StudieS in Mycology 76: 51–167.
available online at www.studiesinmycology.org
The Botryosphaeriaceae: genera and species known from culture
A.J.L. Phillips1*, A. Alves2, J. Abdollahzadeh3, B. Slippers4, M.J. Wingield4, J.Z. Groenewald5, and P.W. Crous5,6,7
1
Centro de Recursos Microbiológicos, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal;
Departamento de Biologia, CESAM, Universidade de Aveiro, 3810-193 Aveiro, Portugal; 3Plant Protection Department, Agriculture Faculty, University of Kurdistan, P.O. Box
416, Sanandaj, Iran; 4Department of Genetics, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, 0002; 5CBS-KNAW Fungal Biodiversity Centre,
Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; 6Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; 7Wageningen
University and Research Centre (WUR), Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
2
*Correspondence: A.J.L. Phillips, alp@fct.unl.pt
Abstract: In this paper we give an account of the genera and species in the Botryosphaeriaceae. We consider morphological characters alone as inadequate to deine genera or
identify species, given the confusion it has repeatedly introduced in the past, their variation during development, and inevitable overlap as representation grows. Thus it seems
likely that all of the older taxa linked to the Botryosphaeriaceae, and for which cultures or DNA sequence data are not available, cannot be linked to the species in this family that
are known from culture. Such older taxa will have to be disregarded for future use unless they are epitypiied. We therefore focus this paper on the 17 genera that can now be
recognised phylogenetically, which concentrates on the species that are presently known from culture. Included is a historical overview of the family, the morphological features
that deine the genera and species and detailed descriptions of the 17 genera and 110 species. Keys to the genera and species are also provided. Phylogenetic relationships
of the genera are given in a multi-locus tree based on combined SSU, ITS, LSU, EF1-α and β-tubulin sequences. The morphological descriptions are supplemented by
phylogenetic trees (ITS alone or ITS + EF1-α) for the species in each genus.
Key words: Botryosphaeriales, canker pathogens, Diplodia, Fusicoccum, Lasiodiplodia, Multi-Locus Sequence Analysis, Sphaeropsis, systematics.
Taxonomic novelties: New species – Neofusicoccum batangarum Begoude, Jol. Roux & Slippers. New combinations – Botryosphaeria fabicerciana (S.F. Chen, D. Pavlic,
M.J. Wingf. & X.D. Zhou) A.J.L. Phillips & A. Alves, Botryosphaeria ramosa (Pavlic, T.I. Burgess, M.J. Wingf.) A.J.L. Phillips & A. Alves, Cophinforma atrovirens (Mehl & Slippers)
A. Alves & A.J.L. Phillips, Cophinforma mamane (D.E. Gardner) A.J.L. Phillips & A. Alves, Dothiorella pretoriensis (Jami, Gryzenh., Slippers & M.J. Wingf.) Abdollahz. & A.J.L.
Phillips, Dothiorella thailandica (D.Q. Dai., J.K. Liu & K.D. Hyde) Abdollahz., A.J.L. Phillips & A. Alves, Dothiorella uruguayensis (C.A. Pérez, Blanchette, Slippers & M.J. Wingf.)
Abdollahz. & A.J.L. Phillips, Lasiodiplodia lignicola (Ariyawansa, J.K. Liu & K.D. Hyde) A.J.L. Phillips, A. Alves & Abdollahz., Neoscytalidium hyalinum (C.K. Campb. & J.L.
Mulder) A.J.L. Phillips, Groenewald & Crous, Sphaeropsis citrigena (A.J.L. Phillips, P.R. Johnst. & Pennycook) A.J.L. Phillips & A. Alves, Sphaeropsis eucalypticola (Doilom, J.K.
Liu, & K.D. Hyde) A.J.L. Phillips, Sphaeropsis porosa (Van Niekerk & Crous) A.J.L. Phillips & A. Alves. Epitypiication (basionym) – Sphaeria sapinea Fries. Neotypiications
(basionyms) – Botryodiplodia theobromae Pat., Physalospora agaves Henn, Sphaeria atrovirens var. visci Alb. & Schwein.
Studies in Mycology
Published online: 30 September 2013; doi:10.3114/sim0021. Hard copy: September 2013.
INTroducTIoN
The Botryosphaeriaceae encompasses a range of morphologically
diverse fungi that are either pathogens, endophytes or saprobes,
mainly on woody hosts. They are found in all geographical and
climatic areas of the world, with the exception of the polar regions.
Their frequent association with plant diseases has stimulated
substantial interest in these fungi, and much of this interest has
been focussed on the systematics of species and genera.
Historical overview
The Botryosphaeriaceae was introduced by Theissen & Sydow
(1918) as a sub-family in the Pseudosphaeriaceae. Although
Theissen (1916) had earlier allocated the Pseudosphaeriaceae
to the Myriangiales, Theissen & Sydow (1917) believed that the
Pseudosphaeriaceae should be united with the Dothideaceae
(Luttrell 1951). Theissen & Sydow (1918) established the sub-class
the Dothideineae to accommodate the order Pseudosphaeriales,
family Botryosphaeriaceae, and the genus Botryosphaeria. Petrak
(1923) rejected Theissen & Sydow’s (1918) classiication and
placed Botryosphaeria in the sub-family Pseudosphaerieae, which
he placed in the Pleosporaceae (Sphaeriales).
Miller (1928) showed that there was a fundamental difference
between the tissues forming the ascoma and those forming the
boundary of the locules. He also showed how these different
tissue types were correlated with features of the ascocarp centrum.
Taxa allocated to the Sphaeriales had true perithecial ascomata
and paraphyses, while those assigned to the Dothideales had
ascostromatic ascomata lacking paraphyses. Thus, Botryosphaeria
species (Pseudosphaeriaceae) were allocated to the Dothideales
because they lacked true perithecial walls (Miller 1928).
Nannfeldt (1932) re-grouped the Euascomycetes into three
orders. The ascostromatic forms, where asci form in cavities in
pre-formed stromata, were accommodated in the Ascoloculares.
The true Sphaeriales, i.e., species in which the asci developed
in a hymenium, were accommodated in the Ascohymeniales.
Although these groups were not widely accepted at the time, they
were consistent with the bitunicate and unitunicate groups later
proposed by Luttrell (1955).
Concepts based on morphological features resulting from the
ontogeny of the perithecial wall and the development of centrum
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51
PhilliPs et al.
tissues were further developed by Miller (1938) and three orders
were recognised. The Sphaeriales had perithecia and paraphyses,
and the Dothideales encompassed ascostromatic forms with
interthecial threads that appeared in the ascomatal cavity before the
asci developed. Miller (1938) retained Botryosphaeria in the family
Pseudosphaeriaceae. Thus, Botryosphaeria was accommodated
in the Pseudosphaeriales and not in the Dothideales.
Luttrell (1951) recognised two major morphological groups in
the pyrenomycetous fungi. He also emphasised the signiicance
of ontogenetic characters of the ascomata in classiication.
The two major groups were those with single-walled asci or the
unitunicate ascomycetes, and the loculoascomycetes, commonly
referred to as the bitunicate ascomycetes (Luttrell 1955). Luttrell
also identiied eight forms of centrum development and highlighted
the taxonomic value of sterile interthecial tissues. Since the type
of the family Pseudosphaeriaceae, and the type of the genus
Pseudosphaeria had been transferred to the Dothideales, the
order Pseudosphaeriales was no longer tenable. Therefore, Luttrell
(1955) replaced the name Pseudosphaeriales with Pleosporales,
based on the most important genus in the group with that type
of centrum development, and assigned Botryosphaeria to the
Pleosporales.
In Barr’s earlier work (1972, 1976), she had not studied
specimens of B. dothidea in which the interthecial tissues were
clearly visible, and despite the clear demonstration by PargueyLeduc (1966) that B. dothidea exhibited a Pleospora centrum
type, she classiied Botryosphaeria in the Dothideales. Later,
however, Barr (1979) acknowledged that Botryosphaeria species
had a centrum typical of the Pleosporales and she concluded that
the genus should reside in this order. This view was retained in
subsequent publications (Barr 1983, 1987).
According to von Arx & Müller (1975) and von Arx (1981,
1987) the orders proposed by Lutrell (1955, 1973) and Barr
(1972, 1987) comprised a collection of unrelated genera
and the taxonomic characters used to separate the orders
overlapped. Moreover, von Arx & Müller (1975) did not support
the placement of closely related genera such as Guignardia
and Botryosphaeria in different orders, i.e. the Dothideales and
Pleosporales respectively (Luttrel 1973). For this reason von Arx
& Müller (1975) placed all bitunicate ascomycetes in the single
order Dothideales, comprising two suborders and 34 families,
including the Botryosphaeriaceae. To complicate matters further,
Sivanesan (1984) placed both Botryosphaeria and Guignardia
in the Dothideales whereas Hawksworth et al. (1995) listed
Botryosphaeria under the Botryosphaeriaceae and Guignardia
under the Mycosphaerellaceae, both in the Dothideales. Hence the
two major systems of classiication were those of Barr (1987) in
which Botryosphaeria is placed in the Pleosporales, and von Arx &
Müller (1975) who placed the genus in the Dothideales. Eriksson
(1981), however, emphasised that Botryosphaeria species have a
centrum typical of the Pleosporales with pseudoparaphyses and
pseudothecia.
The advent of DNA sequencing methods provided taxonomists
with powerful tools to determine phylogenetic relationships
in fungi at various taxonomic levels from species to orders.
Berbee (1996) used gene sequences of the 18S rRNA gene
(SSU) to study phylogenetic relationships amongst genera and
orders of loculoascomycetes. However, the positions of the two
Botryosphaeria species included in that study changed depending
on the analysis used. Thus, in the neighbour-joining trees of Berbee
(1966) these species usually clustered with species of Dothidea
in the Dothideales, but in a single maximum likelihood tree they
52
clustered in the Pleosporales. In a subsequent study of 18S rRNA
sequence data, Silva-Hanlin & Hanlin (1999) could not determine
whether the Botryosphaeria-Guignardia clade corresponded to the
Dothideales or the Pleosporales.
Schoch et al. (2006) constructed a multigene phylogeny based
on SSU, 28S rRNA gene (LSU), translation elongation factor
1-alpha (EF1-α) and RNA polymerase second largest subunit
(RPB2) sequence data for 96 taxa in the Dothideomycetes. Species
of Botryosphaeria and Guignardia formed a clade that could not be
associated with any other order. For this reason they proposed a
new order Botryosphaeriales accommodating the single family, the
Botryosphaeriaceae.
characteristics of the Botryosphaeriaceae
Detailed descriptions of the family Botryosphaeriaceae have
been presented by several authors (von Arx & Müller 1954, 1975,
Hawksworth et al. 1995, Eriksson 1981, Sivanesan 1984, Barr
1987). Members of the family are pathogenic, necrotrophic or
saprobic, especially on woody plants.
The Botryosphaericeae were characterised primarily on the basis
of their large, ovoid to oblong, usually hyaline, aseptate ascospores.
Although this could appear to be an inadequate basis for recognition
of a family, ascospores with this morphology have been considered
as an unusual spore type among loculoascomycetes (Luttrel 1973,
Eriksson 1981, Sivanesan 1984, Barr 1987). More recently, however,
at least six lineages in the family have been recognised as having
pigmented ascospores, and in three of these genera the ascospores
are septate (Phillips et al. 2008). Therefore, this simple circumscription
can no longer be considered suitable for the Botryosphaeriaceae.
Liu et al. (2012) recently provided a comprehensive deinition of
the family in which they considered ascospores to be hyaline and
aseptate, but that could become pigmented and septate with age.
This is an equally unsuitable deinition because ascospores in some
genera become pigmented and 1-septate at an early stage of their
development, long before they can be considered aged. Furthermore,
a circumscription based solely on the sexual state is not suitable
especially since some species are known only from their asexual
state, while in others the sexual state is extremely uncommon. Given
these conditions a modiied circumscription of the family is provided
by Slippers et al. (2013, this volume).
Genera in the Botryosphaeriaceae
When Theissen & Sydow (1918) introduced the Botryosphaeriaceae
they included three genera, namely Botryosphaeria, Phaeobotryon
and Dibotryon. Further genera were included over the years and
the addition of separate generic names for asexual and sexual
morphs resulted in the inclusion of at least 78 genera in the family
(MycoBank, http://www.mycobank.org, accessed May 2013). Many
of these genera have been determined to be synonyms, some new
genera have been introduced, some of the older genera have been
resurrected, yet others have been removed to other families. Liu et
al. (2012) recognised 29 genera of which 17 are known in culture.
The application of DNA sequence analysis and phylogenetic
inference has had a major impact on the systematics of the
Botryosphaeriaceae. Crous et al. (2006) used DNA sequence data
of the 28S rRNA gene to resolve 10 lineages within the family. The
phylogenetic clades correlated with distinct morphological features
and corresponded to separate genera. However, the LSU dataset
that Crous et al. (2006) used could not resolve a large clade
The Botryosphaeriaceae: genera and sPecies known from culTure
that comprised Diplodia, Lasiodiplodia and related genera with
pigmented conidia.
Phillips et al. (2008) attempted to resolve the phylogenetic and
taxonomic status of species of Botryosphaeriaceae with pigmented
ascospores. In a phylogeny based on SSU, the internal transcribed
spacers and intervening 5.8S rRNA gene (ITS) and LSU together with
EF1-α and β-tubulin sequence data they resolved six clades in the
Diplodia/Lasiodiplodia complex and an additional four clades in the
Botryosphaeriaceae. Damm et al. (2007) showed that Aplosporella
represents yet another genus in the Botryosphaeriaceae while Rojas
et al. (2008) determined that Endomelanconiopsis also resides in
this family. Phillips & Alves (2009) considered Melanops to be a
genus in the Botryosphaeriaeae. In a phylogeny based on SSU,
ITS, LSU and RNA polymerase largest subunit (RPB1) sequences,
Minnis et al. (2012) included Kellermania in the Planistromellaceae,
sister to the Botryosphaeriaceae. Furthermore, Wikee et al. (2013,
this volume) reinstated the Phyllostictaceae to accommodate
Phyllosticta (= Guignardia), which they recognised as distinct
from the Botryosphaeriaceae. Finally, Slippers et al. (2013, this
volume) introduced new families to accommodate Saccharata
(Saccharataceae), Melanops (Melanopsaceae), Aplosporella
and Bagnisiella (Aplosporellaceae). Thus, 17 genera can now be
recognised phylogenetically in the Botryosphaeriaceae. We consider
morphological characters alone as inadequate to deine genera
or identify species, given the confusion it has caused in the past.
Slippers et al. (2013, this volume) also illustrates how misleading
some of the prominent conidial and ascospore characters can be
to relect evolutionary origin, given independent origins or losses
of these characters over time. We therefore focus this paper on
the 17 genera that can now be recognised phylogenetically, which
concentrates on the species that are presently known from culture.
circumscription of genera
Characters that are used to differentiate genera in the
Botryosphaeriaceae have largely relied on the morphological
features of the ascospores (Barr 1987, 1989, Hsieh & Chen 1994,
Phillips et al. 2008) and especially the conidial states (Crous et
al. 2006, Phillips et al. 2008). The most informative characters
are conidial features such as pigmentation, wall thickness, and
septation, but other characters such as presence or absence of
paraphyses in the conidiomata can be useful. The phylogenetic
value of these characters can only be meaningfully interpreted,
however, in combination with additional data (e.g. sequence based
molecular data), as illustrated by their misinterpretation in the
past, and the multiple independent origins and losses of shared
characters throughout the evolutionary history of the family (see
Slippers et al. 2013, this volume).
Sexual morph morphology
Ascomata
Ascomata range from uniloculate, discrete structures (Fig. 1A, B)
through to relatively large multiloculate structures (Fig. 1C, D). The
uniloculate forms occur either individually and scattered over the host
(Fig. 1E), or they can be aggregated in botryose clusters (Fig. 1F)
of several hundred ascomata that are often united on a submerged
basal stroma. In the species with multiloculate ascomata, conidiomata
can occur within the same stroma. Sometimes the ascomata develop
at the periphery of a central conidioma (Fig. 1G) and are united with
the conidioma in a single stroma. When cut through horizontally the
www.studiesinmycology.org
contents are typically brilliant white (Fig. 1H). Irrespective of the form
they take, ascomata in Botryosphaeria species are typical of the
loculoascomycetes in which the asci are formed within locules that
develop in a pre-formed stroma. The tissues of the stromata are of
textura angularis and made up of brown, thick-walled cells that turn
blue-black in KOH and red-brown in lactic acid. The thickness of the
stromata varies considerably not only between species but also with
any given species. The walls can be as thin as just 5 or 6 cells layers,
or it can be up to 30 or even more. The locules are lined with thinwalled, hyaline, lattened cells.
The centrum is of the Pleospora type in which the asci are
interspersed with pseudoparaphyses that grow downwards and
fuse at the base of the locule. The form of the ascomata is of little
taxonomic value since even within a species ascomata can vary
from uniloculate with relatively thin walls to complex multiloculate
with thick walls and extensive stromatic tissue. This variation is
probably in response to the substrate or the conditions under which
the ascomata are formed. For example, ascomata in B. dothidea can
be either simple, uniloculate structures scattered individually over
the surface of the host tissue, or they can be aggregated in large
botryose clusters. They can also be formed in large multiloculate
stromata united with conidiomata. Furthermore, there does not
appear to be any correlation between the form of the ascomata and
the asexual genus associated with a particular species.
Asci
Asci are bitunicate of the issitunicate type with a relatively thin
ectotunica and a thick endotunica (Fig. 2A–C). The apex of the
endotunica (Fig. 2D) is modiied to form a well-deined apical
chamber, which results from a displacement of the endotunica by
the cytoplasm within the body of the ascus. No other structures can
be detected in the ascus apex. Asci are clavate to elongate-clavate
approaching cylindrical, but they are never truly cylindrical. They
often have a short, indistinct stipe that terminates in a hoof-shaped
cell attached to the inner wall of the base of the ascoma. Asci arise
from a basal hymenium and grow up through the pseudoparaphyses
(Fig. 2E). Ascospores are discharged forcibly by what has become
known as the “Jack-in-the-box” process whereby the ectotunica
splits transversely near the middle of the ascus and the endotunica
elongates expelling the spores.
Ascospores
Ascospores are arranged within the asci in an irregular, overlapping
biseriate manner (Fig. 2A–C). Typically they are hyaline and
aseptate (Fig. 2H–J), but they can be pale or dark brown (Fig. 2F,
G), sometimes 1-septate, and may have an apiculus at one or both
ends (Fig. 2G). The walls are smooth and in most species they
are usually thin, but in some, notably those species with Diplodia
asexual morphs, it can be moderately thick. Ascospores can be
hyaline or coloured, aseptate or 1–2-septate. In species with
hyaline, aseptate ascospores the spores can become translucent
brown and 1–2-septate with age (Fig. 2K), and the walls may appear
roughened (Fig. 2L) due to the deposition of melanin granules
on the inner surface, giving the spores a somewhat verruculose
appearance. Shapes range from fusiform to ovoid. They are usually
widest in the middle part and the ends are subobtusely rounded.
Pseudoparaphyses
Pseudoparaphyses are hyphal-like, hyaline with thin walls and
frequent septa (Fig. 2E), branched, frequently anatomosing. Often
they are constricted at the septa. As the asci develop and mature,
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PhilliPs et al.
Fig. 1. Ascomata characters. A–D. Vertical sections through ascomata. E. Ascomata erumpent through host bark. F. Botryose clusters of ascomata. G. Transverse section
through a central conidioma surrounded by ascomata. H. Ascomata cut through horizontally revealing the brilliant white contents. Scale bars: A–D, G = 100 μm, E = 1 mm, F,
H = 500 μm.
54
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 2. Asci and ascospores. A–C. Asci. D. Ascus tip showing the apical chamber. E. Pseudoparaphyses. F. Brown, 1-septate ascospores. G. Brown, aseptate ascospore with
an apiculus at either end. H–J. Hyaline, aseptate ascospores. K, L. Pale brown, 2-septate, aged ascospores in two different focal planes to reveal the verruculose inner surface
of the wall. Scale bars: A–C = 20 um, D–L = 10 μm.
the pseudoparaphyses gradually dissolve and only traces can be
found in older ascomata.
Asexual morph morphology
forms. Irrespective of the form, the conidiomata are stromatic, that
is, the pycnidial cavity develops within a preformed stroma (Fig. 3).
The tissues that make up the stromatal and conidiomata walls are
identical to those found in the ascostroma.
Conidiomata
Conidiophores
As with ascomata, conidiomata take on a variety of forms ranging
from thin-walled uniloculate pycnidial to large, complex multiloculate
Conidiophores are not always present in all species. Even within a
species, conidiophores may be present or absent. When present,
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PhilliPs et al.
Fig. 3. Conidiomata. A. Conidiomata covered with mycelium, formed on pine needle in culture. B. Conidiomata erumpent through the host bark. C. Vertical section through
a thin-walled conidioma. D. Section through conidiomata formed in culture. E. Transverse section through a stroma with several pycnidial locules. F. Vertical section through
conidiomata. Scale bars: A = 1 mm, B, D = 500 μm, C, E, F = 50 μm.
they are hyaline, thin-walled and more or less cylindrical. Mostly
they are not branched, but branched, septate conidiophores do
occur.
ampuliform. In species with fusicoccum-like asexual morphs, the
conidiogenous cells are generally smaller and more slender than
the more robust types found in species with diplodia-like asexual
morphs (Fig. 4).
Conidiogenesis
The irst conidia are formed holoblastically at the tips of
conidiogenous cells. Subsequent conidia are formed either
by internal proliferation of the conidiogenous cells resulting in
periclinal thickenings, or they may proliferate percurrently giving
rise to two or three close or widely spaced annellations. Both types
of proliferation can sometimes be seen on a single conidiogenous
cell.
Conidiogenous cells are hyaline with a smooth, thin
wall. Shapes vary from long cylindrical to short lageniform or
56
Conidia
Conidia of the Botryosphaeriaceae display the greatest variation
between genera and species. Although variation between species
is wide, variability within a species can also be quite considerable.
Two basic types of conidia can be distinguished, namely those that
are thin-walled, narrow and fusicoccum-like, and the thick-walled,
wider, diplodia-like conidia. In addition to these two basic types of
conidia, coloured, muriform conidia are found in the Dichomera
synasexual morph of some Botryosphaeria and Neofusicoccum
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 4. Conidiogenous cells and conidia. A–K. Conidiogenous cells with periclinal thickenings (B, K) and annellations (A, C, D), the annellate cell in E has formed a secondary
conidiogenous cell. F. Coloured, aseptate conidium of Diplodia intermedia attached to a conidiogenous cell. G. H, I. Coloured, 1-septate conidia of Dothiorella sp. attached to
conidiogenous cells. L. Paraphyses arising between developing conidia in a Lasiodiplodia species. M. Hyaline, aseptate, thin-walled conida of Botryosphaeria dothidea. N. Hyaline,
aseptate, thin-walled conida of Neofusicoccum arbuti. O. Coloured, septate conidia of B. dothidea. P. Hyaline, aseptate, thick-walled conidia of Diplodia mutila. Q. Hyaline, aseptate,
coloured, 1-septate conidia of Diplodia malorum. R. Coloured, aseptate conidia of Diplodia sapinea. S. Striate, mature and immature conidia of Barriopsis iraniana. T. Striate,
coloured, 1-septate conidia of a Lasiodiplodia species. Scale bars A, G = 10 μm. Scale bar in A applies to B–F, J, K, M–O. Scale bar in G applies to H–I, L, P–T.
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57
PhilliPs et al.
species. Furthermore, arthric chains of dry, powdery conidia are a
prominent feature of Neoscytalidium species.
The thin-walled, fusicoccum-like conidia range from fusiform
to ovoid or elliptical, and typically they are hyaline and aseptate.
However, the wall can become thicker and pale brown, and this
may be related to aging. Other changes can take place just before
germination when the normally hyaline, aseptate conidia can
develop one or two septa and in some species they may become
pale brown. In others, only the central cell becomes pigmented.
The diplodia-like conidia are relatively thick-walled and they
can be hyaline or brown. Furthermore, they may be aseptate or
1-septate, sometimes two or even more septa can form. They
are mostly ovoid with both ends broadly rounded. Externally the
walls are smooth, but melanin deposits on the inner surface of the
walls often give the conidia a verruculose appearance. In some
species, especially those that have been assigned to the genus
Lasiodiplodia, these deposits are arranged in longitudinal rows
giving the conidia a striate appearance.
The timing of the onset of pigmentation varies considerably.
In most Diplodia species, the conidia remain hyaline for a long
time, and indeed they may never become brown. However, if they
do become brown and septate, this occurs only after they have
been discharged from the conidiomata, and in this case large
numbers of brown, 1-septate conidia can be found on the surface
of the host, surrounding the pycnidia. Nevertheless, in the group
of species characterised by their brown, aseptate conidia (such
as D. seriata and D. sapinea) pigmented conidia can be seen
within the pycnidia, and often while the conidia are attached to the
conidiogenous cells. In Lasiodiplodia, the conidia usually remain
hyaline for a long time after they are formed, but they can become
brown and 1-septate whilst enclosed within the pycnidia. Normally,
however, pigmentation and septation happen after they have been
discharged. Furthermore, in Lasiodiplodia the conidia invariably
take on a striate appearance.
Conidia of some diplodia-like species become brown at an
early stage of their development. For example, conidia of D. seriata
become brown before they are discharged from the pycnidia.
This pattern of development is also seen in D. sapinea and its
close relative D. scrobiculata. In these three species (D. sapinea,
D. scrobiculata and D. seriata) the conidia do not form septa,
although one or more can develop at the time of germination. In
one group of diplodia-like species the conidia become brown and
septate at a very early stage, even before they are released from
the conidiogenous cells. The genus Dothiorella was resurrected
to accommodate these species (Phillips et al. 2005) and later
Spencermartinsia was introduced to accommodate species with
apiculate ascospores (Phillips et al. 2008).
Conidia of some species, both those in the diplodia-like
group and the fusicoccum-like group, undergo morphological
changes just before they germinate, and these changes can have
diagnostic value. Thus, normally hyaline, aseptate conidia can
develop one or two septa and become pale translucent brown
just before germination. This pigmentation can be either uniform
over the entire conidium, or one or more cells may be differentially
pigmented. However, this aspect of morphological and colour
changes at the time of germination has not been standardised, nor
has it been studied for all species. Similarly, as the conidia age
they may become darker and some develop septa. The effect of
aging on morphological features of these fungi is even less well
standardised and can be dificult to interpret.
58
Paraphyses
The presence or absence of paraphyses can be a useful character
for differentiating genera in the Botryosphaeriaceae. However, in
practice this can be dificult to apply because paraphyses are not
well-deined. In this work we refer to paraphyses as sterile hyphal
elements that form between and intermingled with conidiogenous
cells. We further regard paraphyses as only those elements
that extend beyond the height of conidiogenous cells and this
helps to distinguish paraphyses from immature, or developing
conidiogenous cells. In working through published descriptions of
species, any mention of paraphyses was critically re-examined and
only those that comply with the above deinition were accepted.
An example where paraphyses are useful taxonomic characters
is in the differentiation of Lasiodiplodia from Neodeightonia. Both
Lasiodiplodia and Neodeightonia have striate conidia, but only
Lasiodiplodia species have paraphyses. Likewise, presence
of paraphyses in Sphaeropsis differentiates this genus from
Diplodia, which does not have pycnidial paraphyses. Length of
the paraphyses and their morphology, especially the presence or
absence of a swelling at the tip can also aid in the differentiation
of species.
Spermatogonia
Spermatial states are common in the Dothideomycetes, and also
known in several species in the Botryosphaeriaceae. However,
where seen they are not consistently formed by all isolates of a
particular species, that is, they can be present or absent. Thus,
their importance in the taxonomy and discrimination of species and
genera is of questionable value.
The aim of the current paper was to consider all the genera
and species in the Botryosphaeriaceae known from culture,
based on their morphological characters and DNA sequence
based phylogenetic relationships. The intention is to provide
a comprehensive and up to date document that can serve as a
foundation on which future descriptions of species and other genera
can build. Of the older taxa linked to the Botryosphaeriaceae, and
for which cultures or DNA sequence data are not available, very
few, if any, can be linked to the current species that are known from
culture. Such older taxa will have to be disregarded for future use
unless they are epitypiied. The current document will serve as a
starting point for that process.
MATerIAlS ANd MeTHodS
Morphology
Fresh collections and type specimens were examined for most
of the species included in this study. However, where the type (or
other suitable specimens) could not be obtained, and no fresh
collections were available, the descriptions were adapted from
the orginal published descriptions. Isolations were made directly
from ascomata or conidiomata on the host whenever possible. The
sporocarps were cut through vertically with a sterile scalpel, one
half was crushed in a drop of sterile water and then spread over
the surface of a plate of 1/2 strength Difco potato-dextrose agar
(PDA; Becton, Dickinson & Co, Sparks, USA). After incubation for
up to 24 h, single germinating spores were transferred to fresh
plates of PDA. The other half of the fruit body was placed in a
drop of water on a microscope slide and the fertile tissues (asci
or conidiogenous layer) were dissected and mounted in 100 %
The Botryosphaeriaceae: genera and sPecies known from culTure
lactic acid for microscopy. This method, when used for ascomata,
allowed unambiguous connection to be established between the
sexual and asexual morph.
Cultures were induced to sporulate by culturing on 2 % water
agar bearing double-autoclaved poplar twigs, or pine needles. After
a suitable period of incubation, ranging from 1–4 wk, conidiomata
were cut through vertically, the conidiogenous layer dissected and
mounted in 100 % lactic acid. Observations on micromorphological
features were made with Leica MZ95 and Leica DMR microscopes
and digital images were recorded with Leica DC300 and Leica
DFC320 cameras, respectively. Measurements were made with
the measurement module of the Leica IM500 image management
system (Leica Micro-systems GmbH, Wetzlar, Germany). Mean,
standard deviation (S.D.) and 95 % conidence intervals were
calculated for asci, ascospores, and conidia. Minimum and
maximum dimensions are given in parenthesis. Cultures were
deposited in the CBS culture collection, taxonomic descriptions
and nomenclature were deposited in MycoBank (www.MycoBank.
org; Crous et al. 2004).
dNA isolation, sequencing and phylogenetic
analyses
was ampliied using the primers ITS1 (White et al. 1990) and NL4
(O’Donnell 1993) as described by Alves et al. (2005). Nucleotide
sequences of the ITS and D1/D2 regions were determined as
described previously (Alves et al. 2004, 2005) using the primers
ITS4 (White et al. 1990) and NL1 (O’Donnell 1993) as internal
sequencing primers.
The primers EF1-688F (Alves et al. 2008) and EF1-986R
(Carbone & Kohn 1999) and Bt2a and Bt2b (Glass & Donaldson
1995) were used to amplify and sequence part of the translation
elongation factor 1-alpha (EF1-α) gene and part of the β-tubulin
gene, respectively. Ampliication and nucleotide sequencing of the
EF1-α and β-tubulin genes were performed as described previously
(Alves et al. 2006, 2008).
The ampliied PCR fragments were puriied with the JETQUICK
PCR Puriication Spin Kit (GENOMED, Löhne, Germany). Both
strands of the PCR products were sequenced at STAB Vida Lda
(Portugal) or GATC Biotech (Germany). The nucleotide sequences
were read and edited with FinchTV v. 1.4.0 (Geospiza Inc.). All
sequences were checked manually and nucleotide arrangements
at ambiguous positions were clariied using both primer direction
sequences
DNA sequencing and phylogenetic analysis
Most of the sequences used in this work were obtained from
GenBank. Methods for DNA isolation, puriication and sequencing
of new sequences are detailed below. New sequences were
deposited in GenBank, and the alignment in TreeBASE. Isolates
and GenBank Accession numbers are listed in Table 1.
dNA isolation
Isolates were grown on PDA plates in darkness at 25 °C until they
completely covered the medium surface. The mycelium was then
scraped off and collected in a 2 mL Eppendorf tube with 50 μL of
autoclaved glass micro spheres (230–320 μm diam). The tubes
were then placed in liquid nitrogen for 5 min and transferred to ice.
To separate organic and aqueous phases, 250 μL of phenol and
250 μL of chloroform were added, together with 500 μL of lysis
buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 %
Triton X-100, 1 % SDS). Tubes were vortexed for 20 min and then
centrifuged (19000 × g, 4 °C, 25 min). The aqueous phase was
transferred to a new 1.5 mL tube and the nucleic acids precipitated
with an equal volume of cold absolute isopropanol. The tubes were
centrifuged again (19 000 × g, 4 °C, 10 min), the supernatants
discarded and the pellets washed with 1 mL of cold 70 % ethanol.
After a further centrifugation (19 000 × g, 4 °C, 5 min), the
supernatants were discarded and the pellets dried at RT with the
tubes open in an inverted position. RNA was digested by incubating
the pellets with 50 μL of TE (10 mM Tris, 1 mM EDTA) + RNAse A
(Sigma®) (50 μg/mL) at 55 °C for 15 min.
dNA sequencing
A portion of the nuclear ribosomal 18S RNA gene (SSU) was
ampliied with primers NS1 and NS4 (White et al. 1990). The
nucleotide sequence was determined using the above primers
along with the internal sequencing primers NS2 and NS3 (White
et al. 1990). The ampliication and sequencing were done as
described by Phillips et al. (2008).
Part of the nuclear rRNA cluster comprising the ITS region plus
the D1/D2 variable domains of the ribosomal 28S RNA gene (LSU)
www.studiesinmycology.org
A phylogenetic analysis based on sequence data from ive loci,
namely SSU, ITS, LSU, EF1-α and β-tubulin, was done to deine
the phylogenetic position of genera in the Botryosphaeriaceae.
Phylogenetic analyses based on ITS or ITS+EF1-α sequences
were done for the species in each of the genera, except where
there are few species in the genus.
Sequences were aligned with ClustalX v. 1.83 (Thompson
et al. 1997), using the following parameters: pairwise alignment
parameters (gap opening = 10, gap extension = 0.1) and multiple
alignment parameters (gap opening = 10, gap extension = 0.2,
transition weight = 0.5, delay divergent sequences = 25 %).
Alignments were checked and manual adjustments were made
where necessary.
Phylogenetic analyses of sequence data were done using PAUP
v. 4.0b10 (Swofford 2003) for Maximum-Parsimony (MP) analyses
and MEGA v. 5 (Tamura et al. 2011) for Maximum-Likelihood
(ML) analyses. The general time reversible model of evolution
(Rodriguez et al. 1990), including estimation of invariable sites and
assuming a discrete gamma distribution with six rate categories
(GTR+Γ+G) was used for the ML analysis. Trees were rooted using
an outgroup and visualised with TreeView (Page 1996).
MP analyses were performed using the heuristic search option
with 1 000 random taxa addition and tree bisection and reconnection
(TBR) as the branch-swapping algorithm. All characters were
unordered and of equal weight and gaps were treated as missing
data. Maxtrees were set to 500, branches of zero length were
collapsed, and all multiple equally most parsimonious trees were
saved. The robustness of the most parsimonious trees was
evaluated from 1 000 bootstrap replications. Other measures used
were consistency index (CI), retention index (RI) and homoplasy
index (HI).
ML analyses were performed on a MP starting tree automatically
generated by the software. Nearest-Neigbour-Interchange (NNI)
was used as the heuristic method for tree inference and 1 000
bootstrap replicates were performed.
59
PhilliPs et al.
Table 1. GenBank and culture collection accession numbers of species treated in the phylogenies.
GenBank accession numbers
cultures
SSu
ITS
lSu
EF1-α
β-tubulin
Barriopsis fusca
CBS 174.26 ex-type
EU673182
EU673330
DQ377857
EU673296
EU673109
Barriopsis iraniana
CBS 124698 ex-type
N/A
FJ919663
N/A
FJ919652
N/A
IRAN 1449C
N/A
FJ919665
N/A
FJ919654
N/A
CBS 134113 ex-type
JX646826
JX646792
JX646809
JX646857
N/A
MFLUCC 11-0657
JX646827
JX646793
JX646810
JX646858
N/A
CBS 133992 ex-neotype
JX646825
JX646791
JX646808
JX646856
JX646841
MFLUCC 10-0051
JX646824
JX646790
JX646807
JX646855
JX646840
CBS 119047 ex-epitype
EU673175
DQ299245
EU673244
EU017539
EU673107
Species
1
Botryobambusa fusicoccum
Botryosphaeria agaves
Botryosphaeria corticis
Botryosphaeria dothidea
Botryosphaeria fabicerciana
Botryosphaeria fusispora
2
ATCC 22927
EU673176
DQ299247
EU673245
EU673291
EU673108
CBS 115476 ex-epitype
EU673173
AY236949
AY928047
AY236898
AY236927
CBS 110302
EU673174
AY259092
EU673243
AY573218
EU673106
CBS 127193 ex-type
N/A
HQ332197
N/A
HQ332213
N/A
CMW 27108
N/A
HQ332200
N/A
HQ332216
N/A
MFLUCC 10-0098 ex-type
JX646823
JX646789
JX646806
JX646854
JX646839
JX646838
MFLUCC 11-0507
JX646822
JX646788
JX646805
JX646853
Botryosphaeria ramosa
CBS 122069 ex-type
N/A
EU144055
N/A
EU144070
N/A
Botryosphaeria schariii
CBS 124703 ex-type
N/A
JQ772020
N/A
JQ772057
N/A
CBS 124702
N/A
JQ772019
N/A
JQ772056
N/A
CBS 124934 ex-type
N/A
FJ888473
N/A
FJ888456
N/A
CBS 124935
N/A
FJ888476
N/A
FJ888457
N/A
MFLUCC 11-0425 ex-type
JX646833
JX646800
JX646817
JX646865
JX646848
MFLUCC 11-0655
JX646834
JX646801
JX646818
JX646866
JX646849
CBS 117444
KF531821
KF531822
DQ377855
KF531801
KF531802
CBS 117450
N/A
EF118051
N/A
GU134937
N/A
CBS 120835 ex-type
N/A
EF445343
N/A
EF445382
N/A
CBS 121104
N/A
EF445344
N/A
EF445383
N/A
CBS 124931 ex-type
N/A
FJ888460
N/A
FJ888444
N/A
CBS 124933 ex-paratype
N/A
FJ888478
N/A
FJ888446
N/A
CBS 130408 ex-type
N/A
JQ239397
JQ239410
JQ239384
JQ239378
CBS 130410 ex-paratype
N/A
JQ239399
JQ239412
JQ239386
JQ239380
CBS 132777 ex-type
N/A
JN693507
N/A
JQ517317
JQ411459
UCROK 1429
N/A
JQ411412
N/A
JQ512121
JQ411443
CBS 124254 ex-type
N/A
GQ923853
N/A
GQ923821
N/A
CBS 124135
N/A
GQ923852
N/A
GQ923820
N/A
CBS 112549 ex-type
EU673206
AY259100
AY928051
AY573227
DQ458853
CBS 112546
EU673207
AY259110
EU673262
DQ458872
EU673117
CBS 168.87 ex-type
EU673209
DQ458893
EU673263
DQ458878
DQ458861
CBS 261.85
EU673210
DQ458894
EU673264
DQ458879
DQ458862
CBS 124462 ex-type
N/A
GQ923858
N/A
GQ923826
N/A
CBS 124134
N/A
HM036528
N/A
GQ923851
N/A
Diplodia malorum
CBS 124130 ex-epitype
N/A
GQ923865
N/A
GQ923833
N/A
CBS 112554
N/A
AY259095
N/A
DQ458870
N/A
Diplodia mutila
CBS 112553
EU673213
AY259093
AY928049
AY573219
DQ458850
CBS 230.30
EU673214
DQ458886
EU673265
DQ458869
DQ458849
Diplodia olivarum
CBS 121887 ex-type
N/A
EU392302
N/A
EU392279
HQ660079
Cophinforma atrovirens
Cophinforma atrovirens
Cophinforma atrovirens
Diplodia africana
Diplodia alatafructa
Diplodia allocellula
Diplodia agrifolia
Diplodia bulgarica
Diplodia corticola
Diplodia cupressi
Diplodia intermedia
Diplodia pseudoseriata
Diplodia quercivora
60
CBS 121886
N/A
EU392297
N/A
EU392274
N/A
CBS 124906 ex-type
N/A
EU080927
N/A
EU863181
N/A
CBS 124907 ex-paratype
N/A
EU080922
N/A
EU863179
N/A
CBS 133852 ex-type
N/A
JX894205
N/A
JX894229
N/A
CBS 133853
N/A
JX894206
N/A
JX894230
N/A
The Botryosphaeriaceae: genera and sPecies known from culTure
Table 1. (Continued).
GenBank accession numbers
Species
1
Diplodia rosulata
Diplodia sapinea
Diplodia scrobiculata
cultures
SSu
ITS
lSu
EF1-α
β-tubulin
CBS 116470 ex-type
EU673211
EU430265
DQ377896
EU430267
EU673132
CBS 116472
EU673212
EU430266
DQ377897
EU430268
EU673131
CBS 393.84 (A) ex-epitype
EU673219
DQ458895
DQ377893
DQ458880
DQ458863
DQ458864
2
CBS 109725 (C)
EU673222
DQ458896
EU673270
DQ458881
CBS 118110 ex-type
N/A
AY253292
N/A
AY624253
AY624258
CBS 109944
EU673218
DQ458899
EU673268
DQ458884
DQ458867
CBS 113423
EU673217
DQ458900
EU673267
DQ458885
DQ458868
Diplodia seriata
CBS 112555 ex-epitype
EU673215
AY259094
AY928050
AY573220
DQ458856
CBS 119049
EU673216
DQ458889
EU673266
DQ458874
DQ458857
Diplodia tsugae
CBS 418.64 ex-isotype
EU673208
DQ458888
DQ377867
DQ458873
DQ458855
Dothiorella americana
CBS 128309 ex-type
N/A
HQ288218
N/A
HQ288262
HQ288297
CBS 128310
N/A
HQ288219
N/A
HQ288263
HQ288298
Dothiorella brevicollis
Dothiorella casuarinae
Dothiorella dulcispinae
Dothiorella iberica
Dothiorella longicollis
Dothiorella moneti
CBS 130411 ex-type
N/A
JQ239403
JQ239416
JQ239390
JQ239371
CBS 130412 ex-paratype
N/A
JQ239404
JQ239417
JQ239391
JQ239372
CBS 120688 ex-type
N/A
DQ846773
N/A
DQ875331
N/A
CBS 120690
N/A
DQ846774
N/A
DQ875333
N/A
CBS 130413 ex-type
N/A
JQ239400
JQ239413
JQ239387
JQ239373
CBS 130414 ex-paratype
N/A
JQ239401
JQ239414
JQ239388
JQ239374
CBS 130415 ex-paratype
N/A
JQ239402
JQ239415
JQ239389
JQ239375
CBS 121764
N/A
EU101299
N/A
EU101344
N/A
CBS 121765
N/A
EU101300
N/A
EU101345
N/A
CBS 115041 ex-type
EU673155
AY573202
AY928053
AY573222
EU673096
CBS 113188
EU673156
AY573198
EU673230
EU673278
EU673097
CAA 005
EU673157
EU673312
EU673231
EU673279
EU673098
CBS 122068 ex-type
N/A
EU144054
N/A
EU144069
N/A
CBS 122067
N/A
EU144052
N/A
EU144067
N/A
MUCC 505 ex-type
N/A
EF591920
EF591937
EF591971
EF591954
MUCC 507
N/A
EF591922
EF591939
EF591973
EF591956
CBS 130404 ex-type
N/A
JQ239405
JQ239418
JQ239392
JQ239376
CBS 130403 ex-paratype
N/A
JQ239406
JQ239419
JQ239393
JQ239377
MUCC 509 ex-type
N/A
EF591924
EF591941
EF591975
EF591958
MUCC 508
N/A
EF591923
EF591940
EF591974
EF591957
IMI 63581b ex-type
EU673158
AY573212
AY928052
AY573235
EU673102
CBS 115038
EU673159
AY573206
DQ377860
AY573223
EU673101
Dothiorella thailandica
CBS 133991 ex-type
JX646829
JX646796
JX646813
JX646861
JX646844
Dothiorella thripsita
BRIP 51876 ex-type
N/A
FJ824738
N/A
N/A
N/A
Dothiorella uruguayensis
CBS 124908 ex-type
N/A
EU080923
N/A
EU863180
N/A
Dothiorella sp.1
CBS 188.87
EU673161
EU673316
DQ377891
EU673283
EU673119
CBS 242.51
EU673162
EU673317
EU673235
EU673284
EU673105
Dothiorella pretoriensis
Dothiorella santali
Dothiorella sarmentorum
Dothiorella sp.2
JL 599
EU673164
EU673314
EU673233
EU673281
EU673099
Dothiorella sp.3
CBS 124723
EU673163
EU673313
EU673232
EU673280
EU673100
Dothiorella sp.4
CBS 124731
EU673170
EU673321
EU673240
EU673288
EU673143
CBS 124730
EU673169
EU673320
EU673239
EU673287
EU673142
CBS 120397 ex-type
N/A
EU683656
EU683629
EU683637
N/A
CBS 122550
N/A
EU683664
EU683634
EU683645
N/A
Endomelanconiopsis endophytica
Endomelanconiopsis microspora
CBS 353.97 ex-type
N/A
EU683655
EU683628
EU683636
N/A
Lasiodiplodia citricola
CBS 124707 ex-type
N/A
GU945354
N/A
GU945340
N/A
CBS 124706
N/A
GU945353
N/A
GU945339
N/A
CBS 118741 ex-type
N/A
DQ103550
N/A
EU673303
N/A
WAC 12534
N/A
DQ103551
N/A
DQ103558
N/A
Lasiodiplodia crassispora
www.studiesinmycology.org
61
PhilliPs et al.
Table 1. (Continued).
