40–70 × 6–7 µm; stipe
extensions septate, straight to flexuous, 180–260 µm
long, 3–4 µm wide at the apical septum, terminating in
a sphaeropedunculate vesicle, 8–13 µm diam; lateral
stipe extensions (90° to main axis) also present.
Conidiogenous apparatus 50–80 µm long, 40–60 µm
wide; primary branches aseptate or 1-septate, 20–30 ×
4–5 µm; secondary branches aseptate, 10–20 × 4–5
µm, tertiary branches aseptate, 10–20 × 4–5 µm, each
terminal branch producing 2–6 phialides; phialides
elongate doliiform to reniform, hyaline, aseptate, 10–
20 × 3–5 µm; apex with minute periclinal thickening
and inconspicuous collarette. Conidia cylindrical,
rounded at both ends, straight, (45–)55–65(–70) ×
(4.5–)5(–6) µm (mean = 58 × 5 µm), 1-septate, lack-
ing a visible abscission scar, held in parallel cylindri-
cal clusters by colorless slime. Megaconidia and
microconidia unknown.
Figs 39–44. Cylindrocladium multiphialidicum (CBS
112678) and Cy. sumatrense (CBS 112829). 39–41. Cy.
multiphialidicum. 39. Conidiophore. 40, 41. Thick-walled
stipe extension (arrow). 42–44. Cy. sumatrense. 42, 43.
Vesicles. 44. Conidia. Scale bars: 39 = 50 µm, 40, 41 = 8
µm, 42, 43 = 6 µm, 44 = 10 µm.
Holotype: Indonesia, Northern Sumatra, from soil collected
under canopies of Eucalyptus trees, 2001, M.J. Wingfield
(holotype herb. CBS 9888, culture ex-type CBS 112829 =
CPC 4518).
Cultural characteristics: Colonies fast growing with
irregular margins and abundant white to cream-
coloured aerial mycelium, which makes the upper
surface white to cream-coloured; reverse with broad
cream-coloured to white outer margin, sienna (13i)
inner region, and rust (7’i) central region. Colonies
reaching 51–66 mm diam after 7 d on MEA in the
dark at 25 °C.
Substrate: Soil.
Distribution: Indonesia.
CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS
Figs 45, 46. Cylindrocladium sumatrense (CBS 112829).
45. Conidiophore and vesicles. 46. Conidia. Scale bar = 10
µm.
Notes: Cylindrocladium sumatrense is similar to Cy.
pacificum in having few conidiophore branches (–3),
and similar conidial dimensions (38–75 × 4–5 µm for
Cy. pacificum, and 45–70 × 4.5–6 µm for Cy.
sumatrense). The two species can be distinguished,
however, in that Cy. pacificum commonly forms
lateral stipe extensions, while this is rarely observed in
Cy. sumatren
427
se.
Additional cultures examined: Indonesia, Northern
Sumatra, soil collected under Eucalyptus forest, 2001, M.J.
Wingfield, CBS 112934 = CPC 4516, CBS 112832 = CPC
4520.
DISCUSSION
The Cy. floridanum species complex has been the
topic of several recent studies (Jeng et al. 1997, Victor
et al. 1997, Kang et al. 2001, Crous 2002), which
have integrated morphological, phylogenetic and
biological species concepts to try and resolve the
various species involved in this complex. Isolates in
this complex have a six nucleotide insertion in their
ITS2 region (Jeng et al. 1997, Risède & Simoneau
2001), which may be indicative of a single insertion
event that occurred in a common ancestor to all these
species. It is surprising, therefore, that in the present
study we have been able to delineate yet another eight
species within this complex. The concordant geneal-
ogy derived from the β-tubulin, calmodulin, elonga-
tion factor 1-α and histone sequence data corroborated
the taxonomic relevance of minor morphological
differences observed among these species. All species
in this complex share similar vesicle morphology, and
are distinguished primarily based on vesicle dimen-
sions, conidiogenous apparatus (size and branching),
conidial morphology, and the ability to produce a
teleomorph in culture. The Calonectria teleomorphs,
however, proved to be relatively similar to one an-
other, and they added little information useful in
species recognition.
The uncertainty surrounding the status of the
Hawaiian isolates of Cy. pacificum, which lacked a
teleomorph, and the homothallic strains from
Thailand, which also have shorter conidia than those
from Hawaii (Kang et al. 2001), is somewhat clarified
with the description of Cy. asiaticum. In the four gene
regions compared, however (Fig. 3), some isolates
clustered within the asiaticum clade only with low
bootstrap support, suggesting that this clade still
contains cryptic elements that will eventually be
resolved following more collections. Cylindrocladium
asiaticum is similar to Cy. sumatrense, but can be
distinguished by having abundant lateral stipe
extensions, and up to 5 branches in its conidiogenous
apparatus. Cylindrocladium sumatrense rarely
produces lateral stipe extensions, and only has up to 3
branches in its conidiogenous apparatus.
Cylindrocladium parasiticum has sphaeropedun-
culate vesicles similar to species in the Cy. floridanum
species complex, but can be distinguished by its
wider, 3-septate macroconidia (45–90 × 4–7 µm).
This species is a major pathogen of peanuts and soy-
bean, causing Cylindrocladium black rot (CBR) on the
former, and red crown rot (RCR) on the latter (Crous
2002). It has been speculated that this pathogen was
introduced into the U.S.A. on Indigofera tinctoria L.,
from where it spread to other crops (Berner et al.
1988). Since it was first described, this pathogen has
been recorded on numerous hosts in many tropical and
subtropical regions of the world. Live cultural proofs
to substantiate these records are, however, only avail-
able for some of these records. A question thus arose
about whether all these reports did in fact represent
the same fungus. As can be seen in Figs 1, 2, isolates
of Cy. parasiticum clustered in two clades. One clade
corresponded chiefly to isolates obtained from Ha-
waii, while the other represented strains from the U.S.
mainland and Indonesia. These clades were not
supported by sufficient bootstrap support or morphol-
ogy, however, to argue that they could represent two
species. The isolates that did cluster apart were those
from soils under Eucalyptus canopies in Colombia,
here described as Cy. colombiense. A reason why
they could have been mistaken for Cy. parasiticum is
that these isolates frequently produce up to 3-septate
macroconidia that are slightly larger than those of Cy.
floridanum and resemble those of Cy. parasiticum.
CROUS ET AL.
428
Cylindrocladium multiphialidicum is quite distinct
from the other species described here because of its
thick-walled stipe extensions and the numerous
branches in its large conidiogenous apparatus. It is
interesting, however, that this species clusters close to
Cy. pseudonaviculatum Crous, J.Z. Groenewald &
C.F. Hill (Jun. 2002) (= Cy. buxicola B. Henricot;
Nov. 2002), which is a pathogen of Buxus semper-
virens L. in New Zealand (Crous et al. 2002), as well
as the U.K. (Henricot & Culham 2002), and Belgium
(Crepel & Inghelbrecht 2003). Cylindrocladium
pseudonaviculatum is distinct from Cy. multiphialidi-
cum in having naviculate vesicles, and in having
conidia larger (50–80 × 4–6 µm) than those of Cy.
multiphialidicum.
Table 1. Isolates of Cylindrocladium (Calonectria) species studied.
Accession number1GenBank accession numbers
(Tub, His, EF, Cal) 3 Host Country Collector
Cy. asiaticum
(Ca. asiatica) CBS 112705 / CPC 3897 AY725612, AY725654, AY725701,
AY725737 Debris Thailand N.L. Hywel-Jones
CBS 112711 / CPC 3898 AY725613, AY725655, AY725702,
AY725738 Debris Thailand N.L. Hywel-Jones
CBS 112938 / CPC 4513 AY725614, AY725656, AY725703,
AY725739 Soil Indonesia M.J. Wingfield
CBS 112952 / CPC 4718 AY725615, AY725657, AY725704,
AY725740 Soil Australia S. Abell
CBS 114073 / CPC 39002AY725616, AY725658, AY725705,
AY725741 Debris Thailand N.L. Hywel-Jones
CPC 682 AF348220, AF348236, AY725706,
AY725742 Soil Thailand M.J. Wingfield
Cy. canadense CBS 110817 / CPC 4992AF348212, AF348228, –,
AY725743 Picea sp. Canada S. Greifenhagen