GenBank accession numbers
Species
1
Lasiodiplodia egyptiacae
Lasiodiplodia gilanensis
Lasiodiplodia gonubiensis
Lasiodiplodia hormozganensis
Lasiodiplodia iraniensis
cultures
SSu
ITS
lSu
EF1-α
β-tubulin
CBS 110492
EU673189
EF622086
EU673251
EF622066
EU673134
CBS 130992 ex-type
N/A
JN814397
N/A
JN814424
N/A
BOT-29
N/A
JN814401
N/A
JN814428
N/A
CBS 124704 ex-type
N/A
GU945351
N/A
GU945342
N/A
CBS 124705
N/A
GU945352
N/A
GU945341
N/A
CBS 115812 ex-type
EU673193
AY639595
DQ377902
DQ103566
DQ458860
CBS 116355
EU673194
AY639594
EU673252
DQ103567
EU673126
CBS 124709 ex-type
N/A
GU945355
N/A
GU945343
N/A
CBS 124708
N/A
GU945356
N/A
GU945344
N/A
CBS 124710 ex-type
N/A
GU945346
N/A
GU945334
N/A
2
CBS 124711
N/A
GU945347
N/A
GU945335
N/A
Lasiodiplodia lignicola
CBS 134112 ex-type
JX646830
JX646797
JX646814
JX646862
JX646845
MFLUCC 11-0656
JX646831
JX646798
JX646815
JX646863
JX646846
Lasiodiplodia margaritacea
CBS 122519 ex-type
N/A
EU144050
N/A
EU144065
N/A
CBS 122065
N/A
EU144051
N/A
EU144066
N/A
CBS 124927 ex-type
N/A
FJ900597
N/A
FJ900643
N/A
CBS 124925 ex-type
N/A
FJ900595
N/A
FJ900641
N/A
CBS 128311 ex-type
N/A
HQ288225
N/A
HQ288267
N/A
CBS 128312
N/A
HQ288226
N/A
HQ288268
N/A
CBS 456.78 ex-type
N/A
EF622083
N/A
EF622063
N/A
CBS 494.78
EU673201
EF622084
EU673258
EF622064
EU673114
Lasiodiplodia mahajangana
Lasiodiplodia missouriana
Lasiodiplodia parva
Lasiodiplodia plurivora
Lasiodiplodia pseudotheobromae
Lasiodiplodia rubropurpurea
Lasiodiplodia theobromae
Lasiodiplodia venezuelensis
Lasiodiplodia viticola
Macrophomina phaseolina
Neodeightonia palmicola
Neodeightonia phoenicum
Neodeightonia subglobosa
CBS 356.59
EU673200
EF622082
EU673257
EF622062
EU673113
CBS 120832 ex-type
N/A
EF445362
N/A
EF445395
N/A
CBS 121103
N/A
AY343482
N/A
EF445396
N/A
CBS 116459 ex-type
EU673199
EF622077
EU673256
EF622057
EU673111
CBS 447.62
EU673198
EF622081
EU673255
EF622060
EU673112
CBS 118740 ex-type
EU673191
DQ103553
DQ377903
EU673304
EU673136
WAC 12536
N/A
DQ103554
N/A
DQ103572
N/A
CBS 164.96 ex-neotype
EU673196
AY640255
EU673253
AY640258
EU673110
CBS 124.13
EU673195
DQ458890
AY928054
DQ458875
DQ458858
CBS 111530
N/A
EF622074
N/A
EF622054
N/A
CAA 006
EU673197
DQ458891
EU673254
DQ458876
DQ458859
CBS 118739 ex-type
EU673192
DQ103547
DQ377904
EU673305
EU673129
WAC 12540
N/A
DQ103548
N/A
DQ103569
N/A
CBS 128313 ex-type
N/A
HQ288227
N/A
HQ288269
HQ288306
CBS 128315
N/A
HQ288228
N/A
HQ288270
HQ288307
CBS 227.33
KF531823
KF531825
DQ377906
KF531804
KF531806
CBS 162.25
KF531824
KF531826
DQ377905
KF531803
KF531805
MFLUCC 10-0822 ex-type
HQ199223
HQ199221
HQ199222
N/A
N/A
MFLUCC 10-0823
HQ199226
HQ199224
HQ199225
N/A
N/A
CBS 122528 ex-type
EU673205
EU673340
EU673261
EU673309
EU673116
CBS 169.34
EU673203
EU673338
EU673259
EU673307
EU673138
CBS 448.91 ex-type
EU673202
EU673337
DQ377866
EU673306
EU673137
MFLUCC 11-0163
N/A
JX646794
JX646811
JX646859
JX646842
Neofusicoccum andinum
CBS 117453 ex-type
N/A
AY693976
N/A
AY693977
N/A
CBS 117452
N/A
DQ306263
N/A
DQ306264
N/A
Neofusicoccum arbuti
CBS 116131 ex-type
KF531814
AY819720
DQ377915
KF531792
KF531793
CBS 117090
KF531813
AY819724
DQ377919
KF531791
KF531794
CMW 6837 ex-type
N/A
AY339262
N/A
AY339270
AY339254
CMW 6853
N/A
AY339263
N/A
AY339271
AY339255
Neofusicoccum australe
62
The Botryosphaeriaceae: genera and sPecies known from culTure
Table 1. (Continued).
GenBank accession numbers
cultures
SSu
ITS
lSu
EF1-α
β-tubulin
Neofusicoccum batangarum
CBS 124924 ex-type
N/A
FJ900607
N/A
FJ900653
FJ900634
CBS 124923
N/A
FJ900608
N/A
FJ900654
FJ900635
Neofusicoccum cordaticola
CBS 123634 ex-type
N/A
EU821898
N/A
EU821868
EU821838
EU821843
Species
1
2
CBS 123635
N/A
EU821903
N/A
EU821873
Neofusicoccum corticosae
CBS 120081 ex-type
N/A
DQ923533
N/A
N/A
N/A
Neofusicoccum eucalypticola
CBS 115679 ex-type
N/A
AY615141
N/A
AY615133
AY615125
CBS 115766
N/A
AY615143
N/A
AY615135
AY615127
CBS 115791 ex-type
N/A
AF283686
N/A
AY236891
AY236920
CMW 10126
N/A
AF283687
N/A
AY236892
AY236921
Neofusicoccum grevilleae
CBS 129518 ex-type
N/A
JF951137
JF951157
N/A
N/A
Neofusicoccum kwambonambiense
CBS 123639 ex-type
N/A
EU821900
N/A
EU821870
EU821840
CBS 123641
N/A
EU821919
N/A
EU821889
EU821859
CBS 110299 ex-type sexual morph
EU673148
AY259091
AY928043
AY573217
DQ458848
CBS 562.92 ex-type asexual morph
N/A
N/A
N/A
N/A
N/A
Neofusicoccum luteum
Neofusicoccum macroclavatum
Neofusicoccum mangiferae
Neofusicoccum mediterraneum
Neofusicoccum nonquaesitum
Neofusicoccum occulatum
CBS 110497
EU673149
EU673311
EU673229
EU673277
EU673092
CBS 118223 ex-type
N/A
DQ093196
N/A
DQ093217
DQ093206
WAC 12445
N/A
DQ093197
N/A
DQ093218
DQ093208
CBS 118531
EU673153
AY615185
DQ377920
DQ093221
AY615172
CBS 118532
EU673154
AY615186
DQ377921
DQ093220
AY615173
CBS 121718 ex-type
N/A
GU251176
N/A
GU251308
GU251836
CBS 121558
N/A
GU799463
N/A
GU799462
GU799461
CBS 126655 ex-type
N/A
GU251163
N/A
GU251295
GU251823
PD 301
N/A
GU251164
N/A
GU251296
GU251824
CBS 128008 ex-type
N/A
EU301030
N/A
EU339509
EU339472
MUCC 286
N/A
EU736947
N/A
EU339511
EU339474
ATCC 58191 ex-type
EU673151
AY236943
AY928045
AY236888
AY236917
CBS 110301
EU673150
AY259098
AY928046
AY573221
EU673095
Neofusicoccum pennatisporum
WAC 13153 ex-type
N/A
EF591925
EF591942
EF591976
EF591959
Neofusicoccum protearum
STE-U 4361 ex-type asexual morph
N/A
AF196295
N/A
N/A
N/A
CBS 114176 ex-type sexual morph
N/A
AF452539
N/A
N/A
N/A
CBS 115475 ex-type
N/A
AY236935
N/A
AY236877
AY236906
CBS 121.26
N/A
AF241177
N/A
AY236879
AY236908
CBS 123645 ex-type
N/A
EU821904
N/A
EU821874
EU821844
CBS 123646
N/A
EU821905
N/A
EU821875
EU821845
CBS 112878 ex-type
N/A
AY343381
N/A
AY343342
N/A
CBS 112977
N/A
AY343380
N/A
AY343341
N/A
CBS 110887 ex-type
N/A
AY343383
N/A
AY343343
N/A
CBS 110880
N/A
AY343382
N/A
AY343344
N/A
CBS 499.66
KF531818
KF531820
DQ377925
KF531798
KF531800
CBS 251.49
KF531817
KF531819
DQ377923
KF531797
KF531799
CBS 145.78 ex-isotype
KF531815
KF531816
DQ377922
KF531795
KF531796
CBS 122071 ex-type
N/A
EF585540
N/A
EF585580
N/A
CBS 122610
N/A
EF585536
N/A
EF585578
N/A
N/A
N/A
N/A
N/A
N/A
CBS 124700 ex-type
N/A
FJ919672
N/A
FJ919661
N/A
IRAN 1458C
N/A
FJ919671
N/A
FJ919660
N/A
CBS 122980 ex-type
EU673184
EU673332
EU673248
EU673298
EU673121
CPC 12442
EU673185
EU673333
DQ377899
EU673299
EU673124
CBS 122055 ex-type
N/A
EF585523
N/A
EF585571
N/A
WAC 12689
N/A
EF585534
EF585554
EF585567
N/A
Neofusicoccum parvum
Neofusicoccum ribis
Neofusicoccum umdonicola
Neofusicoccum viticlavatum
Neofusicoccum vitifusiforme
Neoscytalidium hyalinum
Neoscytalidium novaehollandiae
Phaeobotryon cercidis
Phaeobotryon cupressi
Phaeobotryon mamane
Pseudofusicoccum adansoniae
www.studiesinmycology.org
63
PhilliPs et al.
Table 1. (Continued).
GenBank accession numbers
Species
1
Pseudofusicoccum ardesiacum
Pseudofusicoccum kimberleyense
Pseudofusicoccum olivaceum
Pseudofusicoccum stromaticum
Pseudofusicoccum violaceum
Spencermartinsia viticola
Spencermartinsia sp.1
cultures
SSu
ITS
lSu
EF1-α
β-tubulin
CBS 122062 ex-type
N/A
EU144060
N/A
EU144075
N/A
2
WAC 13294
N/A
GU172405
N/A
GU172437
N/A
CBS 122058 ex-type
N/A
EU144057
N/A
EU144072
N/A
CBS 122059
N/A
EU144056
N/A
EU144071
N/A
CBS 124939 ex-type
N/A
FJ888459
N/A
FJ888437
N/A
CBS 124940
N/A
FJ888462
N/A
FJ888438
N/A
CBS 117448 ex-type
EU673146
AY693974
DQ377931
AY693975
EU673094
CBS 117449
EU673147
DQ436935
DQ377932
DQ436936
EU673093
CBS 124936 ex-type
N/A
FJ888474
N/A
FJ888442
N/A
CBS 124937
N/A
FJ888458
N/A
FJ888440
N/A
CBS 117009 ex-type
EU673165
AY905554
DQ377873
AY905559
EU673104
CBS 302.75
EU673168
EU673319
EU673238
EU673286
EU673135
ICMP 16827
EU673171
EU673322
EU673241
EU673289
EU673144
ICMP 16828
EU673172
EU673323
EU673242
EU673290
EU673145
Spencermartinsia sp.2
CBS 500.72
EU673167
EU673318
EU673237
EU673285
EU673118
Spencermartinsia sp.3
CBS 117006
EU673166
AY905555
EU673236
AY905562
EU673103
Sphaeropsis citrigena
ICMP 16812 ex-type
EU673180
EU673328
EU673246
EU673294
EU673140
ICMP 16818
EU673181
EU673329
EU673247
EU673295
EU673141
CBS 133993 ex-type
JX646835
JX646802
JX646819
JX646867
JX646850
MFLUCC 11-0654
JX646836
JX646803
JX646820
JX646868
JX646851
CBS 110496 ex-type
EU673179
AY343379
DQ377894
AY343340
EU673130
CBS 110574
N/A
AY343378
N/A
AY343339
N/A
CBS 122526 ex-neotype
N/A
EU673324
N/A
EU673292
N/A
CBS 186.97
EU673178
EU673325
DQ377868
EU673293
EU673128
CBS 100163
EU673177
EU673324
DQ377870
EU673292
EU673127
Tiarosporella graminis var. karoo
CBS 118718
KF531827
KF531828
DQ377939
KF531807
KF531808
Tiarosporella madreeya
CBS 532.76
N/A
KC769960
DQ377940
N/A
N/A
Tiarosporella tritici
CBS 118719 ex-type
KF531829
KF531830
DQ377941
KF531809
KF531810
Tiarosporella urbis-rosarum
CBS 130405 ex-type
N/A
JQ239407
JQ239420
JQ239394
JQ239381
CBS 130406 ex-paratype
N/A
JQ239408
JQ239421
JQ239395
JQ239382
Sphaeropsis eucalypticola
Sphaeropsis porosa
Sphaeropsis visci
1
Type species of each genus are given in bold typeface.
Acronyms of culture collections: ATCC: American Type Culture Collection, Virginia, USA; BRIP: Culture collection, Queensland Department of Agriculture and Fisheries,
Queensland, Australia; CAA: Personal culture collection A Alves, University of Aveiro, Portugal; CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CMW:
Tree Patholgy Co-operative Program, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa; ICMP: International Collection of Microorganisms
from Plants, Landcare Research, Aukland, New Zealand; IMI: International Mycological Institute, CBI-Bioscience, Egham, Bakeham Lane, UK; IRAN: Iranian Fungal Culture
Collection, Iranian Research Institute of Plant Protection, Iran; JL: Personal culture collection, J Luque, IRTA, Barcelona, Spain; MFLUCC: Mae Fah Luang University Culture
Collection, Chiang Rai, Thailand; PD: Culture collection, University of California, Davis, USA; STE-U: Culture collection of the Department of Plant Pathology, University
of Stellenbosch, South Africa; UCROK: Culture collection, University of Riverside, California, USA; WAC: Department of Agriculture, Western Australia Plant Pathogen
Collection, South Perth, Western Australia.
2
reSulTS
dNA phylogeny
After alignment the combined ive-locus dataset consisted of 3 362
characters (including alignment gaps) for 94 ingroup taxa and one
outgroup taxon. Of the 3 362 characters, 2 418 were constant
and 159 were variable and parsimony-uninformative. Maximum
parsimony analysis of the remaining 785 parsimony-informative
characters resulted in 16 equally most parsimonious trees of 3 010
steps (CI = 0.499, RI = 0.846, HI = 0.501), one of which is shown
in Fig. 5. The phylogenetic tree resulting from ML analyses using
64
the general time reversible model of DNA evolution (Rodriguez et
al. 1990), including estimation of invariable sites and assuming a
discrete gamma distribution with six rate categories (GTR+Γ+G),
had a topology identical to the MP tree presented.
In both analyses (MP and ML) a clade corresponding to the
family Botryosphaeriaceae received a bootstrap support of 100
%. The genera Saccharata (used as outgroup) and Melanops are
clearly excluded from the family. Within the Botryosphaeriaceae
17 clades corresponding to an equal number of genera could be
readily recognised. All clades received moderate to high bootstrap
support (> 70 %). The only exception was the Dothiorella clade,
which had very low bootstrap support in both MP and ML analyses.
The Botryosphaeriaceae: genera and sPecies known from culTure
D. mu>la CBS 112553
D. mu>la CBS 230.30
100/100
D. rosulata CBS 116470
64/61
D. rosulata CBS 116472
100/100
D. cupressi CBS 168.87
D. cupressi CBS 261.85
76/71
D. tsugae CBS 418.64
73/69
70/62 D. sapinea CBS 393.84
Diplodia
77/85
D. sapinea CBS 109725
100/100
D. scrobiculata CBS 109944
79/82
D. scrobiculata CBS 113423
100/100
D. seriata CBS 112555
99/98 D. seriata CBS 119049
100/100 D. cor>cola CBS 112549
65/60
D. cor>cola CBS 112546
100/100 N. phoenicum CBS 122528
73/74
N. phoenicum CBS 169.34
99/99
Neodeightonia
N. subglobosa CBS 448.91
N. palmicola MFLUCC 10‐0822
89/100
N. palmicola MFLUCC 10‐0823
80/83 L. pseudotheobromae CBS 447.62
94/90
L. pseudotheobromae CBS 116459
100/100 L. lignicola MFLUCC 11‐0435
55/‐‐
L. lignicola MFLUCC 11‐0656
100/100
L. theobromae CBS 124.13
100/100
L. theobromae CAA 006
99/100
L. theobromae CBS 164.96
Lasiodiplodia
‐‐/72
L. parva CBS 356.59
L. parva CBS 494.78
54/‐‐
100/100 L. gonubiensis CBS 115812
L. gonubiensis CBS 116355
56/59
100/100
L. rubropurpurea CBS 118740
97/96
L. venezuelensis CBS 118739
L. crassispora CBS 110492
100/100 L. crassispora CBS 118741
100/100 S. citrigena ICMP 16812
65/65
S. citrigena ICMP 16818
100/100 S. eucalyp>cola MFLUCC 11‐0654
100/100
S. eucalyp>cola MFLUCC 11‐0579
Sphaeropsis
71/76
100/100 S. visci CBS 100163
S. visci CBS 186.97
100/100
73/79
S. porosa CBS 110496
100/100 P. mamane CPC 12264
Phaeobotryon
79/65
P. mamane CPC 12440
B. fusca CBS 174.26
Barriopsis
100/100 B. fusicoccum MFLUCC 11‐0143
Botryobambusa
99/100
B. fusicoccum MFLUCC 11‐0657
95/93
T. graminis CBS 118718
Tiarosporella
T. tri>ci CBS 118719
100/100 B. fusispora MFLUCC 10‐0098
‐‐/71
100/100
B. fusispora MFLUCC 11‐0507
B. cor>cis CBS 119047
94/100
95/99
B. cor>cis ATCC 22927
Botryosphaeria
B. dothidea CBS 115476
100 /100
B. dothidea CBS 110302
74/79
B. agaves MFLUCC 10‐0051
100/100 B. agaves MFLUCC 11‐0125
100/100 M. phaseolina CBS 227.33
78/77
Macrophomina
M. phaseolina CBS 162.25
64/58 C. eucalyp> MFLUCC 11‐0655
100/100
100/100
C. eucalyp> MFLUCC 11‐0425
Cophinforma
C. mamane CBS 117444
64/68 N. dimidiatum CBS 251.49
100/100
Neoscytalidium
100/100
S. hyalinum CBS 145.78
N. dimidiatum CBS 499.66
91/96 D. iberica CBS 115041
90/91 D. iberica CBS 113188
D. sarmentorum IMI 63581b
100/100
Dothiorella
D. sarmentorum CBS 115038
‐‐/51 98/100
Dothiorella sp. CAP 187
100/100
D. thailandica MFLUCC 11‐0438
D. dulcispinae CBS 130413
100/100
95/94 Spencermar>nsia sp. ICMP 16827
94/90 Spencermar>nsia sp. ICMP 16828
77/76
Spencermar>nsia sp. CBS 500.72
Spencermar>nsia
100/100
Spencermar>nsia sp. CBS 117006
S. vi>cola CBS 117009
70/75
100/100 Spencermar>nsia sp. CBS 302.75
100/100 N. parvum CMW 9081
58/66
N. parvum CBS 110301
100/100
N. arbu> CBS 117090
88/89
N. arbu> CBS 116131
100/100
Neofusicoccum
N. mangiferum CBS 118531
100/100
100/100 N. mangiferum CBS 118532
100/100 N. luteum CBS 110299
N. luteum CBS 110497
100/100 E. endophy>ca CBS 120397
99/99
E. endophy>ca CBS 122550
Endomelanconiopsis
66/71
E. microspora CBS 353.97
100/100 P. stroma>cum CBS 117449
Pseudofusicoccum
P. stroma>cum CBS 117448
Melanops tulasnei CBS 116805
Melanops tulasnei CBS 116806
Melanops sp. CBS 118.39
Saccharata proteae CBS 115206
100/100
95/96
Botryosphaeriaceae
100/100
100/100
100/100
10 changes
Fig. 5. One of 16 equally most parsimonious trees obtained from the combined analysis of 5 loci (SSU, LSU, ITS, EF1-α and β-tubulin), for all genera in the Botryosphaeriaceae
that are known from culture. Gaps were treated as missing data. MP/ML values (> 50 %) resulting from 1000 bootstrap replicates are given at the nodes. The tree was rooted
to Saccharata proteae CBS 115206. Clades corresponding to genera within the family Botryosphaeriaceae are highlighted.
www.studiesinmycology.org
65
PhilliPs et al.
Although Liu et al. (2012) included Auerswaldia in the
Botryosphaeriaceae, our analysis of the sequences of their isolates
revealed that A. dothiorella is in fact a species of Dothiorella while
A. lignicola apears to be best placed in Lasiodiplodia. Thus, at this
stage there is no evidence to indicate that Auerswaldia should be
considered as a genus in the Botryosphaeriaceae.
Therefore, we accept 17 genera in the Botryosphaeriaceae.
These genera, deined as clades in the ive-locus phylogeny,
are also supported by morphological characteristics. These
morphological characters provide the basis for the following key
to the genera.
Key to the genera1
1.
1.
Conidia formed within a pycnidium ......................................................................................................................................................... 2
Conida formed as dry powdery arthric chains ................................................................................................................. Neoscytalydium
2.
2.
Conidia hyaline (only rarely turn brown with age) ................................................................................................................................... 3
Conidia brown (can remain hyaline for some time before becoming brown) .......................................................................................... 8
3.
3.
Conidia hyaline, with persistent mucous sheath ..................................................................................................................................... 4
Conidia hyaline, mucous sheath absent ................................................................................................................................................. 5
4.
4.
Conidia fusiform with apical mucoid appendages ................................................................................................................ Tiarosporella
Conidia cylindrical, not reaching 50 μm long ............................................................................................................... Pseudofusicoccum
5.
5.
Conidia thin-walled .................................................................................................................................................................................. 6
Conidia thick-walled ................................................................................................................................................................................ 9
6.
6.
Conidia mostly fusoid to ellipsoidal ......................................................................................................................................................... 7
Conidia cylindrical to cylindro-clavate .............................................................................................................................. Botryobambusa
7.
7.
Most conidia longer than 30 μm ........................................................................................................................................... Cophinforma
Conidia mostly less that 30 μm long ...................................................................................................... Botryosphaeria/Neofusicoccum2
8.
8.
Conidia with a single germ slit .................................................................................................................................. Endomelanconiopsis
Germ slit absent ...................................................................................................................................................................................... 9
9.
9.
Conidia with longitudinal striations ........................................................................................................................................................ 10
Conidia not striate ................................................................................................................................................................................. 12
10. Immature, hyaline conidia striate .............................................................................................................................................. Barriopsis
10. Immature conidia not striate .................................................................................................................................................................. 11
11. Pycnidial paraphyses present .............................................................................................................................................. Lasiodiplodia
11. Pycnidial paraphyses absent ............................................................................................................................................. Neodeightonia
12. Conidia aseptate ................................................................................................................................................................................... 13
12. Conidia with 1 or more septa ................................................................................................................................................................ 15
13. Pycnidial paraphyses present ............................................................................................................................................... Sphaeropsis
13. Pycnidial paraphyses absent ................................................................................................................................................................ 14
14. Conidiogenous cells and conidia enclosed in mucoid sheath ........................................................................................... Macrophomina
14. Mucoid sheath absent .................................................................................................................................................................. Diplodia
15. Conidia become brown and septate only after dehiscence ................................................................................................................... 16
15. Conidia become brown and 1-septate while attached to the conidiogenous cells before dehiscence ...... Dothiorella/Spencermartinsia3
16. Conidia frequently 2-septate .............................................................................................................................................. Phaeobotryon
16. Conidia 1-septate only rarely becoming 2-septate ....................................................................................................................... Diplodia
1
This key is based on morphology of the asexual morph because the sexual morph is not known for some genera, is very uncommon for
others and has not been induced in culture for many of the genera.
2
It is dificult to separate these two genera morphologically but phylogenetically they are distinct.
3
These two genera cannot be separated on the morphology of the conidial states but the presence of apiculi on the ascospores of
Spencermartinsia distinguishes it from Dothiorella.
66
The Botryosphaeriaceae: genera and sPecies known from culTure
Generic and species descriptions
Barriopsis A.J.L. Phillips, A. Alves & Crous, Persoonia 21:
39. 2008. MycoBank MB511712.
Type species: Barriopsis fusca (N.E. Stevens) A.J.L. Phillips, A.
Alves & Crous, Persoonia 21: 39. 2008.
Ascomata pseudothecial, scattered or clustered, brown to black,
wall composed of several layers of textura angularis, ostiole central.
Pseudoparaphyses hyaline, smooth, multiseptate, constricted at
septa. Asci bitunicate, clavate, stipitate, thick-walled with thick
endotunica and well-developed apical chamber. Ascospores
aseptate, ellipsoid to ovoid, brown when mature, without terminal
apiculi. Conidiomata stromatic, pycnidial, supericial, dark brown
to black, un- or multilocular. Ostiole central, circular, non-papillate.
Paraphyses arising from the conidiogenous layer, extending
above the level of developing conidia, thin-walled, hyaline, mostly
aseptate. Conidiophores absent. Conidiogenous cells hyaline, thinwalled, smooth, cylindrical, holoblastic, proliferating at the same
level forming periclinal thickenings. Conidia thick-walled, initially
hyaline, aseptate with longitudinal striations, striations visible on
immature hyaline conidia even while attached to conidiogenous
cells, oval, both ends broadly rounded, becoming brown, aseptate
or 1–3-septate, with prominent longitudinal striations, wall smooth.
Chlamydospores catenate, intercalary, brown, smooth, thickwalled, formed within the agar medium.
Notes: The absence of apiculi on the ascospores differentiate
this genus from Sphaeropsis and Phaeobotryosphaeria. The
aseptate, brown ascospores without apiculi are unique in the
Botryosphaeriaceae, as are the striate immature conidia. The genus
is currently represented by two species that can be distinguished
based on their conidial dimensions. Thus, conidia of B. fusca (20–
28 × 11–16 μm) are smaller than those of B. iraniana (23–30 ×
13–21.5 μm).
Species descriptions
Barriopsis fusca (N.E. Stevens) A.J.L. Phillips, A. Alves &
Crous, Persoonia 21: 39. 2008. MycoBank MB511713. See
Phillips et al. (2008) for illustrations.
Basionym: Physalospora fusca N.E. Stevens, Mycologia 18: 210.
1926.
≡ Phaeobotryosphaeria fusca (N.E. Stevens) Petr., Sydowia 6: 317. 1952.
= Sphaeria disrupta Berk. & M.A. Curtis, Grevillea 4 (no. 32): 149. 1876.
≡ Physalospora disrupta (Berk. & M.A. Curtis) Sacc., Syll. fung. (Abellini)
1: 438. 1882.
≡ Phaeobotryon disruptum (Berk. & M.A. Curtis) Petr. & Syd., Annls
mycol. 23(3/6): 255. 1925.
≡ Botryosphaeria disrupta (Berk. & Curtis) Arx & Müller, Beitr. Kryptl.
Schweiz 11(1): 37. 1954.
Ascomata scattered, immersed, brown to black, separate or
aggregated, wall composed of textura angularis, uniloculate,
ostiole single, central. Pseudoparaphyses hyaline, smooth, 3–4.5
μm wide, multiseptate with septa 14–18 μm apart. Asci bitunicate,
clavate, 8-spored, stipitate, thick-walled with thick endotunica and
well-developed apical chamber, 125–180 × 30–36 μm. Ascospores
biseriate, aseptate, ellipsoid to oval, straight or slightly curved,
apex and base obtuse, without terminal apiculi, wall externally
smooth, internally inely verruculose, brown, widest in the middle,
(30–)31–36.5(–38.5) × (15.5–)16–18.5(–21) μm, 95 % conidence
www.studiesinmycology.org
limits = 32.6–33.4 × 17.0–17.5 μm (av. ± S.D. = 33.0 ± 1.5 × 17.2
± 1.0 μm), L/W ratio = 1.9 ± 0.15.
Type: cuba, Herradura, on twigs of Citrus sp., 15 Jan. 1925, N.E.
Stevens, holotype BPI 599052.
Culture: CBS 174.26 (ex-type).
Host: Citrus sp. (Stevens 1926, pathogenicity not known).
Known distribution: USA; Cuba (Stevens 1926), Florida (BPI
500054 collected by Shear 1923, determined by N.E. Stevens).
Notes: Von Arx & Müller (1954) placed P. fusca as a synonym of
Botryosphaeria disrupta, along with various species in Phaeobotryon
and Phaeobotryosphaeria. However, the broad concept of
Botryosphaeria followed by von Arx & Müller (1954) encompassed
such genera as Phaeobotryon and Phaeobotryosphaeria that
Phillips et al. (2008) showed to be phylogenetically distinct from
Botryosphaeria.
Phillips et al. (2008) could not induce the ex-type culture
to sporulate, no doubt because it had been in culture for more
than 80 years. According to Stevens (1926) the asexual morph
is lasiodiplodia-like and he described it as, “Conidia initially
hyaline, aseptate and thick-walled becoming dark brown and
septate with irregular longitudinal striations, (20–)23–25(–28)
× (11–)12–13(–16) μm”. Stevens (1926) placed this species in
Physalospora, but he was obviously hesitant to do so, judging from
his statement, “To place in the genus Physalospora a fungus with
colored ascospores is of course to do violence to the ideas of that
genus”. On account of the bitunicate asci and brown ascospores
of this species, Physalospora is clearly unsuitable for it. Petrak &
Deighton (1952) transferred this species to Phaeobotryosphaeria
Speg. as Phaeobotryosphaeria fusca (N.E. Stevens) Petr.,
presumably because it has dark ascospores. Phillips et al. (2008)
examined the type species of Phaeobotryosphaeria (P. yerbae)
and found that it had terminal apiculi on the ascospores. Therefore,
Phaeobotryosphaeria was also unsuitable and for that reason they
proposed the new genus Barriopsis for this fungus.
Barriopsis iraniana Abdollahz., Zare & A.J.L. Phillips,
Persoonia 23: 4. 2009. MycoBank MB513235. Fig. 6.
Ascomata not reported. Conidiomata stromatic, pycnidial,
supericial, dark brown to black, covered with dense mycelium, on
pine needles mainly unilocular and up to 600 μm diam, on Populus
twigs mostly multilocular, individual or aggregated, thick-walled,
ostiolate. Ostiole central, circular, non-papillate. Paraphyses
arising from the conidiogenous layer, extending above the level of
developing conidia, up to 70 μm long, 3.5 μm wide, thin-walled,
hyaline, usually aseptate, sometimes becoming up to 2–3-septate,
not constricted at the septa, tip rounded, occasionally branched.
Conidiophores absent. Conidiogenous cells 7–12 × 3–5 μm, hyaline,
thin-walled, smooth, cylindrical, holoblastic, proliferating at the
same level, with visible periclinal thickening. Conidia thick-walled,
initially hyaline, aseptate with longitudinal striations, striations
visible on hyaline conidia even while attached to conidiogenous
cells, oval, both ends broadly rounded, becoming brown, aseptate
or 1–3-septate, with prominent longitudinal striations, wall smooth,
(22.5–)24–30 × (12.8–)14–18(–21.5) μm, 95 % conidence limits
67
PhilliPs et al.
Fig. 6. Barriopsis iraniana. A. Conidiomata on pine needles in culture. B, C. Conidia developing on conidiogenous cells between paraphyses. D. Young, immature conidium
attached to a conidiogenous cell, longitudinal striations are visible on the conidium. E. Hyaline, immature, striate conidia. F–I. Hyaline and brown, striate conidia, 1- and 3-septate
conidia can be seen in F and G. J. Catenulate chlamydospores formed within the agar medium. Scale bars: A = 250 μm, B, C, E–I = 10 μm, D = 5 μm, J = 40 μm.
= 27–27.4 × 16.2–16.6 μm (av. ± S.D. = 27.2 ± 1.8 × 16.4 ± 1.3
μm), L/W ratio = 1.7 ± 0.16. Chlamydospores catenate, intercalary,
brown, smooth, thick-walled, formed within the agar medium.
Culture characteristics: Colonies with appressed mycelial mat
and luffy aerial mycelium in the middle, becoming dull green to
olivaceous-black at the surface, and dull green to grey-olivaceous
at the reverse after 2 wk in the dark at 25 °C. Colonies reaching
45–50 mm diam on MEA after 4 d in the dark at 25 °C. Cardinal
temperatures for growth: min 5 °C, max > 35 °C, opt 25–30 °C.
Type: Iran, Hormozgan Province, Minab, Hajikhademi, on twigs
of Mangifera indica, 27 Feb. 2007, J. Abdollahzadeh & A. Javadi,
holotype IRAN 13939F.
68
Cultures: IRAN 1448C = CBS 124698 (ex-type).
Hosts: Endophytic in stems of Citrus sp., Mangifera indica and Olea
sp. (Abdollahzadeh et al. 2009).
Known distribution: Iran (Hormozgan Province) (Abdollahzadeh et
al. 2009).
Notes: Conidia of Barriopsis iraniana are signiicantly larger than
those reported by Stevens (1926) for B. fusca, the only other species
known in this genus. The only available culture of B. fusca (CBS
174.26, ex-type) has lost its ability to sporulate. According to Stevens
(1926) the asexual morph is lasiodiplodia-like with hyaline conidia that
become dark-brown and septate with irregular longitudinal striations.
However, in contrast to Lasiodiplodia, the conidia of Barriopsis are
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 7. Botryobambusa fusicoccum. A. Asci with ascospores. B. Pseudoparaphyses. C. Ascus with ascospores. Scale bars = 10 μm.
striate at a very early stage of development and the striations are
clearly visible in young, hyaline conidia (Fig. 6). This is an unusual
character not found in any other genus of the Botryosphaeriaceae.
The sexual morph of B. iraniana has not been seen.
Botryobambusa R. Phookamsak, J.K. Liu & K.D. Hyde,
Fungal Divers. 57: 166. 2012. MycoBank MB801313.
Type species: Botryobambusa fusicoccum R. Phookamsak, J.K.
Liu & K.D. Hyde, Fungal Divers. 57: 166. 2012.
Ascomata dark brown to black, immersed under host epidermis to
erumpent, gregarious, multiloculate, locules individual globose to
subglobose or fused, vertical to the host surface, with a central,
papillate, periphysate ostiole. Asci 8-spored, bitunicate, issitunicate,
clavate to cylindro-clavate, pedicellate, with well-developed ocular
chamber. Ascospores hyaline, aseptate, smooth-walled, ellipsoidal
to obovoid, thick-walled, surrounded by mucilagenous sheath.
Conidiomata developing in stromatic clusters, fused, multiloculate,
individually globose to subglobose, wall composed of several
layers of textura angularis, broader at the base, outer layers
dark-brown and thick-walled, inner layers hyaline and thin-walled.
Conidiogenous cells holoblastic, hyaline, cylindrical to ellipsoidal,
smooth. Conidia hyaline, aseptate, cylindrical to cylindro-clavate,
thin-walled.
Notes: Botryobambusa was introduced by Liu et al. (2012) as a
monotypic genus for B. fusicoccum. The genus is distinguished
from the morphologically similar Botryosphaeria by its smaller asci
and ascospores that are surrounded by a mucilagenous sheath.
Phylogenetically the two genera are clearly distinct.
Species description
Botryobambusa fusicoccum R. Phookamsak, J.K. Liu
& K.D. Hyde, Fungal Divers. 57: 166. 2012. MycoBank
MB801314. Fig. 7.
Ascomata 90–152 μm diam, 104–152 μm high, dark brown to
black, immersed under epidermis to erumpent, gregarious, visible
as black dots or paillae on host surface, multiloculate, individual
locules globose to subglobose or fused, vertical to the host surface,
www.studiesinmycology.org
wall 12–20 μm thick, composed of several layers of cells with thick
brown wall. Ostiole central, papillate, persiphystae necks 40–60
μm diam, 30–55 μm long. Pseudoparaphyses frequently septate,
constricted at septum. Asci (45–)55–66(–82) × 14–17(–18) μm,
8-spored, bitunicate, issitunicate, clavate to cylindro-clavate,
pedicellate, apically rounded with well-developed ocular chamber.
Ascospores (8–)11–13(–14) × 5–7 μm, irregularly biseriate, hyaline,
aseptate, ellipsoidal to obovoid, usually wider in the upper third,
thick-walled, surrounded by an irregular mucilagenous sheath.
Conidiomata supericial, clustered in a stroma, multiloculate,
globose to subglobose, wall composed of several layers of textura
angularis, outer layers dark and thick-walled, inner layers hyaline
and thin-walled. Conidiogenous cells (8–)10–14(–16) × 3–5 μm,
holoblastic, cylindrical to ellipsoidal, smooth-walled, hyaline.
Conidia (21–)22–25(–26) × 5–7 μm, hyaline, asepatte, cylindrical
to cylindro-clavate, thin-walled, with rough walls.
Type: Thailand, Lampang Province, Jae Hom District, Mae Yuag
Forestry Plantation, on dead culms of Bambusa sp., 19 Aug. 2010,
R. Phookamsak, holotype MFLU 11-0179.
Cultures: CBS 134113 = MFLUCC 11-0143 (ex-type), MFLUCC 110657.
Host: Bambusa sp. (Liu et al. 2012).
Known distribution: Thailand (Liu et al. 2012).
Notes: The genus Botryobambusa is presently monotypic, and
only known from Bambusa sp. in Thailand. The sexual morph is
characterised by having ascospores surrounded by an irregular
sheath, while the asexual morph is fusicoccum-like in morphology
(Liu et al. 2012).
Botryosphaeria Ces. & De Not., Comm. Soc. crittog. Ital. 1:
211. 1863; emend. Sacc., Michelia 1: 42. 1877. MycoBank
MB635.
=
=
=
=
=
Fusicoccum Corda, in Sturm, Deutschl. Flora, III (Pilze) 2: 111. 1829.
Thuemenia Rehm, Flora 62: 123. 1878.
Coutinia J.V. Almeida & Sousa da Câmara, Rev. Agron. Lisboa 1: 392. 1903.
Cryptosporina Höhn. Öst. bot. Z. 55: 54. 1905.
Amerodothis Theiss. & Syd., Ann. mycol. 13: 295. 1915.
69
PhilliPs et al.
= Epiphyma Theiss., Verh. zool.-bot. Ges. Wien 66: 306. 1916.
= Pyreniella Theiss., Verh. zool.-bot. Ges. Wien 66: 371. 1916.
= Desmotascus F. Stevens, Bot. Gaz. 68: 476. 1919.
= Creomelanops Höhn. Sber. Akad. Wiss. Wien, Math.-naturw. Kl., Abt. 1 129:
146. 1920.
= Macrophomopsis Petr., Ann. mycol. 22: 108. 1924.
= Rostrosphaeria Tehon & E.Y. Daniels, Mycologia 19: 112. 1927.
= Apomella Syd. Annls mycol. 35: 47. 1937.
= Caumadothis Petr., Sydowia 24: 276. 1971.
Type species: Botryosphaeria dothidea (Moug. : Fr.) Ces. & De
Not., Comment. Soc. Crittog. Ital. 1: 212. 1863.
Mycelium immersed, consisting of branched, septate, smooth, hyaline
hyphae. Ascomata eustromatic, scattered, solitary, aggregated or
forming botryose clusters, externally black, uniloculate, with a thick
pseudoparenchymatic wall composed of textura angularis or textura
globosa with the outer layers blackened and their cells more thickened,
ostiolate, embedded in the substrate and partially erumpent at
maturity. Pseudoparaphyses thin-walled, hyaline, frequently septate,
constricted at the septa, deliquescing from the basal parts when the
asci mature. Asci clavate or cylindric-clavate, stipitate, bitunicate,
ectotunica thin, endotunica rather thick, 3-layered (sensu Eriksson
1981), with a prominent apical chamber, 8-spored, developing on a
broad basal hymenial layer. Ascospores irregularly biseriate in the
ascus, hyaline, sometimes becoming pale brown with age, thinwalled, ovoid, fusoid, fusoid-ellipsoid, usually widest in the middle,
straight or inequilateral, smooth, one-celled sometimes becoming
1–2 septate with age, contents smooth or granular, may be guttulate.
Conidiomata stromatic, pycnidial, solitary or aggregated, often
occurring within the same stroma as the ascomata, walls composed
of dark brown, thick-walled textura angularis, becoming thin-walled
and hyaline towards the inner layer. Ostioles indistinct to welldeined, round or irregular. Paraphyses hyaline, cylindrical, tapering
to rounded ends, septate, arising between the conidiophores and
conidiogenous cells. Conidiophores when present hyaline, cylindrical,
branched at the base, smooth, 0–1 septate. Conidiogenous cells
enteroblastic, integrated, hyaline, smooth, cylindrical, irst-formed
conidium holoblastic, determinate or proliferating percurrently with
1–2 indistinct annellations, or proliferating at the same level resulting
in typical phialides (sensu Sutton 1980) with periclinal thickenings.
Conidia hyaline, sometimes becoming olivaceous or darker with age,
thin-walled, smooth, aseptate, occasionally forming one or two septa
with age or before germination, with shapes varying from elliptical to
fusiform or clavate, inely guttulate, apex subobtuse to obtuse, base
conspicuously truncate with a minute marginal basal frill.
Notes: When Cesati & De Notaris (1863) introduced Botryosphaeria
Ces. & De Not. they listed nine species (plus another six that
they did not recombine in the genus) but they did not designate
a type. Subsequently, Saccardo (1877) emended the genus to
exclude hypocreaceous species. Von Höhnel (1909) designated B.
berengeriana De Not. as the type, but this species was not included
in the original description of the genus, although it was published
soon after (De Notaris 1864). Theissen & Sydow (1915) suggested
B. quercuum (Schwein.) Sacc. as the type since it was typical of
Saccardo’s (1877) emendation of Botryosphaeria, and this was
accepted by von Arx & Müller (1954). However, B. quercuum also
was not one of the original species of the genus and therefore is
unsuitable as the type. Barr (1972) proposed B. dothidea (Moug. :
Fr.) Ces. & De Not. as lectotype because it was one of the original
species described, it conforms with Saccardo’s (1877) emendation
and it is an earlier synonym of B. berengeriana, von Höhnel’s (1909)
70
designated type. The proposal of Barr (1972) has been accepted
generally, and Slippers et al. (2004a) provided a revised description
of this species based on the type specimen and fresh collections,
and they designated a neotype and epitype.
Species in Botryosphaeria were described largely on the basis
of the morphology of their ascomata and host associations, and this
led to a proliferation of names. Von Arx and Müller (1954) examined
183 taxa and reduced them to 11 species, with extensive synonymies
under B. dothidea and B. quercuum, together with nine new
combinations. However, because von Arx and Müller (1954) did not
take into account the characters of the asexual morphs and because
species of Botryosphaeria are dificult to separate on the basis of
sexual morph characters, these synonymies have not always been
accepted (Shoemaker 1964, Sivanesan 1984, Slippers et al. 2004a).
The genus Botryosphaeria, based on the type species B. dothidea,
typically has ascospores that are hyaline and aseptate, although they
can become pale brown and septate with age (Shoemaker 1964,
Sivanesan 1984, Denman et al. 2000, Alves et al. 2004, Phillips et al.
2005). Because some species of Botryosphaeria have ascospores
that become brown with age, von Arx & Müller (1954) placed
Dothidea visci with brown ascospores in Botryosphaeria as B. visci,
and later they (von Arx & Müller 1975) also placed the dark-spored
Neodeightonia subglobosa in Botryosphaeria. Since it is the type
species of Neodeightonia, this genus was reduced to synonymy with
Botryosphaeria. In recognising these synonymies, von Arx & Müller
(1954, 1975) broadened the concept of Botryosphaeria to include
species with brown ascospores.
Phillips et al. (2005) resurrected the genus Dothiorella for
species with 1-septate conidia that darken at an early stage of
development, and have sexual morphs with brown, 1-septate
ascospores. Phylogenetically (ITS+EF1-α) the two species studied
by Phillips et al. (2005) fell within Botryospheria as deined by
the broad morphological concept recognised by von Arx & Müller
(1954, 1975). For these reasons, Phillips et al. (2005) described the
sexual morphs of Dothiorella as two new species of Botryosphaeria
with brown, 1-septate ascospores. Subsequently, Luque et al.
(2005) described another dark-spored Botryosphaeria, namely B.
viticola, with a Dothiorella asexual morph.
At least 18 asexual genera have been associated with
Botryosphaeria s. lat. (Denman et al. 2000) including Diplodia,
Dothiorella, Fusicoccum, and Lasiodiplodia. The morphological
diversity of the asexual morphs linked to species of Botryosphaeria,
together with the broad concept of the sexual genus was clear
evidence that Botryosphaeria encompassed several distinct
genera. Thus, through a study of partial sequences of the LSU
gene, Crous et al. (2006) showed that Botryosphaeria s. lat. is
composed of 10 phylogenetic lineages, each of which corresponds
to different asexual genera. To avoid the unnecessary introduction
of new generic names, these authors chose to use existing asexual
generic names for most of the lineages, and restricted the use of
Botryosphaeria to B. dothidea and B. corticis. Seven species are
currently recognised in Botryospheria.
dNA phylogeny
In an ITS phylogeny the ex-type isolate of B. mamane and isolates
previously regarded as B. mamane clustered in Cophinforma
together with C. atrovirens (Fig. 8). Based on combined ITS and
EF1-α sequence data seven species are currently recognised
in Botryosphaeria (Fig. 8). Apart from B. fabicerciana all species
clades are supported by high bootstrap values.
The Botryosphaeriaceae: genera and sPecies known from culTure
95/92
97/99
88/97
B. fabicerciana CBS 127193
B. fabicerciana CMW 27108
B. fusispora MFLUCC 10‐0098
86/84
100/100
B. fusispora MFLUCC 11‐0507
B. cor0cis ATCC 22927
70/64
B. cor0cis CBS 119047
94/96
B. dothidea CBS 110302
74/74
Botryosphaeria
B. dothidea CBS 115476
85/97
100/99
100/100
B. scharifii CBS 124703
B. scharifii CBS 124702
B. ramosa CBS 122069
100/100
B. agaves MFLUCC 11‐0125
B. agaves MFLUCC 10‐0051
68/67
99/99
C. atrovirens MFLUCC 11‐0425
C. atrovirens MFLUCC 11‐0655
C. atrovirens CBS 124935
65/‐‐
100/100
77/73
Cophinforma
C. atrovirens CBS 117450
C. atrovirens CBS 124934
C. atrovirens CBS 117444
100/100
100/100
M. phaseolina CBS 227.33
Macrophomina
M. phaseolina CBS 162.25
100/96
60/62
N. novaehollandiae CBS 122610
N. novaehollandiae CBS 122071
N. hyalinum CBS 145.78
100/100
Neoscytalidium
N. dimidiatum CBS 251.49
N. dimidiatum CBS 499.66
N. parvum CMW 9081
10
N. luteum CBS 110299
Fig. 8. One of 18 equally most parsimonious trees obtained from the combined analysis of ITS and EF1-α sequences from species of the genera Botryosphaeria, Cophinforma,
Macrophomina and Neoscytalidium. The phylogenetic tree resulting from ML analysis using the general time reversible model of DNA evolution and assuming a discrete gamma
distribution (GTR+G) had a topology identical to the MP tree presented. MP/ML values (> 50 %) resulting from 1000 bootstrap replicates are given at the nodes. The tree was
rooted to N. parvum and N. luteum. Clades corresponding to genera and species are highlighted.
www.studiesinmycology.org
71
PhilliPs et al.
Key to Botryosphaeria spp.
1.
1.
Conidia 12–17 μm long ........................................................................................................................................................................... 2
Most conidia longer than 18 μm .............................................................................................................................................................. 3
2.
2.
Average conidial length greater than 15 μm ............................................................................................................................ B. schariii
Average conidial length less than 15 μm ................................................................................................................................ B. ramosa
3.
3.
On Vaccinium species, conidia 23.5–32.5 μm long ................................................................................................................... B. corticis
On hosts other than Vaccinium ............................................................................................................................................................... 4
4.
4.
Conidia 16–22 μm long .......................................................................................................................................................... B. fusispora
Conidia mostly longer than 20 μm ........................................................................................................................................................... 5
5.
5.
Conidial L/W ratio greater than 4.5 ......................................................................................................................................... B. dothidea
Conidial L/W ratio less than 4.0 ........................................................................................................................................ B. fabicerciana
Notes: This key is based only on characters of the asexual morphs, because the sexual morphs are generally uncommon or have not been
induced to form in culture. Botryosphaeria agaves was not included in the key because the asexual morph has never been reported.
Species descriptions
Botryosphaeria agaves (Henn.) E.J. Butler, Ann. Mycol. 9:
415. 1911. MycoBank MB119799. See Liu et al. (2012) for
illustrations.
Basionym: Physalospora agaves Henn., Bot. Jb. 34: 51. 1905.
Ascomata 140–260 μm high × 600–800 μm diam, circular blackened
areas on host tissue, immersed to erumpent on host, uni to
multiloculate, aggregated, individually globose to subglobose, wall
composed of several layers of dark brown walled cells of textura
angularis. Ostiole circular, central, papillate. Pseudoparaphyses
3–5 μm wide, aseptate. Asci 91–122 × 27–38 μm, 8-spored,
bitunicate with a thick endotunica, issitunicate, clavate to cylindroclavate, short pedicellate, with well-developed apical chamber.
Ascospores 21–43 × 8–12 μm, biseriate in the ascus, hyaline,
aseptate, ellipsoidal, fusiform, or inequilateral, usually wider at
the middle, wall rough, surrounded by a mucilaginous sheath.
Conidiomata not reported.