UFV76 AF348224, AF348240, AY725707,
AY725744 Pinus sp. Canada A.C. Alfenas
Cy. chinense CBS 111037 / CPC 1154 AY725617, AY725659, AY725708,
AY725745 Soil Hong Kong M.J. Wingfield
CBS 112744 / CPC 4104 AY725618, AY725660, AY725709,
AY725746 Soil Hong Kong E.C.Y. Liew
CBS 114827 / STEU
41012AY725619, AY725661, AY725710,
AY725747 Soil Hong Kong E.C.Y. Liew
Cy. colombiense
(Ca. colombiensis) CBS 112220 / CPC 7232AF333413, AY725662, AY725711,
AY725748 Eucalyptus grandis Colombia M.J. Wingfield
CBS 112221 / CPC 724 AY725620, AY725663, AY725712,
AY725749 Eucalyptus grandis Colombia M.J. Wingfield
Cy. curvisporum CPC 7652AF333395, AY725664, –, – Soil Madagascar P.W. Crous
Cy. floridanum
(Ca. kyotensis) ATCC 188342AF348215, AF348231, AY725713,
AY725750 Robinia
pseudoacacia Japan T. Terashita
ATCC 188822AF348218, AF348234, AY725714,
AY725751 Prunus persica U.S.A. R.H. Morrison
CBS 413.67 / IMI
2995772AF348219, AF348235, –,
AY725752 Paphiopedilum
callosum Germany W. Gerlach
CBS 114552 / CPC 2350 AF333401, AY725665, AY725715,
AY725753 Soil Hong Kong M.J. Wingfield
Cy. hongkongense
(Ca. hongkongensis) CBS 114711 / CPC 686 AY725621, AY725666, AY725716,
AY725754 Soil Hong Kong M.J. Wingfield
CBS 114828 / STEU
46702AY725622, AY725667, AY725717,
AY725755 Soil Hong Kong E.C.Y. Liew
Cy. indonesiae CBS 112823 / CPC 45082AY725623, AY725668, AY725718,
AY725756 Syzygium
aromaticum
ticum
hylla
hylla
Indonesia M.J. Wingfield
CBS 112834 / CPC 4547 AY725624, AY725669, AY725719,
AY725757 Vanilla sp. Indonesia M.J. Wingfield
CBS 112840 / CPC 4554 AY725625, AY725670, AY725720,
AY725758 Syzygium
aroma Indonesia M.J. Wingfield
Cy. malesianum CBS 112710 / CPC 3899 AY725626, AY725671, AY725721,
AY725759 Debris Thailand N.L. Hywel-Jones
CBS 112752 / CPC 42232AY725627, AY725672, AY725722,
AY725760 Soil Indonesia M.J. Wingfield
Cy. multiphialidicum CBS 1126782AY725628, AY725673, AY725723,
AY725761 Musa sp. Cameroon Abadie
Cy. pacificum A1568 / IMI35428 / CPC
25342AF348222, AF348238, AY725724,
AY725762 Araucaria
heterop Hawaii M. Aragaki
A1569 / IMI35429 / CPC
2535 AF348223, AF348239, AY725725,
AY725763 Araucaria
heterop Hawaii M. Aragaki
CBS 114037 / CPC
10716 AY725629, AY725674, –, – Quercus palustris New
Zealand R. Peers
CBS 114038 / CPC
10717 AY725630, AY725675, –, – Ipomoea aquatica New
Zealand C.F. Hill
Cy. parasiticum
(= Ca. ilicicola) ATCC 46133 / CPC 2381 AF333411, –, –, – Cissus rhombifolia U.S.A. C.L. Schoulties
CBS 190.502AY725631, AY725676, AY725726,
AY725764 Solanum tuberosum Indonesia K.B. Boedijn & J.
Reitsma
CBS 111805 / CPC 2548 AY725632, AY725677, –, – Acacia koa Hawaii M. Aragaki
CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS
429
onaviculatum
CBS 112209 / CPC 491 AY725633, AY725678, –, – Medicago sativa Hawaii M. Aragaki
CBS 112210 / CPC 490 AY725634, AY725679, –, – Mandevilla sp. Hawaii M. Aragaki
CBS 112211 / CPC 489 AY725635, AY725680, –, – Leea guineensis Hawaii M. Aragaki
CBS 112212 / UFV49 /
CPC 495 AY725636, AY725681, –, – Cissus rhombifolia U.S.A. C.R. Semer
CBS 112213 / CPC 488 AY725637, AY725682, –, – Indigofera hirsuta U.S.A. N.E. El-Gholl
CBS 112214 / CPC 467 AY725638, AY725683, –, – Cinnamomum
kanahirai Taiwan M.J. Wingfield
CBS 112215 / CPC 492 AY725639, AY725684, AY725727,
AY725765 Arachis hypogaea U.S.A. Beute
CBS 112216 / CPC 487 AY725640, AY725685, AY725728,
AY725766 Howea forsteriana Hawaii M. Aragaki
CBS 112217 / CPC 486 AY725641, AY725686, –, – Karya sp. K. Rodriques
CBS 112218 / CPC 484 AY725642, AY725687, –, – Caryota sp. Hawaii M. Aragaki
CBS 112219 / CPC 482 AY725643, AY725688, –, – Arachis hypogaea U.S.A. Beute
CBS 112223 / CPC 2549 AY725644, AY725689, –, – Carica papaya Hawaii M. Aragaki
CBS 113783 / CPC 4872 AY725645, AY725690, AY725729,
AY725767 Arachis hypogaea U.S.A. C. Mobley
CBS 113903 / CPC 4871 AY725646, AY725691, AY725730,
AY725768 Arachis hypogaea U.S.A. C. Mobley
CPC 4873 –, AY725692, –, – Arachis hypogaea U.S.A. C. Mobley
CPC 10450 AY725647, AY725693, –, – Echeveria elegans Indonesia C.F. Hill
Cy.