Type: Tanzania, Zanzibar, on leaves of Agave sisalana,
Zimmerman, holotype presumably lost (not in B). Thailand, Chiang
Rai Province, Mae Fah Luang District, Doi Tung, on living and
dead leaves of Agave sp., 16 Jun. 2010, R. Phookamsak, neotype
designated here MFLU 11–0161; MBT176241.
Cultures: MFLUCC 11–0125 = CBS 133992 (ex-neotype), MFLUCC
10-0051.
Host: Agaves sp. (Liu et al. 2012).
Known distribution: Thailand (Liu et al. 2012).
Notes: Liu et al. (2012) proposed a specimen from Agave sp.
collected in Thailand (MFLU 11-0161) to serve as epitype for B.
agaves. However, as they did not cite nor examine the holotype,
their epitypiication is invalid. We have also been unable to trace
the holotype, thus designate the Thailand specimen as neotype to
rectify this situation.
72
Botryosphaeria corticis (Demaree & Wilcox) Arx & E. Müll.,
Beitr. Kryptl. Schweiz 11(1): 43. 1954. MycoBank MB293807.
Figs 9, 10.
Basionym: Physalospora corticis
Phytopathology 32: 1074. 1942.
Demaree
&
Wilcox,
Ascomata abundant, embedded in the host becoming partially
erumpent at maturity, up to 250 μm diam, conical with a dark
brown to black wall composed of up to six cell layers of thick-walled
textura angularis giving way to hyaline, thinner-walled cells lining
the ascomata. Asci hyaline, clavate and stipitate, bitunicate with a
thick endotunica and well-developed apical chamber, eight-spored,
145–165 × 25–28 μm, irregularly biseriate, formed amongst hyaline,
thin-walled, septate pseudoparaphyses. Ascospores ellipsoid to
fusoid, (24–)25.5–33(–34.5) × (9.5–)10–12.5(–13.5) μm, 95 %
conidence limits = 28.5–30.1 × 11.2–11.9 μm (av. ± S.D. of 32
conidia = 29.3 ± 2.4 × 11.6 ± 1.0 μm), L/W = 2.5 ± 0.23. Ascospores
germinate within 24 h at 25 °C and form unbranched germ tubes.
Conidiomata developing in culture on pieces of poplar twigs after
14 d and producing conidia after 28 d, solitary to aggregated,
dark brown to black, globose, up to 450 μm diam. Conidiophores
cylindrical, hyaline, smooth, thin-walled, septate, branched in the
upper parts, 7.5–14 × 3.5–4.5 μm, lining the entire inner surface
of the conidiomata. Conidiogenous cells lageniform, hyaline, thinwalled, smooth, 12.5–17.5 × 2.5–4.5 μm, holoblastic producing a
single conidium at the tip, rarely proliferating at the same level giving
rise to periclinal thickenings. Conidia fusiform, widest in the middle to
upper third, hyaline, thin-walled, smooth, apex acute, base truncate
with a minute marginal frill and persistent mucous sheath, (20.5–)
23.5–32.5(–34.5) × (5.0–)5.5–7(–7.5) μm, 95 % conidence limits =
27.7–30.2 × 6.2–6.7 μm (av. ± S.D. of 26 conidia = 28.9 ± 3.4 × 6.4
± 0.7 μm), L/W = 4.5 ± 0.46. Spermatogonia globose, dark brown to
black. Spermatophores cylindrical, hyaline, branched, 11–14 × 2–3
μm. Spermatogenous cells hyaline, thin-walled, smooth, 14.5–20.5 ×
1.5–2.3 μm, producing conidia at their tips, proliferating internally to
form periclinal thickenings. Spermatia rod-shaped with obtuse ends,
hyaline, thin-walled, smooth, 4–6 × 1.5–2 μm.
Culture characteristics: Colonies on CMA reaching 28–40 mm diam
after 7 d at 25 °C, initially white becoming olive-green with clumps
of loosely aggregated hyphae.
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 9. Botryosphaeria corticis. A. Ascomatal necks emerging through the bark of Vaccinium. B. Section through an ascoma. C. Section through the ascomal wall. D, E. Asci with
ascospores. F. Septate pseudoparaphyses. G, H. Apical chamber at tip of an ascus as seen in interference contrast (G) or phase contrast (H). I, J. Ascospores. Scale bars: A =
0.5 mm, B = 50 μm, C, E = 20 μm, D, F–J = 10 μm.
Type: uSA, North Carolina, Atkinson, Vaccinium corymbosum,
14 Feb. 1940, J.B. Demaree, holotype BPI 598729; New Jersey,
Hammonton, on cankered stems of V. corymbosum, May 2005, P.V.
Oudemans, CBS H-19706 epitype (designated by Phillips et al.
2006a).
Cultures: CBS 119047, CBS 119048 (ex-epitype).
Known distribution: USA (Florida, Georgia, Maryland, Mississippi,
New Jersey, North Carolina) (Phillips et al. 2006, Wright & Harmon
2010).
Notes: This species appears to be restricted to Vaccinium spp.
and has not been reported outside of the continental USA. The
mucilaginous sheath surrounding the conidia is unusual in
Botryosphaeria.
Hosts: Vaccinium species including V. corymbosum, V. ashei, V.
tenellum and V. virgatum (Phillips et al. 2006, Wright & Harmon
2010).
www.studiesinmycology.org
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PhilliPs et al.
Fig. 10. Botryosphaeria corticis. A. Conidiomata formed on poplar twigs in culture. B–E. Conidiogenous cells and paraphyses. F. Conidia with mucous sheath (arrowheads). G–I.
Spermatogenous cells. J. Spermatia. Scale bars: A = 0.5 mm, B–J = 10 μm.
Botryosphaeria dothidea (Moug.: Fr.) Ces. & De Not.,
Comm. Soc. Crittog. Ital. 1: 212. 1863. MycoBank MB183247.
Figs 11, 12.
Basionym: Sphaeria dothidea Moug., In: Fries, Syst. Mycol.
(Lundae) 2(2): 423. 1823.
= Botryosphaeria berengeriana De Not., Sfer. Ital. 82. 1863 [1864].
= Fusicoccum aesculi Corda, In: Sturm, Deutschl. Fl., Abth. 3, 2: 111. 1829.
= Sphaeria coronillae Desm., Annls Sci. Nat., Bot., sér. 2 13: 188. 1840.
≡ Macrophoma coronillae (Desm.) Höhn., Ber. Deutsch. Bot. Ges. 28:
479. 1910.
74
≡ Macrophomopsis coronillae (Desm.) Petr., Annls mycol. 22(1/2): 108.
1924.
≡ Dothiorella coronillae (Desm.) Petr., Sydowia 16(1–6): 188. 1963.
≡ Fusicoccum coronillae (Desm.) Vanev. & Aa, In: van der Aa & Vanev,
A Revision of the Species Described in Phyllosticta (Utrecht): 192. 2002.
= Phyllosticta divergens Sacc., Malpighia 5: 274. 1891.
Ascostroma erumpent, 200−500 μm diam. Ascomata pseudothecial,
forming a botryose aggregate of up to 100, sometimes solitary,
globose with a central ostiole, ¼ to ½ emergent, rarely embedded,
papillate or not, brown to black, pseudothecial wall comprising 5–15
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 11. Botryosphaeria dothidea. A. Botryose clusters of ascomata erumpent through the bark of a poplar twig. B. Transverse section through ascomata revealing brilliant
white contents. C. Section through ascomata. D. Ascus with ascospores. E. Septate pseudoparaphyses. F. Ascospores. G, H. Ascus tip showing apical chamber as seen by
interference contrast (G) or phase contrast (H). I. Ascospores. Scale bars: A, B = 0.5 mm, C = 100 μm, D–I = 10 μm.
layers of textura angularis, outer region of dark brown cells, inner
region of 2–4 layers of hyaline cells lining the locule. Asci bitunicate,
clavate, 63−125 × 16–20 μm, forming between pseudoparaphyses.
Pseudoparaphyses iliform, septate, constricted at the septa, rarely
branched, 2–4 μm wide. Ascospores fusoid to ovoid, sometimes
with tapered ends and appearing spindle-shaped, biseriate in the
ascus, (17–)19–24(–32) × (6–)7–8(–10) μm (av. of 102 ascospores
= 22.7 × 7.8 μm), L/W = 2.9. Conidiomata stromatic, morphologically
indistinguishable from the ascomata. Paraphyses, when present
hyaline, septate, up to 110 μm long, 2.5–6 μm wide at the base
tapering to acutely rounded apices, 2–2.5 μm wide at the tip.
Conidiophores hyaline, smooth, thin-walled, rarely branched at the
base, cylindrical, formed from the cells lining the locule wall, 23–35
× 4–5 μm, or reduced to conidiogenous cells. Conidiogenous cells
holoblastic, hyaline, sub-cylindrical, 6–20 × 2–5 μm, proliferating
percurrently to produce 1–2 annellations or proliferating internally
resulting in periclinal thickenings and typical “phialides” (sensu
Sutton 1980). Conidia narrowly fusiform, or irregularly fusiform,
www.studiesinmycology.org
base subtruncate to bluntly rounded, (17–) 19.5–30(–34) × 4–6(–
7.5) μm, 95 % conidence limits of 350 conidia = 25.8–26.5 × 5.3–
5.4 μm (av. ± S.D. of 350 conidia = 26.2 ± 3.1 × 5.4 ± 0.7 μm), L/W
= 4.9 ± 0.96 with 95 % conidence limits of 4.8–5.0, rarely forming
a septum before germination, smooth with granular contents, in
some isolates becoming dark-walled and septate with age, (9.5–)
10.5–20(–23) × (3–)4–6(–6.5) μm (av. ± S.D. of 150 conidia =
15.5 ± 2.7 × 5.1 ± 0.6 μm). Spermatophores hyaline, smooth,
occasionally branched, cylindrical to subcylindrical septate, 4−15
× 1−3.5 μm. Spermatogenous cells discrete or integrated, hyaline,
smooth, cylindrical, holoblastic or proliferating via phialides with
periclinal thickenings, 7–10 × 2–3 μm. Spermatia unicellular,
hyaline, allantoid to rod-shaped, 3−6 × 1.5−2 μm.
Culture characteristics: Colonies oliveaceous becoming grey with
reverse black. Mycelial mat moderately dense, margin smooth.
Optimum temperature for growth 25(–30) °C, colony on ½ PDA
reaching 50 mm radius after 4 d at 25 °C in the dark.
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PhilliPs et al.
Fig. 12. Botryosphaeria dothidea. A. Botryose cluster of conidiomata erumpent through the bark of a poplar twig. B–E. Conidiogenous cells with periclinal thickenings (black
arrowheads) or annellations (white arrowheads). F. Paraphyses. G, H. Conidia. I. Spermatia. J. Pigmented, thick-walled, septate conidia. Scale bars: A = 0.5 mm, B–J = 10 μm.
Type: France, Rosa sp., 1823, Fries ex Mougeot. Herbarium
S (neotype of Sphaeria dothidea designated by Slippers et al.
2004a). Switzerland, Ticino, Crociisso, Prunus sp., Oct. 2000,
B. Slippers, PREM 57372 (epitype designated by Slippers et al.
2004a). Italy, on branches of Aesculus sp., P.A. Saccardo, PAD,
(neotype of asexual morph designated by Crous & Palm 1999).
Cultures: CBS 115476 = CMW 8000 (ex-epitype).
Hosts: Woody plants in numerous families. Reports of hosts prior
to 2000 are unreliable because the concept of this species was
not clear until Slippers et al. (2004a) redeined it and proposed
neotype, epitype and ex-epitype cultures. It is highly probable that
before that time some of the host associations may have been
of species of Neofusicoccum. However, some recent reports can
conirm the following hosts: Cistus ladanifer (Sánchez et al. 2002),
Fraxinus, Ostrya, Prunus, Populus, (Slippers et al. 2004a), Acacia
rostellifera, Eucalyptus marginata (Taylor et al. 2009) Vitis vinifera,
Olea europaea (Phillips et al. 2005, Lazzizera et al. 2008b), Quercus
76
suber, Q. ilex (Sánchez et al. 2003), Cistus ladanifer (Sánchez et
al., 2002), Juniperus communis, Acer sp., Actinidia deliciosa, C.
limon, Fagus sp., Juglans regia, Mangifera indica, Olea europaea,
Picea sp., Populus nigra, Prunus persica, Quercus sp., Rubus sp.,
Salix sp., V. vinifera (Abdollahzadeh et al. 2013).
Known distribution: Probably worldwide and cosmopolitan.
Notes: The description of S. dothidea (Fries 1823) refers to a
fungus on twigs of Fraxinus sp. According to Slippers et al. (2004a)
the specimen of S. dothidea in the Fries collection that has been
cited as the holotype (von Arx & Müller 1954) is on what appears
to be a Rosa sp. and thus cannot be the holotype. Phillips &
Lucas (1997) and Slippers et al. (2004a) examined the only other
specimen of S. dothidea in the Fries herbarium and found it to be
immature with no spores. Slippers et al. (2004a) designated that
specimen as the neotype and also designated an epitype (PREM
57372) on Prunus sp. collected from Crociisso, Switzerland, with
an ex-epitype culture (CBS 115476 = CMW 8000).
The Botryosphaeriaceae: genera and sPecies known from culTure
Pennycook & Samuels (1985) described two new species of
Botryosphaeria (B. parva and F. luteum) on kiwifruit, and suggested
that B. dothidea may be a complex of species. This suggestion led
to doubts about the earlier identiications of B. dothidea. However,
the name B. dothidea continued to be used in a broad sense. Only
after gene sequence data were used to clarify species concepts
in the genus (e.g. Phillips et al. 2002, Slippers et al. 2004a) it
became apparent that some of the earlier reports of B. dothidea in
association with plant diseases may have been misidentiications.
Thus, the earlier reports of B. dothidea prior to 2004 should be
interpreted with circumspection.
Botryosphaeria fabicerciana (S.F. Chen, D. Pavlic, M.J.
Wingf. & X.D. Zhou) A.J.L. Phillips & A. Alves, comb. nov.
MycoBank MB805457. See Chen et al. (2011) for illustrations.
Basionym: Fusicoccum fabicercianum S.F. Chen, D. Pavlic, M.J.
Wingf. & X.D. Zhou, Plant Pathol. 60: 746. 2011.
Ascomata not reported. Conidiomata developing in culture on pine
needles after 10 d and producing conidia after 14 d, supericial,
solitary to aggregated, dark brown, globose, covered with hyphae,
(245–)346–470(–525) μm, wall composed of three layers: an outer
of thick-walled dark to light brown textura angularis, a middle
layer of thin-walled light brown cells, and an inner layer of thinwalled hyaline cells. Conidiophores absent. Conidiogenous cells
cylindrical to lageniform, hyaline, smooth, thin-walled, holoblastic
producing a single conidium at the tip, rarely proliferating at the
same level giving rise to periclinal thickenings, (6.5–)10.5–13.5(–
16) × (2–)2.5–3.5(–4.5) μm (av. of 50 conidiogenous cells = 12 ×
3 μm). Paraphyses not seen. Conidia hyaline, thin-walled, smooth
with granular contents, unicellular, aseptate, fusiform, widest in
the middle to upper third, apex acute, base truncate with a minute
marginal frill, forming one or two septa before germination, (16.5–)
19.5–24.5(–26) × (4.5–)5–6.5(–7.5) μm (av. of 100 conidia = 22.0
× 5.8 μm), L/W = 3.8.
Culture characteristics: Colonies with luffy mycelium, initially
white turning smoke-grey from the middle of colonies within 4–6
d, with an appressed mycelial mat, sparse to moderately dense.
Cottony aerial mycelium toward the edge of colony, becoming
pale olivaceous-grey, and greenish black (reverse) within 12–16 d.
Optimal temperature for growth 25(–30) ºC, colony covering the 90
mm diam Petri dish after 5 d in the dark at 25 °C.
Type: china, FuJian Province, from senescing twigs of an unknown
Eucalyptus sp., Aug. 2007, M.J. Wingield, holotype PREM 60449.
Cultures: CBS 127193 = CMW 27094 (ex-type).
Hosts: Eucalyptus sp., E. urophylla × E. tereticornis, Eucalyptus
grandis hybrid (Chen et al. 2011).
Known distribution: China (FuJian, HaiNan and GuangXi Provinces)
(Chen et al. 2011).
Notes: Botryosphaeria fabicerciana is morphologically similar to B.
dothidea (size of conidia = 24.5 × 5 μm in culture, 19.5 × 5 μm
on a natural Prunus sp. (Slippers et al. 2004a), but differs from
other species in the genus. Conidia of B. fabicerciana are larger
than those of B. ramosa (av. size of conidia = 13.4 × 5.7 μm in
www.studiesinmycology.org
culture; Pavlic et al. 2008) and B. schariii (av. size of conidia = 15.4
× 5.2 μm; Abdollahzadeh et al. 2013), but smaller than those of
Botryosphaeria corticis (av. size of conidia = 28.9 × 6.4 μm; Phillips
et al. 2006).
Botryosphaeria fusispora Boonmee, J.K. Liu & K.D. Hyde,
Fungal Divers. 57: 171. 2012. MycoBank MB801319. See Liu
et al. (2012) for illustrations.
Ascomata dark brown to black, immersed in the host, becoming
erumpent, clustered, gragarious or scattered, subglobose with
indistinct ostiole, 137–210 μm high × 160–230 μm diam, wall
composed of 3–4 layers of dark brown cells of textura angularis.
Pseudoparaphyses 2.5–5 μm wide, aseptate. Asci 8-spored,
bitunicate, broadly cylindrical, short pedicellate with a welldeveloped apical chamber, 77.5–112.5 × 20–25 μm. Ascospores
biseriate, partially overlapping, hyaline, aseptate, ellipsoidal to
fusiform, smooth-walled, thin-walled, 20–27.5 × 10–12.5 μm.
Conidiomata stromatic, solitary, semi-immersed, dark brown to
black, 140–180 × 160–210 μm, walls composed of thick-walled
dark brown cells of textura angularis, beconming thinner-walled
and hyaline towards the inner region. Conidiophores hyaline,
septate, cylindrical, smooth, 2–4.5 μm wide. Conidiogenous cells
holoblastic, hyaline, cylindrical, integrated, producing a single
apical conidium. Conidia hyaline, thin-walled, aseptate, fusiform to
ellipsoidal, sometimes irregular ellipsoidal, smooth, apex obtuse,
base truncate or bluntly rounded, 16–22 × 4–5.5 μm.
Culture characteristics: Colonies on MEA growing rapidly, reaching
9 cm diam within 7 d at room temperature, aerial mycelium at irst
white becoming dark grey to black.
Type: Thailand, Chiang Rai, Doi Tung, on dried bark of Entada sp.,
10 Jun. 2009, S. Boonmee, holotype MFLU 10-0028.
Culture: MFLUCC 10-0098 (ex-type).
Hosts: Caryota sp., Entada sp. (Liu et al. 2012).
Known distribution: Thailand (Liu et al. 2012).
Notes: The shorter conidia separate this species from B. corticis,
B. dothidea and B. fabicerciana. Conidia of B. fusispora are larger
than those of B. ramosa and B. schariii.
Botryosphaeria ramosa (Pavlic, T.I. Burgess, M.J. Wingf.)
A.J.L. Phillips & A. Alves, comb. nov. MycoBank MB805458.
Fig. 13.
Basionym: Fusicoccum ramosum Pavlic, T.I. Burgess & M.J.
Wingf., Mycologia 100: 861. 2008.
Ascomata not reported. Conidiomata semi-immersed, solitary,
globose, papillate, chestnut, covered by hyphal hairs, up to 510
μm diam, sometimes with a neck to 1.7 mm long, arising from
the substrate. Conidiophores reduced to conidiogenous cells.
Conidiogenous cells smooth, cylindrical to subcylindrical, hyaline,
the irst conidium produced holoblastically and subsequent conidia
enteroblastically, (6–)7.5–10(–11) × (2–)2–3(–3.5) μm. Conidia
fusiform to ellipsoid to oval, rounded at apex, base truncate,
77
PhilliPs et al.
Fig. 13. Botryosphaeria ramosa. A–C. Conidia developing on conidiogenous cells. Scale bars = 10 μm.
smooth with ine granular contents, hyaline, thin-walled, aseptate,
(11–)12–15(–16) × (4.7–)5–6(–7) μm, L/W ratio = 2.3.
Culture characteristics: Colonies initially white turning greyolivaceous from the middle of colonies within 5–7 d, with appressed
mycelial mat and white moderately dense, cottony aerial mycelium
toward the edge of colony, becoming smoke grey to olivaceousgrey (surface) and iron grey (beneath) within 10–14 d. Optimum
growth at 25 °C, covering the 9 cm diam Petri dish after 4 d in the
dark.
Type: Australia, Western Australia, Bell Gorge, on Eucalyptus
camaldulensis, Jul. 2006, T.I. Burgess, holotype PREM 59846.
Cultures: CBS 122069 = CMW 26167 (ex-type).
Host: Asymptomatic branches of Eucalyptus camaldulensis (Pavlic
et al. 2008).
Known distribution: Western Australia (Pavlic et al. 2008).
Notes: No sexual morph has been reported, but phylogenetically
this is clearly a species of Botryosphaeria. Only one culture of
B. ramosa is known. Although Pavlic et al. (2008) reported long,
branched conidiophores, we could not ind such structures in the
holotype. No Dichomera synasexual morph was reported by Pavlic
et al. (2008). The conidia of B. ramosa are signiicantly shorter than
those of any other species in this genus, although they are of a
similar length to B. schariii.
Botryosphaeria schariii Abdollahz., Zare, A.J.L. Phillips,
Mycologia 105: 213. 2013. MycoBank MB564800. Fig. 14.
Ascomata not reported. Conidiomata stromatic, pycnidial, produced
on pine needles on WA within 2–4 wk, solitary or aggregated, dark
brown to black, globose, up to 760 μm diam, supericial, mostly
Fig. 14. Botryosphaeria schariii. A. Conidiomata on pine needles in culture. B. Conidia developing on conidiogenous cells. C. Conidiogenous cells with periclinal thickenings.
D, E. Conidiogenous cells and conidiophores. F. Conidia. Scale bars: A = 1 mm, B–E = 5 μm, F = 10 μm.
78
The Botryosphaeriaceae: genera and sPecies known from culTure
uniloculate, thick-walled, non-papillate with a central ostiole.
Conidiophores cylindrical, hyaline, smooth, thin-walled, septate,
branched at apex, 7.5–33.5 × 2–4.5 μm, lining the entire inner surface
of the conidiomata. Conidiogenous cells cylindrical to lageniform,
hyaline, thin-walled, smooth, 7–15 × 1.5–3.5 μm, holoblastic, phialidic
with periclinal thickening. Conidia fusiform, unicellular, hyaline, thinwalled, smooth, apex obtuse, base subtruncate to bluntly rounded,
(11.5–)13–17(–19) × 4–6.5 μm, 95 % conidence limits = 15.2–15.6 ×
5.2–5.4 μm (av. ± S.D. = 15.4 ± 1.4 × 5.2 ± 0.5 μm), L/W ratio = 2.7.
95/95 C. mamane 97‐59
C. mamane 97‐58
C. atrovirens CBS 124935
C. atrovirens CBS 117444
C. atrovirens CBS 117447
100/96
Culture characteristics: Colonies with abundant aerial mycelium
reaching to the lid of Petri dishes, aerial mycelium becoming smokegrey to olivaceous-grey at the surface and greenish olivaceous to
dull green at the reverse after 2 wk in the dark at 25 °C. Colonies
reaching 84 mm on MEA after 3 d in the dark at 25 °C. Cardinal
temperatures for growth: min ≤ 5 °C, max ≥ 35 °C, opt 25 °C.
C. atrovirens CBS 117450
100/100
Notes: Botryosphaeria schariii is phylogenetically closely related to
B. ramosa. Conidia of B. schariii and B. ramosa are considerably
shorter than all other species in the Botryosphaeria clade.
However, the slightly longer conidia of B. schariii distinguish it
from B. ramosa. This speces was found on twigs of mango trees in
Hormozgan Province (Minab) and from mango fruits, imported from
Pakistan, in Kurdistan Province (Sanandaj) and Tehran.
Cophinforma Doilom, J.K. Liu & K.D. Hyde, Fungal Divers.
57: 174. 2012. MycoBank MB801315.
Type species: Cophinforma atrovirens (Mehl & Slippers) A. Alves &
A.J.L. Phillips, comb. nov.
Ascomata initially immersed under host epidermis, becoming semiimmersed to erumpent, gregarious and fused, uniloculate, globose
to subglobose ostiolate. Ostiole central, papillate, periphysate.
Asci 8-spored, bitunicate, issitunicate, clavate to cylindro-clavate,
pedicellate, apex rounded with well-developed ocular chamber.
Ascospore overlapping uniseriate to biseriate, hyaline, aseptate,
smooth-walled, ellipsoidal to obovoid, slightly wider above the
centre. Conidiomata indistinguishable from ascomata. Paraphyses
absent. Conidiogenous cells enteroblastic, integrated, hyaline,
smooth, cylindrical, irst-formed conidium holoblastic, proliferating
at the same level resulting in typical phialides (sensu Sutton 1980)
with periclinal thickenings. Conidia hyaline, thin-walled, smooth,
aseptate, fusiform. Spermatophores reduced to conidiogenous
cells, occurring intermingled among conidiogenous cells in same
conidioma, subcylindrical, hyaline, smooth. Spermatia hyaline,
smooth, granular, subcylindrical.
www.studiesinmycology.org
C. atrovirens CBS 117917
C. atrovirens MFLUCC 11‐0425
C. atrovirens CBS 124934
91/88 B. dothidea CBS 110302
98/91
Cultures: CBS 124703 = IRAN 1529C (ex-type).
Known distribution: Iran (Hormozgan and Kurdistan Provinces and
Tehran) (Abdollahzadeh et al. 2013).
C. atrovirens CBS 117445
C. atrovirens CBS 117446
Type: Iran, Tehran, on fruits of Mangifera indica imported from
Pakistan, Aug. 2006, J. Abdollahzadeh, holotype IRAN 14275F.
Host: On twigs and fruits of Mangifera indica (Abdollahzadeh et al.
2013).
C. atrovirens MFLUCC 11‐0655
B. dothidea CBS 115476
100/99
B. cor2cis CBS 119047
B. cor2cis ATCC 22927
M. phaseolina CBS 227.33
M. phaseolina CBS 162.25
1 change
Fig. 15. Single most parsimonious tree obtained from the analysis of ITS
sequences from species of the genera Botryosphaeria and Cophinforma. The
phylogenetic tree resulting from ML analysis using the general time reversible
model of DNA evolution and assuming a discrete gamma distribution with invariant
sites (GTR+G+I) had a topology identical to the MP tree presented. MP/ML values
(> 50 %) resulting from 1000 bootstrap replicates are given at the nodes. The tree
was rooted to M. phaseolina.
Notes: Cophinforma was introduced by Liu et al. (2012) as a
monotypic genus for C. eucalypti. Here we show that two species
previously included in Botryosphaeria are better accommodated in
Cophinforma. Conidia of the two known species of Cophinforma
are longer than any known species in Botryosphaeria. In all
other aspects the two genera are morphologically similar but are
phylogenetically distinct. Two species are currently recognised in
Cophinforma.
dNA phylogeny
The irst 87 bases of the ITS sequences of the two C. atrovirens
isolates appear to have many sequencing errors and were excluded
from the analyses. In the ITS + EF1-α phylogeny (Fig. 8) the two
isolates of C. atrovirens clustered with C. eucalypti and since the
sequences were identical we consider this to represent a single
species. The oldest epithet is atrovirens, thus C. eucalypti becomes
a synonym. Unfortunately, no EF-α sequences are available for C.
mamane and no cultures are extant, and thus we could not include
C. mamane in the combined ITS + EF-α phylogeny. Nevertheless,
in the ITS phylogeny (Fig. 15), 3 bp differences separate C.
mamane from C. atrovirens and for this reason we consider these
to represent two distinct species.
79
PhilliPs et al.
Key to Cophinforma spp.
The two species are morphologically very similar, with signiicant overlap in conidial dimensions, suggesting that they can only clearly be
distinguished based on DNA data.
1. Conidia 30–40 × 8–9 μm ............................................................................................................................................................ C. mamane
1. Conidia 31–36 × 7–10 μm ........................................................................................................................................................ C. atrovirens
Species descriptions
Cophinforma atrovirens (Mehl & Slippers) A. Alves & A.J.L.
Phillips, comb. nov. MycoBank MB805459. Fig. 16.
Basionym: Fusicoccum atrovirens Mehl & Slippers, Mycologia 103:
543. 2011.
= Cophinforma eucalypti Doilom, J.K. Liu & K.D. Hyde, Fungal Divers. 57: 174.
2012.
Ascomata not reported. Conidiomata on pine needles and host
material pycnidial, supericial, multilocular, dark brown to black,
eustromatic, complex, effuse, globose, covered with hyphae; wall
composed of three layers, an outer of thick-walled dark to light brown
textura angularis, a middle layer of thin-walled light brown cells, and an
inner layer of thin-walled hyaline cells, (180–)215–275(–285) μm diam.
Conidiomata indistinguishable from ascomata. Conidiophores absent.
Conidiogenous cells hyaline, holoblastic, smooth, discrete, cylindrical,
proliferating percurrently to form one or two distinct annellations,
or proliferating at the same level giving rise to periclinal thickenings
(10.5–)13.5–19(–22) × (2–)3.5–4.5(–5.5) μm (av. of 50 conidiogenous
cells = 16.3 × 3.8 μm). Paraphyses absent. Conidia hyaline, thinwalled, unicellular, aseptate, rarely becoming septate on germination,
granular, ellipsoid to obovoid, (27–)31–36(–40) × (6–)7–10(–12)
μm (av. of 50 conidia = 33.5 × 8.5 μm). Spermatophores reduced to
Spermatogenous cells, occurring intermingled among conidiogenous
cells in same conidioma, subcylindrical, hyaline, smooth, 5–20 × 3–5
μm. Spermatia hyaline, smooth, granular, subcylindrical, straight or
slightly curved, apex obtuse, base truncate, 5–8 × 3–4 μm.
Culture characteristics: Colonies luffy, initially white to olivaceous
in the center, edges becoming olivaceous to greenish black with
age. Submerged mycelia (reverse) initially white to dark amber
on the edges to olivaceous in the center, becoming olivaceous to
greenish black with age. Optimum temperature for growth 30 °C.
Type: South Africa, Mpumalanga Province, Mawewe Nature
Reserve, from an asymptomatic branch of Pterocarpus angolensis,
Dec. 2005, J.W.M. Mehl & J. Roux, holotype PREM 60341;
paratype PREM 60342.
Cultures: CBS 124934 = CMW 22674 (ex-holotype), CBS 124935
= CMW 22682 (ex-paratype).
Hosts: Asymptomatic branches and twigs of Pterocarpus angolensis
(Mehl et al. 2011), on dead branch of Eucalyptus sp. (Liu et al.
2012) as C. eucalypti.
Known distribution: South Africa (Mehl et al. 2011), Thailand (Liu
et al. 2012).
Notes: Morphologically this species is closely related to C. mamane
but the highly divergent ITS phylogeny and several morphological
80
characters separate the two species. Conidia can be 1- or
2-septate in C. mamane (Mohali et al. 2007) but remain aseptate
until germination in C. atrovirens.
Cophinforma mamane (D.E. Gardner) A.J.L. Phillips &
A. Alves, comb. nov. MycoBank MB805460. See Gardner
(1997) for illustrations.
Basionym: Botryosphaeria mamane D.E. Gardner, Mycologia 89:
299. 1997.
Stromata erumpent through host tissue, black, 0.5−1.25 mm diam,
multiloculate, locules spherical to ovoid, ostiolate, 100−200 μm
diam. Ascomata, conidiomata and Spermatogonia distinct but often
formed in the same stroma. Ascomata with a short neck, opening
through a nonperiphysate ostiolar canal. Asci bitunicate, clavate,
8-spored, 100−180 × 25−35 μm, associated with ilamentous
pseudoparaphyses. Ascospores aseptate, hyaline, with granular
or reticulately textured contents, oval to broadly fusiform, 25−39 ×
15−20 μm. Conidiogenous cells simple, uniformly lining the locule
wall. Conidia at irst produced holoblastically, later enteroblastically,
hyaline, 1-celled, fusiform, with truncate base when newly formed,
(19−)30−44(−55) × (7−)8−9(−10) μm. Spermatia hyaline, rod-like to
allantoid, 3−9 × 2−4 μm.
Type: uSA, Hawaii, Hawaii Island, Hawaii Volcanoes National Park,
Kipuka Ki, on bark of a swollen branch of Sophora chrysophylla, 1
May 1996, D.E. Gardner, holotype BISH 644614; isotype BISH
737731; paratypes BPI 737732, BPI 737733.
Cultures: No ex-type cultures are known to be extant. CBS 117444
and CBS 117450 are reportedly B. mamane but they were isolated
from Eucalyptus and Acacia in Venezuela and their ITS sequences
differ by 3 bp from the ex-type isolate of B. mamane collected by
Gardner in 1996 and thus represent a different species.
Hosts: Sophora chrysophylla (Gardner 1997).
Diseases: Witch’s brooms (Gardner 1997).
Known distribution: USA (Hawaii) (Gardner 1997).
Notes: Originally reported from Hawaii, this species is thought to
be restricted to Sophora chrysophylla. Mohali et al. (2007) reported
what they considered to be B. mamane on Acacia mangium (CBS
117445/CBS 117450) and Eucalyptus urophylla (CBS 117444/
CBS 117917) in Venezuela. They based this conclusion on an ITS
phylogeny and similarity in conidial characters and dimensions
of their isolates with those of the ex-type strains of B. mamane.
Unfortunately, the ex-type isolates of B. mamane no longer exist.
Apparently D.E. Gardner sent sub-cultures to G. Stanosz at the
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 16. Cophinforma atrovirens. A, B. Conidiomata formed on pine needles in culture. Conidia are oozing from the conidiomata in B. C–E. Conidiogenous cells. F, G. Conidia.
H–J. Spermatogenous cells. K. Spermatia. Scale bars: A = 0.5 mm, C = 10 μm. Scale bar in A applies to B. Scale bar in C applies to D–K.
University of Wisconsin and these were given the codes 97-58 and
97-59. Zhou & Stanosz (2001) sequenced ITS of these two strains
and the sequences are available in GenBank as AF246929 and
AF246930. Unfortunately no other sequences were generated and
these two isolates have since been lost.
In the ITS phylogeny generated by Mohali et al. (2007)
isolates from E. urophylla and A. mangium clustered with the two
ex-type isolates of B. mamane. However, three base pairs in ITS
separate the ex-type isolates of C. mamane from the Venezuelan
isolates. Furthermore, ITS sequences of the Venezuelan isolates
of C. mamane are exactly the same as the ITS sequence of C.
atrovirens. Therefore we consider the Venezuelan isolates to
represent C. atrovirens.
Diplodia Fr., In: Mont., Ann. Sci. Nat. Bot., sér. 2, 1: 302.
1834. MycoBank MB8047.
Type species: Diplodia mutila Fr., In: Mont., Ann. Sci. Nat. Bot., sér.
2, 1: 302. 1834.
www.studiesinmycology.org
Ascomata unilocular, solitary or clustered, immersed, partially
erumpent when mature, dark brown to black, thick-walled, wall
composed of outer layers of thick-walled, dark brown textura
angularis, inner layers of thin-walled, hyaline textura angularis.
Ostiole central, circular, papillate, periphysate. Pseudoparaphyses
hyaline, branched, septate. Asci clavate, stipitate, bitunicate,
containing eight, biseriate ascospores. Ascospores fusiform,
hyaline, thin-walled, smooth, aseptate, rarely becoming light brown
and 1–2-septate with age. Mycelium immersed or supericial,
branched, septate, melanised, dark brown. Conidiomata pycnidial,
ostiolate, formed in uni- or multiloculate stromata, immersed,
becoming erumpent at maturity. Ostiole central, circular, papillate.
Paraphyses lacking. Conidiophores (when present) hyaline,
simple, occasionally septate, rarely branched, cylindrical, arising
from the cells lining the pycnidial cavity. Conidiogenous cells
holoblastic, hyaline, cylindrical, determinate or proliferating at the
same level giving rise to periclinal thickenings, or proliferating
percurrently and forming two or three annellations. Conidia initially
hyaline, aseptate, thick-walled, becoming 1–2-septate and pale
81
PhilliPs et al.
transluscent brown after discharge from the pycnidia, but the
colouration is often delayed or never occurs, in some species the
conidia become pigmented while still enclosed in the conidioma
and in these species the conidia rarely become septate.
Notes: Two distinct conidial morphologies are seen in Diplodia
species. In one type the conidia are initially hyaline and aseptate
and later become pale to dark brown and 1-septate. Pigmentation
is often delayed and in some species dark conidia are never seen.
In the other type, the conidia become pigmented at an early stage
of development, even while they are still enclosed within the
pycnidia. These conidia only rarely become septate. These two
morphological groups are supported by two distinct phylogenetic
lineages.
Key to Diplodia spp.
1.
1.
Conidia hyaline and aseptate, becoming brown and 1-septate only with age ........................................................................................ 2
Conidia dark brown and aseptate before discharge from pycnidia ....................................................................................................... 11
2.
2.
Av. conidial length greater than 29 μm .................................................................................................................................................... 3
Av. conidial length less than 29 μm ......................................................................................................................................................... 5
3.
3.
Conidia 18–22 μm wide ............................................................................................................................................................. D. tsugae
Conidia not more than 16 μm wide ......................................................................................................................................................... 4
4.
4.
On Quercus, av. conidial length 29.9 × 13.5 μm ..................................................................................................................... D. corticola
On hosts other than Quercus, colonies rosulate ..................................................................................................................... D. rosulata
5.
5.
On Malus, conidia pale brown ................................................................................................................................................ D. bulgarica
Conidia hyaline, becoming pigmented and 1-septate with age ............................................................................................................... 6
6.
6.
On Cupressus or Juniperus spp. ............................................................................................................................................. D. cupressi
On other hosts ......................................................................................................................................................................................... 7
7.
7.
Av. conidial length 28 μm or longer ......................................................................................................................................................... 8
Av. conidial length less than 28 μm ......................................................................................................................................................... 9
8.
8.
Conidia up to 17 or more μm wide ......................................................................................................................................... D. malorum
Conidia never reach 17 μm wide ........................................................................................................................................................... 10
9.
9.
On Quercus ......................................................................................................................................................................... D. quercivora
On other hosts ......................................................................................................................................................................... D. africana
10. Av. conidial length greater than 27 μm (27.7 μm) ..................................................................................................................... D. agrifolia
10. Av. conidial length less than 27 μm ....................................................................................................................................................... 11
11. Av. conidial size 24.5 × 12.5 μm, on Olea ............................................................................................................................... D. olivarum
11. Av. conidial size 25.5 × 13.5 μm, on other hosts ......................................................................................................................... D. mutila
12. Av. conidial length greater than 35 μm .................................................................................................................................................. 13
12. Av. conidial length less than 35 μm ....................................................................................................................................................... 14
13. Conidial length exceeding 50 μm (up to 54 μm) ....................................................................................................................... D. sapinea
13. Conidial length never exceeding 50 μm (up to 41.5 μm) ................................................................................................... D. scrobiculata
14. Av. conidial length greater than 28 μm ................................................................................................................................ D. intermedia
14. Av. conidial length less than 28 μm ....................................................................................................................................................... 15
15. Av. conidial length greater than or equal to 25 μm ................................................................................................................................ 16
15. Av. conidial length less than 25 μm ...................................................................................................................................... D. allocellula
16. Conidial length never exceeding 30 μm .................................................................................................................................... D. seriata
16. Conidial length exceeding 30 μm ........................................................................................................... D. alatafructa/D. pseudoseriata1
1
These two species cannot be distinguished based on their morphology.
82
The Botryosphaeriaceae: genera and sPecies known from culTure
98/92 CBS 112553
CBS 230.30
‐‐/50
100/99
UCROK1429
CBS 132777
98/98
CBS 121887
CAP 301
61/‐‐
CBS 120835
98/96
96/77
CBS 121104
100/100 CBS 116472
CBS 116470
72/‐‐
CBS 112554
100/100
100/58
100/100
CBS 124130
CBS 124135
CBS 124254
A
63/85
D. mu&la
D. agrifolia
D. olivarum
D. africana
D. rosulata
D. malorum
D. bulgarica
CBS 261.85
100/100
CBS 168.87
CBS 418.64
D. cupressi
D. tsugae
96/96 CBS 109944
97/89
CBS 113423
CBS 118110
66/82
CBS 109725
86/78
55/77
CBS 393.84
75/90
CBS 124462
CBS 124134
CBS 112555
52/‐‐
89/98
CBS 119049
57/58
98/95
98/99
D. pinea
D. intermedia
D. seriata
D. alatafructa CBS124931
54/60
100/100
B
D. scrobiculata
D. pseudoseriata CBS124906
D. alatafructa CBS124933
D. pseudoseriata/
D. alatafructa
D. pseudoseriata CBS124907
100/100 CBS 130408
CBS 130410
100/100
C
CBS 112547
CBS 112549
100/100
100/100 CBS 133852
CBS 133853
D. allocellula
D. cor&cola
D. quercivora
Lasiodiplodia theobromae CBS 164.96
10
Lasiodiplodia gonubiensis CBS 115812
Fig. 17. One of 75 equally most parsimonious trees (tree length = 371, CI = 0.752, RI = 0.673, HI = 0.248) obtained from the combined analysis of ITS and EF1-α sequences
from Diplodia species. Phylogenetic information contained in alignment gaps was incorporated into the phylogenetic analyses using simple indel coding as implemented by
GapCoder (Young & Healy 2003). The phylogenetic tree resulting from ML analysis using the general time reversible model of DNA evolution and assuming a discrete gamma
distribution (GTR+G) had a topology identical to the MP tree presented. MP/ML values (> 50 %) resulting from 1000 bootstrap replicates are given at the nodes. The tree was
rooted to Lasiodiplodia theobromae CBS 164.96 and Lasiodiplodia gonubiensis CBS 115812. Clades corresponding to the 17 recognised species within the genus Diplodia
are highlighted.
dNA phylogeny
Phylogenetic analysis based on combined ITS and EF1-α
sequences revealed three major clades, A, B and C (Fig. 17).
Most of the species in clade A have hyaline conidia that become
pigmented and 1-septate only some time after discharge from the
pycnidia. The exception is D. bulgarica, which has pale brown
www.studiesinmycology.org
conidia, but these become more darkly pigmented and 1-septate
with time. Eleven species can be distinguished in this clade and all
are supported by moderate to high bootstrap values. In clade B the
species all have conidia that become pigmented soon after they
have formed, sometimes while still attached to the conidiogenous
cell and usually before discharge from the pycnidia. Only rarely do
they become septate. Bootstrap support for some of the species,
83
PhilliPs et al.
such as D. pinea and D. intermedia, is quite low. Diplodia alatafructa
and D. pseudoseriata could not be separated clearly because none
of the polymorphisms between isolates in this clade are ixed or
consistent within a species. Clade C contains only two species,
D. corticola and D. quercivora, and the conidia of these species
have a morphology similar to that found in clade A, but they tend
to be larger.
Species descriptions
Diplodia africana Damm & Crous, Mycologia 99: 671.
2008. MycoBank MB501323. See Damm et al. (2007) for
illustrations.
Ascomata not reported. Conidiomata pycnidial, stromatic,
produced on pine needles on SNA in 2–4 wk, solitary, globose to
ovoid, dark brown, up to 500 μm wide, semi-immersed to erumpent,
unilocular, sometimes multilocular in vitro, with a short neck and a
central ostiole, wall 6–8 cell layers thick, outer layers composed
of dark-brown textura angularis, becoming thin-walled and hyaline
toward the inner region. Conidiophores 1–2 celled, hyaline, 10–25
× 3.5–6 μm. Conidiogenous cells holoblastic, hyaline, cylindrical,
sometimes ampulliform, proliferating percurrently near the apex,
sometimes with periclinical thickening, 3–15 × 3–6 μm. Conidia
aseptate, hyaline, thick-walled, smooth, subcylindrical to oblongelliptical, sometimes slightly curved, with rounded ends, hyaline
after discharge from pycnidia, a few of them becoming brown,
septate and inely verruculose with age, (17–)25.5–33(–34) × (10–)
12–14(–15) μm (av. ± SD = 29.2 ± 3.6 × 13 ± 1.1 μm), L/W ratio
= 2.2.
Culture characteristics: Colonies on PDA in the dark: mycelium
pale olivaceous-grey, surface pale olivaceous-grey to dark greyolivaceous, reverse olivaceous-black, umbonate with irregular
zonation and lobate edges. Colonies under near ultraviolet:
mycelium and surface greenish olivaceous to dark grey-olivaceous;
reverse greenish olivaceous to olivaceous-black. Colonies reaching
26.8 mm diam after 2 d, reaching the edge the Petri dish within 5 d.
Cardinal temperatures for growth: min 5 °C, max 35 °C, opt 20 °C.
Type: South Africa, Western Cape Province, Paarl, from wood
section close to pruning wound of Prunus persica, 10 Jun. 2004, U.
Damm, holotype CBS H-19843.
Cultures: CBS 120835 = STE-U 5908 (ex-type), STE-U 6289.
Host: Prunus persica (Damm et al. 2007).
Known distribution: South Africa (Western Cape Province) (Damm
et al. 2007).
Notes: Conidia of D. africana are hyaline and thick-walled even
after discharge from conidiomata and only a few conidia become
brown and septate with age. It shares these features with D. mutila,
D. corticola, D. cupressi, D. rosulata, D. quercivora and D. tsugae.
Diplodia agrifolia S.C. Lynch, A. Eskalen, Mycologia 105:
135. 2013. MycoBank MB800443. See Lynch et al. (2013)
for illustrations.
84
Ascomata not reported. Conidiomata single or in groups, immersed
to erumpent when mature, black and globose, 189 × 171–836 ×
721 μm, wall composed of three layers; an outer layer of dark, thickwalled cells, middle layer with dark brown, thin-walled cells, and
an inner layer of thin-walled hyaline cells. Ostiole central, circular,
apapillate to papillate. Conidiophores absent. Conidiogenous cells
holoblastic, discrete, cylindrical, hyaline, smooth, indeterminate,
proliferating at the same level giving rise to periclinal thickenings or
proliferating percurrently to form one to two indistinct annellations,
18.0 ± 7.4 × 8.1 ± 2.4 μm. Conidia in equal proportions hyaline,
aseptate and pale to dark brown and 1-septate before and after
discharge, smooth, thick-walled, oblong to ovoid, straight, both
ends broadly rounded, (21.5–)27–36.5 × (12–)14.5–18 μm (av. ±
S.D. = 27.7 ± 2.2 × 14.7 ± 1.2 μm), L/W = 1.9.
Type: uSA, California, San Diego County, Mataguay Scout Camp,
on cankered branch of Quercus agrifolia, 23 Feb. 2010, S.C. Lynch
& A. Eskalen, holotype BPI 884095 (dried culture of D. agrifolia).
Cultures: CBS 132777 = ATCC MYA-4895 = UCROK 732 (ex-type).
Hosts: Quercus agrifolia and Q. kelloggii (Lynch et al. 2013).
Known distribution: uSA, Coast range of north-central California
southward to northern Baja California, with Q. kelloggii extending
as far north as Eugene, Oregon (Lynch et al. 2013).
Notes: Phylogenetically, D. agrifolia is distinct from but closely
related to D. mutila. Diplodia agrifolia differs from D. mutila in
the conidia that are longer and wider than D. mutila. Conidia of
D. agrifolia are hyaline and aseptate, but most become dark brown
and 1-septate before discharge from pycnidia, whereas conidia of
D. mutila are hyaline, aseptate, rarely becoming pale brown and
1-septate with age.
Diplodia alatafructa Mehl & Slippers, Mycologia 103: 542.
2011. MycoBank MB513498. See Mehl et al. (2011) for
illustrations.
Ascomata not reported. Conidiomata on both pine needles and
host material stromatic, supericial, unilocular, dark brown to
black, mostly solitary, more or less globose/circular, covered with
mycelium/hyphae, wall composed of three layers; an outer thickwalled dark brown textura angularis, a middle layer of light brown to
reddish brown thin-walled cells, and an inner layer of hyaline thinwalled cells, (114–)130–155(–160) μm diam (av. of 50 conidioma
= 141.4 μm). Ostiole central, circular. Conidiophores absent.
Conidiogenous cells holoblastic, hyaline, discrete, spherical to
cylindrical, proliferating percurrently to form two or three distinct
annellations, or proliferating at same level giving rise to periclinal
thickenings, (10–)12.5–18(–23) × (8–)11–14(–15.5) μm (av. of 40
conidiogenous cells = 15.4 × 12.5 μm). Conidia initially hyaline
becoming pigmented and dark brown with age, unicellular, rarely
septate or biseptate, rarely striate, ellipsoid to obovoid, thick-walled,
granular, rounded at apices, eguttulate, smooth, (22.5–)24.5–29(–
33) × (9.5–)11–14(–16) μm (av. of 50 conidia = 26.9 × 12.4 μm).
Culture characteristics: Colonies with luffy mycelium, initially white
to amber in the centre turning dark amber within 7 d and becoming
white to dark amber, almost olivaceous with age; submerged
The Botryosphaeriaceae: genera and sPecies known from culTure
mycelium (reverse) same except becoming white to dark amber,
almost olivaceous, at the periphery, and olivaceous in the centre
with age. Optimum temperature for growth 25 °C.
Type: South Africa, Mpumalanga Province, Sudwala Caves area,
from a stem wound on P. angolensis, Dec. 2005, J.W.M. Mehl & J.
Roux, holotype PREM 60337.
percurrently to form 1−5 annellations, 9−18 × 2−5 μm. Conidia
aseptate, smooth, thick-walled, oblong to ovoid, straight, both ends
broadly rounded, (22.5−)24−27(−28) × (14.5−)15.5−18(−18.5) μm,
95 % conidence limits = 25.0−25.7 × 16.6–17.0 μm (av. ± S.D. of
50 conidia = 25.4 ± 1.2 × 16.8 ± 0.7 μm), L/W ratio = 1.5.
Culture: CBS 124931 (ex-type).
Type: Bulgaria, Plovdiv, on dead twigs of Malus sylvestris, 2005,
S.G. Bobev, holotype CBS H-20189 (a dried culture of CBS
124254 grown on pine needles).
Host: Pterocarpus angolensis (Mehl et al. 2011).
Culture: CBS 124254 (ex-type).
Known distribution: South Africa (Mehl et al. 2011).
Hosts: Malus spp. (Phillips et al. 2012).
Note: See notes to D. pseudoseriata.
Known distribution: Bulgaria, Iran (Phillips et al. 2012).
Diplodia allocellula Jami, Gryzenh., Slippers & M.J. Wingf.,
Cryptogamie Mycol. 33: 257. 2012. MycoBank MB564140.
See Jami et al. (2012) for illustrations.
Notes: This species is morphologically distinct from all other
Diplodia species reported from apples. Conidia are shorter and
wider than both D. malorum and D. intermedia. Furthermore, the
conidia are distinctive in that they become pale brown soon after
they are formed. Phylogenetically this species is closely related to
D. cupressi and D. tsugae.
Ascomata not reported. Conidiomata immersed on MEA, solitary,
globose, brown, up to 100 μm diam. Conidiogenous cells holoblastic,
smooth, unicellular, cylindrical to sub-cylindrical, hyaline (4–)4.5–
5(–5.5) × (10.5–)13.5–23.5(–27.5) μm. Conidia ovoid to ellipsoid,
smooth with ine granular contents, apex rounded, base truncate,
thick-walled, aseptate, initially hyaline, becoming dark brown,
aseptate (20–)21.5–25(–30) × (9–)10–12.5(–14.5) μm.
Type: South Africa, Gauteng Province, Pretoria, from branch of
Acacia karroo with dieback, Nov. 2009, M. Gryzenhout & F. Jami,
holotype PREM 60701.
Cultures: CBS 130408 (ex-type) CBS 130409, CBS 130410
(paratype).
Host: Acacia karroo (Jami et al. 2012).
Known distribution: South Africa (Gauteng Province) (Jami et al.
2012).
Notes: Phylogenetically D. allocellula falls within the group of
species with conidia that become brown and aseptate at an early
stage of their development. Morphologically it is most similar to D.
seriata and D. alatafructa/D. pseudoseriata but can be distinguished
from these species on account of its generally smaller conidia.
Diplodia bulgarica A.J.L. Phillips, J. Lopes & S.G. Bobev,
Persoonia 29: 33. 2012. MycoBank MB19632. Fig 18.
Ascomata not reported. Conidiomata pycnidial, stromatic, produced
on pine needles on WA after 7–21 d, solitary, immersed, partially
erumpent when mature, dark brown to black, globose to ovoid,
up to 600 μm diam and 700 μm high, wall composed of an outer
layer of dark brown, thick-walled textura angularis, a middle layer
of dark brown thin-walled cells, an inner layer of thin-walled hyaline
cells. Ostiole central, circular, papillate. Conidiophores absent.
Conidiogenous cells hyaline, cylindrical, holoblastic, forming a single
conidium at the tip, discrete, smooth, indeterminate, proliferating
internally giving rise to periclinal thickenings, or proliferating
www.studiesinmycology.org
Diplodia corticola A.J.L. Phillips, A. Alves & J. Luque,
Mycologia 96: 603. 2004. MycoBank MB488568. Figs 19, 20.
= Botryosphaeria corticola A.J.L. Phillips, A. Alves & J. Luque, Mycologia 96:
603. 2004.
Pseudothecia stromatic, immersed, partially erumpent when
mature, dark brown to black, more or less circular, up 1 mm
diam, multiloculate, individual locules 200−300 μm diam, thickwalled, wall composed of outer layers of thick-walled, dark
brown textura angularis, inner layers of thin-walled, hyaline
textura angularis. Ostiole circular, central, papillate, periphysate.
Pseudoparaphyses hyaline, branched, septate, 2−3 μm wide.
Asci clavate, stipitate, bitunicate, containing eight, biseriate
ascospores, 160−250 × 30−35 μm (including stipe). Ascospores
broadly fusiform to rhomboid, widest in the middle, both ends
obtuse, hyaline, moderately thick-walled (ca. 1 μm), smoothwalled, aseptate, rarely becoming light brown and 1–2-septate
with age, (28.5−)30−38(−40.5) × (13−)14−18.5(−19) μm, 95 %
conidence limits = 33.6–35 × 15.3–16.2 μm (av. ± S.D. of 90
ascospores = 34.3 ± 2.4 × 15.8 ± 1.5 μm), L/W ratio = 2.2.
Conidiomata eustromatic, immersed, partially erumpent when
mature, dark brown to black, more or less circular, up to 1 mm
diam, multiloculate, individual locules 200−300 μm diam, wall
composed of three layers, an outer of dark brown, thick-walled
textura angularis, a middle layer of dark brown thin-walled cells,
and an inner layer of thin-walled hyaline cells. Ostiole central,
circular, papillate. Conidiophores reduced to conidiogenous
cells. Conidiogenous cells holoblastic, discrete, cylindrical,
hyaline, smooth, indeterminate, proliferating at the same level
giving rise to periclinal thickenings, or proliferating percurrently
to form one or two indistinct annellations, 12−19(−24) × 4−6
μm. Conidia hyaline, aseptate, eguttulate or sometimes with a
large central guttule, contents granular, smooth, thick-walled,
oblong to cylindrical, straight, both ends broadly rounded, rarely
becoming brown and septate when aged, (23.5−)26−34.5(−46)
× (9−)12−16(−18.5) μm, 95 % conidence limits = 29.6–30.3 ×
13.4–13.8 μm (av. ± S.D. of 250 conidia = 29.9 ± 2.5 × 13.6 ± 1.4
μm), L/W ratio = 2.2.
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PhilliPs et al.
Fig. 18. Diplodia bulgarica. A. Culture grown on PDA. B. Conidiomata developing on pine needles in culture. C. Conidioma on pine needles oozing conidia. D–G. Conidiogenous
cells with developing conidia. H. Pale brown, aseptate conidia. I. Pale brown, aseptate conidia and one 2-celled conidium. J, K. Brown conidium in two focal planes showing the
inely verruculose inner surface of the wall. Scale bars: B = 0.5 mm, C = 200 μm, D–I = 10 μm, J, K, = 5 μm.
Culture characteristics: Colonies reaching 36−44 mm diam on PDA
after 4 d in the dark at 25 °C. Cardinal temperatures for growth:
min 5 °C, max < 35 °C, opt 20−25 °C.
Type: Portugal, Beira Littoral, Requeixo near Aveiro, on dead
branches of Quercus suber, Feb. 2002, A. Alves, holotype LISE
94839.
Culture: CBS 112549 (ex-type).
Hosts: Quercus spp. (Alves et al. 2004).
86
Known distribution: Iberian Peninsula, Italy, N. America (Alves et
al. 2004).
Notes: Conidia of this species are larger than in any other species
of Diplodia. Phylogenetically D. corticola groups with D. quercivora
(also an oak pathogen) in a distinct clade. It is responsible for
dieback and cankers on Q. suber and Q. ilex and has been
implicated as contributing to the general decline of cork oaks in the
Iberian Peninsula and other regions of the Mediterranean.
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 19. Diplodia corticola. A. Ascomata partially erumpent through the host bark. B. Multilocular ascoma cut through horizontally revealing the brilliant white contents. C. Vertical
section through an ascoma showing the thick wall and three locules opening through periphysate ostioles. D, E. Ascus tip as seen by interference contrast (D) and phase
contrast (E) showing the well-developed apical chamber. F. Mature ascus containing ascospores, several immature asci and pseudoparaphyses. G. Pseudoparaphyses. H–J.
Ascospores. K, L. Brown, 2-septate ascospores. Scale bars: A = 1 mm, B = 500 μm, C = 100 μm, D, E, G = 10 μm, F = 20 μm, H–L = 5 μm.
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87
PhilliPs et al.
Fig. 20. Diplodia corticola. A. Sectioned conidiomata showing thick wall and three locules. B, C. Percurrently proliferating conidiogenous cells in surface view (B) and optical
section (C) with annellations arrowed. D. Phialide with periclinal thickenings. E. Conidia. F. Brown and septate conidia. Scale bars: A = 100 μm, B–F = 10 μm.
Diplodia cupressi A.J.L. Phillips & A. Alves, Fungal Divers.
23: 9. 2006. MycoBank MB510136. Fig. 21.
Ascomata not reported. Conidiomata up to 300 μm diam, solitary,
separate, uniloculate, dark brown to black, globose, ostiolate, wall
composed of thick-walled textura angularis, becoming thin-walled
and hyaline toward the inner region. Conidiophores reduced
to conidiogenous cells. Conidiogenous cells hyaline, smooth,
holoblastic forming conidia at their tips, proliferating internally
giving rise to periclinal thickenings or proliferating percurrently with
1–4 close or widely spaced annellations, formed from the inner wall
of the pycnidium, 12.5–20 × 4–4.5 μm. Conidia thick-walled, wall
up to 2 μm wide, ovoid with both ends rounded, aseptate, hyaline
and remaining so for a long time, becoming brown and 1-septate
after discharge from the pycnidia, (21.5–)23.5–28.5(–30.5) ×
(12–) 13.5–15(–16) μm, 95 % conidence limits = 24.4–25.4 ×
13.9–14.5 μm, (av. ± S.D. of 50 conidia = 24.9 ± 1.9 × 14.2 ± 0.9
μm), L/W = 1.76. Spermatophores hyaline, smooth, cylindrical, up
to 10 μm long, 2.5–3 μm wide. Spermatogenous cells discrete or
integrated, hyaline, smooth, cylindrical, holoblastic or proliferating
via determinate phialides with periclinal thickening, 10–14 × 2–2.5
μm. Spermatia hyaline, smooth, aseptate, rod-shaped with rounded
ends, 4–5 × 1.5 μm.
Type: Israel, Bet Dagan, dried culture from cankered stems of
Cupressus sempervirens, 1986, Z. Solel, holotype IMI 303475.
88
Culture: CBS 168.87 (ex-type).
Hosts: Cupressus and Juniperus spp. (Alves et al. 2006, Solel et
al. 1987).
Known distribution: Cyprus, Greece, Israel, Italy, Morocco, South
Africa, Tunisia, USA (De Wet et al. 2009, Alves et al. 2006, Solel
et al. 1987).
Notes: Solel et al. (1987) considered this fungus to be a sub-population
of Diplodia pinea and named it Diplodia pinea f. sp. cupressi. Swart et
al. (1993) challenged this assumption and showed that D. pinea f. sp.
cupressi differed morphologically from D. pinea in terms of conidial
dimensions, shape, colouration and this was supported by isozyme
proiles. The observations of Swart et al. (1993) were supported by
ITS sequence data by Zhou & Stanosz (2001). Finally, Alves et al.
(2006) introduced the name D. cupressi for the Cypress pathogen.
This species is morphologically similar to D. mutila but the conidia of
D. cupressi are wider than are typical for D. mutila (Alves et al. 2004).
Diplodia intermedia A.J.L. Phillips, J. Lopes & A. Alves,
Persoonia 29: 33. 2012. MycoBank MB19633. Fig. 22.
Ascomata unilocular, solitary or clustered, immersed, partially
erumpent when mature, globose, up to 400 μm diam, dark brown to
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 21. Diplodia cupressi. A. Conidiomata on host bark. B–E. Conidiogenous cells. F. Spermatogenous cells. G. Spermatia. H. Hyaline, aseptate conidia. I. Mature dark-walled,
1-septate conidia. Scale bars: A = 1 mm, B, H = 10 μm. Scale bar of B applies to C–G. Scale bar of H applies to I.
black, thick-walled, wall composed of outer layers of thick-walled,
dark brown textura angularis, inner layers of thin-walled, hyaline
textura angularis. Ostiole central, circular, nonpapillate, periphysate.
Pseudoparaphyses hyaline, branched, septate, constricted at the
septum, 2–3 μm wide. Asci clavate, stipitate, bitunicate, containing
eight ascospores biseriate in the ascus, 85–160 × 22–28 μm.
Ascospores fusiform, widest in the upper third, hyaline, thinwalled, smooth, aseptate, 32–37(–40) × 6–8 μm. Conidiomata
pycnidial, stromatic, solitary or clustered, immersed in the host,
partially erumpent at maturity, dark brown to black, ostiolate,
nonpapillate, thick-walled, outer and inner layers composed of
dark brown and thin-walled hyaline textura angularis, respectively.
Conidiogenous cells hyaline, thin-walled, smooth, cylindrical,
swollen at the base, discrete, producing a single conidium at the
tip, indeterminate, proliferating internally giving rise to periclinal
thickenings or proliferating percurrently forming 2–3 annellations.
Conidia aseptate, ovoid, widest in the middle, with obtuse apex and
truncate or rounded base, initially hyaline, becoming dark brown
before release from the pycnidia, wall moderately thick, externally
smooth, internally roughened, (24.5–)29–33.5(–37) × (10–)11–16(–
17.5) μm, with 95 % conidence limits = 30.2–31.1 × 13–13.6 μm
(av. ± S.D. of 150 conidia = 30.6 ± 1.9 × 13.3 ± 1.8 μm), L/W =
2.3. Spermatia hyaline, aseptate, smooth, oblong, ends rounded,
5.5–9.5 × 4–6.5 μm. Spermatogenous cells not seen.
Type: Portugal, Setúbal, Monte da Caparica, dead twigs of Malus
sylvestris, Mar. 2006, A.J.L. Phillips, holotype CBS H-20190.
www.studiesinmycology.org
Culture: CBS 124462 (ex-type).
Hosts: Cydonia, Malus (Phillips et al. 2012).
Known distribution: Portugal (Phillips et al. 2012).
Notes: Phylogenetically this species is very closely related to D.
sapinea. However, on account of its smaller conidia, apparent
preference for Rosaceae hosts, and the distinct clade it forms in
the ITS + EF1-α phylogenies, Phillips et al. (2012) considered it to
represent a distinct and separate species.
Diplodia malorum Fuckel, Jb. Nassau. Ver. Naturk. 23–24:
395. 1870. MycoBank MB 246351. Fig. 23.
Ascomata not reported. Conidiomata pycnidial, stromatic, immersed,
erumpent, dark brown to black, aggregated, internally white,
ostiolate, ostiole circular, central, short papilla. Conidiophores absent.
Conidiogenous cells cylindrical, thin-walled, hyaline, holoblastic,
indeterminate, proliferating at the same level to produce periclinal
thickenings, or proliferating percurrently giving rise to 2–3 indistinct
annellations. Conidia oblong with broadly rounded ends, smoothwalled, thick walled, hyaline, eguttulate, aseptate, becoming dark
brown and 1-septate soon after release from the pycnidium, (24–)26–
32(–36) × (12–)13–17.5(–18.5) μm, 95 % conidence limits = 28.0–28.3
× 14.3–14.5 μm (av. ± S.D. = 28.1 ± 2.4 ×14.4± 1.4 μm), L/W = 1.9.
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PhilliPs et al.
Fig. 22. Diplodia intermedia. A. Culture grown on PDA. B. Conidiomata developing on pine needles in culture. C. Asci. D, E. Ascus, ascospores and pseudoparaphyses. F–I.
Conidiogenous cells. J, K. Conidia in two focal planes to show inely verruculose inner surface of the wall. L, M. Conidia. N, O. Spermatia. Scale bars: B = 0.5 mm, C, D = 20
μm, E–M = 10 μm, N, O = 5 μm.
Type: Germany, Rhineland, on Malus sp., 1870, J. Fuckel,
Fuckel, Fungi rhenani Nº 1706, holotype in G, isotypes K and
M. Portugal, Setúbal, Monte da Caparica, Malus sylvestris, Feb.
2006, A.J.L. Phillips, epitype CBS H-201888.
The conidia are larger than those of D. mutila and they frequently
become brown and 1-septate soon after discharge from the
conidioma.
Culture: CBS 124130 (ex-epitype).
Diplodia mutila (Fr.) Mont., Ann. Sci. nat., sér. 2, 1: 302.
1834. MycoBank MB201741. Fig. 24.
Hosts: Malus spp. (Phillips et al. 2012).
Known distribution: Germany, Portugal (Phillips et al. 2012).
Notes: Since the time that it was introduced by Fuckel (1870), the
name D. malorum has been used infrequently, while the name D.
mutila was applied to the apple pathogen. However, D. malorum
is morphologically and phylogenetically distinct from D. mutila.
90
Basionym: Sphaeria mutila Fr., Syst. Mycol. (Lundae) 2: 424. 1823.
≡ Physalospora mutila (Fr.) N.E. Stevens, Mycologia 28: 333. 1936.
= Botryosphaeria stevensii Shoemaker, Canad. J. Bot. 42: 1299. 1964.
Further synonyms are given by Stevens (1933).
Ascomata unilocular, solitary or clustered, immersed, partially
erumpent when mature, globose, up to 300 μm diam, dark brown to
black, thick-walled, wall composed of outer layers of thick-walled,
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 23. Diplodia malorum. A. Culture growing on PDA. B. Pycnidia formed on pine needles. C–E. Conidiogenous cells. F. Hyaline aseptate conidia. G. Hyaline and 1-septate
brown conidia. H, I. Brown conidia at two different planes of focus to show the inely verruculose inner surface of the wall. Scale bars: B = 500 μm, C–I = 10 μm.
dark brown textura angularis, inner layers of thin-walled, hyaline
textura angularis. Ostiole central, circular, papillate, periphysate.
Pseudoparaphyses hyaline, branched, septate, 2−3 μm wide. Asci
clavate, stipitate, bitunicate, containing eight, biseriate ascospore,
100−160 × 14−22 μm (including stipe). Ascospores fusiform, widest
in the middle, both ends obtuse, hyaline, thin-walled, smooth,
aseptate, rarely becoming light brown and 1–2-septate with age,
(24.5−)28–35(−36) × (9.5−)10−13(−13.5) μm, 95 % conidence
www.studiesinmycology.org
limits = 30.8–32.1 × 11.2–11.7 μm (av. ± S.D. of 50 ascospores
= 31.5 ± 2.3 × 11.4 ± 0.9 μm), L/W = 2.8. Conidiomata solitary or
aggregated in clusters of up to ive or more, immersed, partially
erumpent when mature, dark brown to black, more or less globose,
up to 600 μm diam, wall composed of three layers, an outer of
dark brown, thick-walled textura angularis, a middle layer of dark
brown thin-walled cells, an inner layer of thin-walled hyaline
cells. Ostiole central, circular, papillate. Conidiophores absent.
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PhilliPs et al.
Fig. 24. Diplodia mutila. A. Sectioned ascoma. B. Immature asci and pseudoparaphyses. C, D. Asci with ascospores. E, F. Ascospores. G. Conidiomata partially erumpent
through host. H. Sectioned conidioma. I–L. Conidiogenous cells. M–P. Conidia. M. Hyaline, aseptate conidia of CBS 112553. N. Pale brown, 1-septate conidia of CBS 112553.
O. Hyaline, aseptate conidia of BPI 599153. P. Hyaline, aseptate conidia of K(M) 99664. Scale bars: A = 100 μm, B = 10 μm, E, F = 10 μm, G = 500 μm, H = 100 μm, I, L = 10
μm, M = 10 μm. Scale bar in B applies to C, D. Scale bar in I applies to J, K. Scale bar in M applies to N–P.
92
The Botryosphaeriaceae: genera and sPecies known from culTure
Conidiogenous cells holoblastic, discrete, cylindrical, hyaline,
smooth, indeterminate, proliferating at the same level giving rise
to periclinal thickenings, or proliferating percurrently to form one
or two indistinct annellations, 11−15 × 4−5 μm. Conidia hyaline,
aseptate, smooth, thick-walled, oblong to ovoid, straight, both ends
broadly rounded, rarely becoming pale brown and septate when
aged, (23.5−)24.5−27(−27.5) × (12.5−)13−14(−14.5) μm, 95 %
conidence limits = 25.1–25.7 × 13.2–13.5 μm (av. ± S.D. of 50
conidia = 25.4 ± 1.0 × 13.4 ± 0.5 μm), L/W ratio = 1.9.
Type: of Physalospora mutila (designated by Alves et al. 2004):
uK, England, Cornwall, Saltash, on bark of Malus sp., 22 Aug.
1935, N.E. Stevens, lectotype BPI 599153. Of Diplodia mutila:
France, Ardenne, Sedan, on bark of Populus nigra, date unknown,
Montagne sp., isotype K(M)99664.
Cultures: No ex-type, or authentic cultures of either state are
known. CBS 112553 has been regarded, unoficially, as a standard
isolate of D. mutila (Alves et al. 2004, Damm et al. 2008).
Hosts: While Farr et al. (2013) list 55 hosts for D. mutila it is now
clear that many of the earlier reports of this fungus could be
misidentiications (Alves et al. 2004, Alves et al. 2006, Lazzizera
et al. 2008, Phillips et al. 2012). The following are conirmed hosts:
Chamaecyparis lawsoniana, Fraxinus, Malus, Populus, Taxus
baccata, Vitis vinifera.
Known distribution: England, France, Italy, Portugal, South Africa,
USA (California).
Notes: The taxonomic history of D. mutila and the controversy
surrounding the characters that deine this fungus have been
explained by Sutton (1980) and Alves et al. (2004). However,
in the interests of presenting a comprehensive analysis, these
explanations are repeated here.
Fries (1823) described Sphaeria mutila and distributed two
exsiccati under that name as Scler. Suec. 164 and 385. Alves et
al. (2004) examined material of these two exsiccati in STR and
found both to be devoid of spores. Stevens (1933) and Sutton
(1980) also reported that these two exsiccati in BPI and K had no
spores. Sutton (1980) reported that 164 was an ascomycete of the
Botryosphaeria type and pointed out that Sphaeria mutila should
be adopted for the ascomycetous element it represents. Montagne
sent Fries a fungus that was identiied as S. mutila. The record was
listed under S. mutila Fr. by Montagne (1834) with the note that
this species would become the type of a new genus, Diplodia, later
characterised by Fries (1849). Therefore, the name of the pycnidial
fungus dates from Montagne (1834); it is typiied by his material
and the correct citation is Diplodia mutila Fr. in Montagne (1834).
Montagne distributed this fungus in his exsiccatus No. 498.
According to Françoise Deluzarche of the Institut de Botanique,
Strasbourg, France, no material of this could be found in STR (Alves
et al. 2004). However, according to Alves et al. (2004), Montagne’s
specimen of D. mutila in Kew, K(M)99664 (isotype), agrees in all
aspects with Stevens’ (1933) account of Montagne’s exs. 498 but
differs from the description given by Sutton (1980). While Sutton
(1980) referred to the conidia as initially hyaline with a large central
guttule, later becoming dark brown and medianly one eusepate,
Alves et al. (2004) reported that the vast majority of conidia in
K(M)99664 are hyaline and aseptate, although pale brown and oneor two-septate conidia are seen rarely. The conidia usually have a
large central guttule. Furthermore, the dimensions that Sutton (1980)
www.studiesinmycology.org
reported (27–31 × 12–13.5 μm) are somewhat larger than Alves et al.
(2004) found (23.5–27.5 × 12–14 μm). Stevens (1933) reported the
conidia as (20–)25–27 × 10–12(–16) μm.
In the original description, Montagne (1834) described the
conidia as “Asci [conidia] elliptico-oblongi, didymi, sporidiis binis
referti.” Stevens (1933) studied slides of Montagne’s exsiccatus
in STR and described the conidia as hyaline and aseptate with a
thick smooth, glassy wall, although pale brown, 1-septate conidia
sometimes were present. Both Shoemaker (1964) and Laundon
(1973) agreed with Stevens’ concept. Sutton (1980), however,
described the conidia as hyaline at irst but becoming dark brown
and 1-septate when mature. In his illustration of this species he
depicts a predominance of dark conidia. Alves et al. (2004) reexamined the isotype in K and concluded that the conidia are
predominantly hyaline, although some are dark and 1-septate. The
consensus was that conidia of D. mutila are (20–)25–27.5 × 10–12
μm (Stevens 1933, Shoemaker 1964, Laundon 1973, Sivanesan
1984), but Sutton (1980) considered they can be up to 31 μm long.
Stevens (1936) reported on the sexual morph of D. mutila that
he found on apple and ash in England. The connection between
this fungus and D. mutila was established through single ascospore
isolations and Stevens applied the name Physalospora mutila (Fr.)
N.E. Stevens. Shoemaker (1964) considered this to be a species of
Botryosphaeria and applied the new name B. stevensii Shoemaker
because the name Botryosphaeria mutila was already taken.
Stevens (1936) referred to a specimen on cut sticks of Fraxinus
excelsior as the type.
When Alves et al. (2004) examined the type specimen of P. mutila
in BPI 599151 they could ind no ascomycete. There was, however,
ample material of the sexual morph on BPI 599153, which is a
specimen of P. mutila on apple collected by Stevens from the same
locality and at the same time that he collected BPI 599151. Since
this specimen conformed in all ways with the protologue, Alves et
al. (2004) designated this specimen as lectotype. Unfortunately, no
ex-type cultures exist. The type host of P. mutila is Fraxinus excelsior
whereas the type host of Diplodia mutila is a Populus sp.
Diplodia olivarum A.J.L. Phillips, Frisullo & Lazzizera,
Fungal Divers. 31: 67. 2008. MycoBank MB511402. Fig. 25.
Ascomata not reported. Conidiomata pycnidial, stromatic, produced
on pine needles on WA after 7–14 d, solitary, globose to ovoid, dark
brown to black, up to 150 μm wide, wall composed of dark brown,
thick-walled textura angularis, becoming thin-walled and hyaline
towards the inner region, semi-immersed to erumpent, unilocular,
with a short neck. Ostiole circular, central. Conidiophores hyaline,
cylindrical, 10–15 × 3.5–5 μm. Conidiogenous cells hyaline,
cylindrical, holoblastic forming a single conidium at the tip,
proliferating internally to form periclinal thickenings or proliferating
percurrently giving rise to 2–3 annellations, 8–12 × 3–6 μm. Conidia
hyaline, aseptate, smooth, thick-walled, oblong to oval, widest
in the middle, apex broadly rounded, base rounded or truncate,
rarely becoming pale brown, internally verruculose, 1-septate after
discharge from the pycnidia, (21.5–)22–27.5(–28.5) × (10–)11–
13.5(–14.5) μm, 95 % conidence limits = 23.9–24.8 × 12.2–12.7
μm, av. ± S.D. = 24.4 ± 1.6 × 12.4 ± 1 μm), L/W = 2.0.
Type: Italy, Puglia, Lecce, Scorrano, Basco Belvedere, on rotting
drupes of Olea europaea, Dec. 2004, S. Frisullo, holotype CBS
H-19914.
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PhilliPs et al.
Fig. 25. Diplodia olivarum. A–C. Conidia developing on conidiogenous cells. D. Hyaline, aseptate conidia. E. Dark pigmented, one-septate conidia. Scale bars = 10 μm.
Culture: CBS 121887 (ex-type).
Culture: CBS 124906 (ex-type).
Host: Olea europaea (Lazzizera et al. 2008).
Hosts: Acca sellowiana, Blepharocalyx salicifolius, Eugenia
unilora, Eugenia involucrata, Hexachlamis edulis, Myrceugenia
euosma, Myrciaria tenella, Myrcianthes cisplatensis (Pérez et al.
2010).
Known distribution: Italy (Lazzizera et al. 2008), Spain (Gramaje
et al. 2012).
Notes: This species is similar to D. mutila but the two can be
distinguished based on minor differences in the dimensions of their
conidia. Although the ranges of dimensions overlap considerably,
mean dimensions of conidia of D. olivarum are smaller than D. mutila.
Diplodia pseudoseriata C.A. Pérez, Blanchette, Slippers
& M.J. Wingf., Fungal Divers. 41: 63. 2010. MycoBank
MB513545. Fig. 26.
Ascomata not reported. Conidiomata (formed in culture on sterilised
pine needles) semi-immersed or supericial, solitary, globose, black,
covered by mycelium, up to 430 μm diam. Conidiogenous cells
cylindrical, discrete, producing a single conidium at the tip, with no
evident annellations. Conidia initially hyaline becoming dark brown,
wall externally smooth, roughened on the inner surface, sometimes
1-septate, ovoid, apex obtuse, base truncate, (23–)25.5–26.5(–
30.5) × (10–)11.5–12(–14) μm.
Type: uruguay, Paysandu, Guaviyu, isolated from asymptomatic
twig of Blepharocalyx salicifolius, Aug. 2006, C. Pérez, holotype
PREM 60264.
94
Known distribution: Uruguay (Pérez et al. 2010).
Notes: Diplodia pseudoseriata was described from native
Myrtaceae trees in Uruguay (Pérez et al. 2010) while D. alatafructa
was described from Pterocarpus angolensis in South Africa (Mehl
et al. 2011). In the phylogeny constructed by Phillips et al. (2012)
and in the present work, isolates of both of these species formed
a cluster suggesting that they represent several phylogenetic
species. Nevertheless, sequences of the ex-type isolates are
divergent and indicate two separate species. Thus, it seems likely
that either cultures or sequences of the other isolates of these two
species have been mislabelled. Furthermore, the isolates in this
cluster should be studied in detail to determine if they represent a
complex of species.
Diplodia quercivora Linaldeddu & A.J.L. Phillips, Mycologia
105: 1269. 2013. MycoBank MB801757. Fig 27.
Ascomata not reported. Conidiomata pycnidial, stromatic,
produced on poplar twigs on PDA within 14 d, supericial, dark
brown to black, mostly uniloculate, solitary, globose, thick-
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 26. Diplodia pseudoseriata. A, B. Conidiogenous cells. C–G. Conidia. The conidium in C, D is shown at two different focal planes revealing the ornamentation on the inner
surface of the conidium wall. Scale bar A = 10 μm. Scale bar in A applies to B–G.
walled, non-papillate with a central ostiole. Paraphyses not seen.
Conidiogenous cells hyaline, smooth, cylindrical, sometimes
slightly swollen at the base, holoblastic forming conidia at their
tips, proliferating internally giving rise to periclinal thickenings,
9.1–13.5 × 3.5–6 μm. Conidia hyaline, aseptate, smooth, thickwalled, subcylindrical to oblong-elliptical, widest at the middle,
both ends broadly rounded, rarely becoming brown and 1-septate
with age, (23–)28(–30.5) × (11.5–)14(–14.5) μm, 95 % conidence
limits = 27.7–28.5 × 12.9–13.2 μm (av. ± S.D. of 50 conidia = 28.1
± 1.4 × 13.8 ± 0.6 μm), L/W = 2.16.
Culture characteristics: Cardinal temperatures for growth: min < 5 ºC,
max > 35 ºC and opt 20–25 ºC. All isolates failed to grow at 40 ºC, but
mycelium resumed growth when plates were moved to 25 ºC.
Type: Tunisia, Tabarka, isolated from branch cankers of Quercus
canariensis, 20 Sep. 2006, B.T. Linaldeddu, holotype LISE 96110
(a dried culture sporulating on holm oak twigs).
Culture: CBS 133852 (ex-type).
Host: Quercus canariensis (Linaldeddu et al. 2013).
Known distribution: North-west Tunisia (Linaldeddu et al. 2013).
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Note: Diplodia quercivora is similar to D. corticola but the two
species are readily distinguishable by conidial shape and size.
Diplodia rosulata Gure, Slippers & Stenlid, Mycol. Res. 109:
1010. 2005. MycoBank MB344348. Fig. 28.
Ascomata not reported. Conidiomata (formed on WA on sterilised
pine needles and seeds after 45 d), pycnidial, stromatic, erumpent,
solitary, globose with a central ostiole, papillate, wall composed of
outer layers of thick-walled, dark brown textura angularis, becoming
thin-walled and hyaline towards the inner layers. Conidiogenous
cells holoblastic, hyaline, cylindrical, proliferating percurrently with
indistinct annellations, 8–12 × 2–4 μm. Conidia oval to ellipsoid or
ovoid, ends obtuse, initially hyaline, aseptate, granular contents,
wall 1.5–2 μm thick and smooth, often turning light brown and
1-septate after discharge, (21–)25–32(–36) × (10–)11–17.5(–19.5)
μm (av. size of 106 conidia = 28 × 14.5 μm ), L/W ratio = 1.93.
Culture characteristics: Colonies initially beige to whitish (upper
surface), becoming greenish grey from above, bluish-grey with
whitish centre from below, cultures partially translucent after 2 wk,
becoming opaque after 3 wk. Colony margin forming a concentric
ring after 3–4 d with smooth margins, followed by additional rings
forming as small sectors along the circumference of the colony,
creating a lobed rosette appearance after 4–5 d. Mycelium dense,
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PhilliPs et al.
Fig. 27. Diplodia quercivora. A–H. Conidiogenous cells with developing conidia, arrows in D–H indicate periclinal thickenings. I. Hyaline, aseptate conidia. J. Hyaline, aseptate
conidia and one pale brown conidium. K. Hyaline, aseptate conidium and one pale brown, one-septate conidium. Scale bar A = 5 μm. Scale bar in A applies to B–K.
forming an appressed mat, average growth rate approximately 7
and 8.5 mm illing the 9 cm Petri dishes within 12 and 10 d at 20 °C
and 25 °C, respectively.
Type: ethiopia, Southeastern Oromia, Gambo, MunessaShashamane Forest Enterprise, from seeds of Prunus africana, 20
Jul. 2001, A. Gure, holotype CBS H-12357.
Culture: CBS 116470 (ex-type).
Host: Prunus africana (Gure et al. 2005).
Known distribution: Ethiopia (Gure et al. 2005).
Notes: Diplodia rosulata has a distinct rosulate colony morphology,
which separates it from all other Diplodia spp. including the closely
related D. africana and D. olivarum. Iranian isolates of D. bulgarica
have also rosulate colonies, but the conidia of D. rosulata (28 ×
14.5 μm, L/W = 1.93) are longer and narrower than those of D.
bulgarica (25.4 × 16.8 μm, L/W = 1.5).
Diplodia sapinea (Fr.) Fuckel, J. nassau. Ver. Naturk. 23–
24: 393. 1870. MycoBank MB146913. Fig. 29.
Basionym: Sphaeria sapinea Fr., Syst. Mycol. 2: 491. 1823.
96
Synonyms see Sutton & Dyko (1989).
Ascomata not reported. Conidiomata pycnidial, stromatic,
globose, immersed, sometimes appearing supericial, separate
or aggregated, dark brown to black, unilocular, 0.3–0.5 mm diam,
wall 6–8 layers, 30–60 μm thick, outer wall of dark brown thickwalled textura angularis, cells darker around the the ostiole. Ostiole
central, circular single. Conidiophores absent. Conidiogenous cells
lageniform to cylindrical, occasionally proliferating percurrently,
discrete, indeterminate, hyaline, smooth, arising from the inner wall
of the locule, 8.5–15 × 4–7.5 μm. Conidia oblong to clavate, straight
to slightly curved, at irst aseptate, sometimes much later becoming
1-euseptate, walls 0.5–1 μm thick, outer surface of wall smooth, or
appearing pitted, apex obtuse, base truncate, (25.5–)30.5–52.5(–
54) × (10–)12.5–20(–21) μm (av. ± S.D. of 200 conidia = 40.8 ± 4.9
× 15.5 ± 2.1 μm).
Type: Sweden, Suecia Smaland, Femsjo, on Pinus sp., E. Fries,
Scleromyceti Sueciae Exsiccati No 126, Sphaeria sapinea Fries,
lectotype: B, isotypes: G, K, E, UPS, C, BR, FH. The Netherlands,
Gelderland, Schovenhorst, Putten, Pinetum, on cones of Pinus
nigra, June 1984, H.A. van der Aa. epitype designated here CBS
H-18340; MBT176178, culture ex-epitype CBS 393.84.
Cultures: CBS 393.84 (ex-epitype), CBS 109725.
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 28. Diplodia rosulata. A, B. Conidiogenous cells. C, D. Hyaline, aseptate conidia. E–H. Brown, one-septate conidia. Scale bar A = 10 μm. Scale bar in A applies to B–H.
Hosts: Host range includes Abies, Larix, Picea, Thuja, Pseudotsuga,
and 33 species of Pinus (Palmer et al. 1987).
Known distribution: Worldwide wherever pines are grown (Palmer
et al. 1987).
Notes: The history of this species has been explained by Sutton &
Dyko (1989). Briely, the pine pathogen was known for many years
as Diplodia pinea (Desm.) Kickx. and later as Sphaeropsis sapinea
(Fr.) Dyko & Sutton. According to Sutton & Dyko (1989) S. sapinea
is based on Sphaeria sapinea Fr. and they proposed the specimen
of Fries exsiccata in B as lectotype. Sutton & Dyko (1989) give
extensive synonymies for S. sapinea including Diplodia pinea. We
examined Sphaeria pinea Desm. (Desmaziéres No 1277 in PC)
and found that the conidia are smaller (25–32 × 12–15 μm) than
those reported by Sutton & Dyko (1989) for the type of S. sapinea
and thus they represent two distinct species. Furthermore, average
conidial dimensions of the common pine pathogen fall within the
range of 33–39 × 11.5–13 μm (Palmer et al. 1987, Swart et al.
1991), thus corresponding to S. sapinea. Therefore we consider
that the correct name to apply to the common pine pathogen is
Diplodia sapinea based on Sphaeria sapinea.
Differences in colony appearance and growth rate were
reported for isolates of D. sapinea from the north central
United States and these two colony types were referred to as
morphotypes A and B (Palmer et al. 1987). Isolates of the A
morphotype were described as producing luffy, white to greygreen mycelium and faster growth on PDA than isolates of the
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B morphotypes which produced white to black mycelium closely
appressed to the agar (Palmer et al. 1987). Other differences
between the two morphotypes have been suggested including
differences in radial growth, conidial dimensions and conidial
septation (Palmer et al. 1987), and texture of the conidium wall
(Wang et al.1985). However, each of these differences has
been shown to vary substantially within each group, or to be
similar for each group. Nevertheless, according to Palmer et al.
(1987) conidia of type A isolates are larger than conidia of type
B isolates. They also considered that although conidia of both
morphotypes were mostly aseptate, when septa were present the
type B isolates had up to three septa while the type A isolates only
ever formed a single septum.
De Wet et al. (2002) used RAPD markers and morphological
characters to distinguish a third morphotype, which they referred to
as the C morphotype. The C morphotypes had considerably larger
conidia than the A morphotypes (De Wet et al. 2002) and were
signiicantly more virulent than the A and B morphotypes (De Wet
et al. 2002). Also see the notes for D. scrobiculata below.
Diplodia scrobiculata J. de Wet, Slippers & M.J. Wingf.,
Mycol. Res. 107: 562. 2003. MycoBank MB372427. Fig. 30.
Ascomata not reported. Conidiomata pycnidial, stromatic, covered
in mycelium, dark, immersed in pine needles or in the agar, single,
papillate ostiole, (100–)150(–250) μm diam. Conidiogenous cells
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PhilliPs et al.
Fig. 29. Diplodia sapinea. A. Annellate conidiogenous cells. B–E. Conidia developing on conidiogenous cells. F–I. Conidia, the ones in H have up to 2 septa. J, K. Conidium in
two different focal planes to show verruculose inner side of the wall. Scale bars = 10 μm. Scale bar in A applies to B. Scale bar in C applies to D–K.
Fig. 30. Diplodia scrobiculata. A, B. Conidiogenous layer with developing conidia. C. Conidia. Scale bar = 10 μm. Scale bar in A applies to B and C.
98
The Botryosphaeriaceae: genera and sPecies known from culTure
discrete, dark, smooth, 10 mm in diameter, holoblastic with limited
percurrent proliferation forming a small number of annellations.
Conidia clavate to truncate, dark mouse grey, (37.5–)39.5(–41.5)
× (13–)14(–15.5) μm, 1–3 septa, thick, pitted walls (Wang et al.
1985).
Culture characteristics: Colonies pale mouse-grey to mouse-grey
viewed from the top of the Petri dish, dark mouse-grey to fuscous
black viewed from the bottom of the Petri dish, colonies with sinuate
edges. Optimal growth at 25 °C, covering the medium surface (9
cm Petri dishes) in 8 d. Mycelium dark, septate, appressed to the
agar surface.
Type: uSA, Wisconsin, Jackson County, Pinus banksiana, 1987,
M.A. Palmer, holotype PREM 57461.
Cultures: CMW 189 = CBS 118110 (ex-type). Other authentic
culture CBS 117836.
Hosts: Pinus banksiana, P. resinosa, and P. greggii (De Wet et al.
2003).
Known distribution: Europe (France, Italy), Mexico and, USA
(California, Minnesota, Wisconsin) (De Wet et al. 2003).
Notes: The differences in morphology and behaviour of the various
morphotypes of D. sapinea were considered insuficient to justify
separation into distinct species. However, De Wet et al. (2003)
showed that differences in partial sequences of six protein coding
genes and six microsatellite markers were consistent between the
A and B morphotypes and they considered this to be suficient
evidence to consider them as two distinct species. On this basis
they described the B morphotypes as Diplodia scrobiculata, while
the A and C morphotypes were regarded as Diplodia pinea, now
treated as D. sapinea.
Diplodia seriata De Not., Micr. Ital. Dec. 4: 6. 1942.
MycoBank MB180468. Fig. 31.
= Sphaeria obtusa Schwein., Trans. Amer. Phil. Soc. II, 4: 220. 1832.
≡ Physalospora obtusa (Schwein.) Cooke, Grevillea 20: 86. 1892.
≡ Botryosphaeria obtusa (Schwein.) Shoemaker, Canad. J. Bot. 42:
1298. 1964.
= Diplodia pseudodiplodia Fuckel, Jb. Nassau. Ver. Naturk. 23–24: 393. 1870.
= Physalospora cydoniae G. Arnaud, Annals d’École National d’Agric. de
Montpellier, Série 2, 12(1): 9. 1911.
= Physalospora malorum Shear, N.E. Stevens & Wilcox, J. Agric. Res. 28:
596. 1924.
= Diplodia profusa De Not., Micr. Ital. Dec. 4: No 8. 1842.
Ascomata stromatic, immersed, solitary to botryose up to 3 mm
wide. Asci bitunicate, issitunicate, clavate, 90–120 × 17μm.
Pseudoparaphyses hyaline, branched, septate, 2−3 μm wide. Asci
clavate, stipitate, bitunicate, containing eight, biseriate ascospores,
95−100 × 15−20 μm (including stipe). Ascospores irregularly
biseriate in the ascus, broadly fusoid, widest in the middle, smooth,
hyaline, aseptate, 25–33 × 7–12 μm. Conidiomata stromatic,
separate or aggregated and conluent, immersed in the host,
partially emergent at maturity, dark brown to black, ostiolate, nonpapillate, thick-walled, outer layers composed of dark brown textura
angularis, inner layers of thin-walled hyaline textura angularis.
Conidiogenous cells hyaline, thin-walled, smooth, cylindrical,
swollen at the base, discrete, producing a single conidium at the
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tip, indeterminate, proliferating internally giving rise to periclinal
thickenings or proliferating percurrently forming 2–3 annellations,
3–5.5 × 7–10(–15) μm. Conidia initially hyaline, becoming dark
brown, moderately thick-walled (ca. 0.5 μm thick), wall externally
smooth, roughened on the inner surface, aseptate, ovoid, widest
in the middle, apex obtuse, base truncate or rounded, (21.5–)22–
27(–28) × (11–)11.5–14.5(–15.5) μm, 95 % conidence limits =
24.3–25.4 × 12-6–13.2 μm (av. ± S.D. of 50 conidia = 24.9 ± 1.9 ×
12.9 ± 1.1 μm), L/W = 1.9.
Type: Italy, on dead stems of Jasminium sp., 18 Aug. 1837, De
Notaris, holotype HERB RO. Portugal, Montemor-o-Novo, on
dead stems of Vitis vinifera, 31 Jul. 1997, A.J.L. Phillips, epitype
CBS H-19809.
Culture: CBS 112555 (ex-epitype).
Hosts: Apparently plurivorous.
Known distribution: Apparently worldwide.
Notes: The connection between the sexual and asexual morph was
established by Hesler (1916) and conirmed by Shear, Stevens and
Wilcox (1925) and Stevens (1936). When Shoemaker transferred
this name to Botryosphaeria he decided not to apply a name to the
asexual morph and for many years it was referred to as B. obtusa.
After Crous et al. (2005) transferred this species to Diplodia, no
valid name was available.
A great deal of controversy has surrounded the correct name
for this fungus. Peck (1881) found what he considered to be
the conidial state of this species in New York, and reported it as
Sphaeropsis malorum (Berk.) Berk. According to Stevens (1933),
S. malorum (Berk.) Berk. is a synonym of Diplodia mutila Fr., which
has hyaline conidia. Stevens (1933) studied Peck’s collection and
conirmed that the conidia are dark and aseptate.
This fungus has also been referred to as S. malorum Peck.
This name came about when Saccardo (1884) transferred S.
malorum (Berk.) Berk. to the genus Phoma on account of its
hyaline conidia. Because Peck’s collection had brown conidia,
Saccardo considered it not the same as Berkley’s collection, and
used the name S. malorum Peck. Thus, Peck did not name a new
species and even if he had proposed the name S. malorum in 1880,
it would be an illegitimate later homonym of S. malorum (Berk.)
Berk. (1860). Since S. malorum Peck is illegitimate and S. malorum
(Berk.) Berk. is a synonym of D. mutila, neither of these names can
be used for this species.
Slippers et al. (2007) initially regarded Diplodia malorum
Fuckel to be a more appropriate name for this fungus. However,
after studying the type specimen in G (Fungi rhenani 1706) they
rejected this possibility. Therefore, D. malorum is not the asexual
morph of “Botryosphaeria” obtusa. Finally, through a study of type
specimens Phillips et al. (2007) determined that D. seriata was the
oldest name available for the asexual morph of what had been
referred to as“B.” obtusa.