pseud CPC 3570 AF449453, AY725694, AY725731,
AY725769 Buxus sempervirens New
Zealand –
Cy. sumatrense CBS 112757 / CPC 4501 AY725648, AY725695, AY725732,
AY725770 Soil Indonesia M.J. Wingfield
CBS 112829 / CPC 45182AY725649, AY725696, AY725733,
AY725771 Soil Indonesia M.J. Wingfield
CBS 112832 / CPC 4520 AY725650, AY725697, AY725734,
AY725772 Soil Indonesia M.J. Wingfield
CBS 112934 / CPC 4516 AY725651, AY725798, AY725735,
AY725773 Soil Indonesia M.J. Wingfield
1 CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CPC: Pedro Crous working collection housed at CBS; IMI: International
Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, U.K.; ATCC: American Type Culture Collection, Virginia, U.S.A.; UFV: Univeridade
Federal de Viçosa, Brazil.
2 Ex-type cultures.
3 Tub = β-tubulin, His = Histone H3, EF = Elongation factor 1-α, Cal = Calmodulin, - = not sequenced.
Key to Cylindrocladium species with sphaeropedunculate vesicles and 1-septate conidia (To be inserted in
Crous 2002, p. 60, couplet no. 21)
21. Macroconidiophore branches –8; conidiogenous apparatus up to 100 µm long and wide....................................A
21. Macroconidiophore branches –6; conidiogenous apparatus up to 90 µm long and wide.....................................22
A. Stipe thick-walled; conidia (45–)48–55(–65) × (4–)4.5(–5) µm, mean = 53 × 4.5 µm .......Cy. multiphialidicum
A. Stipe thin-walled; conidia (38–)45–48(–55) × 4(–4.5) µm, mean = 46.5 × 4 µm....................................................
....................................................................................................... Cy. hongkongense (teleo. Ca. hongkongensis)
22. Macroconidiophore branches 4–6..........................................................................................................................a
22. Macroconidiophore branches –3............................................................................................................................e
a. Lateral stipe extensions absent; macroconidia (40–)45–55(–60) × 3(–4) µm, mean = 50.5 × 4 µmCy. indonesiae
a. Lateral stipe extensions present..............................................................................................................................b
b. Macroconidia up to 55 µm, mean length less than 50 µm......................................................................................c
b. Macroconidia longer than 55 µm, mean length exceeding 50 µm .........................................................................d
c. Macroconidia (35–)45–50(–55) × 3–4(–5) µm, mean = 40 × 3.5 µm; teleomorph readily formed.........................
......................................................................................................................Cy. floridanum (teleo. Ca. kyotensis)
c. Macroconidia (34–)45–52(–55) × (3–)4 µm, mean = 47.5 × 4 µm; teleomorph not observed.... Cy. malesianum
d. Primary vesicles 7–12 µm diam; macroconidia 1(–3)-septate ........... Cy. colombiense (teleo. Ca. colombiensis)
d. Primary vesicles 12–17 µm diam; macroconidia 1-septate.............................. Cy. asiaticum (teleo. Ca. asiatica)
e. Conidial mean length 50 µm or shorter.................................................................................................................. f
e. Conidial mean length above 50 µm........................................................................................................................g
CROUS ET AL.
f. Vesicles pyriform to sphaeropedunculate; (38–)48–55(–65) × 4(–5) µm, mean = 50 × 4 µm....... Cy. canadense
f. Vesicles sphaeropedunculate; (38–)41–48(–56) × (3.5–)4(–4.5) µm (mean = 45 × 4 µm)................Cy. chinense
g. Lateral stipe extensions common ......................................................................................................Cy. pacificum
g. Lateral stipe extensions rare ............................................................................................................Cy. sumatrense
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