Diplodia tsugae (A. Funk) A.J.L. Phillips & A. Alves,
Persoonia 29: 35. 2012. MycoBank MB801409. See Funk
(1964) for illustrations.
Basionym: Botryosphaeria tsugae A. Funk, Canad. J. Bot. 42: 770.
1964.
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PhilliPs et al.
Fig. 31. Diplodia seriata. A–C. Asci with ascospores. D. Sectioned conidioma. E, F. Conidia developing on conidiogenous cells, one conidium in F is starting to become coloured.
G, H. Brown, aseptate conidia. Scale bars: A = 20 μm, B, C = 10 μm, D = 50 μm, E–H = 10 μm.
Ascomata pseudothecial, black, globose or subglobose, immersed,
uniloculate, with a short apical beak which becomes ostiolate
and breaks through the periderm, 360−540 μm diam, wall
pseudoparenchymatous, large-celled, 60−70 μm thick. Asci clavate,
short-stalked, bitunicate, formed between pseudoparaphyses,
140−180 × 30−36 μm. Ascospores ellipsoid to fusoid-ellipsoid,
sometimes inequilateral, one-celled, hyaline, 42−47 × 13−18 μm.
Conidiomata pycnidial, stromatic, black, immersed, globose or
subglobose, uniloculate, with a short papilla which breaks through
100
the periderm, 400−540 μm diam, wall pseudoparenchymatous,
35−45 μm thick. Conidiophores simple, bearing a single conidium
at the tip. Conidia oblong to ovoid, one-celled, hyaline, 36−41 ×
18−22 μm.
Type: canada, British Columbia, near Coola (Snootli Creek), on
branches of Tsuga heterophylla, 11 Sep. 1963, A. Funk, holotype
DAVFP 15485. Lake Cowichan, 1 Nov. 1962, A. Funk, isotype CBS
H-6790.
The Botryosphaeriaceae: genera and sPecies known from culTure
Culture: CBS 418.64 = IMI 197143 (ex-isotype).
Host: Tsuga heterophylla (Funk 1964).
Known distribution: Canada (British Columbia) (Funk 1964).
Notes: When Funk (1964) introduced B. tsugae he did not name
the asexual morph, but referred to it as a species of Macrophoma.
However, morphologically and phylogenetically it is undoubtedly
a species in Diplodia and for this reason Phillips et al. (2012)
transferred it to Diplodia as D. tsugae.
Dothiorella Sacc., Michelia 2: 5. 1880. MycoBank MB8098.
Type species: Dothiorella pyrenophora Sacc., Michelia 2: 5. 1880.
Ascomata immersed becoming erumpent, inally appearing
supericial, usually aggregated, often in rows or in small rounded
groups, at times connected at sides, globose, sphaeroid or ovoid,
medium sized, rarely small; apex rounded, with short or well
developed papilla, often opening widely by rounded ostiole, lined
with hyaline cells; surface smooth or roughened with protruding
cells or bearing short to elongate hyphal appendages; peridium
wide, composed externally of rows of large, brown-walled,
pseudoparenchymatous cells, often blackened over surface,
internally of more compressed rows of pallid cells, at times wedgeshaped groups of cells extending from lower sides, or basal portion
of peridium thickened and hypostromatic, hyphae dark brown,
coarse, forming slight or well-developed subiculum beneath and
connecting ascomata. Asci bitunicate, basal, clavate or oblong,
endotunica thickened. Pseudoparaphyses cellular, usually wide.
Ascospores dull brown or dark reddish brown, ellipsoid, fusoid,
obovoid, ends obtuse or somewhat acute, straight, inequilateral or
slightly curved, one- to two-septate, infrequently one-celled, not or
slightly constricted at septum; contents minutely granular; wall thick,
smooth or verruculose at times; overlapping biseriate in the ascus.
Conidiomata stromatic, ostiolate, individual or in loose clusters
of up to 10 conidiomata, immersed, breaking through the bark
when mature. Ostiole circular, central, non-papillate or papillate.
Paraphyses absent. Conidiophores absent. Conidiogenous cells
holoblastic, hyaline, smooth-walled, cylindrical and slightly swollen
at the base, determinate or indeterminate and proliferating at
the same level to form periclinal thickenings, rarely proliferating
percurrently to produce two or three indistinct annellations, borne
directly on the cells lining the pycnidial cavity. Conidia initially
hyaline, becoming dark brown and one-euseptate within the
pycnidial cavity often while still attached to the conidiogenous
cell, ellipsoid to ovoid, thick-walled, externally smooth or striate,
internally verruculose.
Notes: The genus Dothiorella has been the source of much
confusion in the past and the name has been used in more than
one sense. Dothiorella has been used for asexual morphs with
hyaline, aseptate conidia similar to those normally associated with
Fusicoccum and Neofusicoccum. Presumably this confusion started
when Petrak (1922) transferred F. aesculi to Dothiorella, citing the
species as the conidial state of B. berengeriana (Sutton 1980).
In later years, Dothiorella was used for fusicoccum-like asexual
morphs with multiloculate conidiomata (Grossenbacher & Duggar
1911, Barr 1987, Rayachhetry et al. 1996). Sivanesan (1984)
confused matters further by reducing Dothiorella pyrenophora to
synonymy with Dothichiza sorbi, which has small, hyaline, aseptate
conidia and is the asexual morph of Dothiora pyrenophora (Fr.)
Fr. However, he was referring to Dothiorella pyrenophora Sacc.
(1884), which is a later homonym of Dothiorella pyrenophora Sacc.
1880 (Sutton 1977). The taxonomic history of Dothiorella has
been explained by Sutton (1977) and Crous & Palm (1999), and is
illustrated by Crous & Palm (1999).
Dothiorella was reduced to synonymy under Diplodia by Crous
& Palm (1999), who used a broad morphological concept for
Diplodia. Phillips et al. (2005) re-examined the type of Dothiorella
pyrenophora Sacc. (K 54912) and found that it differed from
Diplodia by having conidia that are brown and 1-septate early in
their development, while they are still attached to the conidiogenous
cells. In Diplodia conidial darkening and septation takes place after
discharge from the conidiomata. Crous et al. (2006) re-examined
the types of both Diplodia and Dothiorella and conirmed these
morphological differences.
Sexual morphs of Dothiorella have pigmented, septate
ascospores. Phillips et al. (2005) and Luque et al. (2005) broadened
the concept of Botryosphaeria to include species with brown,
1-septate ascospores. Their reasons for doing this were based on
the fact that ITS phylogenies placed D. sarmentorum and D. iberica
within the boundaries of Botryosphaeria as it was circumscribed
at that time. In a phylogeny based on partial sequences of the
LSU gene Crous et al. (2006) revealed that Botryosphaeria sensu
lato is composed of a number of distinct lineages that represent
different genera. They suggested that the species with dark brown,
1-septate ascospores should be accommodated in Dothidotthia.
Phillips et al. (2008) showed that Dothidotthia symphoricarpa (the
type species of Dothidotthia) belongs in a distinct family within
the Pleosporales while D. sarmentorum, D. iberica and D. viticola
fall within two separate genera in the Botryosphaeriaceae and a
new genus, Spencermartinsia was introduced to accommodate D.
viticola.
More than 350 species names exist in Dothiorella, but presently
cultures are available for only 17 species in fungal collections.
Of these, ten species are known in Dothiorella, two species
introduced in Spencermatinsia should be transferred to Dothiorella,
Auerswaldia dothiorella is re-combined here as D. thailandica
and the other four species remain unnamed. All of these, except
D. sarmentorum, have been introduced since 2005. Considering
the earlier problems surrounding the circumscription of this genus
especially the confusion with Diplodia, it is likely that many more
species will be found. The sexual stage of the species is rarely
encountered in nature and under experimental conditions and no
ascomata have been observed for any of the species, except for D.
sarmentorum and D. iberica. Therefore, differentiation of species
has mostly been done based on asexual morphs and cultural
characteristics.
Key to Dothiorella species
1.
1.
Conidiomata papillate .............................................................................................................................................................................. 2
Conidiomata non-papillate ...................................................................................................................................................................... 6
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PhilliPs et al.
2.
2.
Conidiomata with long necks (up to 1.5 mm) ........................................................................................................................ D. longicollis
Conidiomata with short necks (less than 0.5 mm) .................................................................................................................................. 3
3.
3.
Conidia length not exceeding 22 μm (16–22 × 7–10 μm) .................................................................................................. D. dulcispinae
Conidial length exceeding 22 μm (up to 33 μm) ..................................................................................................................................... 4
4.
4.
Conidial width less than 12 μm (conidia fed by thrips) ............................................................................................................. D. thripsita
Conidial length greater than 12 μm (up to 14 μm) ................................................................................................................................... 5
5.
5.
Colony growth rate on MEA in the dark at 25 ºC > 20 mm/d .............................................................................................. D. pretoriensis
Colony growth rate on MEA in the dark at 25 ºC < 20 mm/d ................................................................................................ D. brevicollis
6.
6.
Conidial length less than 16 μm (av. length 15 μm) ............................................................................................................ D. americana
Conidial length 16 μm or more (av. length > 18 μm) ............................................................................................................................... 7
7.
7.
Average width of conidia greater than 10 μm .......................................................................................................................................... 8
Average width of conidia less than 10 μm ............................................................................................................................................... 9
8.
8.
Conidia 23–31 × 9–11 μm (av. 27.1 × 10.8 μm) ..................................................................................................................... D. casuarini
Conidia 23–23.4 × 10.8–11 μm (av. 23.2 × 10.9 μm) ................................................................................................................. D. iberica
9.
9.
Average length of conidia greater than 20 μm ...................................................................................................................................... 10
Average length of conidia less than 20 μm ........................................................................................................................................... 11
10. Conidia 21.4–21.9 × 9.7–9.9 μm (L/W ratio 2.2) ............................................................................................................ D. sarmentorum1
10. Conidia 22–22.5 × 9–9.5 μm (L/W ratio 2.4) .................................................................................................................. D. uruguayensis1
11. Conidia with slight undulating striations on the surface ...................................................................................................... D. thailandica
11. Conidia smooth ..................................................................................................................................................................................... 12
12. Conidial L/W ratio 2 .................................................................................................................................................................... D. santali
12. Conidial L/W ratio 2.4 ................................................................................................................................................................ D. moneti
1
It is dificult to distinguish these two species in terms of morphology but phylogenetically they are distinct.
dNA phylogeny
Phylogenetic analyses revealed two main clades representing the
two distinct genera Dothiorella and Spencermartinsia. These two
genera cannot be separated based on ITS sequence data and it is
necessary to combine the ITS with EF1-α or other protein coding
genes. The phylogeny based on ITS and EF1-α sequence data
revealed 16 subclades representing 16 distinct species in Dothiorella.
Most of these sub-clades received high bootstrap support (BS) in the
MP analysis. But, these values are quite low for some internal nodes
that can be improved with more sampling and gene loci (Fig. 32).
It is important to note that all of the known species of Dothiorella
in culture and studied here can be separated based solely on ITS,
although bootstrap support values for some of the internal nodes are
quite low (Fig. 33). Based on multi-gene phylogenies, Auerswaldia
dothiorella, a species recently described by Liu et al. (2013) was
found to reside in Dothiorella closely related to D. dulcispinae and
a new name is introduced here. Spencermartinsia pretoriensis and
S. uruguayensis, two recently described species were also found to
reside in Dothiorella, and are treated below.
Species descriptions
Dothiorella americana Úrbez-Torres, Peduto & Gubler,
Fungal Divers. 52: 184. 2011. MycoBank MB519956. See
Úrbez-Torres et al. (2011) for illustrations.
Ascomata not reported. Conidiomata pycnidial, stromatic,
produced on PDA within 2 wk, solitary, globose, black, covered with
moderate mycelium, up to 650 μm wide, thick-walled, unilocular,
with a central ostiole. Conidiophores absent. Conidiogenous cells
holoblastic, hyaline, cylindrical to subcylindrical 7–16 × 4–6 μm.
Conidia initially hyaline, unicellular, becoming light brown to dark
brown and 1-septate while still attached to the conidiogenous cells,
light to dark brown, thin-walled, oval to ovoid, round apex and
truncate base, (13.5–)14–156(–17) × (5–)5.5–6.5(–8) μm (av. of 60
conidia = 15 × 6.1), L/W ratio = 2.4.
Culture characteristics: Colonies on PDA suppressed, initially
olivaceous buff in the centre of the colony and white at the edge,
becoming olivaceous within 7 d, turning dark green within 28 d on
the surface, violaceous grey at the reverse after 28 d. Colonies
reaching 90 mm diam on PDA after 5 d in the dark at 25 °C.
Cardinal temperatures for growth: min 10 °C, max 35 °C, opt
20–25 °C.
Type: uSA, Missouri, Purdy, on diseased interspeciic grape
cultivar Vignoles (Ravat51), R.K. Striegler & G.M. Leavitt, holotype
UCD2252MO.
Cultures: CBS 128309 (ex-type), CBS 128310.
Hosts: Vitis spp. (Úrbez-Torres et al. 2011).
Known distribution: USA (Missouri) (Úrbez-Torres et al. 2011).
102
The Botryosphaeriaceae: genera and sPecies known from culTure
86
68
50
D. juglandis CBS 188.87
D. coryli CBS 242.51
Dothiorella sp.1
Dothiorella sp. JL599
Dothiorella sp.2
CBS 124723
Dothiorella sp.3
CBS 113188
D. iberica
CBS 115041
57
CAA 005
CBS 128309
98
D. americana
98 CBS 128310
IMI 63581b
D. sarmentorum
84 CBS 115038
CMW 36460
70
CBS 121764
99
CMW 36462
D. dulcispinae
CBS 121765
99
CMW 36461
95
99
100
D. thailandica
CMW 26165
CMW 26166
D. longicollis
CMW 36463
75
CMW 36464
MUCC 509
72
MUCC 508
77
99
100
100
100
100
CMW 4857
CMW 4855
99
100
ICMP 16824
ICMP 16819
UY672
89
ICMP 16827
96
D. brevicollis
D. santali
D. mone8
D. pretoriensis
D. casuarini
Dothiorella sp.4
D. uruguayensis
Spencermar8nsia sp.1
ICMP 16828
D. medicaginis CBS 500.72
100
CMW 36480
CMW 36481
60
75
92
MUCC 505
MUCC 506
59
CBS 110299
CBS 110497
CBS 133991
Spencermar8nsia sp.2
CBS 117006
Spencermar8nsia sp.3
CBS 302.75
CBS 117009
S. vi8cola
N. luteum
10 changes
Fig. 32. Single most parsimonious tree obtained from combined ITS and EF-1α sequence data, for species in Dothiorella and Spencermartinsia. MP bootstrap values are given
based on 1000 pseudoreplicates on the nodes. The tree is rooted to Neofusicoccum luteum (CBS 110299, CBS 110497).
Notes: Based on ITS and EF1-α sequence data, D. americana is
closely related to D. iberica and D. sarentorum. But, morphologically
conidia of this species are smaller than those in any other in
Dothiorella sp. and obviously is a distinct species. Úrbez-Torres
et al. (2011) considered this species to be a weak pathogen on
grapevines.
www.studiesinmycology.org
Dothiorella brevicollis Jami, Gryzenh., Slippers & M.J.
Wingf., Cryptog. Mycol. 33: 260. 2012. MycoBank MB564142.
See Jami et al. (2012) for illustrations.
Ascomata not reported. Conidiomata pycnidial, stromatic, produced
on Acacia karroo twigs on MEA within 2–4 wk, brown, solitary, up
103
PhilliPs et al.
CMW 36460
CMW 36461
69
D. dulcispinae
CMW 36462
CBS 121765
96
CBS 121764
59
CBS 133991
CMW 36463
98
73
CMW 36464
CMW 4857
100
D. thailandica
D. brevicollis
D. casuarini
CMW 4855
MUCC 509
85
61
MUCC 508
71
92
58
MUCC 505
D. mone1
MUCC 506
81
100
CMW 36480
CMW 36481
77
54
BRIP 51876
99
CMW 26165
CMW 26166
100
ICMP 16824
ICMP 16819
97
64 D. juglandis CBS 188.87
D. coryli CBS 242.51
54
57
D. pretoriensis
D. thripsita
D. longicollis
Dothiorella sp.4
D. uruguayensis
UY672
100
D. santali
Dothiorella sp.1
Dothiorella sp. JL599
Dothiorella sp.2
CBS 124723
Dothiorella sp.3
59
IMI 63581b
D. sarmentorum
51 CBS 115038
84
62
CBS 128309
CBS 128310
D. americana
CBS 113188
CBS 115041
D. iberica
CAA 005
CBS110299
CBS110497
N. luteum
10 changes
Fig. 33. Single most parsimonious tree obtained from ITS sequence data for species in Dothiorella. MP bootstrap values are given based on 1000 pseudoreplicates on the
nodes. The tree is rooted to Neofusicoccum luteum (CBS 110299, CBS 110497).
104
The Botryosphaeriaceae: genera and sPecies known from culTure
to 200 μm wide, semi-immersed, unilocular, globose, papillate with
a short neck, wall 5–7 cell layers, outer layers composed of darkbrown textura angularis, becoming thin-walled and hyaline toward
the inner region. Conidiophores absent. Conidiogenous cells
holoblastic, hyaline, cylindrical, (3–)3.5–7.5(–9) × (3–)3.5–4 μm.
Conidia initially hyaline and aseptate, becoming dark brown and
1-septate, with 2 cells of equal length, thick-walled, ovoid, smooth
with ine granular content, rounded apices, (20–)21.5–26(–27) ×
(8–)9–12(–13) μm.
Culture characteristics: Colonies on MEA appressed, conidiomata
emerging after 9–10 d under near UV light, becoming pale
olivaceous-grey to dark olivaceous-grey at the surface, and
olivaceous-black to iron-grey at the reverse, with irregular edges.
Colonies reaching 90 mm diam on PDA after 6 d (17.6 mm/d) in
the dark at 25 °C. Cardinal temperatures for growth: min 5 °C, max
35 °C, opt 25 °C.
Type: South Africa, Gauteng Province, Pretoria, from healthy
wood section of Acacia karroo, Nov. 2009, F. Jami, holotype
PREM 60704.
Cultures: CBS 130411 = CMW 36463 (ex-type), CBS 130412 =
CMW 36464.
Host: Acacia karroo (Jami et al. 2012).
Known distribution: South Africa (Gauteng Province) (Jami et al.
2012).
Notes: Phylogenetically this species is closely related to D. longicollis
and D. dulcispinae and in terms of morphology it resembles D.
thripsita and D. dulcispinae. All of these species produce papillate
conidiomata. Dohiorella longicollis differs from the other three
species by having very long necks (up to 1.5 mm). Moreover,
conidia of D. longicollis (20.4 × 8.7 μm) are smaller than those of
D. brevicollis (21.5–26 × 9–12 μm) and longer than those of D.
dulcispinae (16–22 × 7–10 μm). Conidia of D. brevicollis are clearly
larger (21.5–26 × 9–12 μm) than those of D. dulcispinae (16–22 ×
7–10 μm). It is dificult to distinguish D. brevicollis from D. thripsita
(av. size of conidia 20–25 × 8.5–11.5 μm) but phylogenetically,
based on ITS sequence data, they are distinct (Fig. 33).
Dothiorella casuarini J. de Wet, Slippers & M.J. Wingf.,
Mycologia 101: 505. 2009. MycoBank MB510856. See De
Wet et al. (2009) for illustrations.
Ascomata not reported. Conidiomata pycnidial, stromatic, few
produced on pine needles, black, globose, solitary, scattered
and immersed in water agar, ostiolate. Conidiophores absent.
Conidiogenous cells emerging directly from cells lining the
pycnidial cavity, holoblastic, hyaline, smooth-walled, subcylindrical,
determinate or indeterminate and proliferating at the same
level resulting in periclinal thickening, very rarely proliferating
percurrently to produce two or three indistinct annellations. Conidia
initially aseptate and hyaline, becoming brown to dark brown or
sepia and 1-septate within the conidiomata, rarely 2–3-septate,
ellipsoid to ovoid, rarely narrow ellipsoid, apex obtuse, base
truncate, (22–)23–31(−38) × (8–)9–12 (−13.5) μm (av. of 60 conidia
= 27.1 × 10.8 μm).
www.studiesinmycology.org
Culture characteristics: Colonies smooth to luffy, pale greenish grey
to greenish grey from above, becoming lighter or white around the
edges, light bluish or sky grey from below, colony margins irregular,
rosette-like. Mycelium thick-walled, branched, septate, melanised,
pale to dark brown, with strings of dark brown chlamydospore-like
hyphal swellings.
Type: Australia, Canberra, Cotter River, on Casuarina sp., 2000,
M.J. Wingield, holotype PREM 59650.
Cultures: CBS 120688 = CMW 4855 (ex-type), CBS 120690 =
CMW 4857.
Host: Casuarina sp. (De Wet et al. 2009).
Known distribution: Australia (Canberra) (De Wet et al. 2009).
Note: Phylogenetically this species formed a distinct highly
supported clade and morphologically conidia of D. casuarini are
longer (27.1 × 10.8 μm) than those of any other Dothiorella species.
Dothiorella dulcispinae Jami, Gryzenh., Slippers & M.J.
Wingf., Cryptogam. Mycol. 33: 258. 2012. MycoBank
MB564141. See Jami et al. (2012) for illustrations.
Ascomata not reported. Conidiomata pycnidial, stromatic, produced
on Acacia karroo twigs on MEA within 2–4 wk, solitary, dark brown,
up to 200 μm wide, semi-immersed, unilocular, globose papillate
with a short neck (100–300 μm), wall 6–8 cell layers, outer layers
composed of dark-brown textura angularis, becoming thin-walled
and hyaline toward the inner region. Conidiophores absent.
Conidiogenous cells 1–2-celled, holoblastic, hyaline, cylindrical,
proliferating percurrently. Conidia initially hyaline and aseptate,
becoming dark brown or sepia and 1-septate, with 2 cells of
unequal length, thick-walled, ovoid, smooth with ine granular
content, rounded apices, (14–)16–22(–24) × (6–)7–10(–11) μm.
Culture characteristics: Colonies on MEA developing dense aerial
mycelium with age, becoming pale olivaceous-grey to olivaceousblack at the surface, and olivaceous black at the reverse, umbonate
with irregular zonation and lobate edges. Colonies reaching 90 mm
diam on PDA after 5 d (17.9 mm/d) in the dark at 25 °C. Cardinal
temperatures for growth: min 5 °C, max 35 °C, opt 25 °C.
Type: South Africa, Gauteng Province, Pretoria, from die-back
wood section of Acacia karroo, Nov. 2009, F. Jami, holotype
PREM 60706.
Cultures: CBS 130413 = CMW 36460 (ex-type), CBS 130414 =
CMW 36461, CBS 130415 = CMW 36462, CBS 121764, CBS
121765.
Host: Acacia karroo (Jami et al. 2012).
Known distribution: South Africa (Gauteng Province) (Jami et al.
2012).
Notes: See notes for D. brevicollis.
105
PhilliPs et al.
Dothiorella iberica A.J.L. Phillips, J. Luque & A. Alves,
Mycologia 97: 524. 2005. MycoBank MB344530. Fig. 34.
= Botryosphaeria iberica A.J.L. Phillips, J. Luque & A. Alves, Mycologia 97:
524. 2005.
Ascomata dark brown to black, globose pseudothecial, up to
350 μm diam, submerged in the substrate, partly erumpent at
maturity, ostiole circular, central, papillate, wall up to 50 μm thick,
composed of dark brown thick-walled textura angularis, cells
8–17 × 6–10 μm and lined with thinner-walled, hyaline, textura
angularis. Pseudoparaphyses thin walled, hyaline, frequently
septate, slightly constricted at the septum, 2.5–3.5(–4) μm wide.
Asci 100–125 × 18–25 μm, stipitate, arising from the base of the
ascoma, clavate, thick-walled, bitunicate with a well-developed
apical chamber, stipitate, (4–)8-spored, irregularly biseriate.
Ascospores oblong, ovate to sub-clavate, (0–)1-septate, slightly
constricted at the septum, dark brown, moderately thick-walled,
inely verruculose on the inner surface, straight or inequilateral,
widest in the lower 1/3 to middle of the apical cell, basal cell
tapering towards the rounded end, (17.5–)22.5–23.5(–29) ×
(8.5–)10–10.5(–12.5) μm (av. ± S.D. of 50 ascospores = 23.1 ±
2.1 × 10.2 ± 0.8 μm). Conidiomata pycnidial, stromatic, solitary,
globose, up to 450 μm wide, thick walled, composed of dark brown
thick-walled textura angularis, becoming thin-walled and hyaline
towards the inner region. Conidiophores absent. Conidiogenous
cells lining the pycnidial cavity, holoblastic, hyaline, subcylindrical,
8–15 × 3–5(–6.5) μm, proliferating at the same level giving rise to
periclinal thickenings, or rarely proliferating percurrently forming
one or two indistinct annellations. Conidia initially hyaline,
becoming dark brown and one-euseptate often while still attached
to the conidiogenous cell, ovoid with a broadly rounded apex and
truncate base, brown walled, 1-septate, slightly constricted at the
septum, (17–)23–23.5(–28.5) × (8–)10.5–11(–16) μm (av. ± S.D.
of 400 conidia = 23.2 ± 1.9 × 10.9 ± 1.2 μm), L/W ratio = 2.2.
Spermatia not seen. Cardinal temperatures for growth: min 5 °C,
max < 35 °C, opt 20–25 °C.
Type: Spain, Zaragoza province, Aragon, Tarazona, on dead twigs
of Quercus ilex, Dec. 2002, J. Luque, holotype LISE 94944.
Cultures: CBS 115041 (ex-type), CBS 113188.
Hosts: Cupressus (Azouaoui-Idjer et al. 2012), Juniperus communis
(Alves et al. 2013), Malus (Phillips et al. 2005), Persea (Mcdonald
& Eskalen 2011), Pistacia (Phillips et al. 2008), Quercus (Phillips et
al. 2005, 2008, Lynch et al. 2013), Vitis (Úrbez-Torres et al. 2007,
Qiu et al. 2011, Baskaratevan et al. 2012) and probably many more.
Known distribution: Algeria (Azouaoui-Idjer et al. 2012), Australia
(Qiu et al. 2011), Italy (Phillips et al. 2005), New Zealand
(Baskaratevan et al. 2012), Portugal (Alves et al. 2013), Spain
(Phillips et al. 2005, 2008), USA (Phillips et al. 2008, Úrbez-Torres
et al. 2007, Mcdonald & Eskalen 2011, Lynch et al. 2013).
Notes: This species is similar to D. sarmentorum but can be
distinguished on characteristics of the asci, ascospores and
conidia. Thus, in D. iberica the asci are shorter and more clavate,
the ascospores characteristically taper towards the base, and
on average the conidia are slightly longer. Also see notes for D.
americana.
106
Dothiorella longicollis Pavlic, T.I. Burgess & M.J. Wingf.,
Mycologia 100: 859. 2008. MycoBank MB512053. See Pavlic
et al. (2008) for illustrations.
Ascomata not reported. Conidiomata semi-immersed, mostly
solitary, with globose base (up to 550 μm diam), papillate with
long neck (sometimes branching) up to 1.5 mm, arising from the
substrate. Conidiophores absent. Conidiogenous cells holoblastic,
cylindrical to subcylindrical, hyaline, the irst conidium produced
holoblastically and subsequent conidia enteroblastically, (5–)6–8(–
10) × (2.5–)3–4(–4.5) μm (av. of 30 conidiogenous cells = 7.3 ×
3.4 μm). Conidia initially hyaline, unicellular, becoming cinnamon
to sepia and 1-septate while still attached to conidiogenous cells,
oval to ovoid, apices rounded and base truncate, (17–)19–22(–23)
× (7–)8–9.5(–10.5) μm (av. of 50 conidia = 20.4 × 8.7 μm), L/W
ratio = 2.3.
Culture characteristics: Colonies initially white to olivaceous buff,
becoming greenish olivaceous to citrine from the middle of colonies
within 7 d, iron-grey (surface) and black (reverse) with age, with
suppressed, moderately luffy mycelium, edges smooth appearing
sinuate as the colony darkens with age. Conidiomata readily formed
from the middle of colony within 7–10 d, covering the entire surface
of the colony and immersed in the medium (seen as round black
structures on the reverse side of Petri dishes) 14 d after incubation.
Optimum growth at 25 °C, covering 90 mm diam Petri dishes after
4 d in the dark.
Type: Australia, Western Australia, Tunnel Creek National Park,
on healthy branches of Lysiphyllum cunninghamii, Jul. 2006, T.I.
Burgess, holotype PREM 59485.
Cultures: CMW 26166 = CBS 122068 (ex-type), CMW 26165 =
CBS 122067.
Hosts: Asymptomatic branches of L. cunninghamii (Caesalpiniaceae)
and Terminalia sp. (Combretaceae) (Pavlic et al. 2008).
Known distribution: Australia (Western Australia) (Pavlic et al.
2008).
Notes: This species differs from all othe Dothiorella species by
having papillate pycnidia with very long necks (up to 1.5 mm). Also
see notes for D. brevicollis.
Dothiorella moneti K. Taylor, Barber, G.E. Hardy & T.I.
Burgess, Mycol. Res. 113: 342. 2009. MycoBank MB511825.
See Taylor et al. (2009) for illustrations.
Ascomata not reported. Conidiomata pycnidial, stromatic, supericial,
dark brown-grey, cylindrical, mostly solitary, covered in mycelium,
0.5–1.5 mm in length and 0.1–0.5 mm in diam. Conidiophores
absent. Conidiogenous cells holoblastic, hyaline, cylindrical to lask
shaped, (4–)6–12(–16) × 2–4(–5) (av. of 150 conidiogenous cells =
8.4 × 2.6 μm). Conidia initially hyaline and aseptate becoming dark
brown and 1-septate sometimes while still attached to conidiogenous
cell, ellipsoid, smooth-walled, apex obtuse, frequently base truncate,
often strongly constricted at the septum, usually widest at the middle
of apical cell, (13–)17–22(–32) × (6–)7–10(–11) μm (av. of 300
conidia = 19.8 × 8.4 μm), L/W ratio = 2.4.
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 34. Dothiorella iberica. A. Vertical section through an ascoma. B. Ascus with brown, 1-septate ascospores. C. Immature asci and one ascus with four ascospores. D. Details
of the ascoma wall. E. Pseudoparaphyses. F. Ascospores. G. Ascospore. H. Young conidiogenous cells. I. Conidiogenous cells with developing conidia. J, K. Conidia viewed at
two different focal planes to show verruculose inner surface of the wall. L, M. Conidia. N. Germinating conidia. Scale bars: A = 50 μm, B–N = 10 μm.
www.studiesinmycology.org
107
PhilliPs et al.
Culture characteristics: Colonies composed of appressed mycelial
mat with diffuse irregular edges, initially white, edge remaining
white, centre turning olive-grey to dark greenish grey and entire
culture becoming dark olive-grey by day 8 and very dark greenish
grey with age. Conidiomata produced profusely in the centre of
culture within 8 d. Cardinal temperatures for growth: min 5 °C, max
< 35 °C, opt 25 °C.
Type: Australia, Western Australia, Yalgorup National Park,
from healthy stem of Acacia rostellifera, Jun. 2005, K.M. Taylor,
holotype PERTH 07692978.
Cultures: MUCC 505 = WAC 13154 (ex-type), MUCC 506.
Host: Acacia rostellifera (Taylor et al. 2009).
Known distribution: Australia (Western Australia) (Taylor et al.
2009).
Notes: In the original description, Taylor et al. (2009) mention
that pycnidial paraphyses are very rare, but they did not provide
an illustration of these structures although they do show young
conidiogenous cells. Since pycnidial paraphyses have not been
reported in any other Dothiorella species, other than D. santali,
it is possible that Taylor et al. (2009) were referring to immature
conidiogenous cells rather than paraphyses. Morphologically and
phylogenetically, D. moneti is closely related to D. santali. This
species is quite different in nucleotide sequences from D. santali
(6–7 substitutions in ITS, 11 substitutions and 9 insertions/deletions
in EF1-α) and thus are easily separated based on ITS sequence
data (Fig. 33). Moreover, it can be distinguished by having longer
conidia (19.8 × 8.4 μm, L/W ratio = 2.4) compare with D. santali
(18.2 × 9 μm, L/W ratio = 2).
Dothiorella pretoriensis (Jami, Gryzenh., Slippers & M.J.
Wingf.) Abdollahz. & A.J.L. Phillips, comb. nov. MycoBank
MB803995. See Jami et al. (2012) for illustrations.
Basionym: Spencermartinsia pretoriensis Jami, Gryzenh., Slippers
& M.J. Wingf., Cryptogam. Mycol. 33(3): 261. 2012.
Conidiomata (on sterile twigs of Acacia karroo) pycnidial, up
to 200 μm diam, semi-immersed, unilocular, with short necks;
wall of 5–7 layers of thick, dark-brown cells of textura angularis.
Conidiophores reduced to conidiogenous cells, or a supporting
cell. Conidiogenous cells 1–2-celled, hyaline, subcylindrical,
proliferating percurrently near apex, (3–)3.5–7.5(–9) × (3–)3.5–4
μm. Conidia ovoid, smooth, granular, apices rounded, thickwalled, initially hyaline, aseptate, becoming dark brown and
1-septate, apex obtuse, base bluntly rounded, (18–)20–28(–33)
× (6.5–)7–14(–11) μm (Jami et al. 2012).
Culture characteristics: Colonies on MEA appressed; surface pale
olivaceous to dark greenish olivaceous; reverse olivaceous-black,
with regular zonation and lobate margins. Colonies growing at 5–25
°C, reaching up to 22.5 mm / d at 25 °C.
Type: South Africa, Gauteng, Pretoria, from wood of Acacia karroo
with die-back symptoms, Nov. 2009, F. Jami, holotype PREM
60709.
Cultures: CMW 36481 = CBS 130404 (ex-type).
108
Host: Acacia karroo (Jami et al. 2012).
Known distribution: South Africa (Gauteng Province) (Jami et al.
2012).
Note: Dothiorella pretoriensis induced dieback when inoculated
into healthy branches of A. karroo, suggesting that it is a pathogen
of this host (Jami et al. 2012).
Dothiorella santali K. Taylor, Barber & T.I. Burgess, Mycol.
Res. 113: 345. 2009. MycoBank MB511828. See Taylor et al.
(2009) for illustrations.
Ascomata not reported. Conidiomata pycnidial, stromatic, mostly
supericial, dark brown to black, globose, solitary, occasionally
covered in mycelium, 100–600 μm in length and 50–650 μm in
diam. Conidiophores absent. Conidiogenous cells holoblastic,
hyaline, cylindrical to lask-shaped, (4–)6–12(–17) × 2–3(–4) (av. of
50 conidiogenous cells = 8.6 × 2.4 μm). Conidia initially hyaline and
aseptate becoming pigmented brown and 1-septate often while still
attached to conidiogenous cell, ellipsoid, apex obtuse, sometimes
base truncate, sometimes slightly constricted at the septum, usually
widest at the middle of apical cell, (15–)16–20(–22) × 7–11(–13) μm
(av. of 100 conidia = 18.2 × 9.0 μm), L/W ratio = 2.0.
Culture characteristics: Colonies initially white, appressed mycelial
mat, within 8 d turning greenish to dark greenish grey and luffy,
becoming very dark greenish grey to black with age. Conidiomata
produced on the agar. Cardinal temperatures for growth: min 5 ºC,
max < 35 ºC, opt 25 ºC.
Type: Australia, Western Australia, Yalgorup National Park, from
healthy stem of Santalum acuminatum, Jun. 2005, K.M. Taylor,
holotype PERTH 07693028.
Cultures: MUCC 509 = WAC 13155 (ex-type), MUCC 508.
Host: S. acuminatum (Taylor et al. 2009).
Known distribution: Australia (Western Australia) (Taylor et al.
2009).
Note: See notes for D. moneti.
Dothiorella sarmentorum (Fr.) A.J.L. Phillips, J. Luque & A.
Alves, Mycologia 97: 522. 2005. MycoBank MB501403. Fig.
35.
Basionym: Sphaeria sarmentorum Fr., K. svenska Vetensk-Acad.
Handl. 39: 107. 1818.
≡ Diplodia sarmentorum (Fr.) Fr., Summ. veg. Scand. (Stockholm) 2: 417.
1849.
= Diplodia pruni Fuckel, Jahrb. Nassauischen Vereins Naturk., 23–24: 169.
1870 [1869].
= Botryosphaeria sarmentorum A.J.L. Phillips, J. Luque & A. Alves, Mycologia
97: 522. 2005.
Ascomata dark brown to black, globose pseudothecial, 350–400 μm
diam, submerged in the substrate, partially erumpent at maturity,
ostiolate; ostiole circular, central, papillate; wall 50–75 μm thick,
composed of dark brown thick-walled textura angularis, cells 10–
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 35. Dothiorella sarmentorum. A. Vertical section through an ascoma. B. Cylindrical to clavate asci bearing eight brown ascospores. C. Details of ascus tip and ascospores.
D. Conidiogenous layer with developing conidia. E. Dark brown, 1-septate conidia. Scale bars: A, B = 50 μm, C–E = 10 μm.
17 × 6–9 μm, lined with thinner-walled, hyaline, textura angularis.
Pseudoparaphyses thin-walled, hyaline, frequently septate, often
constricted at the septa, 3–4 μm wide. Asci 140–210 × 17–24 μm,
stipitate, arising from the base of the ascoma, cylindric-clavate,
bitunicate, endotunica thick-walled, with a well-developed apical
chamber, 4–6(–8)-spored, obliquely uniseriate or irregularly biseriate.
Ascospores oblong to ovate, widest in the middle part, straight,
(0–)1-septate, slightly constricted at the septum, dark brown,
moderately thick-walled, surface smooth, inely verruculose on the
inner surface, (21–)24.5–25.5(–30.5) × (10–)11.5–12.5(–14) μm (av.
± S.D. = 25.0 ± 2.0 × 12.1 ± 0.9 μm). Conidiomata pycnidial, stromatic,
solitary, globose, up to 450 μm wide, wall 5–8 cell layers thick,
composed of dark brown thick-walled textura angularis, becoming
thin-walled and hyaline towards the inner region. Conidiophores
absent. Conidiogenous cells lining the pycnidial cavity, holoblastic,
hyaline, subcylindrical, 7–15 × 3–7 μm, proliferating at the same level
giving rise to periclinal thickenings, or rarely proliferating percurrently
to form one or two close, indistinct annellations. Conidia initially
hyaline and aseptate becoming pigmented brown and 1-septate
often while still attached to conidiogenous cell, brown walled, slightly
constricted at the septum, ovoid with a broadly rounded apex and
truncate base, (17.5–)21.5–22(–25) × (8–)9.5–10(–11.5) μm (av. ±
S.D. = 21.6 ± 1.5 × 9.8 ± 0.9 μm), L/W ratio = 2.2. Spermatogenous
cells discrete or integrated, hyaline, smooth, cylindrical, holoblastic
or proliferating via phialides with periclinal thickenings, 7–10 × 2–3
μm. Spermatia hyaline, smooth, aseptate, rod-shaped with rounded
www.studiesinmycology.org
ends, 4–5.5 × 2 μm. Cardinal temperatures for growth: min 5 °C, max
< 35 °C, opt 20–25 °C.
Type: Of the sexual morph; uK, England, Warwickshire, on
Ulmus sp., Aug. 1956, E.A. Ellis, holotype IMI 63581b (as Otthia
spiraeae); of the asexual morph; Sweden, Lund, Botanical Garden,
on Menispermum canadense, 1818, E.M. Fries Scleromyc. Suec.
18, holotype UPS-FRIES (as Sphaeria sarmentorum); isotype of
the asexual morph, K(M) 104852.
Cultures: IMI 63581b (ex-type), CBS 115038.
Hosts: Dothiorella sarmentorum is a plurivorous species and has
been isolated from 34 different host species including Malus,
Menispermum, Prunus, Pyrus, Ulmus, etc.
Known distribution: This species is cosmopolitan distributed
worldwide and has been found across six continents.
Notes: In proposing 145 species as synonyms of D. sarmentorum,
Wollenweber (1941) reported a wide range of dimensions for
the conidia, namely, (15–)20–24(–35) × (7–)7.4–11.5(–15) μm.
Some species in Dothiorella are separated by minor differences
in conidium dimensions. It is therefore possible that some of
Wollenweber’s synonyms are in fact distinct species. Also see
notes for D. americana and D. iberica.
109
PhilliPs et al.
Dothiorella thailandica (D.Q. Dai., J.K. Liu & K.D. Hyde)
Abdollahz., A.J.L. Phillips & A. Alves, comb. nov. MycoBank
MB805461. See Liu et al. (2012) for illustrations.
Basionym: Auerswaldia dothiorella D.Q. Dai., J.K. Liu & K.D. Hyde,
Fungal Divers. 57: 162. 2012.
Saprobic on dead bamboo. Ascomata not reported. Conidiomata
pycnidial, 400–800 μm wide, 200–250 μm high, 250–500 μm diam,
immersed in the host tissue and becoming erumpent at maturity,
globose, coriaceous, dark brown in the erumpent part. Conidiomata
wall 15–50 μm wide, with brown to dark brown outer layers and
hyaline to light brown inner layers, comprising several layers with
cells of textura angularis, cells 3–9.5 × 2–6 μm. Conidiophores
reduced to conidiogenous cells which are 2–5.5 × 1.5–4.5 μm,
holoblastic, discrete, hyaline, cylindrical to ellipsoidal, smooth,
straight or curved, formed from cells lining the innermost layer of
the pycnidium. Conidia 15–20 × 6.5–8 μm (av. 20 conidia = 18.5 ×
7 μm), initially hyaline and aseptate, becoming brown at maturity,
1–septate, slightly constricted at the septa, oblong to ellipsoidal,
ends rounded, with slight undulating striations on the surface,
occasionally curved, lower cell smaller, thick-walled.
Culture characteristics: Colonies on PDA, slow growing, 15 mm
diam after 45 d at 23–25 °C, circular, with uneven margin, greyish
brown after 7 d, becoming cottony and brown at the centre and dark
brown towards the edge. Chlamydospores produced after 30 d.
Culture characteristics: Colonies on 10 % potato-dextrose agar
(Difco) reaching to 65 mm diam after 5 d in the dark at 23 °C,
covered the entire plate after 3 wk in the dark followed by 5 d under
black light, and were olivaceous-black to charcoal with sparse aerial
mycelium, reverse greyish black to charcoal. Abundant conidia
produced on Sachs’ agar supporting sterilised pieces of maize leaf.
Type: Australia, Queensland, Tallegalla, on dead stems and
phyllodes of Acacia harpophylla, Mar. 2008, D.J. Tree & C.E.C.
Tree, holotype BRIP 51876.
Culture: BRIP 51876 (ex-type).
Host: Acacia harpophylla (Shivas et al. 2009).
Known distribution: Australia (Queensland) (Shivas et al. 2009).
Notes: Larvae and adults of the thrips Mecynothrips hardyi feed
almost exclusively on conidia of D. thripsita (Shivas et al. 2009).
Only ITS sequence data are available for the single isolate of this
species. Based on ITS sequence data D. thripsita constitutes a
completely distinct clade from all other species in Dothiorella (Fig.
33). In morphology it resembles D. brevicollis.
Type: Thailand, Chiang Rai Province, Doi Pui, on dead bamboo
culm, 1 Sep. 2011, D.Q. Dai, holotype MFLU 12–0751.
Dothiorella uruguayensis (C.A. Pérez, Blanchette, Slippers
& M.J. Wingf.) Abdollahz. & A.J.L. Phillips, comb. nov.
MycoBank MB803999. For illustrations see Pérez et al.
(2010).
Culture: MFLUCC 11-0438 = CBS 133991 (ex-type).
Basionym: Spencermartinsia uruguayensis C.A. Pérez, Blanchette,
Slippers & M.J. Wingf., Fungal Divers. 41: 65. 2010.
Host: Bamboo (Liu et al. 2012).
Known distribution: Thailand (Liu et al. 2012).
Notes: This species is phylogenetically closely related to D.
dulcispinae. Furthermore, in terms of morphology it resembles D.
santali and D. moneti. But D. thailandica can easily be separated
from those three species by its striate conidia.
Dothiorella thripsita R.G. Shivas & D.J. Tree, Fungal Planet
No. 32. 2009. MycoBank MB513166. See Shivas et al. (2009)
for illustrations.
Ascomata not reported. Conidiomata pycnidial, stromatic, solitary,
immersed, partially erumpent when mature, dark brown, globose
to ellipsoidal, papillate with a central ostiole, up to 300 × 200
μm diam, uniloculate, wall composed of an outer layer of dark
brown, thick-walled textura angularis, and an inner layer of thinwalled hyaline cells. Conidiophores absent. Conidiogenous cells
10–15 × 3–6 μm, holoblastic, discrete, cylindrical, hyaline, smooth,
indeterminate. Conidia initially hyaline, becoming dark brown and
1-euseptate often while still attached to the conidiogenous cell,
aseptate and pale brown when young, becoming septate and
brown when mature, often with a guttule in each cell, cylindrical to
clavate, straight, both ends broadly rounded, 20–25 × 8.5–11.5 μm,
conidial wall densely and minutely verruculose, proile smooth in
LM, verruculose in SEM.
110
Conidiomata (on PNA) pycnidial, supericial, solitary, globose,
black, non-papillate, covered with mycelium, up to 350 μm diam.
Conidiogenous cells hyaline, subcylindrical. Conidia (17–)22–
22.5(–26.5) × (7–)9–9.5(–12) μm, dark brown, 1-septate, slightly
constricted at septum, ovoid with broadly rounded apex and
truncate base (from Pérez et al. 2010).
Type: uruguay, Paysandu, Tres Bocas, endophytic on twigs of
Hexachlamis edulis, Aug. 2006, C.A. Pérez, holotype PREM
60268.
Cultures: UY672 = CMW 26763 = CBS 124908 (ex-type).
Host: Hexachlamis edulis (Pérez et al. 2010).
Known distribution: Uruguay (Pérez et al. 2010).
Notes: Inoculation results suggest that D. uruguayensis is a weak
pathogen on Hexachlamis edulis. It also proved to be uncommon
in the area, and not pathogenic to Eucalyptus (Pérez et al. 2010).
Endomelanconiopsis Rojas & Samuels, Mycologia 100:
770. 2008. MycoBank MB511837.
Type species: Endomelanconiopsis endophytica Rojas & Samuels,
Mycologia 100: 770. 2008.
Mycelium immersed, branched, septate, hyaline to pale brown.
Conidiomata stromatic, immersed, peridermal to subepidermal,
The Botryosphaeriaceae: genera and sPecies known from culTure
separate, irregularly multilocular, walls composed of small-celled,
pale brown, thin-walled textura angularis, becoming hyaline towards
the conidiogenous region. Dehisence irregular. Conidiophores
absent. Conidiogenous cells holoblastic, determinate, discrete,
cylindrical, tapered markedly or gradually towards the apices,
hyaline, smooth, thin-walled, formed from the walls of the locules.
Conidia aseptate, pyriform to limoniform, dark brown, thick-walled,
smooth, base often protruding and papillate, often with a central
guttule and a single germ slit.
Notes: Endomelanconiopsis was introduced by Rojas et al. (2008)
for E. endophytica and E. microspora. The genus is similar to
Endomelanconium Petrak but belongs to the Botryosphaeriaceae
and the conidia are non-papillate. Only two species are currently
known in culture and the main difference between them is that
chlamydospores are abundant in E. microspora but absent in E.
endophytica.
Species descriptions
Endomelanconiopsis endophytica Rojas & Samuels,
Mycologia 100: 770. 2008. MycoBank MB511838. See Rojas
et al. (2008) for illustrations.
Conidiomata stromatic, scattered throughout colony, varying
from globose to cylindrical, 1–3 cylindrical necks, supericial or
immersed in the agar; often cylindrical papillae protruding from
the agar in groups of a few; wall composed of pale brown and
black angular cells, becoming hyaline and more hyphal toward
the conidiogenous cells; locule convoluted, completely lined
with conidiogenous cells. Conidiogenous cells formed from the
inner cells all over the conidiomata wall, discrete, determinate,
cylindrical, tapered toward the apex, hyaline, holoblastic, rarely
with a single percurrent proliferation, 7.5–23.5 × 1–3.5 μm at
apex, 1.5–4 μm at base (av. = 14.2 × 1.6 μm at apex, 14.2 × 2.2
μm at base). Conidia ellipsoidal to limoniform, apex rounded, base
lat to rounded, aseptate, hyaline when immature, dark brown
with a single longitudinal slit three-quarters of the length of the
conidia when mature, (4.5–)5.5–7.5(–10) × (3–)3.5–4.5(–6) μm.
Spermatia forming in the same locules as conidia from densely
arranged, enteroblastic, phialidic conidiogenous cells, appearing
to arise from the inner cells of the conidioma wall, ellipsoidal to
allantoid, formed on PDA and SNA, 2–7(–10) × (1–2(–3) μm.
Chlamydospores not observed.
Culture characteristics: Colonies at irst colourless with hyaline
immersed hyphae, after 4 d colonies olivaceous in center and
concentric rings with irregular shape, after 10 d aerial mycelium
dense dark olivaceous or grey or shiny black with little aerial
mycelium. Optimum temperature at 30–37 °C; colony radius 43–55
mm after 5 d on PDA.
Type: Panama, Nombre de Dios, isolated from leaves of Theobroma
cacao, 2000, E. Rojas, L. Mejía & Z. Maynard, holotype BPI
878370.
Culture: CBS 120379 (ex-type).
Hosts: Heisteria concinna, Theobroma cacao (Rojas et al. 2008).
Distribution: Panama (Rojas et al. 2008).
www.studiesinmycology.org
Notes: The germ slit in the conidia of E. endophytica and E.
microspora is an unusual feature in the Botryosphaeriaceae. While
Neodeightonia subglobosa was reported to have conidia with germ
slits (Punithalingam 1969), and these were interpreted by Crous et
al. (2006) as striations similar to those seen in Lasiodiplodia.
Endomelanconiopsis microspora (Verkley & Aa) E.I.
Rojas & Samuels, Mycologia 100: 772. 2008. MycoBank
MB511839.
Basionym: Endomelanconium microsporum Verkley & Aa,
Mycologia, 89: 967. 1997.
Conidiomata stromatic, solitary and globose to subglobose, or
convoluted with merging cavities, supericial or immersed in the
agar, at irst pale olivaceaous, later black, glabrous, often with an
apical papilla but seldom a functional ostiole, mostly dehiscing
by bursting or partial dissolution of upper wall tissue, 200–500
μm diam. Conidiomatal wall composed of two layers, an outer
layer of brown to olivaceous textura epidermoidea-angularis, and
an inner layer variable in thickness of hyaline textura angularisglobulosa. Conidiogenous cells formed from the inner cells all
over the conidiomatal wall, discrete, determinate, cylindrical,
but tapering towards the apex, hyaline, holoblastic, rarely with
a single percurrent proliferation, mostly 6–10 × 5–7 μm. Conidia
ellipsoidal to pyriform, apex rounded, base with an inconspicuous
scar, aseptate, smooth, hyaline when liberated, soon becoming
dark brown with a single longitudinal hyaline slit, containg one
large and a few smaller oil droplets, (4.5–)5.5–6.5(–7) × (3.5–
)4–4.5) μm. Chlamydospores abundant in immersed mycelium,
intercalary and terminal, when interclary, subglobose to fusiform,
single or catenate (2–5), when terminal, globose to clavatepyriform, occasionally with a small basal, apophysis-like cell or an
apical papilla, thick-walled, brown, often verruculose, illed with
oil droplets, mostly 9–17 × 6–10 μm. In older cultures additional
chlamydospores forming in basipetal succession behind the
terminal ones.
Type: Papua New Guinea, Central Province, 22 km E of Port
Moresby, Varirata National Park near Varirata Lookout, soil in dry
secondary forest with Casuarina and Eucalyptus, and conglomerate
rock outcrops, 23 Oct. 1995, A. Aptroot, H.A. van der Aa 12183 (a
dried culture on oatmeal agar), holotype CBS H-12183.
Culture: CBS 353.97 (ex-type).
Substrate: Soil (Verkley & van der Aa 1997).
Known distribution: Papua New Guinea (Verkley & van der Aa
1997).
Note: Endomelanconiopsis microspora is characterised by having
stromatic conidiomata that give rise to brown, aseptate conidia, and
abundant terminal, and intercalary chlamydospore-like structures
that are formed in culture (Verkley & van der Aa 1997).
Lasiodiplodia Ellis & Everh., Bot. Gaz. 21: 92. 1896.
MycoBank MB8708.
Type species: Lasiodiplodia theobromae (Pat.) Griff. & Maubl., Bull.
trimest. Soc. Mycol. Fr. 25: 57. 1909.
111
PhilliPs et al.
Mycelium immersed or supericial, branched, septate, dark brown.
Ascomata eustromatic, dark brown to black, uniloculate with thick
pseudoparenchymatic wall, ostiolate, embedded in the substrate
and partially erumpent at maturity. Pseudoparaphyses hyaline,
septate. Asci bitunicate with thick endotunica and well-developed
apical chamber, clavate, stipitate, 8-spored. Ascospores irregularly
biseriate, initially hyaline, becoming dark brown, aseptate.
Conidiomata stromatic, immersed or supericial, separate or
aggregated and conluent, globose, dark brown, uni- or multilocular;
wall of dark brown, thick-walled textura angularis, paler and
thinner-walled towards the conidiogenous region, often with dark
brown supericial hyphae over the surface. Ostiole central, single,
papillate. Conidiophores often reduced to conidiogenous cells,
if present hyaline, simple, sometimes septate, rarely branched,
cylindrical, arising from the inner layers of cells lining the locules.
Conidiogenous cells hyaline, smooth, cylindrical to subobpyriform,
holoblastic, discrete, determinate or indeterminate and proliferating
percurrently with one or two distinct annellations, or proliferating
at the same level giving rise to periclinal thickenings, formed from
cells lining the inner wall of the conidiomata. Conidia hyaline when
young, later becoming medianly 1-euseptate, dark brown with
longitudinal striations, thick-walled, oblong to ellipsoid, straight,
broadly rounded at the apex, base truncate. Paraphyses hyaline,
cylindrical, septate.
Notes: Lasiodiplodia was introduced by Ellis in 1894 with L.
tubericola as the type species. Although Ellis did not describe it,
Clendenin (1896) provided a description of the genus and the
species, attributing both to Ellis and Everhardt. Grifin & Maublanc
(1909) considered that on account of the pycnidial paraphyses, the
cocoa pathogen, Botryodiplodia theobromae, was more suitably
accommodated in Lasiodiplodia. Since the epithet theobromae
(1892) is older than tubericola (1896), L. theobromae should be
regarded as the type species of Lasiodiplodia. Unfortunately,
neither Patouillard (1892) nor Clendenin (1896) referred to any
type or other specimens of the genus or species. Pavlic et al.
(2004) could not locate the types, and they also could not ind any
specimens from the original hosts or origins.
It has been thought that Lasiodiplodia could represent a
possible synonym of Diplodia (Denman et al. 2000). However,
phylogenetic studies by Zhou & Stanosz (2001), Slippers et al.
(2004) and Phillips et al. (2008) show that it clusters separately
from Diplodia. On account of the phylogenetic and morphological
differences there is no reason to consider the two as synonymous.
Morphologically the two genera are also clearly distinct. Thus,
striations on the conidia distinguish Lasiodiplodia from Diplodia, the
conidiomatal paraphyses distinguish it from Neodeightonia, which
also has striate conidia. Although Barriopsis has striate conidia,
they are unique in the Botryosphaeriaceae because they are also
present on immature, hyaline conidia. The sexual morph has been
reported only for L. theobromae, but the connection with the asexual
morph has not been conirmed (see notes under L. theobromae).
While 27 species names are listed in MycoBank, only 18 species
are currently known in culture and all, except L. theobromae, have
been introduced since 2004. Species can be differentiated based
on conidial morphology (especially dimensions) and morphology of
the paraphyses.
Key to Lasiodiplodia spp.
1.
1.
Conidia sub-globose, L/W ratio less than 1.5 .......................................................................................................................................... 2
Conidia ellipsoidal to ovoid, L/W ratio greater than 1.5 ........................................................................................................................... 3
2.
2.
Conidia 13.5–21.5 × 10–14 um (av. length 17.5 μm) ...................................................................................................... L. mahajangana
Conidia 12–19 × 10–12.5 μm (av. length 15.3 μm) .......................................................................................................... L. margaritacea
3.
3.
L/W ratio greater than 2.0 ....................................................................................................................................................................... 4
L/W ratio less than 2.0 ............................................................................................................................................................................ 5
4.
4.
Conidia 26–33 × 12–15 μm (av. length 28.4 μm) ............................................................................................................ L. venezuelensis
Conidia 17–23 × 8–11 μm (av. length 19.5 μm) .......................................................................................................................... L. viticola
5.
5.
Longest paraphyses more than 100 μm long .......................................................................................................................................... 6
Longest paraphyses less than 100 μm long ............................................................................................................................................ 9
6.
6.
Average conidial length less than 25 μm ................................................................................................................................................ 7
Average conidial length greater than 25 μm ........................................................................................................................................... 8
7.
7.
Average conidial width = 13 μm ............................................................................................................................................. L. iraniensis
Conidia average width = 11.5 μm ................................................................................................................................................. L. parva
8.
8.
Conidia 22–35 μm long (av. 29.6 μm), L/W ratio = 1.9 ............................................................................................................ L. plurivora
Conidia 20–31 μm long (av. 24.5 μm), L/W ratio = 1.6 .............................................................................................................. L. citricola
9.
9.
Average width of conidia less than 16 μm ............................................................................................................................................. 10
Average width of conidia 16 μm or more ............................................................................................................................................... 15
10. Conidia small, mostly less than 25 μm long .......................................................................................................................................... 11
10. Conidia large, mostly longer than 25 μm, up to 30 μm or more ............................................................................................................ 14
112
The Botryosphaeriaceae: genera and sPecies known from culTure
11. Average width of conidia less than 10 μm ............................................................................................................................................. 12
11. Average width of conidia greater than 10 μm ........................................................................................................................................ 13
12. Length of paraphyses up to 15, conidia up to 17.5 μm ............................................................................................................ L. lignicola
12. Length of paraphyses up to 55, conidia up to 21 μm ........................................................................................................ L. missouriana
13. Paraphyses up to 55 μm long, conidial L/W ratio = 2 .......................................................................................................... L. egyptiacae
13. Paraphyses up to 85 μm long, conidial L/W ratio = 1.7 .............................................................................................. L. hormozganensis
14. Conidiomata dark brown to black ....................................................................................................................................... L. theobromae
14. Conidiomata reddish-purple ........................................................................................................................................... L. rubropurpurea
15. Conidia not exceeding 35 μm long ........................................................................................................................................................ 16
15. Conidia up to 39 μm long ...................................................................................................................................................................... 17
16. Paraphyses mostly septate ................................................................................................................................................ L. crassispora
16. Paraphyses mostly aseptate .................................................................................................................................. L. pseudotheobromae
17. Paraphyses up to 95 μm long ............................................................................................................................................... L. gilanensis
17. Paraphyses not exceeding 65 μm, never reaching 95 μm ................................................................................................. L. gonubiensis
dNA phylogeny
Combined analysis of ITS and EF1-α separates the 18 species
currently recognised in this genus (Fig. 36). Some of the
species, such as L. citricola / L. parva / L. hormozganensis, are
distinguishable mainly from differences in their EF1-α sequences.
Furthermore, bootstrap support for some of the inner branches is
quite low. This would suggest that a reappraisal of the species in
Lasiodiplodia based on more gene loci should be undertaken.
Lasiodiplodia citricola Abdollahz., Javadi & A.J.L. Phillips,
Persoonia 25: 4. 2010. MycoBank MB16777. Fig. 37.
Ascomata not reported. Conidiomata stromatic, produced on pine
needles on WA within 2–4 wk, supericial, dark brown to black,
covered with dense mycelium, mostly uniloculate, up to 2 mm diam,
solitary, globose, thick-walled, non-papillate with a central ostiole.
Paraphyses hyaline, cylindrical, thin-walled, initially aseptate,
becoming up to 1–5 septate when mature, occasionally branched,
rounded at apex, occasionally basal, middle or apical cells
swollen, up to 125 μm long, 3–4 μm wide. Conidiophores absent.
Conidiogenous cells holoblastic, discrete, hyaline, smooth, thinwalled, cylindrical, proliferating percurrently with 1–2 annellations,
11–16 × 3–5 μm. Conidia initially hyaline, aseptate, ellipsoid to
ovoid, with granular content, both ends broadly rounded, wall
< 2 μm, becoming pigmented, verruculose, ovoid, 1-septate with
longitudinal striations, (20–)22–27(–31) × (11–)12–17(–19) μm, 95
% conidence limits = 24.1–24.9 × 15–15.7 μm (av. ± S.D. = 24.5 ±
0.2 × 15.4 ± 1.8 μm, L/W ratio = 1.6).
Culture characteristics: Colonies with abundant aerial mycelia
reaching to the lid of Petri plate, aerial mycelia becoming smoke
grey to olivaceous-grey or iron-grey at the surface and greenish
grey to dark slate blue at the reverse after 2 wk in the dark at
25 °C. Colonies reaching 85 mm on MEA after 2 d in the dark at 25
°C. Cardinal temperatures for growth: min ≤ 10 °C, max ≥ 35 °C,
opt 25–30 °C.
www.studiesinmycology.org
Type: Iran, Gilan Province, Chaboksar, on twigs of Citrus sp., Jun.
2007, J. Abdollahzadeh & A. Javadi, holotype IRAN 14270F.
Cultures: IRAN 1522C = CBS 124707 (ex-type), IRAN 1521C =
CBS 124706.
Hosts: Citrus sp. (Abdollahzadeh et al. 2010), Juglans regia (Chen
et al. 2013).
Known distribution: Iran (Chaboksar, Gilan Province; Sari,
Mazandaran Province; Northern Iran) (Abdollahzadeh et al. 2010),
USA (California) (Chen et al. 2013).
Notes: Phylogenetically, Lasiodiplodia citricola is closely related to
L. parva, but conidia of L. citricola, (20–)22–27(–31) × (11–)12–
17(–19) μm, are longer and wider than those of L. parva (15.5–)16–
23.5(–24.5) × (10–)10.5–13(–14.5) μm. In terms of morphology it
resembles L. plurivora but on average the conidia of L. citricola (av.
length = 24.5 μm) are shorter than those of L. plurivora (av. length
= 29.6 μm). This species produces a pink pigment in PDA cultures
at 35 °C.
Lasiodiplodia crassispora T.I. Burgess & Barber, Mycologia
98: 425. 2006. MycoBank MB500235. Fig. 38.
Ascomata not reported. Conidiomata stromatic, supericial, mostly
solitary, conical, smooth, iron grey, 0.5–1 mm diam. Paraphyses
cylindrical, septate, hyaline (21–)30–62(–66) × 2–3.5(–4) μm
(av. of 50 paraphyses = 45.7 × 2.7 μm). Conidiophores absent.
Conidiogenous cells holoblastic, hyaline, subcylindrical to cylindrical
to ampulliform, proliferating percurrently, (6–)8–16(–19) × 3–7 μm
(av. of 50 conidiogenous cells = 11.8 × 5 μm). Conidia produced
in culture initially hyaline, unicellular, ellipsoid to obovoid, thickwalled (2–3 μm, av. of 50 conidia = 2.6 μm) with granular content,
round at apex, occasionally truncate at base, becoming pigmented
with one septum when mature or before germination, developing
longitudinal striations when mature, 27–30(–33) × 14–17 μm (av. ±
S.D. = 28.8 × 16.0 μm, L/W ratio = 1.8).
113
PhilliPs et al.
73
CBS 111530
74
CBS 164.96
CBS 124927
CMW27801
99
60
L. mahajangana
UCD2604MO
UCD2553AR
100
62
L. theobromae
L. vi1cola
CBS 124710
100
L. iraniensis
CBS 124711
60
95
UCD2199MO
L. missouriana
UCD2193MO
76
CBS 124704
78
L. gilanensis
76 CBS 124705
100
78
CBS 120832
CBS 121103
L. plurivora
72 CBS 494.78
L. parva
CBS 456.78
51
98
CBS 124706
L. citricola
CBS 124707
BOT29
74
L. egyp1cae
CBS 130992
76
CBS 447.62
95
L. pseudotheobromae
CBS 116459
CBS 124709
83
99
L. hormozganensis
CBS 124708
CBS 122065
L. margaritacea
CBS 122519
CMW15207
97
L. rubropurpurea
CBS 118740
78
75
100
68
100
CMW13512
CBS 118739
L. venezuelensis
CMW13488
L. crassispora
CBS 118741
100
1/100/100
CBS 115812
L. gonubiensis
CBS 116355
100
CBS 134112
MFLUCC 11‐0656
L. lignicola
Diplodia mu1la CBS 112553
Diplodia seriata CBS 112555
10 changes
Fig. 36. One of the two equally most parsimonious trees obtained from combined ITS and EF-1α sequence data for species in Lasiodiplodia. MP bootstrap values are given
based on 1000 pseudoreplicates on the nodes. The tree is rooted to Diplodia mutila (CBS 112553) and D. seriata (CBS 112555).
Culture characteristics: Colonies moderately dense, with appressed
mycelial mat, initially white to buff turning pale olivaceous-grey
within 7 d and darkening with age. After 7 d the submerged
114
mycelia are olivaceous-grey, becoming black with age. Optimum
temperature for growth 30 °C, reaching 74 mm on PDA after 3 d at
30 °C in the dark.
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 37. Lasiodiplodia citricola. A. Conidiomata on pine needles in culture. B. Conidia developing on conidiogenous cells. C. Annellations on conidiogenous cell. D. Conidia
developing on conidiogenous cells between paraphyses. E. Septate paraphyses. F. Hyaline, immature conidia. G, H. Mature conidia in two different focal planes to show the
longitudinal striations. Scale bars: A = 1 mm, B, C = 5 μm, D–H = 10 μm.
Fig. 38. Lasiodiplodia crassispora. A. Hyaline, aseptate conidia. B, C. Dark brown, 1-septate conidia in two focal planes to show the longitudinal striations. Scale bar A = 10 μm.
Scale bar in A applies to B and C.
www.studiesinmycology.org
115
PhilliPs et al.
Fig. 39. Lasiodiplodia egypticae. A, B. Conidiogenous layer with conidia developing on conidiogenous cells between paraphyses. C. Hyaline, aseptate conidia. D. Dark-walled,
1-septate conidia. Scale bar A = 10 μm. Scale bar in A applies to B–D.
Type: Australia, Western Australia, Kununurra, from canker on
Santalum album, Dec. 2003, T.I. Burgess, holotype MURU 407.
Cultures: WAC 12533 = CMW 14691 (ex-type), CMW 13448.
Hosts: Santalum album (Burgess et al. 2006), Eucalyptus urophylla
(Perez et al. 2010), Vitis vinifera (Úrbez-Torres et al. 2010, van
Niekerk et al. 2010).
Known distribution: Australia (Western Australia) (Burgess et al.
2006), South Africa (van Niekerk et al. 2010), Uruguay (Perez et al.
2010), USA (California) (Úrbez-Torres et al. 2010).
Notes: This species is phylogenetically closely related to
L. rubropurpurea and L. venezuelensis, but can be distinguished
from L. rubropurpurea by the absence of red-purple conidiomata.
Furthermore, conidia of L. crassispora (av. = 28.8 × 16 μm) are
wider than those of L. venezuelensis (av. = 28.4 × 13.5 μm). In terms
of morphology L. crassispora resembles L. pseudotheobromae
and the only feature that distinguishes the two species is that in
L. crassispora the pseudoparaphyses are mostly septate, while in
L. pseudotheobromae they are mostly aseptate.
Lasiodiplodia egyptiacae A.M. Ismail, L. Lombard & Crous,
Australas. Plant Path. 41: 655. 2012. MycoBank MB564516.
Fig. 39.
Ascomata not reported. Conidiomata stromatic, mostly solitary,
dark-grey to black, globose to subglobose. Paraphyses hyaline,
subcylindrical, aseptate, up to 57 μm long, 2–3 μm wide.
Conidiophores absent. Conidiogenous cells holoblastic, hyaline,
cylindrical, proliferating percurrently, 5–11 × 3–5 μm. Conidia
initially hyaline, smooth, thick-walled, aseptate, obovoid to ellipsoid,
granular, mostly somewhat tapered at apex, and rounded at base,
becoming brown, 1-septate, with longitudinal striations when
mature, (17–)20–24(−27) × 11–12(−13) μm (av. ± S.D. = 22 ± 2 ×
12 ± 1 μm, L/W ratio = 2).
Culture characteristics: Colonies on PDA with moderately dense,
raised mycelium mat, initially white to smoke-grey, turning greenish
116
grey on the surface and greenish grey in reverse, becoming dark
slate-blue with age. Cardinal temperatures for growth: min 15 °C,
max 35 °C, opt 25 °C.
Type: egypt, Sharkia Province, El Menayar, from M. indica leaf,
Feb. 2010, A.M. Ismail, holotype CBS H-20736.
Cultures: BOT-10 = CBS 130992 (ex-type), BOT-29.
Host: Mangifera indica (Ismail et al. 2012).
Known distribution: Brazil (Marques et al. 2013), Egypt (Ismail et
al. 2012).
Notes: This species is morphologically and phylogenetically
closely related to L. citricola, L. hormozganensis, L. parva and
L. pseudotheobromae, but can be distinguished based on the
dimensions of conidia and paraphyses.
Lasiodiplodia gilanensis Abdollahz., Javadi & A.J.L.
Phillips, Persoonia 25: 5. 2010. MycoBank MB16778. Fig. 40.
Ascomata not reported. Conidiomata stromatic, produced on
pine needles on WA within 2–4 wk, supericial, dark brown to
black, covered with dense mycelium, mostly uniloculate, up to
940 μm diam, solitary, globose, thick-walled, non-papillate with
a central ostiole. Paraphyses, hyaline, cylindrical, thin-walled,
initially aseptate, becoming up to 1–3 septate when mature,
rarely branched, rounded at apex, up to 95 μm long, 2–4 μm
wide. Conidiophores absent. Conidiogenous cells holoblastic,
discrete, hyaline, smooth, thin-walled, cylindrical, 11–18 × 3–5 μm.
Conidia initially hyaline, aseptate, ellipsoid to ovoid, with granular
content, rounded at apex, base mostly truncate, wall < 2 μm,
becoming pigmented, verruculose, ellipsoid to ovoid, 1-septate with
longitudinal striations, (25–)28–35(–39) × (14.5–)15–18(–19) μm,
95 % conidence limits = 30.6–31.4 × 16.5–16.7 μm (av. ± S.D. =
31 ± 2.4 × 16.6 ± 1 μm, L/W ratio = 1.9).
Culture characteristics: Colonies with abundant aerial mycelia
reaching to the lid of Petri plate, aerial mycelia becoming smokegrey to olivaceous-grey at the surface and greenish grey to dark
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 40. Lasiodiplodia gilanensis. A. Conidiomata on pine needles in culture. B. Conidia developing on conidiogenous cells. C. Conidia developing on conidiogenous cells
between paraphyses. D. Paraphyses. E. Hyaline, immature conidia. F, G. Mature conidia in two different focal planes to show the longitudinal striations. Scale bars: A = 1 mm,
B = 5 μm, C–G = 10 μm.
slate blue at the reverse after 2 wk in the dark at 25 °C. Colonies
reaching 80 mm on MEA after 2 d in the dark at 25 °C. Cardinal
temperatures for growth: min ≤ 10 °C, max ≥ 35 °C, opt 25–30 °C.
Lasiodiplodia gonubiensis Pavlic, Slippers & M.J. Wingf.,
Stud. Mycol. 50: 318. 2004. MycoBank MB500079. See
Pavlic et al. (2004) for illustrations.
Type: Iran, Gilan Province, Rahimabad-Garmabdost, on twigs of
unknown woody plant, Jun. 2007, J. Abdollahzadeh & A. Javadi,
holotype IRAN 14272F.
Ascomata not reported. Conidiomata stromatic, formed on WA
on sterilised pine needles within 7–21 d, semi-immersed, solitary,
globose, papillate, leaden-black, covered by mycelium, up to 460
μm diam. Paraphyses cylindrical, aseptate, hyaline, (14–)26.5–47(–
65) × (1.5–)2–2.5(–3) μm. Conidiophores absent. Conidiogenous
cells holoblastic, cylindrical, hyaline, (6.5–)10–15(–18) × (1–)2–4(–
4.5) μm. Conidia initially hyaline, unicellular, ellipsoid to obovoid,
thick-walled with granular content, rounded at apex, occasionally
truncate at base becoming cinnamon to sepia with longitudinal
striations, forming one to three septa, (28–)32–36(–39) × (14–)16–
18.5(–21) μm (av. of 100 conidia = 33.8 × 17.3 μm, L/W ratio = 1.9).
Cultures: IRAN 1523C = CBS 124704 (ex-type), IRAN 1501C =
CBS 124705.
Hosts: Unknown (isolated from twigs of an unknown woody plant).
Known distribution: Rahimabad-Garmabdost, Gilan Province,
Northern Iran (Abdollahzadeh et al. 2010).
Notes: Phylogenetically, L. gilanensis is closely related to L. plurivora
and L. missouriana, but the three species can be distinguished
on average conidial dimensions. Moreover, the paraphyses of L.
gilanensis are up to 95 μm long and 4 μm wide, whereas paraphyses
of L. plurivora are up to 130 μm long and 10 μm wide (Damm et al.
2007). Also, the 1–3 basal cells of L. plurivora paraphyses often
are broader than the apical cells whereas, in L. gilanensis they are
the same as the apical cells. In terms of morphology, L. gilanensis
is similar to L. gonubiensis, but paraphyses of L. gilanensis (up
to 95 μm) are longer than those of L. gonubiensis (up to 65 μm).
Moreover, conidia of L. gilanensis (av. ± S.D. = 31 × 16.6 μm) are
slightly shorter than in L. gonubiensis (av. ± S.D. = 33.8 × 17.3 μm).
This species produces a pink pigment in PDA cultures at 35 °C.
www.studiesinmycology.org
Culture characteristics: Colonies initially white to smoke-grey with
luffy, aerial mycelium, becoming olivaceous-grey on the surface
after 3–4 d, with dense aerial mycelium, margins slightly irregular;
reverse side of the colonies dark slate-blue. Optimum temperature
for growth 25 °C, covering the medium surface (90 mm Petri dish)
after 5 d in the dark. Isolates growing at 35 °C produced a coral
red pigment within 4 d.
Type: South Africa, Eastern Cape Province, Gonubie, isolated
from Syzygium cordatum, Jul. 2002, D. Pavlic, holotype PREM
58127 (conidiomata on needles of Pinus sp. on WA).
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PhilliPs et al.
Fig. 41. Lasiodiplodia hormozganensis. A. Conidiomata on pine needles in culture. B, C. Conidia developing on conidiogenous cells between paraphyses. D, E. Septate and
aseptate paraphyses. F. Hyaline immature conidia. G, H. Mature conidia in two different focal planes to show the longitudinal striations. Scale bars: A = 1 mm, B, C = 5 μm,
D–H = 10 μm.
Cultures: CMW 14077 = CBS 115812 (ex-type), CMW 14078 =
CBS 116355.
Hosts: Syzygium cordatum (Pavlic et al. 2004).
Known distribution: South Africa (Gonubie, Eastern Cape Province)
(Pavlic et al. 2004).
Notes: Phylogenetically this species is clearly distinct from all
other Lasiodiplodia species. In terms of morphology, conidia of L
gonubiensis are larger than those of any other species presently
known in the genus.
Lasiodiplodia hormozganensis Abdollahz., Zare & A.J.L.
Phillips, Persoonia 25: 6. 2010. MycoBank MB16779. Fig 41.
Ascomata not reported. Conidiomata stromatic, produced on pine
needles on WA within 2–4 wk, supericial, dark brown to black, covered
118
with dense mycelium, mostly uniloculate, up to 950 μm diam, solitary,
globose, thick-walled, non-papillate with a central ostiole. Paraphyses,
hyaline, cylindrical, thin-walled, initially aseptate, becoming up to
1–7-septate when mature, rarely branched, occasionally basal, middle
or apical cells swollen, rounded at apex, up to 83 μm long, 2–4 μm wide.
Conidiophores absent. Conidiogenous cells holoblastic, discrete, hyaline,
smooth, thin-walled, cylindrical, 9–15 × 3–5 μm. Conidia initially hyaline,
aseptate, ellipsoid to cylindrical, with granular contents, rounded at apex,
base round or truncate, wall < 2 μm, becoming pigmented, verruculose,
ellipsoid to ovoid, 1-septate with longitudinal striations, (15.5–)18–24(–
25) × 11–14 μm, 95 % conidence limits = 21.2–21.7 × 12.4–12.6 μm (av.
± S.D. = 21.5 ± 1.9 × 12.5 ± 0.8 μm, L/W ratio = 1.7).
Culture characteristics: Colonies with abundant aerial mycelia
reaching to the lid of Petri dish, aerial mycelia becoming smoke
grey to olivaceous-grey at the surface and greenish grey to dark
slate blue at the reverse after 2 wk in the dark at 25 °C. Colonies
reaching 83 mm on MEA after 2 d in the dark at 25 °C. Cardinal
temperatures for growth: min ≤ 10 °C, max ≥ 35 °C, opt 25–30 °C.
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 42. Lasiodiplodia iraniensis. A. Conidiomata on pine needles in culture. B. Conidia developing on conidiogenous cells. C, D. Conidia developing on conidiogenous cells
between paraphyses. E. Paraphyses. F. Hyaline, immature conidia. G, H. Mature conidia in two different focal planes to show the longitudinal striations. Scale bars: A = 1 mm,
B, C = 5 μm, D–H = 10 μm.
Type: Iran, Hormozgan Province, Rodan, on twigs of Olea sp., Jun.
2007, J. Abdollahzadeh & A. Javadi, holotype IRAN 14271F.
Cultures: IRAN 1500C = CBS 124709 (ex-type), IRAN 1498C =
CBS 124708.
Hosts: Mangifera indica, (Abdollahzadeh et al. 2010, Marques et al.
2013), Olea sp. (Abdollahzadeh et al. 2010).
Known distribution: Iran (Hormozgan Province) (Abdollahzadeh et
al. 2010), Brazil (Marques et al. 2013).
Notes: Phylogenetically and morphologically, this species is
closely related to L. citricola, L. egyptiacae, L. parva and L.
pseudotheobromae, but can be distinguished based on average
conidial dimensions and paraphyses length. This species does not
produce a pink pigment in PDA cultures at 35 °C.
www.studiesinmycology.org
Lasiodiplodia iraniensis Abdollahz., Zare & A.J.L. Phillips,
Persoonia 25: 8. 2010. MycoBank MB16780. Fig 42.
Ascomata not reported. Conidiomata stromatic, produced on pine
needles on WA within 2–4 wk, supericial, dark brown to black,
covered with dense mycelium, mostly uniloculate, up to 980 μm
diam, solitary, globose, thick-walled, non-papillate with a central
ostiole. Paraphyses, hyaline, cylindrical, thin-walled, initially
aseptate, becoming up to 1–6 septate when mature, rarely
branched, occasionally basal, middle or apical cells swollen,
rounded at apex, up to 127 μm long, 2–4 μm wide. Conidiophores
absent. Conidiogenous cells holoblastic, discrete, hyaline,
smooth, thin-walled, cylindrical, 9–16 × 3–5 μm. Conidia initially
hyaline, aseptate, subglobose to subcylindrical, with granular
content, both ends rounded, wall < 2 μm, becoming pigmented,
verruculose, ellipsoid to ovoid, 1-septate with longitudinal
striations, (15.5–) 17–23(–29.5) × 11–14 μm, 95 % conidence
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PhilliPs et al.
limits = 20.6–20.8 × 13–13.1 μm (av. ± S.D. = 20.7 ± 2 × 13 ± 0.9
μm, L/W ratio = 1.6).
Culture characteristics: Colonies with abundant aerial mycelia
reaching to the lid of Petri dish, aerial mycelia becoming smoke
grey to olivaceous-grey at the surface and greenish grey to dark
slate blue at the reverse after 2 wk in the dark at 25 °C. Colonies
reaching 80 mm on MEA after 2 d in the dark at 25 °C. Cardinal
temperatures for growth: min ≤ 10 °C, max ≥ 35 °C, opt 25–30 °C.
Type: Iran, Hormozgan Province, Bandar Abbas, Geno mountain,
on twigs of Salvadora persica, Mar. 2007, J. Abdollahzadeh & A.
Javadi, holotype IRAN 14268F.
Cultures: IRAN 1520C = CBS 124710 (ex-type), IRAN 1519C.
Hosts: Citrus sp., Eucalyptus sp., Juglans sp., Mangifera indica,
Salvadora persica, Terminalia catapa (Abdollahzadeh et al. 2010).
Known distribution: Brazil (Marques et al. 2013), Iran (Hormozgan
& Golestan Provinces) (Abdollahzadeh et al. 2010).
Notes: Phylogenetically this species is closely related to L.
mahajangana, L. theobromae and L. viticola. This species can
be easily separated from the irst two species based on conidial
dimensions. Conidia of L. iraniensis (av. = 20.7 × 13 μm) are larger
and smaller than those of L. mahajangana (av. = 17.5 × 11.5 μm)
and smaller than L. theobromae (av. = 26.2 × 14.2 μm). Conidia
of L. viticola (av. = 19.5 × 9.5 μm) are shorter and narrower than
those of L. iraniensis. Furthermore, the paraphyses in L. iraniensis
are longer than 100 μm, while they are less than 100 μm in L.
viticola. Although, conidial dimensions of L. iraniensis are similar to
those of L. parva, the average width of conidia of L. iraniensis (13
μm) is greater than in L. parva (av. width = 11.5 μm). This species
produces a pink pigment in PDA cultures at 35 °C.
Lasiodiplodia lignicola (Ariyawansa, J.K. Liu & K.D. Hyde)
A.J.L. Phillips, A. Alves & Abdollahz., comb. nov. MycoBank
MB805462. Fig. 43.
Basionym: Auerswaldia lignicola Ariyawansa, J.K. Liu & K.D. Hyde,
Fungal Divers. 57: 161. 2012.
Saprobic on dead wood. Ascomata 0.5–0.75 mm diam, 0.75–1
mm high, dark brown to black, developing on host tissue, semiimmersed, globose to subglobose, coriaceous, multiloculate, with 4–5
locules, with individual ostioles, cells of ascostromata brown-walled
textura angularis. Locules 100–130 × 110–130 μm, with individual
papillate ostioles. Peridium of locules 30–60 μm diam, thick-walled,
wall composed of outer layers of thick-walled, dark brown cells of
textura angularis, inner layers of thin-walled cells of textura angularis.
Pseudoparaphyses not observed. Asci bitunicate, issitunicate, clavate
to broadly clavate, with short and narrow pedicel, rounded at the apex
with an ocular chamber, 80–90 × 15–25 μm. Ascospores uniseriate or
partially overlapping, reddish brown to dark brown, aseptate, fusiform
to ellipsoid with narrowly rounded ends, smooth-walled, 15–20 × 8–10
μm (av. of 40 ascospores = 19 × 9 μm). Conidiomata indistinguishable
from ascomata. Paraphyses aseptate, thin-walled, with slightly
bulbous tip up to 15 μm long. Conidiophores hyaline, thin-walled,
cylindrical, 6–12 × 2.5–3 μm. Conidiogenous cells hyaline, thin-walled,
smooth, cylindrical, forming a single conidium at the tip, holoblastic,
120
proliferating at the same level giving rise to periclinal thickenings, 10–
15 × 2.5–3.5 μm. Conidia hyaline, smooth, thick-walled, globose to
ovoid, becoming dark brown with longitudinal striations, (15–)16–17.5
× (8–)8.5–10.5(–11) μm, L/W ratio = 1.7.
Culture characteristics: Colonies growing slowly on MEA, reaching
3 mm after 5 d at 27 °C, effuse, velvety, with entire to slightly
undulate edge, dark brown to black.
Type: Thailand, Chiang Rai Province, Muang District, Bandu,
on dead wood, 30 Sep. 2011, A.D. Ariyawansa, holotype MFLU
12–0750.
Cultures: MFLUCC 11-0435 = CBS 134112 (ex-type), MFLUCC 110656.
Hosts: Dead wood of unknown host (Liu et al. 2012).
Known distribution: Thailand (Liu et al. 2012).
Notes: This species was introduced by Liu et al. (2012) under
Auerswaldia lignicola. However, in the phylogenenies presented
here, it is obviously a distinct species in Lasiodiplodia and formed
a clade as a group basal to all other species. This is one of the
few species in which the asexual morph and sexual have been
deinitively linked, and the dark brown ascospores (Liu et al. 2012)
are assumed to be a typical feature of the genus.
Lasiodiplodia mahajangana Begoude, Jol. Roux & Slippers,
Mycol. Prog. 9: 110. 2010. MycoBank MB514012. See Didier
Begoude et al. (2010) for illustrations.
Ascomata not reported. Conidiomata stromatic, produced on
pine needles on MEA within 2 wk, up to 300 μm diam, solitary
and covered by mycelium, supericial, conical, unilocular, with
long necks (up to 200 μm) and single ostioles at the tips, locule
walls thick, consisting of two layers: an outer dark brown textura
angularis, lined with inner thin-walled, hyaline cells. Paraphyses
rare, cylindrical, hyaline, aseptate 1-celled, (27.5–)33.5–52.5(–66)
× (2–)2.5–3.5(–5) μm, (av. of 50 paraphyses = 43 × 3 μm), rounded
at the tips, unbranched. Conidiophores absent. Conidiogenous
cells holoblastic, discrete, hyaline, cylindrical, (10–)10.5–18(–26)
× (3–)3.5–5.5(–6) μm (av. of 50 conidiogenous cells = 14.5 × 4.5
μm, L/W ratio = 3.2). Conidia initially aseptate, hyaline, ellipsoid
to ovoid, thick-walled (< 2.5 μm), granular content, becoming
1-septate and pigmented after release, vertical striations observed
at maturity, (13.5–)15.5–19(–21.5) × (10–)11.5–13(–14) μm (av. of
50 conidia = 17.5 × 11.5 μm, L/W ratio = 1.4).
Culture characteristics: Colonies initially white, luffy with abundant
aerial mycelium, becoming pale olivaceous-grey after 4 d, with
the reverse sides of the colonies olivaceous-grey. Optimum
temperature for growth 25–30 °C, covering a 90 mm Petri dish after
3 d on MEA in the dark, no growth observed at 10 °C.
Type: Madagascar, Mahajanga, isolated from healthy branches of
Terminalia catappa, Oct. 2007, J. Roux, PREM 60288 holotype
(a dry culture of CMW 27801 = CBS 124925 on pine needles);
isolated from healthy branches of Terminalia catappa, Oct. 2007, J.
Roux, paratype PREM 60289.
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 43. Lasiodiplodia lignicola. A. Conidiomata developing on pine needles in culture. B–D. Conidiogenous cells. E, F. Brown, striate conidia. Scale bars: A = 500 μm, B = 10
μm. Scale bar in B applies to C–F.
Cultures: CMW 27820 = CBS 124927, CMW 27801 = CBS 124925
(ex-type).
Host: Terminalia catappa (Didier Begoude et al. 2010).
Known distribution: Madagascar (Mahajanga) (Didier Begoude et
al. 2010).
Notes: Conidia of L. mahajangana are smaller than those of its
closest relative, L. theobromae. Paraphyses of L. mahajangana
are aseptate while those of L. theobromae are septate. In terms
of morphology it is similar to L. margaritacea and the two can
be distinguished only on the average lengths of their conidia (L.
mahajangana = 17.5 μm, L. margaritacea = 15.3 μm).
Lasiodiplodia margaritacea Pavlic, T.I. Burgess & M.J.
Wingf., Mycologia 100: 860. 2008. MycoBank MB512052.
See Pavlic et al. (2008) for illustrations.
Ascomata not reported. Conidiomata stromatic, semi-immersed,
solitary, globose, papillate, black, covered by hyphal hairs, up to
www.studiesinmycology.org
520 μm diam. Paraphyses cylindrical, 1–2-septate, hyaline, (19–)
28–46(–54) × (1.5–)2–2.5(–3) μm (av. = 37.1 × 2.2 μm), formed
among conidiogenous cells. Conidiophores absent. Conidiogenous
cells holoblastic, cylindrical to subcylindrical, hyaline, the irst
conidium produced holoblastically and subsequent conidia
enteroblastically, (6–)10–11(–19.5) × (2–)3–4(–4.5) μm (av. = 10.3
× 3.3 μm). Conidia globose to subglobose to obovoid, (12–)14–
17(–19) × (10–)11–12(–12.5) μm (av. of 50 conidia = 15.3 × 11.4
μm, L/W ratio = 1.3), with granular content, thick-walled (1–2 μm),
initially unicellular, hyaline, becoming cinnamon to sepia, forming
one septum and longitudinal striations with maturation.
Culture characteristics: Colonies initially white to smoke grey with
woolly aerial mycelium, becoming pale olivaceous-grey within 5–7 d,
olivaceous-grey to iron-grey with age, margins regular. Submerged
mycelium dense, reverse grey olivaceous to olivaceous-black after
7 d, becoming black with age. Optimum growth at 30 °C, covering
the 90 mm Petri dish after 3 d in the dark.
Type: Australia, Western Australia, Tunnel Creek Gorge, on
Adansonia gibbosa, Jul. 2006, T.I. Burgess, holotype PREM
59844 (a dry culture of CMW 26162 on pine needles).
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PhilliPs et al.
Cultures: CMW 26162 = CBS 122519 (ex-type), CMW 26163 =
CBS 122065.
Host: Asymptomatic branches of Adansonia gibbosa (Pavlic et al.
2008).
Known distribution: Australia (Western Australia) (Pavlic et al.
2008).
Notes: The small sub-globose conidia clearly distinguish this
species from all species other than L. mahajangana, and these
two can be separated morphologically only on average conidial
lengths (L. mahajangana = 17.5 μm, L. margaritacea = 15.3 μm).
Phylogenetically, however, they are clearly two distinct species.
Lasiodiplodia missouriana Úrbez-Torres, Peduto & Gubler,
Fungal Divers. 52: 181. 2012. MycoBank MB519954. See
Úrbez-Torres et al. (2012) for illustrations.
Ascomata not reported. Conidiomata stromatic, supericial, formed
on PDA within 2–3 wk, black, covered with mycelium, up to 320 μm
diam, globose to ovoid, thick-walled, unilocular, with a central ostiole,
often oozing conidia. Paraphyses hyaline, cylindrical, aseptate,
not branched, round at apex, up to 55 μm long, 2–3 μm wide.
Conidiophores absent. Conidiogenous cells holoblastic, hyaline,
smooth, cylindrical. Conidia produced in culture initially hyaline,
unicellular, ellipsoid to ovoid, thick-walled (1–2 μm), contents granular,
becoming dark brown, 1-septate, with longitudinal striations while still
inside the conidiomata, (16–)17.5–19.5(–21) × (8–)9–10.5(–11.5) μm
(av. of 60 conidia ± 18.5 × 9.8 μm, L/W ratio = 1.9).
Culture characteristics: Colonies on PDA with moderately dense
aerial mycelium, initially white becoming pale olivaceous-grey within
7 d and turning iron grey to greenish black within 28 d; reverse dark
slate blue after 28 d. Colonies covering the dish on PDA after 48 h
in the dark at 25 °C. Cardinal temperatures for growth: min 10 °C,
max 35 °C, opt 25–30 °C.
Type: uSA, Saint James, on Vitis vinifera × V. labrusca hybrid
cv. Catawba, Jun. 2006, R.K. Striegler & G.M. Leavitt, holotype
UCD2193MO.
Cultures: UCD2193MO = CBS 128311 (ex-type), UCD2199MO =
CBS128312.
Hosts: Vitis spp. (Úrbez-Torres et al. 2012).
Known distribution: USA (Missouri) (Úrbez-Torres et al. 2012)
Notes: The small conidia of this species distinguish it morphologically
from all others except L. hormozganensis and these two can
be distinguished only by small differences in conidial widths
(L. missouriana = 8–12 μm, L. hormozganensis = 11–14 μm).
Nevertheless, phylogenetically they are clearly two distinct species.
Lasiodiplodia parva A.J.L. Phillips, A. Alves & Crous, Fungal
Divers. 28: 9. 2007. MycoBank MB510942. Fig. 44.
Ascomata not reported. Conidiomata stromatic, formed on poplar
twigs in culture, uniloculate, dark brown to black, immersed in
122
the host becoming erumpent when mature. Paraphyses hyaline,
cylindrical, septate, ends rounded, up to 105 μm long, 3–4 μm wide
arising amongst the conidiogenous cells. Conidiophores absent.
Conidiogenous cells hyaline, smooth, cylindrical, slightly swollen at
the base, holoblastic, proliferating percurrently to form one or two
annellations, or proliferating at the same level giving rise to periclinal
thickenings. Conidia ovoid, apex broadly rounded, base rounded or
truncate, widest in the middle or upper third, thick-walled, initially
hyaline and aseptate and remaining so for a long time, becoming
1-septate and dark-walled only some time after release from the
conidiomata, with melanin deposits on the inner surface of the wall
arranged longitudinally giving a striate appearance to the conidia,
(15.5–)16–23.5(–24.5) × (10–)10.5–13(–14.5) μm, 95 % conidence
limits = 19.8–20.5 × 11.4–11.7 μm (av. ± S.D. = 20.2 ± 1.9 × 11.5 ±
0.8 μm, L/W ratio = 1.8).
Type: colombia, Dep. Meta, Villavicencio, cassava ield soil, 1978,
O. Rangel, holotype CBS H-19915.
Cultures: CBS 456.78 (ex-type), CBS 494.78.
Hosts: Cassava-ield soil, Theobroma cacao (Alves et al. 2008).
Known distribution: Colombia, Sri Lanka (Alves et al. 2008).
Notes: This species can be separated from its closest
relatives, L. citricola, L. egypticae, L. hormozganensis and L.
pseudotheobromae based on conidial and paraphyses dimensions.
In terms of morphology it is similar to L. iraniensis and the two
species can be separated only on the average width of conidia, but
phylogenetically they are clearly distinct.
Lasiodiplodia plurivora Damm & Crous, Mycologia 99:
674. 2007. MycoBank MB501322. See Damm et al. (2007)
for illustrations.
Ascomata not reported. Conidiomata stromatic, produced on pine
needles on SNA within 2–4 wk, solitary, globose to ovoid, dark
brown, up to 400 μm diam, embedded in needle tissue, semiimmersed, unilocular, with a central ostiole; wall 4–7 cell layers
thick, outer layers composed of dark brown textura angularis,
becoming thin-walled and hyaline toward the inner region.
Conidiophores absent. Conidiogenous cells holoblastic, discrete,
hyaline, cylindrical, proliferating percurrently several times near
the apex, 8–13 × 4–7 μm. Paraphyses hyaline, cylindrical,
2–7-celled, the 1–3 basal cells often broader than the apical cells,
apical cell with rounded tip, sometimes branched, up to 130 μm
long, 2–5 μm broad at the upper part and up to 10 μm broad at
the lower part (basal cells). Conidia initially aseptate, thick-walled
(< 3 μm), hyaline, ellipsoidal to obovate, sometimes somewhat
irregular, with granular content, becoming 1-septate after release,
brown, obovate, verruculose and with longitudinal striations,
(22–)26.5–32.5(–35) × (13–)14.5–17(–18.5) μm (av. ± S.D. =
29.6 ± 2.9 × 15.6 ± 1.2 μm, L/W ratio = 1.9).
Culture characteristics: Colonies on PDA in the dark: mycelium and
surface white to pale olivaceous-grey, reverse pale olivaceous-buff
to pale grey-olivaceous, lat with undulate margins. Under nearultraviolet light: mycelium and surface white to pale mouse-grey,
reverse pale olivaceous-buff to smoke-grey. Colonies 76 mm after 2
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 44. Lasiodiplodia parva. A. Conidiogenous layer with paraphyses and developing conidia. B. Percurrently proliferating conidiogenous cells. C. Hyaline, aseptate conidia and
dark-walled, septate conidia. D, E. Mature conidia at two different focal planes showing the striations on the inner side of the conidial wall. Scale bars = 10 μm.
d, reaching the edge the Petri dish after 3 d. Cardinal temperatures
for growth: min 10 °C, max ≥ 35 °C, opt 30 °C.
Type: South Africa, Western Cape Province, Stellenbosch, from
V-shaped necrotic lesion of P. salicina, May 2004, U. Damm,
holotype CBS H-19844.
Cultures: CBS 120832 = STE-U 5803 (ex-type), CBS 121103 =
STE-U 4583.
Hosts: Prunus salicina, Vitis vinifera (Damm et al. 2007).
Known distribution: South Africa (Western Cape Province) (Damm
et al. 2007).
Notes: Phylogenetically this species is close to L. gilanensis and
L. missouriana, but it can be separated from that species based
on conidial dimensions and paraphyses length and shape. Conidia
of L. gilanensis (av. = 29.6 × 15.6 μm) are larger than those of L.
missouriana (av. = 18.5 × 9.8 μm), but compared to L. gilanensis
they are slightly shorter. Moreover, paraphyses of L. plurivora (up
to 130 μm) are longer than 100 μm, while in L. gilanensis and L.
missouriana they are consistently less than 100 μm. In terms of
morphology it is close to L. citricola, but conidia of L. citricola (av.
= 24.5 × 15.4 μm) are quite small compared with L. plurivora (av. =
29.6 × 15.6 μm).
Lasiodiplodia pseudotheobromae A.J.L. Phillips, A. Alves
& Crous, Fungal Divers. 28: 8. 2007. MycoBank MB510941.
Fig. 45.
Ascomata not reported. Conidiomata stromatic, formed on poplar
twigs in culture, uniloculate, dark brown to black, immersed in
the host becoming erumpent when mature. Paraphyses hyaline,
cylindrical, mostly aseptate, sometimes branched, ends rounded,
up to 58 μm long, 3–4 μm wide arising amongst the conidiogenous
cells. Conidiophores absent. Conidiogenous cells hyaline, smooth,
cylindrical, slightly swollen at the base, holoblastic, proliferating
percurrently to form one or two closely spaced annellations.
Conidia ellipsoidal, apex and base rounded, widest at the middle,
thick-walled, initially hyaline and aseptate and remaining so for
a long time, becoming 1-septate and dark brown only some time
after release from the conidiomata, with melanin deposits on the
inner surface of the wall arranged longitudinally giving a striate
appearance to the conidia, (22.5–)23.5–32(–33) × (13.5–)14–18(–
20) μm, 95 % conidence limits = 27.5–28.5 × 15.5–16.5 μm (av. ±
S.D. = 28.0 ± 2.5 × 16.0 ± 1.2 μm, L/W ratio = 1.7).
Type: costa rica, San Carlos, on Gmelina arborea, J. CarranzaVelazquez, holotype CBS H-19916.
Cultures: CBS 116459 (ex-type), CBS 447.62.
Hosts: Acacia mangium, Citrus aurantium, Coffea sp., Gmelina
arborea, Rosa sp. (Alves et al. 2008).
www.studiesinmycology.org
123
PhilliPs et al.
Fig. 45. Lasiodiplodia pseudotheobromae. A. Conidiogenous layer with developing conidia and paraphyses. B. Paraphyses. C. Conidium developing on an annellidic
conidiogenous cell. D. Immature, hyaline conidia. E, F. Mature, dark-walled, one-septate, striate conidia in two different focal planes to show the striations on the inner side of
the wall. Scale bars = 10 μm.
Known distribution: Costa Rica, Netherlands, Suriname, Zaire
(Alves et al. 2008).
Notes: This species can be separated from its closest relatives,
L. citricola, L. egypticae, L. hormozganensis and L. parva and as
previously mentioned under L. plurivora. In terms of morphology
it is close to L. crassispora but the two species differ in that the
pseudparaphyses of L. crassispora are mostly septate, while in
L. pseudotheobromae they are mostly aseptate.
Lasiodiplodia rubropurpurea Burgess, Barber & Pegg,
Mycologia 98: 431. 2006. MycoBank MB500236. See
Burgess et al. (2006) for illustrations.
Ascomata not reported. Conidiomata stromatic, supericial,
globose, red to dark vinaceous, mostly solitary, 0.5–1.5 mm diam
and covered with mycelium. Paraphyses cylindrical, aseptate,
hyaline (30–)32–52(–58) × 1.5–3.5 μm (av. of 50 paraphyses
= 42.4 × 2.6 μm). Conidiophores reduced to conidiogenous
cells. Conidiogenous cells holoblastic, hyaline, subcylindrical to
ampulliform, 7–13(–15) × 3–5 μm (av. of 50 conidiogenous cells
= 10.2 × 4 μm), proliferating percurrently with a single annellation.
Conidia initially hyaline, unicellular, ellipsoid to obovoid, thickwalled (1 μm) with granular contents, rounded at apex, occasionally
truncate at base, initially hyaline and unicellular, becoming
pigmented with one septum when mature or before germination,
longitudinal striations observed at maturation, 24–33 × 13–17 μm
(av. of 100 conidia = 28.2 × 14.6 μm, L/W ratio = 1.9).
124
Culture characteristics: Colonies moderately dense, with
appressed mycelial mat, colonies initially white to buff turning to
pale olivaceous-grey within 7 d and becoming darker with age.
After 7 d submerged mycelia olivaceous-grey, becoming black with
age. Optimum temperature for growth 25–30 °C, reaching 76 mm
on PDA after 3 d at both 25 °C and 30 °C in the dark.
Type: Australia, Queensland, Tully, from canker on Eucalyptus
grandis, May 2003, T.I. Burgess, holotype MURU 409.
Cultures: WAC12535 = CMW 14700 = CBS 118740 (ex-type),
WAC12536 = CMW 15207.
Host: Eucalyptus grandis (Burgess et al. 2006).
Known distribution: Australia (Queensland) (Burgess et al. 2006).
Note: The red-purple conidiomata of L. rubropurpurea are unique
in this genus and distuinguish it from all other species (Burgess et
al. 2006).
Lasiodiplodia theobromae (Pat.) Griff. & Maubl., Bull. Soc.
Mycol. Fr. 25: 57. 1909. MycoBank MB188476. Fig. 46.
Basionym: Botryodiplodia theobromae Pat., Bull. Soc. Mycol. Fr.
8: 136. 1892.
≡ Diplodia theobromae (Pat.) W. Nowell, Diseases of Crop Plants in the
Lesser Antiles: 158. 1923.
= Sphaeria glandicola Schwein., Trans. Am. phil. Soc., Ser. 2 4(2): 214. 1832.
≡ Physalospora glandicola (Schwein.) N.E. Stevens, Mycologia 25: 504.
1933.
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 46. Lasiodiplodia theobromae (A–D from holotype of Sphaeria rhodina). A, B. Asci. C, D. Ascospores. E, I. Conidiogenous layer with conidiogenous cells and paraphyses.
F. Paraphyses. G. Immature hyaline conidia. H. Developing conidia. J, K. Mature, dark-walled, one-septate, striate conidia in two different focal planes. Scale bars = 10 μm.
= Physalospora rhodina Berk. & M.A. Curtis, Grevillea 17: 92. 1889.
≡ Botryosphaeria rhodina (Berk. & M.A. Curtis) Arx, Gen. Fungi Sporul.
Cult. (Lehr): 143. 1970.
= Diplodia gossypina Cooke, Grevillea 7: 95. 1879.
= Macrophoma vestita Prill. & Delacr., Bull. Soc. Mycol. Fr. 10: 165. 1894.
= Diplodia cacaoicola Henn., Bot. Jb. 22: 80. 1895.
= Lasiodiplodia tubericola Ellis & Everh., Bot. Gaz. 21: 92. 1896.
≡ Diplodia tubericola (Ellis & Everh.) Taubenh., Am. J. Bot. 2: 328. 1915.
≡ Botryodiplodia tubericola (Ellis & Everh.) Petr., Ann. Mycol. 21: 332.
1923.
= Botryodiplodia gossypii Ellis & Barth., J. Mycol. 8: 175–176. 1902.
= Botryodiplodia elasticae Petch., Ann. R. Bot. Gdns Peradeniya 3: 7. 1906.
= Diplodia arachidis Petch., Ann. R. Bot. Gdns Peradeniya 3: 6. 1906.
= Chaetodiplodia grisea Petch., Ann. R. Bot. Gdns Peradeniya 3: 6. 1906.
= Lasiodiplodia nigra Appel & Laubert, Arbeiten Kaiserl. Biol. Anst. Ld.-u.
Forstw. 5: 147. 1907.
= Diplodia rapax Massee, Bull. Misc. Inf., Kew: 3. 1910.
= Diplodia natalensis Pole-Evans Transvaal Dept. of Agricult. Sci. Bull. 4: 15.
1911 (1910).
= Diplodia manihoti Sacc. (as “maniothi”), Ann. Mycol. 12: 310. 1914.
≡ Botryodiplodia manihoti (Sacc.) Petr. (as “maniothi”), Ann. Mycol. 22:
83. 1924.
= Botryodiplodia manihotis Syd. & P. Syd., Ann. Mycol. 14: 202. 1916.
= Diplodia corchori Syd. & P. Syd., Ann. Mycol. 14: 196. 1916.
www.studiesinmycology.org
= Diplodia musae Died., Ann. Mycol. 14: 200. 1916.
= Lasiodiplodia trilorae B.B. Higgins, Bull. Georgia Exp. Stn 118: 16. 1916.
= Diplodia ananassae Sacc., Atti Acad. Sci. Ven.-Tren.-Istr. 10: 75. 1917.
≡ Botryodiplodia ananassae (Sacc.) Petr., Ann. Mycol. 27: 365. 1929.
= Physalospora gossypina N.E. Stevens, Mycologia 17: 198. 1925.
= Botryodiplodia manihoticola Petr., In: Petrak & Syd., Feddes Repert., Beih.
42: 143. 1926.
Ascomata dark brown to black, aggregated, thick-walled, wall
composed of dark brown, thick-walled textura angularis, becoming
thinner and hyaline towards the inner layers, 250–400 μm diam.
Asci bitunicate, clavate, stipitate, 8-spored, 90–120 μm long.
Ascospores irregularly biseriate, hyaline, aseptate (24–)30–
35(–42) × (7–)11–14(–17) μm. Conidiomata stromatic, simple
or aggregated, immersed in the host becoming erumpent when
mature, dark brown, unilocular, thick- or thin-walled, wall formed of
dark brown thick-walled textura angularis, frequently setose, up to
5 mm wide, ostiole central, single, papillate. Paraphyses hyaline,
cylindrical, septate, occasionally branched, ends rounded, up to
55μm long, 3–4 μm wide. Conidiophores hyaline, simple, sometimes
septate, rarely branched, cylindrical, arising from the inner layers of
125
PhilliPs et al.
cells lining the locules. Conidiogenous cells hyaline, thin-walled,
smooth, cylindrical to sub-obpyriform, holoblastic, discrete,
determinate or indeterminate and proliferating percurrently with
one or two distinct annellations, or proliferating at the same level,
giving rise to periclinal thickenings. Conidia subovoid to ellipsoidovoid, apex broadly rounded, tapering to truncate base, widest
in middle to upper third, thick-walled, contents granular, initially
hyaline and aseptate, remaining hyaline for a long time, becoming
dark brown and 1-septate only a long time after discharge from the
conidiomata, with melanin deposits on the inner surface of the wall
arranged longitudinally giving a striate appearance to the conidia,
(19–)21.5–31.5(–32.5) × (12–)13–17 (–18.5) μm, 95 % conidence
limits = 26.2–27 × 14–14.4 μm (av. ± S.D. = 26.2 ± 2.6 × 14.2 ± 1.2
μm, L/W ratio = 1.9).
Type: ecuador, on Theobroma cacao, Lagerheim, holotype not
found, and presumably lost. Papua New Guinea, Madang, Jais
Aben, from unidentiied fruit along coral reef coast, No. 1995, A.
Aptroot, CBS H-21411, neotype designated here; MBT176098,
culture ex-neotype CBS 164.96.
Cultures: CBS 164.96 (ex-neotype), CBS 111530.
Hosts: Punithalingam (1976) refers to a wide host range.
Considering that the original concept of L. theobromae now refers
to a complex of species (Alves et al. 2008), many of the older
records of this fungus are unreliable.
Known distribution: Widely distributed in tropical and subtropical
regions (Punithalingham 1976).
Notes: Botryodiplodia theobromae was originally described from
Theobroma cacao in Ecuador. In spite of searching through
literature and many herbaria, we have been unable to locate the
holotype specimen. In recent years numerous new species have
been described, but in spite of this, the generic application of the
name, L. theobromae, has not been resolved. To address this issue,
we thus designate CBS 164.96 as ex-neotype culture, and have
deposited a dried specimen as neotype. Although this isolate, from
an unidentiied fruit on a coral reef coast in Papua New Guinea,
is from neither the type locality (Equador) nor the type substrate
(cocoa plant), it has long been regarded as a reference strain for
L. theobromae. For this reason we consider that it best serves to
stabilise this species by continuing to use this isolate as a reference
strain and to elevate its status to ex-neotype.
The connection between L. theobromae and its sexual
morph has not been proven conclusively. Stevens (1925) made
single ascospore cultures from a fungus that he referred to as
Physalospora gossypina on cotton stems in Florida, and from
Hicoria, Ilex, Liquidambar, Quercus and Vitis. In all cases the
conidia formed in these cultures were morphologically identical
to those of L. theobromae. Stevens (1926) then determined
that the fungus he called P. gossypina was in fact Physalospora
rhodina Cooke, which was later transferred by von Arx (1970) to
Botryosphaeria as B. rhodina (Cooke) Arx. However, there have
been no subsequent reports to conirm this connection, leaving
some doubts about its authenticity. Thus the connection between
the sexual morph and asexual morph has not been established
beyond all doubt and the value of the above description of the
sexual morph is questionable. Phylogenetically this species is close
to L. mahajangana, but it is easily separated by its larger conidia
(av. = 26.2 × 14.2 μm) compared with L. mahajangana (av. = 17.5
126
× 11.5 μm). In terms of morphology L. theobromae is similar to L.
rubropurpurea, but it differs from L. rubropurpurea by the absence
of red-purple conidiomata. Moreover, conidial length of this species
(av. length = 26.2 μm) is slightly shorter than in L. rubropurpurea
(av. length = 28.2 μm).
Lasiodiplodia venezuelensis T.I. Burgess, Barber &
Mohali, Mycologia 98: 432. 2006. MycoBank MB500237. See
Burgess et al. (2006) for illustrations.
Ascomata not reported. Conidiomata stromatic, supericial,
smooth, cylindrical, mostly solitary, 0.5–1 mm diam, often oozing
immature conidia. Paraphyses cylindrical, septate, hyaline (12–)
16–41(–45) × (1.5–)2–5 μm (av. of 50 paraphyses = 28.3 × 3.5 μm).
Conidiophores absent. Conidiogenous cells holoblastic, hyaline,
subcylindrical to cylindrical to ampulliform, (5–)7–14(–15) × 3–4.5(–
5), proliferating percurrently. Conidia initially hyaline, unicellular,
ellipsoid to obovoid, thick-walled (1.5–)2.5(–3) μm, av. of 50 conidia
= 1.96 μm) with granular contents, rounded at apex, occasionally
truncate at base, becoming pigmented with one septum when
mature or before germination, developing longitudinal striations
when mature, 26–33 × 12–15 μm (av. of 75 conidia = 28.4 × 13.5
μm, L/W ratio = 2.1).
Culture characteristics: Colonies moderately dense, with appressed
mycelial mat, initially white to buff turning pale olivaceous-grey
within 7 d and becoming darker with age. After 7 d submerged
mycelia olivaceous-grey, becoming black with age. Optimum
temperature for growth 25 °C, reaching 75 mm on PDA after 3 d at
25 °C in the dark.
Type: Venezuela, Estado Portuguesa, Acarigua, from wood of
living Acacia mangium, Oct. 2003, S. Mohali, holotype MURU 413.
Cultures: WAC12539 = CMW 13511 = CBS 118739 (ex-type),
WAC12540 = CMW 13512.
Host: Acacia mangium (Burgess et al. 2006).
Known distribution: Venezuela (Burgess et al. 2006).
Notes: Phylogenetically, this species is closely related to L.
crassispora and L. rubropurpurea, but can be distinguished from
L. rubropurpurea by the absence of red-purple conidiomata.
Furthermore, conidia of L. venezuelensis are narrower (av. = 28.4
× 13.5 μm) than those of L. crassispora (av. = 28.8 × 16 μm). In
terms of morphology this species is similar to L. viticola, but conidia
of L. venezuelensis (av. = 28.4 × 13.5 μm) are considerably larger
than those of L. viticola (av. = 19.5 × 9.5 μm).
Lasiodiplodia viticola Úrbez-Torres, Peduto & Gubler,
Fungal Divers. 52: 183. 2011. MycoBank MB519966. See
Úrbez-Torres et al. (2010) for illustrations.
Ascomata not reported. Conidiomata stromatic, solitary, formed
on PDA within 3–4 wk, black, covered with moderately dense
mycelium, up to 900 μm wide, globose to ovoid, thick-walled,
unilocular, with a central ostiole, often oozing conidia. Paraphyses
hyaline, cylindrical, aseptate, not branched, round at apex, up to
The Botryosphaeriaceae: genera and sPecies known from culTure
60 μm long, 2–3 μm wide. Conidiophores absent. Conidiogenous
cells holoblastic, hyaline, smooth, cylindrical. Conidia produced
in culture initially hyaline, unicellular, ellipsoidal, base rounded or
truncate, thick-walled (1–2 μm), granular content, becoming dark
brown, 1-septate, with longitudinal striations while still inside the
conidiomata, (16.5–)18–20.5(–23) × (8–)9–10.1(–10.5) μm (av. of
60 conidia = 19.5 × 9.5 μm, L/W ratio = 2.05). Colonies on PDA
with dense aerial mycelium, mycelium initially white becoming pale
olive-buff within 7 d and turning iron grey to greenish black within
28 d, reverse dark slate blue after 28 d, reaching 90 mm on PDA
after 48 h in the dark at 25 °C. Cardinal temperatures for growth:
min 10 °C, max 35 °C, opt 25–30 °C.
Type: uSA, Arkansas, Altus, on interspeciic hybrid grape
Vignoles cv. Ravat 51R, D. Cartwright & W. D. Gubler, holotype
UCD2553AR.
Cultures: UCD2553AR = CBS 128313 (ex-type), UCD2604MO =
CBS 128314.
Hosts: Vitis hybrids (Úrbez-Torres et al. 2010).
Known distribution: USA (Arkansas and Missouri) (Úrbez-Torres et
al. 2010).
Note: Phylogenetically this species is closely related to L.
mahajangana, L. theobromae and L. iraniensis, but can be easily
distinguished based on conidial and paraphyses dimensions (see
notes for L. iraniensis).
Macrophomina Petr. Ann. Mycol. 21: 314. 1923. MycoBank
MB8814.
Type species: Macrophomina phaseolina (Tassi) Goid., Annali
Sper. agr. N.S. 1: 457. 1947.
Mycelium supericial or immersed, brown to hyaline, branched,
septate, often dendroid in culture. Ascomata not reported.
Conidiomata pycnidial, stromatic, separate, globose, dark brown,
immersed, unilocular, thick-walled, wall consisting of an outer layer of
dark brown thick-walled textura angularis, becoming hyaline towards
the inside. Ostiole central, circular, papillate. Conidiophores absent.
Conidiogenous cells enteroblastic, phialidic, determinate, discrete,
lageniform to doliiform, hyaline, smooth, with wide aperture and
minute collarette, formed from the inner cells of the pycnidial wall.
Conidia hyaline, aseptate, obtuse at each end, straight, cylindrical
to fusiform, thin-walled, smooth, guttulate. Sclerotia black, smooth,
hard, formed of dark brown, thick-walled cells.
Note: Of the ive species listed in MycoBank, only one (M.
phaseolina) is known in culture.
Macrophomina phaseolina (Tassi) Goid., Annali Sper. agr.
N.S. 1: 457. 1947. MycoBank MB300023. See Crous et al.
(2006) for illustrations.
Basionym: Macrophoma phaseolina Tassi, Bull. Lab. Ort. bot.
Siena 4: 9. 1901.
≡ Tiarosporella phaseolina (Tassi) Aa, In: von Arx, Gen. Fungi Sporul.
Cult., Edn 3 (Vaduz): 208. 1981.
Additional synonyms listed by Holliday & Punithalingam (1988).
www.studiesinmycology.org
Sclerotia occurring in host tissue or in soil, black, smooth, hard,
100–1000 μm diam. Ascomata not reported. Conidiomata pycnidial,
stromatic, dark brown to black, solitary or gregarious, up to 200
μm diam, opening by a central ostiole, wall multilayered, cells dark
brown, thick-walled. Conidiophores reduced to conidiogenous cells
that are arranged along the inner lining of the conidioma, hyaline,
short obpyriform to subcylindrical, proliferating several times
percurrently near the apex, 6–12 × 4–6 μm, young conidiogenous
cells having a mucous layer that extends over the apex of the
developing conidium. Conidia ellipsoid to obovoid, (16–)20–24(–
32) × (6–)7–9(–11) μm; immature conidia hyaline, enclosed in
a mucous sheath that upon dehiscence encloses the top half of
the conidium, becoming two lateral tentaculiform, apical mucoid
appendages (type C, Nag Raj 1993); mature conidia becoming
medium to dark brown, with a granular outer layer that in some
cases appears pitted, without any mucoid appendages; conidium
hilum frequently with a marginal frill.
Cultures: Niger, Vigna minima, M. Ndiaye, CPC 11052, 11070.
Senegal, soil, M. Ndiaye, CPC 11079, 11085, 11106, 11108.
uganda, Eucalyptus sp., Jan. 1925, CBS 162.25; unknown, Zea
mays, Jun. 1933, S.F. Ashby, CBS 227.33.
Hosts: Plurivorous.
Known distribution: Cosmopolitan.
Notes: Although Macrophomina phaseolina can have apical mucoid
appendages as found in Tiarosporella (Sutton & Marasas 1976), it
is distinguished by having percurrently proliferating conidiogenous
cells, which are not seen in any species of Tiarosporella sensu Nag
Raj (1993), nor in those investigated by Crous et al. (2006), and
conidia that become dark brown at maturity, and the presence of
microsclerotia. Based on these differences (and in the absence
of authentic cultures of T. paludosa), Crous et al. (2006) chose to
retain the genus Macrophomina and the name M. phaseolina.
Neodeightonia Booth, in Punithalingam, Mycol. Pap. 19: 17.
1970 [1969]. MycoBank MB3450.
Type species: Neodeightonia subglobosa Booth, in Punithalingam,
Mycol. Pap. 119: 19. 1970 [1969].
Ascostromata immersed, dark brown to black, with a single
aparaphysate locule, wall composed of pseudoparenchymatic cells
many layers thick, asci developing amongst partially disintegrating
sterile thin-walled tissue in locule. Neck of ascostromata narrow,
opening by an apical ostiole, formed by the disintegration of the
central thin-walled cells. Pseudoparaphyses hyphae-like, septate,
constricted at the septa. Asci parallel, more or less separated
from one another by stromatic tissue, clavate to cylindric-clavate,
8-spored, bitunicate with a thick endotunica. Ascospores biseriate,
initially hyaline, brown when mature, oval to broadly ellipsoidal with
a single transverse septum, surrounded or not by a mucilagenous
sheath. Conidiomata brown to black, solitary or aggregated,
sometimes intermixed with ascomata, globose, uni- to multilocular,
stromatic, wall composed of dark-brown thick-walled textura
angularis. Paraphyses absent. Conidiogenous cells holoblastic,
hyaline, aseptate, cylindrical to sub-cylindrical. Conidia spherical
to globose, initially hyaline, pale to dark brown when mature, thickwalled, smooth to inely rough-walled with ine striations.
127
PhilliPs et al.
Notes: Neodeightonia was introduced by Booth (Punithalingam
1969). Von Arx & Müller (1975) transferred N. subglobosa
to Botryosphaeria, and because this is the type species of
the genus, they reduced Neodeightonia to synonymy under
Botryosphaeria. However, morphologically (based on the dark,
1-septate ascospores) and phylogenetically (Phillips et al. 2008),
this genus is distinguishable from Botryosphaeria, and the genus
was reinstated by Phillips et al. (2008). Punithalingam (1969)
referred to germ slits in the conidia. Crous et al. (2006) suggested
that these were in fact striations on the conidial wall, and that
more than one could occur per conidium, a feature conirmed
by Phillips et al. (2008). The striate walls suggest an afinity to
Lasiodiplodia. Nevertheless, Neodeightonia can be distinguished
from Lasiodiplodia by the absence of conidiomatal paraphyses.
Thus, conidial striations distinguish Neodeightonia from Diplodia,
and the absence of conidiomatal paraphyses distinguishes it from
Lasiodiplodia.
dNA phylogeny
N. phoenicum CBS 122528
87
68
86
N. phoenicum CBS 123168
N. phoenicum CBS 169.34
N. phoenicum HQ443209
N. subglobosa CBS 448.91
100
N. palmicola MFLUCC10‐0822
N. palmicola MFLUCC10‐0823
Botryosphaeria dothidea CBS 115476
Botryosphaeria cor5cis ATCC 22927
10 changes
The three species fall in three clades with N. palmicola distantly
related to the other two known species (Fig. 47).
Fig. 47. The single most parsimonious tree obtained from ITS sequences of
Neodeightonia species. Bootstrap values from 1000 replicates are given at the nodes.
Key to Neodeightonia spp.
The three species known in culture can be separated on conidial length:
1.
1.
Conidia less than 15 μm long, 9–12 μm long ..................................................................................................................... N. subglobosa
Conidia longer than 15 μm ...................................................................................................................................................................... 2
2.
2.
Conidia 15.5–21.5 μm long ................................................................................................................................................. N. phoenicum
Conidia never shorter than 17 μm, 17.5–24.5 μm long ......................................................................................................... N. palmicola
Species descriptions
Neodeightonia palmicola J.K. Liu, Phookamsak & K.D.
Hyde, Sydowia 62: 268. 2010. MycoBank MB518804. Fig.
48.
Ascomata uniloculate, immersed to erumpent in host tissue,
globose to subglobose, brown to dark brown, rounded at the base,
180–230 μm high (excluding the neck), 270–420 μm diam. Ostiole
circular, central, papillate. Peridium 26–55 μm wide, comprising
several layers of brown-walled cells, the outer stratum of 1–3
cells comprising thick, dark brown walls, the inner layer 3–5 cells,
textura angularis comprising pale brown to hyaline, thin-walled
cells. Pseudoparaphyses thin-walled, hyaline, frequently septate,
often constricted at the septa, up to 3–5 μm diam. Asci 8-spored,
bitunicate, issitunicate, with thick endotunica, clavate to cylindricalclavate, stipitate, apically rounded, with a well-developed ocular
apical chamber, arising from the base of ascoma, (80–)110–210(–
225) × 17–22.5(–24) μm (av. = 154.2 × 20.5 μm). Ascospores
obliquely uniseriate or irregularly biseriate, ellipsoidal-fusiform or
fusiform, widest in the middle, both ends obtuse, 1-celled, aseptate,
hyaline, smooth, thin-walled, with bipolar germ pores, surrounded
by a wing-like hyaline sheath, 23–31.5 × 8.5–12.5 μm (av. = 27 ×
10 μm). Conidioma (formed on WA on sterilised pine needles within
21–28 d) uniloculate, semi-immersed, solitary, globose, covered by
128
mycelium, up to 240 μm wide, wall 4–8 cell layers thick, composed
of dark brown thick-walled textura angularis, becoming thinwalled and hyaline toward the inner region. Conidiogenous cells
holoblastic, cylindrical to subcylindrical, hyaline, 9–20 × 3–6 μm.
Conidia initially hyaline, unicellular, ellipsoid to obovoid, thick-walled
with granular content, rounded at apex, occasionally truncate at
base, aging conidia becoming cinnamon to sepia, forming a single
septum, 17.5–24.5 × 9.5–12.5 μm (av. of 50 conidia = 21.5 × 11.0
μm).
Type: Thailand, Chiang Rai, Muang District, Khun Korn Waterfall,
on dead leaves of Arenga westerhoutii, 18 Dec. 2009, Jian-Kui Liu,
holotype MFLU10 0407.
Culture: MFLUCC10 0822 = CBS 136074 (ex-type).
Host: Arenga westerhoutii (Liu et al. 2010).
Known distribution: Thailand (Liu et al. 2010).
Notes: This species is unusual in having ascospores surrounded
by a mucilagenous sheath and pycnidial paraphyses, features not
seen in other species of Neodeightonia. Furthermore, there are no
striations on the conidia and it is also phylogenetically somewhat
divergent from other Neodeightonia species.
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 48. Neodeightonia palmicola. A–C. Asci. D–F Ascospores with apiculi at either end. Scale bars = 10 μm.
Neodeightonia phoenicum A.J.L. Phillips & Crous,
Persoonia 21: 43. 2008. MycoBank MB511708. Fig. 49.
= Macrophoma phoenicum Sacc., Annuar. R. Ist. Bot. Roma 4: 195. 1890.
≡ Diplodia phoenicum (Sacc.) H.S. Fawc. & Klotz, Bull. Calif. Agric. Exp.
Sta. 52: 8. 1932.
≡ Strionemadiplodia phoenicum (Sacc.) Zambett., Bull. trimest. Soc.
mycol. Fr. 70: 235. 1955 (1954).
Ascomata not reported. Conidiomata formed on pine needles
in culture pycnidial, stromatic, multiloculate, dark brown to
black, immersed in the host becoming erumpent when mature.
Paraphyses absent. Conidiogenous cells hyaline, smooth,
cylindrical, swollen at the base, holoblastic, proliferating
percurrently to form one or two annellations, or proliferating at the
same level giving rise to periclinal thickenings. Conidia ovoid to
ellipsoid, apex and base broadly rounded, widest in the middle to
upper third, thick-walled, initially hyaline and aseptate, becoming
dark brown and 1-septate some time after discharge from the
pycnidia, with melanin deposits on the inner surface of the wall
arranged longitudinally giving a striate appearance to the conidia,
(14.5–)15.5–21.5(–24) × (9–)10–12(–14) μm, 95 % conidence
limits = 18.6–19.5 × 11.2–11.8 μm (av. ± S.D. = 19.1 ± 1.7 × 11.5
± 1.1 μm), L/W ratio = 1.7.
Type: Spain, Catalonia, Tarragona, Salou, on Phoenix sp., F.
Garcia, holotype CBS H-20108.
Cultures: CBS 122528 (ex-type), CBS 123168, CBS 169.34.
Hosts: Phoenix spp. (Phillips et al. 2008).
Known distribution: Spain, USA (California) (Phillips et al. 2008).
Neodeightonia subglobosa C. Booth, Mycol. Pap. 119: 19.
1970 (1969). MycoBank MB318601. Fig. 50.
≡ Botryosphaeria subglobosa (C. Booth) Arx & E. Müll., Stud. Mycol. 9:
15. 1975.
= Sphaeropsis subglobosa Cooke, Grevillea 7: 95. 1879.
≡ Macrodiplodia subglobosa (Cooke) Kuntze, Revis. gen. pl. 3: 492.
1898.
≡ Coniothyrium subglobosum (Cooke) Tassi, Bulletin Labor. Orto Bot. de
R. Univ. Siena 5: 25. 1902.
www.studiesinmycology.org
Ascomata immersed, up to 300 μm wide, dark brown to black
with a single locule, aparaphysate, locule illed with disintegrating
sterile thin-walled tissue, amongst which the asci develop, neck
narrow, cone-shaped, opening by an apical ostiole. Asci bitunicate,
clavate, with well-developed apical chamber, 110–140 × 16–20
μm, 8-spored. Ascospores hyaline, aseptate, becoming brown
and 1-septate, ovoid to broadly ellipsoidal, smooth or with a inely
roughened surface, 20–26 × 7–10 μm. Conidiomata brown to black,
solitary or aggregated, sometimes intermixed with ascomata,
globose, uni- to multilocular, stromatic, up to 200 μm broad.
Paraphyses absent. Conidiogenous cells holoblastic, simple,
hyaline. Conidia spherical to globose, pale to dark brown when
mature, smooth to inely rough-walled, 9–12 × 6–9 μm.
Type: Sierra leone, Njala (Kori), on dead culms of Bambusa
arundinacea, 17 Aug. 1954, F.C. Deighton, holotype IMI 57769(f).
Culture: CBS 448.91 (ex-type).
Host: Bambusa arundinacea (Punithalingam 1969).
Known distribution: Sierra Leone (Punithalingam 1969).
Notes: According to Phillips et al. (2008) the type specimen of
Neodeightonia subglobosa contains only immature asci with hyaline
ascospores. However, Punithalingam (1969) clearly described and
illustrated the ascospores as brown and 1-septate. According to
Punithalingam (1969) this species is homothallic and forms asci
in culture.
Neofusicoccum Crous, Slippers & A.J.L. Phillips, Stud.
Mycol. 55: 247. 2006. MycoBank MB500870.
Type species: Neofusicoccum parvum (Pennycook & Samuels)
Crous, Slippers & A.J.L. Phillips, Stud. Mycol. 55: 248. 2006.
Synasexual morph: Dichomera-like.
Notes: Neofusicoccum was introduced by Crous et al. (2006) for
species that are morphologically similar to, but phylogenetically
distinct from Botryosphaeria and thus could no longer be
accommodated in that genus. Morphologically Neofusicoccum
129
PhilliPs et al.
Fig. 49. Neodeightonia phoenicum. A. Conidiogenous layer. B–E. Conidiogenous cells. F. Hyaline, aseptate conidia. G, H. Brown, 1-septate conidia with longitudinal striations.
Scale bars = 10 μm.
Fig. 50. Neodeightonia subglobosa. A. Globose conidiomata. B, C. Conidiogenous cells. D. Hyaline conidia. E. Mature, brown conidia with faint striations. Scale bars: A = 250
μm, B–E = 10 μm.
resembles Botryosphaeria and it can be dificult to separate
the two genera. The presence of a Dichomera synasexual
130
morph in Neofusicoccum has been used to differentiate it from
Botryosphaeria. However, not all Neofusicoccum species, or even
The Botryosphaeriaceae: genera and sPecies known from culTure
all isolates of any given species form such a synasexual morph, and
some isolates of B. dothidea have been reported to form dichomeralike conidia (Phillips et al. 2005, Barber et al. 2005). Paraphyses
have not been reported in conidiomata of any Neofusicoccum
species, but have been seen in most of the currently accepted
Botryosphaeria species. However, the similarity of paraphyses
to developing conidiogenous cells makes this feature dificult to
apply as a general rule to separate the two genera. Conidial L/W
ratios of the fusicoccum-like state are normally less than 4 and the
condidia are more ellipsoidal than in the deinitely fusiform ones of
Fusicoccum s. str.
Currently 22 species are recognised in Neofusicoccum and
they have been separated on the basis of conidial dimensions
and pigmentation, pigment production in culture media and
ITS sequence data, although taxonomic signiicance of some of
these characters have recently been questioned (Abdollahzadeh
et al. 2013). Species in some of the species complexes are
morphologically indistinguishable and are deined almost
exclusively on sequence of ITS often together with loci of other
genes. In some cases, multi-gene sequence data are essentail to
unambiguously identify the species.
Species in Neofusicoccum appear to be evolving quite rapidly
and this is relected in the appearance of distinct groups of isolates
in various geographic regions with ixed nucleotide differences
in ITS and EF1-α and other regions of the genome. Some have
already been described as new species (Pavlic et al. 2009) while
others are regarded as local variants (e.g. Lazzizera et al. 2008,
Spagnolo et al. 2010). Many of the species in Neofusicoccum are
morphologically similar and can be very dificult to distinguish from
one another. Neofusicoccum species are notoriously variable and
the full range of variability within species has not been determined
for most of the species. Nevertheless, an attempt has been made
to differentiate all species in the key presented here, but it must be
stressed that the outcome should be checked carefully against the
description of that species. Host association has been used in this
key for some species that have thus far not been found on any other
host. However, it must be borne in mind that this apparent host
specialisation may not be absolute. For example, N. vitifusiforme
was originally considered to be restricted to Vitis (van Niekerk et al.
2004), but was later isolated from rotting olive drupes in Southern
Italy (Lazzizera et al. 2008) and shown to be pathogenic on that
host. Some species may well be truly host speciic, such as N.
arbuti (Farr et al. 2005) and N. protearum (Denman et al. 2003),
which have not yet been found on any other host since they were
irst described.
Some species can be determined relatively easily. For
example, the conidia of N. macroclavatum and N. pennatosporum
are far longer than any other species in the genus and these two
species are easily differentiated on the shape and dimensions of
their conidia.
Key to Neofusicoccum spp.
1.
1.
Average length of conidia 30 μm or more ............................................................................................................................................... 2
Average length of conida less than 30 μm .............................................................................................................................................. 3
2.
2.
Conidia fusiform, up to 50 μm long .............................................................................................................................. N. pennatisporum
Conidia clavate-fusiform, length not exceeding 41 μm ................................................................................................ N. macroclavatum
3.
3.
Average length of conidia 25 μm or more ............................................................................................................................................... 4
Average length of conidia less than 25 μm ............................................................................................................................................. 7
4.
4.
Average conidial width less than 6 μm ................................................................................................................................... N. andinum
Average conidial width 7 μm or more ...................................................................................................................................................... 5
5.
5.
On Eucalyptus spp. ......................................................................................................................................................... N. eucalypticola
On hosts other than Eucalyptus .............................................................................................................................................................. 6
6.
6.
On Grevillea spp., conidial length not exceeding 32 μm ....................................................................................................... N. grevilleae
On hosts other than Grevillea, conidial length up to 40 μm ................................................................................................. N. protearum
7.
7.
Average length of conidia 20 μm or more ............................................................................................................................................... 8
Average length of conidia less than 20 μm ........................................................................................................................................... 15
8.
8.
Conidial L/W ratio 4 or more ................................................................................................................................................................... 9
Conidial L/W ratio less than 4 ............................................................................................................................................................... 11
9.
9.
Average conidial width 6 μm, L/W ratio 4 ..................................................................................................................... N. mediterraneum
Average conidial width less than 6, L/W ratio greater than 4 ................................................................................................................ 10
10. No yellow pigment, on Syzygium cordatum ........................................................................................................................ N. cordaticola
10. Yellow pigment on PDA, on hosts other than Syzygium ......................................................................................................... N. australe
11. Average conidial width 7 μm or more .................................................................................................................................................... 12
11. Average conidial width less than 7 μm .................................................................................................................................................. 13
12. Conidial width less than 11 μm ..................................................................................................................................... N. nonquaesitum1
12. Conidial width up to 12 μm ............................................................................................................................................ N. eucalyptorum1
www.studiesinmycology.org
131
PhilliPs et al.
13. Broad host range, average conidial width less than 6 μm .............................................................................................................. N. ribis
13. Narrow host range, average conidial width greater than 6 μm .............................................................................................................. 14
14. On Syzygium cordatum, from South Africa ........................................................................................................... N. kwambonambiense
14. On hosts other than Syzygium, from outside Africa .................................................................................................................... N. arbuti
15. Average conidial length less than 15 μm ........................................................................................................................... N. mangiferae
15. Average conidial length greater than 15 μm ......................................................................................................................................... 16
16. Conidial L/W ratio less than 3 ............................................................................................................................................................... 17
16. Conidial L/W ratio greater than 3 .......................................................................................................................................................... 18
17. Conidia fusoid to ovoid, L/W ratio 2.9 ............................................................................................................................... N. batangarum
17. Conidia ellipsoid to clavate, L/W ratio 2.4 ......................................................................................................................... N. viticlavatum
18. Average conidial length less than 18 μm ................................................................................................................................. N. parvum
18. Average conidial length greater than 18 μm ......................................................................................................................................... 19
19. Yellow pigment on PDA ............................................................................................................................................................. N. luteum
19. No yellow pigment ................................................................................................................................................................................. 20
20. Conidia L/W ratio 3.3 ......................................................................................................................................................... N. vitifusiforme
20. Conidia L/W ratio 3.5 ............................................................................................................................................................................. 21
21. Conidia fusiform to oval, average length greater than 19 μm ............................................................................................ N. umdonicola
21. Conidia fusiform to ellipsoid, average length less than 19 μm ............................................................................................. N. occulatum
1
Morphologically it is very dificult to separate these two species, but phylogenetically they are clearly distinct.
Notes: In this key we have used conidial morphology and
dimensions, cultural characteristics, host association and
geographic distribution to separate all the 22 described
Neofusicoccum species. But, it is important to consider that there is
overlap between species in some of those characters. Furthermore,
some characters are not stable between populations or individuals
of a given species. For example, not all isolates of N. luteum and
N. australe produce a yellow pigment in culture media and recently
we found this pigment production in some isolates of N. parvum.
Thus, deinitive identiication of most of the species is dependent
on the use sequence data for the ITS region alone, or more often in
combination with EF1-α sequence data.
dNA phylogeny
Phylogenetic analyses were done using ITS alone and ITS combined
with EF1-α. No EF1-α sequences are available for N. protearum,
N. corticosae and N. grevilleae. Thus, the phylogenetic position of
these species was deduced based on ITS phylogeny. Phylogenetic
analysis using ITS sequence data revealed 21 Neofusicoccum
species (Fig. 51). With the exception of N. ribis and N. occulatum,
all of the species in the N. ribis / N. parvum species complex can
be separated based on ITS alone (Fig. 51). However, the bootstrap
support was quite low for most of them. In the ITS phylogeny, D.
eucalypti and N. corticosae were grouped with N. vitifusiforme in
a single clade but with only 63 % support. On the other hand, in
the phylogenetic analysis based on ITS and EF1-α, D. eucalypti
was grouped with N. vitifusiforme (Fig. 52), which suggests that
D. eucalypti is a synasexual morph of N. vitifusiforme. Despite the
absence of N. corticosae in the ITS/EF1-α phylogeny, in the ITS
phylogenetic tree it is clear that N. corticosae is a synonym of N.
vitifusiforme.
132
Species descriptions
Neofusicoccum andinum (Mohali, Slippers & M.J. Wingf.)
Mohali, Slippers & M.J. Wingf., Stud. Mycol. 55: 247.
2006. MycoBank MB500871. See Mohali et al. (2006) for
illustrations.
Basionym: Fusicoccum andinum Mohali, Slippers & M.J. Wingf.,
Mycol. Res. 110: 408. 2006.
Ascomata not reported. Conidiomata stromatic, supericial,
produced abundantly on the surface of MEA at 25 °C, oozing
conidia after 30 d at 25 °C on MEA, solitary or botryose, globose,
(331–)374–597(–740) × (302–)339–557(–671) μm (av. of 50
conidiomata = 486 × 448 μm); conidiomata wall, composed of
brown textura angularis, 6–8 cell layers thick. Conidiogenous cells
holoblastic, hyaline, smooth, cylindrical, producing a single apical
conidium, proliferating enteroblastically, (8–)11–17(–23) × (1.5–)2–
2.5(–3) μm. Conidia hyaline, granular, clavate to slightly navicular,
apex obtuse and base truncate, 0–1 septa, (19–)23–31(–40) ×
(4–)5–6(–8) μm (av. of 50 conidia = 27 × 5.5 mm), L/W ratio = 4.84.
Dichomera synasexual morph not reported.
Culture characteristics: Colonies on MEA at 25 °C in darkness for
15 d luffy and lat becoming pale olivaceous-grey (surface) and
olivaceous buff (reverse), producing columns of mycelium reaching
the Petri dish lid after 30 d at 25 °C, reaching 80 mm diam on MEA
after 4 d in the dark at 25 °C. Cardinal temperatures for growth:
min 15 °C (reaching an average 24 mm diam), max < 35 °C, opt
20–30 °C.
Type: Venezuela, Mérida State, Merida, Mucuchies (3140 m),
Cordillera of Los Andes, on branches of Eucalyptus sp., Feb. 2003,
S. Mohali, holotype PREM 58238.
The Botryosphaeriaceae: genera and sPecies known from culTure
ATCC 58191
50
CBS
85
N. parvum
CBS 123634
N. corda5cola
CBS
CBS 123639
N. kwambonambiense
CBS
90
CBS 124924
CBSa
58
N. batangarum
CBS
CBS 115475
MUCC286
CBS 128008
70
N. ribis
N. occulatum
CBS
CBS 123645
N. umdonicola
N. grevilleae
CBS 129518
CBS 118531
CBS 118532
98
N. mangiferae
N. cor5cosae CPC 12926
N. vi5fusiforme CBS 110887
51
57
N. vi5fusiforme CBS 110880
63
N. vi5fusiforme
D. eucalyp5 WAC12401
D. eucalyp5 WAC12402
98
97
65
CBS 117453
CBS 117452
N. andinum
CBS 126655
PD301
N. nonquaesitum
CBS 116131
CBS 117090
100
N. arbu5
STE‐U 4361
CBS 114176
N. protearum
MUCC510
98
CBS 118223
WAC12445
CBS 121558
CBS 121718
60
N. vi5clavatum
85 CMW 6837
CMW 6853
89
CBS 110299
76 CAP037
97
CBS 115791
CMW 10126
100
88
N. macroclavatum
N. mediterraneum
CBS 112878
88 CBS 112977
100
N. penna5sporum
CBS 115766
CBS 115679
N. australe
N. luteum
N. eucalyptorum
N. eucalyp5cola
CBS 115476
B. dothidea
CBS 119047 B. cor5cis
Fig. 51. Single most parsimonious tree obtained from ITS sequence data of Neofusicoccum species. MP bootstrap values from 1000 pseudoreplicates are given at the nodes.
The tree is rooted to Botryosphaeria dothidea (CBS 115476) and B. corticis (CBS 119047).
www.studiesinmycology.org
133
PhilliPs et al.
CBS 121.26
100
CBS 115475
CBS 124924
54
N. batangarum
CBS 124923
CBS 123646
90
N. umdonicola
CBS 123645
CBS 123639
89
N. kwambonambiense
CBS 123641
CBS 123634
89
100
N. corda5cola
CBS 123635
ATCC 58191
N. parvum
CBS 110301
82
MUCC286
CBS 128008
72
N. ribis
N. occulatum
80
97 CBS 117453
N. andinum
CMW 13446
90
98
CBS 116131
CBS 117090
N. arbu5
CBS 126655
N. nonquaesitum
98 PD301
80
CBS 118223
WAC12445
98
N. macroclavatum
99 CBS 115791
CMW 10126
100
100
CBS 115766
CBS 115679
N. eucalyptorum
N. eucalyp5cola
88
CBS 118531
100
87
CBS 118532
CBS 121558
100
CBS 121718
82
100
CBS 112878
CBS 112977
N. mangiferae
N. mediterraneum
N. vi5clavatum
76
59
N. vi5fusiforme CBS 110887
N. vi5fusiforme CBS110880
100
100
75
D. eucalyp5 WAC12401
N. vi5fusiforme
D. eucalyp5 WAC12402
CMW 6837
100
CMW 6853
98
99
CBS 110299
CAP037
MUCC510
CBS 115476
N. australe
N. luteum
N. penna5sporum
B. dothidea
CBS 119047 B. cor5cis
10 changes
Fig. 52. Single most parsimonious tree obtained from combined ITS and EF-1α sequence data of Neofusicoccum species. MP bootstrap values from 1000 pseudoreplicates are
given at the nodes. The tree is rooted to Botryosphaeria dothidea (CBS 115476) and B. corticis (CBS 119047).
134
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 53. Neofusicoccum arbuti. A, B. Conidiogenous cells. C. Conidia. Scale bar A = 10 μm. Scale bar in A applies to B and C.
Cultures: CBS 117453 = CMW 13445 (ex-type), CBS 117452 =
CMW 13446.
Host: Eucalyptus sp. (Mohali et al. 2006).
Known distribution: Venezuela (Mohali et al. 2006).
Notes: Neofusicoccum andinum was introduced by Mohali et al.
(2006) for isolates from Eucalyptus sp. in Venezuela. There have
been no subsequent reports of this species. Based on phylogenetic
inference, (ITS, EF1-α) it is most closely related to N. arbuti and
N. nonquaesitum. The clavate to slightly navicular conidia of N.
andinum separate it from N. arbuti, which has obovoid to fusiform
conidia. Conidia of N. andinum are longer and narrower (27 × 5.5
μm) than those of N. nonquaesitum (23 × 7.5 μm).
Neofusicoccum arbuti (D.F. Farr & M. Elliott) Crous, Slippers
& A.J.L. Phillips, Stud. Mycol. 55: 247. 2006. MycoBank
MB500872. Fig. 53.
Basionym: Fusicoccum arbuti D.F. Farr & M. Elliott, Mycologia 97:
731. 2005.
Ascomata not reported. Conidiomata black, scattered, uniloculate
to multiloculate, 0.5–1.5 × 1.5–3 mm, becoming clumped irregular
in shape, papillate, stromata in longitudinal section of dark
brown textura intricata, locule walls of several layers of thickwalled, dark-brown textura angularis, becoming hyaline towards
conidiogenous region. Conidiophores reduced to conidiogenous
cells. Conidiogenous cells holoblastic, cylindrical to subobpyriform,
hyaline, discrete, determinate, occasionally indeterminate and
proliferating percurrently resulting in periclinal thickenings or rarely
indistinct annellations, lining inner wall of pycnidium, 9–16.5 ×
2.5–3.5 μm. Conidia obovoid, fusiform, base truncate, apex obtuse
to subobtuse, hyaline, guttulate, non-spetate, older conidia may
become brownish and septate before germination, on sterile twig
18.5–27.5 × 5.5–7.5 μm (av. of 235 conidia = 22.8 × 6.4 μm), L/W
ratio = 3.6. Spermatia cylindric to allantoid, lexuous or somewhat
dumbbell-shaped, hyaline, smooth, aseptate, 3.4–6.3 × 1–1.5
μm, av. of 37 spermatia = 4.3 ± 0.6 × 1.2 ± 0.14 μm. Dichomera
synasexual morph not reported.
www.studiesinmycology.org
Culture characteristics: Mycelium immersed, of branched, septate,
smooth, hyaline hyphae, becoming brown, constricted with age,
forming sparse, brown, thick-walled, intercalary, serial chlamydospores.
Colonies on PDA at 25 ºC in darkness for 8 d, light yellow to olive-grey
or olive-brown, darkest around plug, pigmentation extending about
2/3 of the colony width, outer area of colony white, reverse same,
surface mycelium cottony except around plug where the mycelium is
appressed, obscurely zonate, margin irregular, not producing yellow
pigments diffusing into the agar. Cardinal temperatures for growth:
opt. 25 °C, max. < 35 °C (25 mm at 15 °C, 63 mm at 20 °C, 70 mm at
25 °C, 37 mm at 30 °C, no growth at 35 °C).
Type: uSA, Washington, King Co., Seattle, Magnolia Bluffs,
isolated from cankers of Arbutus menziesii, Oct. 2003, collected by
M. Elliott, isolated by A. Rossman, holotype BPI 843970.
Cultures: AR 4036 = CBS 116131 (ex-type), CBS 117090 = UW 13.
Hosts: Arbutus menziesii (Paciic madrone) (Farr et al. 2005),
Vaccinium spp. (Espinoza et al. 2009).
Known distribution: Western USA and Canada from British
Columbia to California (Farr et al. 2005), Chile (Espinoza et al.
2009).
Notes: This species is phylogenetically most closely related to
N. andinum and N. nonquasetinum. The three species can be
distinguished on the shapes and dimensions of their conidia. See
notes for N. andinum.
Neofusicoccum australe (Slippers, Crous & M.J. Wingf.)
Crous, Slippers & A.J.L. Phillips, Stud. Mycol. 55: 248. 2006.
MycoBank MB500873. Fig. 54.
Basionym: Fusicoccum australe Slippers, Crous & M.J. Wingf.,
Mycologia 96: 1035. 2004.
= Botryosphaeria australis Slippers, Crous & M.J. Wingf., Mycologia 96: 1035.
2004.
Ascostromata erumpent through the host bark, 1.2 mm diam.
Ascomata pseudothecial, forming botryose aggregates of 2−10,
135
PhilliPs et al.
Fig. 54. Neofusicoccum australe. A, B. Asci with ascospores. C. Conidiomata on pine needles in culture. D, E. Conidiogenous cells. F. Conidia. Scale bars: A, B, D–F = 10 μm,
C = 1 mm.
sometimes solitary, globose with a central ostiole, papillate or
not, embedded with only the papilla emerging up to 2/3 emergent,
black, 100−300 μm; pseudothecial wall comprising 5−8 layers of
textura angularis, outer region of dark brown or brown cells, inner
region 3−6 layers of hyaline cells lining the locules. Asci bitunicate,
clavate, 8-spored, 60−125 × 16−26 μm. Pseudoparaphyses iliform,
septate, rarely branched, 3−4 μm wide. Ascospores fusoid to ovoid,
unicellular, hyaline, smooth with granular contents, biseriate in the
ascus, 20−23(−25) × 7−8(−9) μm (av. of 50 ascospores = 21.9 × 7.6
μm), L/W = 2.9. Conidiomata stromatic, supericial, globose, mostly
solitary. Conidiogenous cells holoblastic, hyaline, subcylindrical,
phialidic with periclinal thickenings or proliferating percurrently
with 1−4 annellations, 10−14 × 2−3 μm. Conidia hyaline, fusiform,
base subtruncate to bluntly rounded, non-septate, rarely forming
a septum before germination, smooth with granular contents,
(18−)23−26(−30) × 5−6(−7.5) μm (av. of 240 conidia = 24.7 × 5.1
μm), L/W ratio = 4.8. Spermatia not seen. Dichomera synasexual
morph: Conidia subglobose, obpyriform or obovoid, apex obtuse,
base truncate to bluntly rounded, (9.5–)10.5–14.5(–17.5) × (7–)
9–11 μm, pale brown when immature with 1–2 transverse septa,
0–1 longitudinal septa, and 0–2 oblique septa, becoming dark
brown and muriform when mature with 1–3 transverse septa, 1–4
longitudinal septa, and 0–3 oblique septa.
Culture characteristics: Colonies buff to light primrose, light
yellowish pigment diffusing into the medium, most noticeably at
15–20 °C in the dark, becoming olivaceous buff to olivaceous-grey
after 5–6 d with sparse to moderately dense, appressed mycelial
mat in centre with sparse tufts of aerial mycelium around the edges,
136
margin smooth. Optimum temperature for growth 25 °C, colony
reaching 48 mm diam on PDA after 4 d at 25 °C in the dark.
Type: Australia, Victoria, Batemans Bay, Acacia sp., M.J. Wingield,
holotype PREM 57589.
Cultures: CMW 6837 (ex-type), CMW 6853.
Hosts: Acacia sp. (Slippers et al. 2004c), Acacia cochlearis, Acacia
rostellifera, Agonis lexuosa (Dakin et al. 2010), Allocasuarina
fraseriana, Banksia grandis, Callitris preissii, Citrus sp. (Adesemoye
& Eskalen 2011), Chamaecyparis lawsoniana, Picea abies, Pinus
pinaster, P. pinea, Sequoia sempervirens, Taxus baccata, Thuja
plicata, Thujopsis dolabrata (Alves et al. 2013) Elaeocarpus
holopetalus (Cunnington et al. 2007), Eucalyptus gomphocephala,
Eucalyptus marginata, Santalum acuminatum (Taylor et al. 2009),
Eucalyptus globulus (Burgess et al. 2005, 2006), Eucalyptus
diversicolor (Barber et al. 2005), Malus domestica, Prunus
domestica, Prunus dulcis, Prunus persica, Prunus salicina, Pyrus
communis (Damm et al. 2007, Slippers et al. 2007, Gramaje et al.
2012), Olea europaea (Lazzizera et al. 2008), Persea americana
(McDonald et al. 2009, Auger et al. 2013), Phoenix canariensis
(Cunnington et al. 2007), Pistacia vera (Armengol et al. 2008),
Protea cynaroides, Protea sp. (Denman et al. 2003 (as N. luteum),
Marincowitz et al. 2008), Quercus robur (Barradas et al. 2013),
Rubus sp. (Phillips et al. 2006), Salix sp. (Cunnington et al. 2007),
Syzygium cordatum (Pavlic et al. 2007), Vaccinium corybosum
(Cunnington et al. 2007, Espinoza et al. 2009); Vitis vinifera (van
Niekerk et al. 2004, Úrbez-Torres et al. 2006b, Úrbez-Torres &
The Botryosphaeriaceae: genera and sPecies known from culTure
Gubler 2009, Martin et al. 2011, White et al. 2011, Besoain et al.
2013), Widdringtonia nodilora (Slippers et al. 2005b).
Known distribution: Australia (Slippers et al. 2004c, Barber et al.
2005, Burgess et al. 2005, Cunnington et al. 2007, Taylor et al.
2009), Chile (Espinosa et al. 2009, Auger et al. 2013, Besoain et
al. 2013), Italy (Lazzizera et al. 2008), Portugal (van Niekerk et al.
2004, Phillips et al. 2006, Alves et al. 2013, Barradas et al. 2013),
South Africa (Damm et al. 2007, Denman et al. 2003, Slippers et al.
2005b, Pavlic et al. 2007, Slippers et al. 2007, White et al. 2011),
Spain (Armengol et al. 2008, Marincowitz et al. 2008, Martin et al.
2011, Gramaje et al. 2012), Spain (Tenerife) (Marincowitz et al.
2008), Uraguay (Perez et al. 2010), USA (California) (Úrbez-Torres
et al. 2006b, McDonald et al. 2009, Úrbez-Torres & Gubler 2009,
Adesemoye & Eskalen 2011).
Notes: This is a sister species to N. luteum and the two differ
mainly in the intensity of the yellow pigment produced in culture,
although conidia of N. australe are generally larger (24.7 × 5.1 μm,
L/W ratio = 4.8) than those of N. luteum (19.7 × 5.6 μm, L/W ratio =
3.6). Slippers et al. (2004) irst reported this species from Australia
and South Africa, and mentioned a single isolate from pistachio in
Italy. Nevertheless, they regarded this as a species restricted to the
Southern Hemisphere. In their study of “Botryosphaeria” species
on grapevines, van Niekerk et al. (2004) included an isolate of
N. australe from Robinia pseudoacacia collected in Portugal. An
isolate of a “Botryosphaeria” from Rubus sp., also in Portugal
was also identiied as N. australe (Phillips et al. 2006) and it has
been isolated frequently from Oleae europaea in southern Italy
(Lazzizera et al. 2008). These reports thus suggest that N. australe
is a widespread and plurivorous species. Interestingly, N. australe
is the dominant associate of natural woody vegetation in the southwest of Western Australia, while N. parvum, a species commonly
isolated elsewhere in the world, is only found associated with dying
trees in the peri-urban landscape. Isolates from olives in southern
Italy consistently differ from typical isolates of N. australe by 1 bp in
their ITS and 3 bp in their EF1-α sequences (Lazzizera et al. 2008).
Neofusicoccum batangarum Begoude, Jol. Roux &
Slippers, sp. nov. MycoBank MB514013. See Didier
Begoude et al. (2010) for illustrations.
≡ Neofusicoccum batangarum Begoude, Jol. Roux & Slippers, Mycol.
Prog. 9: 113. 2010. Nom. inval., Art 37.7.
Ascomata not reported. Conidiomata stromatic produced on pine
needles within 14 d, solitary and covered by mycelium, initially
embedded, 3/4 erumpant through the pine needles at maturity,
obpyriform to ampulliform with a central and circular ostiole at the
neck, unilocular, locule wall thick consisting of two layers: an outer
layer of dark brown textura cells, lined with an inner layer of of
thin-walled, hyaline cells. Conidiophores reduced to conidiogenous
cells. Conidiogenous cells holoblastic, hyaline, smooth, cylindrical,
proliferating percurrently, sometimes forming a periclinal thickening,
(11–)12.5–19(–27) × (2–)2.5–3 (–3.5) μm. Conidia non-septate,
hyaline, smooth, fusoid to ovoid, thin-walled, (12–)14–17.5(–20) ×
(4–)4.5–6(–6.5) μm (av. of 50 conidia = 15.5 × 5.5 μm), L/W ratio
= 2.9. Spermatia not reported. Dichomera synasexual morph not
reported.
Culture characteristics: Colonies on MEA forming concentric rings,
mycelium white and immersed at the leading edge, becoming
www.studiesinmycology.org
smokey grey to grey-olivaceous from the old ring after 5 d on MEA.
Cardinal temperatures for growth: opt 25 °C (covering the 90 mm
diam Petri plate after 4 d on MEA in the dark), little growth observed
at 10 and 35 °C.
Type: cameroon, Kribi, Beach, isolated from healthy branches
of Terminalia catappa, Dec. 2007, D. Begoude & J. Roux, a dry
culture on pine needles, holotype PREM 60285.
Cultures: CMW 28363 = CBS 124924 (ex-type), CMW 28320 =
CBS 124923.
Hosts: Terminalia catappa (Didier Begoude et al. 2010), Schinus
terebinthifolius (Shetty et al. 2011).
Known distribution: Kribi, Cameroon (Didier Begoude et al. 2010),
Florida, USA (Shetty et al. 2011).
Notes: The original description of N. batangarum is invalid, as no
type specimen was designated, only an “ex-paratype specimen”,
which was in fact a typing error, as it should have read “holotype”.
This issue is now addressed, and the name validly published.
Based on ITS and EF1-α sequence data, N. batangarum is
most closely related to N. ribis and can be distinguished from it
based only on four ixed unique single nucleotide polymorphisms
(SNPs) in four gene regions (ITS, EF1-α, β-tubulin and BOTF15).
It can be discriminated from other species in the N. ribis / N.
parvum complex by the formation of concentric rings on MEA, a
characteristic that has not been observed in any other species of
the complex. Furthermore, the small conidia (15.5 × 5.5 μm, L/W
ratio = 2.9) clearly distinguish this species from all other species
in the N. ribis / N. parvum complex. Shetty et al. (2011) isolated
N. batangarum from seeds of Schinus terebinthifolius and showed
that it is an aggressive pathogen and potential biocontrol agent of
this invasive exotic tree.
Neofusicoccum cordaticola Pavlic, Slippers & M.J. Wingf.,
Mycologia 101: 643. 2009. MycoBank MB512498. See Pavlic
et al. (2009) for illustrations.
Ascomata not reported. Neofusicoccum cordaticola is
morphologically similar to other species in the N. parvum / N. ribis
species complex. Conidia hyaline, unicellular, narrowly fusiform
to oval, apices rounded, 18–28 × 4.5–7 μm (av. of 150 conidia
= 23.3 × 5.3 μm), L/W = 4.3. It differs from other species in the
N. parvum / N. ribis complex by uniquely ixed nucleotides in ive
nuclear loci: ITS (EU821898) position 141 (C), 372 (G) and 416
(C); EF1-α (EU821868) positions 58 (C) and 221 (C); β-tubulin
(EU821838) position 32 (T), 96 (T) and 316 (G); locus BotF15
(EU821802) position 121 (T) and 122 (C); RNA polymerase II
subunit (EU821928) positions 100 (A), 112 (T), 265 (A) and 409
(C).
Type: South Africa, Kwazulu-Natal Province, Sodwana Bay, on
symptomless branches and leaves, dying branches and pulp of ripe
fruits of Syzygium cordatum, Mar. 2002, D. Pavlic, a dry culture on
pine needles holotype PREM 60066.
Cultures: CMW 13992 = CBS 123634 (ex-type), CMW 14056 =
CBS 123635.
137
PhilliPs et al.
Host: Syzygium cordatum (Pavlic et al. 2009).
Known distribution: South Africa (Pavlic et al. 2009).
Notes: Although variation in conidial dimensions is evident in the N.
parvum / N. ribis complex, it is dificult to separate all the species in
this complex. Furthermore, precise identiication of these species is
dependent on DNA sequence comparisons.
Neofusicoccum eucalypticola (Slippers, Crous & M.J.
Wingf.) Crous, Slippers & A.J.L. Phillips, Stud. Mycol. 55:
248. MycoBank MB500874. See Slippers et al. (2004) for
illustrations.
Basionym: Fusicoccum eucalypticola Slippers, Crous & M.J.
Wingf., Stud. Mycol. 50: 351. 2004.
= Botryosphaeria eucalypticola Slippers, Crous & M.J. Wingf., Stud. Mycol.
50: 351. 2004.
Ascomata pseudothecia, mostly solitary, sometimes forming
a botryose aggregate of 2–3 structures, globose with a central
ostiole, papillate, embedded with 1/3–2/3 emerging, black,
160–340 μm diam pseudothecial wall comprising 5–8 layers of
textura angularis, outer region of dark or medium brown cells,
inner region of hyaline cells lining the locule. Asci bitunicate,
clavate, 8-spored, 70–110 × 20–25 μm. Pseudoparaphyses
iliform, septate, rarely branched in the upper parts, 2–4 μm wide.
Ascospores fusoid to ovoid, unicellular, hyaline, smooth with
granular contents, biseriate in the ascus, 20–22(–23.5) × 7–8 μm
(av. of 50 ascospores = 21.7 × 7.6 μm), L/W 2.8. Conidiomata
formed on WA on sterilised pine needles within 7–21 d, stromatic,
supericial, globose, mostly solitary, and covered by mycelium.
Conidia produced in culture fusiform to rod-shaped, often bent
or irregularly shaped, apex obtuse, bases subtruncate to bluntly
rounded, hyaline, unicellular, sometimes forming 1–2 transverse
septa before germination, smooth with inely granular contents,
(20–)25–27(–35) × (5–)7–9(–10) μm (av. of 135 conidia = 26.3
× 7.2 μm), L/W = 3.6. Spermatia not reported. Dichomera
synasexual morph not reported.
Culture characteristics: Colonies white to buff or olivaceous-grey,
sometimes becoming olivaceous-black at the centre after 7 d,
with a dense mat of aerial mycelium, edges smooth to crenulate,
sometimes not reaching the edge of the plate. Optimum temperature
for growth 25 °C, reaching 34–43 mm radius on PDA after 4 d at
25 °C in the dark.
Type: Australia, Victoria, Orbost, on Eucalyptus grandis, 2001,
M.J. Wingield, holotype PREM 57848.
Culture: CBS 115679 = CMW 6539 (ex-type), CBS 115766 = CMW
6217.
Hosts: Eucalyptus spp. (Slippers et al. 2004, Burgess et al. 2006).
Known distribution: Australia (Slippers et al. 2004).
Notes: Neofusicoccum eucalypticola is phylogenetically most
closely related to N. eucalyptorum, and the two species can be
separated on the shapes and dimensions of their conidia in culture.
Thus, conidia of N. eucalypticola are fusiform and longer (25–27
μm) than the ovoid to clavate conidia of N. eucalyptorum, which are
138
20–23 μm long. Slippers et al. (2004) found that N. eucalyptorum
was the dominant species collected from Eucalyptus species in
eastern Australia.
Neofusicoccum eucalyptorum (Crous, H. Sm. ter. & M.J.
Wingf.) Crous, Slippers & A.J.L. Phillips, Stud. Mycol. 55:
248. 2006. MycoBank MB500875. See Smith et al. (2001)
for illustrations.
Basionym: Fusicoccum eucalyptorum Crous, H. Sm. ter. & M.J.
Wingf., Mycologia 93: 280. 2001.
= Phoma australis Cooke, Grevillea 15: 17. 1886.
≡ Idiocercus australis (Cooke) H.J. Swart, Trans. Brit. Mycol. Soc. 90:
283. 1988.
= Botryosphaeria eucalyptorum Crous, H. Sm. ter. & M.J. Wingf., Mycologia
93: 280. 2001.
Ascomata embedded in host tissue, up to 300 μm diam, becoming
erumpent, solitary or botryose, stromatic, dark brown to black, with
central, black ostioles. Asci clavate, 8-spored, bitunicate with a welldeveloped apical chamber, 70−140 × 15−21 μm. Pseudoparaphyses
iliform. Ascospores irregularly biseriate, hyaline, aseptate, granular
contents, becoming light brown with age, prominantly inequilateral
when young, less so when mature, fusoid, widest in the middle,
base obtuse, apex obtuse or subobtuse, (20−)23−26(−28) ×
(7−)8−9(−11) μm. Conidiomata embedded in host tissue, solitary
or botryose, stromatic, globose, up to 450 μm diam, wall 6−8 layers
thick, composed of brown textura angularis, becoming hyaline
towards the inner region. Conidiogenous cells holoblastic, hyaline,
subcylindrical, proliferating percurrently with 1−3 annellations, or
proliferating at the same level with minute periclinal thickenings,
10−25 × 3.5−6 μm. Conidia hyaline, granular, ovoid to slightly
clavate, apex obtuse, tapering towards a subtruncate or bluntly
rounded base, sometimes with a minute marginal frill visible on
younger conidia, (20−)22−25(−28) × (6−)7−8(−9) μm in vivo,
(18−)20−23(−25) × 7−8(−12) μm in vitro. Spermatia not reported.
Dichomera synasexual morph not reported.
Culture characteristics: Colonies on MEA iron-grey (reverse) and
olivaceous-grey (surface) with extensive grey aerial mycelium,
and smooth margins, attaining a radius of 21–24 mm after 4 d in
darkness at 25 °C. Cardinal temperatures for growth: min > 5 °C,
max < 35 °C, opt 25 °C.
Type: of sexual morph: South Africa, Mpumalanga, Sabie,
Eucalyptus grandis, 1995, H. Smith, holotype PREM 56603; of
asexual morph: South Africa, Mpumalanga, Sabie, E. grandis,
1995, H. Smith, holotype PREM 56604.
Cultures: The ex-type isolate was not designated in the original
publication and could not be traced. Slippers et al. (2004b) regarded
the following as representatives CBS 115791 = CMW10125, CMW
10126.
Hosts: Eucalyptus spp. (Burgess et al. 2006, Smith et al. 2001,
Slippers et al. 2004b, Perez et al. 2010), Myrceugenia glaucescens,
Myrrhinium atropurpureum var. octandrum, Blepharocalyx
salicifolius (Perez et al. 2010).
Known distribution: Australia (Slippers et al. 2004b), South Africa
(Smith et al. 2001), Uraguay (Perez et al. 2010).
The Botryosphaeriaceae: genera and sPecies known from culTure
Notes: Neofusicoccum eucalyptorum is a sister species to N.
eucalypticola and the two can be separated on the shapes
and dimensions of conidia formed in culture. See notes for N.
eucalypticola.
Neofusicoccum grevilleae Crous & R.G. Shivas, Persoonia
26: 117. 2011. MycoBank MB560162. See Crous et al. (2011)
for illustrations.
Leaf spots medium brown, situated along leaf margins, surrounded
by a dark red-brown border, spots extending to the midrib, up to 7 mm
diam, and up to 2 cm long. Conidiomata amphigenous, stromatic,
up to 200 μm diam (on sterilised pine needles). Wall consisting of
3–5 layers of brown textura angularis. Conidiophores lining the
inner layer of conidioma, hyaline, smooth, 0–1-septate, 15–30 ×
3–5 μm. Conidiogenous cells holoblastic, integrated, doliiform to
subcylindrical, phialidic, proliferating 2–3 times percurrently near
apex, 15–25 × 3–4 μm. Conidia hyaline, smooth, thin-walled, with
granular cytoplasm, fusoid-ellipsoidal, widest in middle or in upper
third of conidium, apex subobtuse, base truncate, (20–)25–28(–32)
× (6–)7–8(–10) μm (av. size of conidia = 25.7 × 7.5 μm), L/W = 3.4.
Culture characteristics: Colonies after 14 d at 25 °C in darkness
lat, spreading, with abundant, grey aerial mycelium, covering the
dish after 7 d, on PDA, OA and MEA iron-grey, sporulating poorly
on water agar supplemented with sterile pine needles. Spermatia
not reported. Dichomera synasexual morph not reported.
Type: Australia, Queensland, Brisbane, on leaves of Grevillea
aurea, 14 Jul. 2009, P.W. Crous & R.G. Shivas, holotype CBS
H-20578.
Cultures: CBS 129518 = CPC 16999 (ex-type).
Host: Grevillea aurea (Crous et al. 2011).
Known distribution: Australia, Western Australia (Crous & Shivas
2011).
Notes: Based on ITS sequence data, N. grevilliae is most closely
related to the N. ribis / N. parvum complex, but conidia of N.
grevilliae (25.7 × 7.5 μm) are larger than those of all seven species
in that complex.
Neofusicoccum kwambonambiense Pavlic, Slippers & M.J.
Wingf., Mycologia 101: 643. 2009. MycoBank MB512499.
See Pavlic et al. (2009) for illustrations.
Ascomata not reported. Neofusicoccum kwambonambiense is
morphologically similar to other related species in the N. parvum /
N. ribis species complex. Conidia hyaline, unicellular, fusiform to
ellipsoid, apices rounded 16–28 × 5–8 mm (av. 140 conidia 22.3 ×
6.3 μm), L/W 3.6. It differs from other species in the N. parvum / N.
ribis complex by uniquely ixed nucleotides in four nuclear loci: ITS
(EU821900) position 163 (T) and 173 (G); β-tubulin (EU821840)
position 175 (T), 235 (A) and 251 (A); locus BotF15 (EU821804)
position 87, and 172; RNA polymerase II subunit (EU821930)
positions 49 (G), 382 (A), 421 (A) and 526 (C). Spermatia not
reported. Dichomera synasexual morph not reported.
www.studiesinmycology.org
Type: South Africa, Kwazulu-Natal Province, Kwambonambi, on
symptomless branches and leaves, dying branches and pulp of ripe
fruits of Syzygium cordatum, Mar 2002, D. Pavlic, a dry culture on
pine needles, holotype PREM 60067.
Cultures: CMW 14023 = CBS 123639 (ex-type), CMW 14140 =
CBS 123641.
Host: Syzygium cordatum (Pavlic et al. 2009).
Known distribution: South Africa (Pavlic et al. 2009).
Note: See notes for N. cordaticola.
Neofusicoccum luteum (Pennycook & Samuels) Crous,
Slippers & A.J.L. Phillips, Stud. Mycol. 55: 248. 2006.
MycoBank MB500876. Fig. 55.
Basionym: Fusicoccum luteum Pennycook & Samuels, Mycotaxon
24: 456. 1985.
= Botryosphaeria lutea A.J.L. Phillips, Sydowia 54: 70. 2002.
Ascomata initially immersed, later becoming erumpent through
the host tissue, black, < 0.5 mm diam, uni- or multilocular, locules
spherical to ovoid, 150–200 μm diam, ascomata and conidiomata
often formed in the same stroma, opening through a nonperiphysate
ostiole, with a short neck, wall consisting of 8–12 layers of dark
brown to black, thick-walled cells, forming pseudoparenchymatic
textura angularis, up to 60 μm thick, with 3–4 layers of thinwalled, hyaline cells lining the cavity. Asci bitunicate, cylindrical,
to clavate, stipitate, 84–176 × 16–24 μm, 8-spored, associated
with ilamentous pseudoparaphyses. Pseudoparaphyses hyaline,
septate, branched, 2–3.5 μm wide. Ascospores irregularly biseriate
in the ascus, hyaline, guttulate, smooth, aseptate, oval to broadly
fusiform, widest in the middle or upper third of the ascospore,
tapering to the obtuse base and apex 18–22.5(–24) × 7.5–12
μm. Conidiomata frequently formed on the same stromata as the
ascomata, stromatic, separate or conluent, dark brown to black,
uni- or multilocular immersed in the host, sub-peridermal, locules
up to 150 μm diam, walls consisting of a dark brown textura
angularis, becoming smaller, thinner-walled and hyaline towards the
conidiogenous region. Ostioles papillate, circular. Conidiophores
hyaline, smooth, thin-walled, rarely branched at the base,
cylindrical, formed from the cells of the inner locule wall, 8–19 × 3–4
μm. Conidiogenous cells holoblastic, discrete, integrated, hyaline,
smooth, cylindrical, producing the irst conidium holoblastically and
subsequent conidia enteroblastically, proliferating percurrently with
2–3 indistinct percurrent proliferations, or proliferating internally
forming typical phialides (sensu Sutton, 1980) and periclinal
thickening, (6–)8–16(–18) × (2.5–)3–4(–4.5) μm. Conidia hyaline,
thin-walled, aseptate, smooth, ellipsoidal, widest in the middle
or upper third of the conidium, apex subobtuse, base truncate
(15–)16.5–22.5(–24) × 4.5–6(–7.5) μm, 95 % conidence limits of
242 conidia = 19.4–19.9 μm (av. ± S.D. of 242 conidia = 19.7 ±
1.8 × 5.6 ± 0.6 μm), L/W ratio = 3.6 ± 0.5 with 95 % conidence
limits = 3.5, often with a minute basal frill. Spermatia hyaline, rodshaped to reniform with either truncate or rounded ends 3–5 × 1
μm. Dichomera synasexual morph not reported.
Type: of sexual morph: Portugal, Estremadura, Oeiras, Quinta
do Marquês, on cane of Vitis vinifera cv. Galego Dourado, Mar.
139
PhilliPs et al.
Fig. 55. Neofusicoccum luteum. A, B. Cultures of N. luteum on PDA after 2 days (A) and 4 days (B) of incubation at 25 ºC. A pale yellow pigment is produced at irst (A) that
later becomes violaceous (B). C. Vertical section through an ascoma. D. Asci stained with cotton blue. E. Ascus with eight ascospores. F. Conidiogenous cells. G. Conidia.
Scale bars: C = 100 μm, D = 50 μm, E–G = 10 μm.
1996, A.J.L. Phillips, holotype LISE 94070; of asexual morph:
New Zealand, Bay of Plenty, Te Puke, No 1 Road, DSIR Research
Orchard, from lesions on ripe fruit of Actinidia deliciosa, 6 Oct.
1982, S.R. Pennycook, holotype PDD 45400.
Cultures: PDDCC 8004 = ATCC 58193 (ex-type of asexual morph) /
CBS 110299 (ex-type of sexual morph), CAP037.
Hosts: Plurivorous including Actinidia chinensis, Actinidia deliciosa
(Gadgil et al. 2005, Pennycook & Samuels 1985), Banksia
sp., Buckinghamia sp. (Denman et al. 2003), Chamaecyparis
lawsoniana, Cupressus sempervirens, C. lusitanica, Juniperus
communis, Pinus pinea, Sequoia sempervirens, Thujopsis dolabrata,
Thuja plicata (Alves et al. 2013), Chrysanthemoides monilifera
(Cunnington et al. 2007), Crataegus mexicana (Adesmoye et al.
2013), Diospyros kaki (Gadgil et al. 2005), Eucalyptus sp. (Denman
et al. 2003), Ficus microcarpa (Mayorquin et al. 2012), Fraxinus
angustifolia (Phillips et al. 2002), Malus domestica (Gadgil et al.
2005), Olea europaea (Sergeeva et al. 2009), Persea americana
(McDonald & Eskalen 2011), Protea cynaroides (Denman et al.
2003), Pyrus communis (Gadgil et al. 2005), Pyrus pyrifolia (Gadgil
et al. 2005), Quercus robur (Barradas et al. 2013), Rhododendron
sp. (Varela et al. 2011), Salix fragilis (Cunnington et al. 2007), Salix
magniica (Gadgil et al. 2005), Sophora japonica (Phillips et al.
2002), Syzygium cordatum (Pavlic et al. 2007), Vitis vinifera (van
Niekerk et al. 2004, Úrbez-Torres et al. 2006b).
Known distribution: eastern Australia (Denman et al. 2003,
Cunnington et al. 2007, Sergeeva et al. 2009), USA (California)
140
(Úrbez-Torres et al. 2006b, McDonald & Eskelen 2011, Mayorquin
et al. 2012), Italy (Lazzizera et al. 2008), New Zealand (Gadgil et al.
2005, Pennycook & Samuels 1985), Portugal (Phillips et al. 2002,
Alves et al. 2013, Barradas et al. 2013), South Africa (Denman et
al. 2003, van Niekerk et al. 2004, Pavlic et al. 2007), Spain (Varela
et al. 2011), Uruguay (Peréz et al. 2010).
Notes: The morphology of the conidiomata varies depending on
the substrate on which this species is found. Thus, Phillips et al.
(2002) reported that on grapevine canes they were thick-walled
and eustromatic while on leaves they were thin-walled and
globose. Phylogenetically it groups with N. australe. See notes for
N. australe.
Neofusicoccum macroclavatum (T.I. Burgess, Barber &
Hardy) T.I. Burgess, Barber & Hardy, Stud. Mycol. 55: 248.
2006. MycoBank MB500877. See Burgess et al. (2005) for
illustrations.
Basionym: Fusicoccum macroclavatum T.I. Burgess, Barber &
Hardy, Austral. Pl. Pathol. 34: 562. 2005.
Ascomata not reported. Conidiomata stromatic, formed on water
agar on sterilised pine needles within 21 d, supericial, globose,
mostly solitary, 1–2 mm diam, covered with mycelium, single or
multiloculate. Conidiogenous cells holoblastic, hyaline, subcylindrical to cylindrical to ampuliform, proliferating percurrently
with up to 2 annellations, (4.5–)5.5–10.5(–13) × 2–3.5(–4.5) μm.
The Botryosphaeriaceae: genera and sPecies known from culTure
Conidia produced in culture on pine needles elongate-clavate to
fusiform, base subtruncate to bluntly rounded, hyaline, unicellular,
occasionally 1–4-septate when mature or before germination,
smooth wall with ine granular contents, (19–)25–35(–41) × (5–)
6–8(–10) μm (av. of 125 conidia = 30.3 × 7.1 μm), L/W = 4.2.
Spermatia observed in culture hyaline, cylindrical, sub-cylindrical
or clavate, base truncate with rounded apex, 4.5–9.5(–13) ×
2–3.5(–4.5) μm (av. of 50 spermatia = 7.7 × 2.6 μm). Dichomera
synasexual morph not reported.
Culture characteristics: Colonies on half strength PDA initially white
to buff turning olivaceous-grey within 7 d and becoming black with
age, moderately dense, appressed mycelial mat with irregular
very dense aerial aggregations. Optimum temperature for growth
25 °C, reaching 53 mm in diameter on half strength PDA after 4 d
at 25 °C in the dark.
Cultures: Cultures linked to the type could not be located and
probably do not exist. Slippers et al. (2005) regarded the following
as representatives: CBS 118531 = CMW7024, CBS 118532 =
CMW7797.
Host: Mangifera indica (Slippers et al. 2005).
Known distribution: Australia, India (Slippers et al. 2005).
Notes: Phylogenetically this species is closely related to N.
eucalypticola and N. eucalyptorum, but the conidia of N.
mangiferae are distinct from all other Neofusicoccum spp. in their
shorter average length (~13–14 mm) and smaller length/width ratio
(2–2.5). The conidia often become 1- or 2-septate, light brown with
distinctly darker middle cells. This feature is shared with N. parvum
and N. mediterraneum, but is not seen in all isolates of these two
latter species.
Type: Australia, Western Australia, Denmark, from wood of living
Eucalyptus globulus, Oct. 2002, T.I. Burgess, holotype MURU 400.
Cultures: WAC 12444 = CBS 118223 (ex-type), WAC 12445 =
CMW 15948.
Hosts: Eucalyptus globulus, E. saligna (Burgess et al. 2005).
Known distribution: Western Australia (Burgess et al. 2005).
Notes: Phylogenetically N. macroclavatum is closely related to N.
andinum, N. nonquaesitum and N. arbuti. It can be distinguished
from all other species in Neofusicoccum on the characteristic shape
of its conidia that are considerably larger than most other known
species in this genus; only N. pennatisporum has longer conidia.
Neofusicoccum mangiferae (Syd. & P. Syd.) Crous, Slippers
& A.J.L. Phillips, Stud. Mycol. 55: 248. 2006. MycoBank
MB500878. See Slippers et al. (2005) for illustrations.
Basionym: Dothiorella mangiferae Syd. & P. Syd., Ann. Mycol., 14:
192. 1916.
≡ Nattrassia mangiferae (Syd. & P. Syd.) B. Sutton & Dyko, Mycol. Res.
93: 484. 1989.
≡ Fusicoccum mangiferae (Syd. & P. Syd.) Johnson, Slippers & M.J.
Wingf., Mycologia 97 (1): 106. 2005.
≡ Fusicoccum mangiferae (Syd. & P. Syd.) G.I. Johnson, Slippers & M.J.
Wingf. (as “mangiferum”), Mycologia 97 (1): 106. 2005.
= Hendersonula cypria Nattrass, A irst list of Cyprus fungi: 43. 1937.
= Exosporina fawcettii E.E. Wilson, Hilgardia 17 (12): 427. 1947.
Neofusicoccum mediterraneum Crous, M.J. Wingf. &
A.J.L. Phillips, Fungal Planet No. 19: 2. 2007. MycoBank
MB504461. Fig. 56.
Ascomata not reported. Conidiomata amphigenous, stromatic,
brown, up to 450 μm diam on pine needles, ostiolate, exuding
conidia in a white mucoid mass, wall consisting of 3–5 layers of
brown textura angularis. Conidiophores lining the inner layer of
the conidioma, hyaline, smooth, 0–1-septate, 15–40 × 3–5 μm.
Conidiogenous cells holoblastic, hyaline, integrated, phialidic,
subcylindrical, rarely ampulliform, proliferating several times
percurrently near apex, rarely with minute periclinal thickening,
15–30 × 3–5 μm. Conidia hyaline, smooth, thin-walled, fusoidellipsoidal, widest in the middle or in the upper third, apex
subobtuse, base subtruncate, somewhat lattened with minute
marginal frill, with granular cytoplasm, (19–)22–26(–27) × (5.5–)6(–
6.5) μm in vitro (av. size of conidia = 24 × 6 μm), L/W = 4. Spermatia
not reported. Dichomera synasexual morph not reported.
Culture characteristics: Colonies on 2 % MEA luffy, iron-grey, with
abundant grey aerial mycelium, fertile on water agar overlaid with
autoclaved pine needles.
Type: Greece, Rhodes, Rhodos Palace Hotel parking lot, on
branches and leaves of Eucalyptus sp., 12 Jun. 2006, collected
by P.W. Crous, M.J. Wingield & A.J.L. Phillips, holotype CBS
H-19921.
Ascomata not reported. Conidiomata stromatic, erumpent, dark
brown to black, uni- to multi-loculate; walls composed of thickwalled, brown textura angularis, locules opening by means of
separate ostioles; spherical, 150–400 μm diam. Conidiophores
absent. Conidiogenous cells lageniform to ampulliform, hyaline,
discrete, arising from the inner wall of the stroma, producing a
succession of conidia at one level, collarette absent, periclinal
thickening and cytoplasmic channel wide, 6.5–14 × 2.5–4 μm.
Conidia holoblastic, ellipsoid to nearly fusiform, at irst aseptate,
then becoming 1–2 euseptate, central cell dark brown, end cells
hyaline to pale brown, smooth (11–)12–15(–17.5) × 5–6.6 μm (av.
of 54 conidia = 13.6 × 5.4 μm).
Hosts: Citrus sp. (Inderbitzin et al. 2010, Abdollahzadeh et al. 2013),
Ficus microcarpa (Mayorquin et al. 2012), Fortunella sp., Fraxinus
sp., Juniperus sp., Persea americana, Pistacia vera, Prunus dulcis,
Rubus sp., Sequoiadendron giganteum (Inderbitzin et al. 2010),
Eucalyptus (Crous et al. 2007, Inderbitzin et al. 2010), Juglans
regia (Inderbitzin et al. 2010, Trouillas et al. 2010), Mangifera
indica (Abdollahzadeh et al. 2013), Olea europaea (Lazzizera et
al. 2008), Vitis vinifera (Úrbez-Torres et al. 2010, Inderbitzin et al.
2010, Martin et al. 2011, Pintos Varela et al. 2011).
Type: India, Lucknow, on Mangifera indica, F. Bahadur (E.J. Butler
1724), 22 Oct. 1908, holotype HCIO.
Known distribution: USA (California) (Úrbez-Torres et al. 2010,
Trouillas et al. 2010, Inderbitzin et al. 2010, Mayorquin et al. 2012),
www.studiesinmycology.org
Cultures: CBS 121718 (ex-type), CBS 121558.
141
PhilliPs et al.
Fig. 56. Neofusicoccum mediterraneum. A. Conidiomata formed in culture on poplar twig. B–E. Conidiogenous cells. F. Hyaline, aseptate conidia. G. Coloured, septate conidia.
H. Spermatogenous cells. I. Spermatia. Scale bars: A = 500 μm, B, F–H = 10 μm, C–E, I = 5 μm.
Greece (Crous et al. 2007), Iran (Abdollahzadeh et al. 2013), Italy
(Lazzizera et al. 2008), Spain (Martin et al. 2011, Pintos Varela et
al. 2011).
Notes: Neofusicoccum mediterraneum is phylogenetically most
closely related to N. viticlavatum and N. vitifusiforme, but it can be
separated by having larger conidia (24 × 6 μm) than those of N.
viticlavatum (16–18 × 6.5–7.5 μm) and N. vitifusiforme (19–21 ×
5.5–6.5 μm). Conidia in some isolates become septate, light brown
with distinctly darker middle cells; a feature seen in N. mangiferum
and N. parvum, but can be distinguished from these two species in
having larger conidia.
A search of GenBank revealed a wide range of variation amongst
the ITS sequences for isolates of N. mediterraneum. Furthermore,
in the six-locus phylogeny of Inderbitzin et al. (2010), two distinct
clades were resolved for this species. Therefore, as mentioned by
Abdollahzadeh et al. (2013), it seems that N. mediterraneum is a
complex of species that should be examined in more detail using
greater numbers of isolates and additional gene loci.
Neofusicoccum nonquaesitum Inderb., Trouillas, Bostock
& Michailides, Mycologia 102: 1360. 2010. MycoBank
MB518135. See Inderbitzin et al. (2010) for illustrations.
Ascomata not reported. Conidiomata stromatic, single or in groups,
immersed or immersed-erumpent, lenticular to subglobose, 200–
500 × 150–400 μm, sometimes with a short neck, wall up to 50
μm wide, three-layered, outer layer composed of dark, thick-walled
cells, intermediate layer lighter pigmented, cells smaller, inner layer
hyaline, cells thin-walled. Conidiophores short, undifferentiated,
originating from the inner pycnidial wall, branching at times, up to 30
142
μm long, 1.5–2 μm wide, bearing single, unbranched conidiogenous
cells, of similar dimensions as conidiophores. Conidiogenous cells
holoblastic proliferating percurrently with up to ive proliferations.
Conidia hyaline, fusiform to oval, base truncate, rarely 1–3-septate,
sometimes becoming pigmented, 17–29 × 5.5–10.5 μm (av. size of
conidia = 23.2 × 7.6 μm), L/W ratio = 3.1. Spermatia when present
most abundant in upper part of pycnidium, cylindrical, with rounded
or truncate apices, curved at times, 4–10 × 2–4 μm, rarely up to 15
× 5 μm. Dichomera synasexual morph not reported.
Culture characteristics: Colonies on half strength PDA plate with
cork oak or pistachio leaf after 12 d under continuous light on a
laboratory bench white to olive-brown or olivaceous-black, reverse
white to olivaceous-black, conidioma forming mainly on leaf, black,
some covered by mycelium, immersed-erumpent, up to 600 μm
diam and of variable shape, conidia and spermatia present.
Type: uSA, California, Napa County, St Helena, on cankered
branch of Umbellularia californica, 12 Nov. 2004, F.P. Trouillas,
holotype UC1946389 (dried branch of U. californica inoculated
with PD484).
Cultures: CBS 126655 = PD484 (ex-type), PD301.
Hosts: Umbellularia californica, Prunus dulcis (Inderbitzin
et al. 2010), Vaccinium corymbosum (Espinoza et al. 2009)
Sequoiadendron giganteum (Rooney-Latham et al. 2012).
Known distribution: USA (California) (Inderbitzin et al. 2010), Chile
(Espinoza et al. 2009), North America (Rooney-Latham et al. 2012).
Note: See notes for N. andinum.
The Botryosphaeriaceae: genera and sPecies known from culTure
Neofusicoccum occulatum Sakalidis & T.I. Burgess, Mol.
Phylogenet. Evol. 60: 340. 2011. MycoBank MB518777. See
Sakalidis et al. (2011) for illustrations.
Ascomata not reported. Conidiomata on Populus sp. twigs
stromatic, solitary often or in groups, rapidly covered with
mycelium, supericial, conical or spherical or obpyriform, unilocular.
Conidiogenous cells holoblastic, hyaline, oval to fusiform, 4–14
× 0.5–2.5 μm (av. size = 8 × 1 μm). Conidia hyaline, unicellular,
fusifom to ellipsoid to cymbiform, apices obtuse, base truncate,
sometime both apices taper, aseptate, smooth-walled, 14–22
× 3.5–7.5 μm (av. size of conidia = 18.3 × 5.2 μm), L/W = 3.5.
Dichomera synasexual morph: Conidiogenous cells holoblastic,
hyaline, globose to turbinate 11.5 × 1.5 μm. Conidia two forms
observed “irregular long” and “irregular round” both brown and
muriform “irregular round” 1–3 transverse septa, 0–1 long septa
and 0–3 oblique septa, 7.5–13.5 × 5.5–8.5 μm (av. size of conidia =
9.8 × 7 μm), L/W = 1.4, rarely found “irregular long” 1–5 transverse
septa, 0–2 oblique septa, 11.5–20.5 × 4–7.5 μm (av. of 20 conidia
= 15.5 × 5.8 μm), L/W = 2.7.
Culture characteristics: Colonies white, lattened with tufts of white
mycelium, becoming very to dark greenish grey colour after 14
d with the reverse side of the colonies greenish black. Optimal
temperature for growth 30 ºC, covering a 90 mm Petri dish on MEA
in 3–4 d, limited growth occurred at 4 ºC and 10 ºC.
Type: Australia, Queensland, Karanda, symptomless branches of
Eucalyptus grandis hybrid, Mar 2002, T.I. Burgess, dried culture
sporulating on Populus sp. twigs, holotype MURU467.
Cultures: MUCC 227 = CBS 128008 (ex-type), MUCC 286 = WAC
12395.
Host: Eucalyptus (Sakalidis et al. 2011).
Known distribution: Australia (Sakalidis et al. 2011).
Notes: A pale yellowish pigment was observed once in the media of
three isolates MUCC 270 and MUCC 296 and MUCC 232 (Sakalidis
et al. 2011). Neofusicoccum occulatum is morphologically similar to
other closely related species in the N. parvum / N. ribis species
complex and differs from other species in the complex by one
uniquely ixed nucleotide difference in partial EF1-α (EU339509)
position 164 (A). See notes for N. cordaticola.
Neofusicoccum parvum (Pennycook & Samuels) Crous,
Slippers & A.J.L. Phillips, Stud. Mycol. 55: 248. 2006.
MycoBank MB500879. Fig. 57.
Basionym: Fusicoccum parvum Pennycook & Samuels, Mycotaxon
24: 455. 1985.
= Botryosphaeria parva Pennycook & Samuels, Mycotaxon 24: 455. 1985.
Ascomata forming botryose clusters 2–5 mm diam, each comprising
up to 100 ascomata, erumpent through the bark, globose, with a
short, conical papilla, dark brown to black, smooth, thick-walled, wall
composed of dark brown thick-walled cells, lined with thin-walled,
hyaline cells, locules 150–250 μm diam, contents conspicuously
white when dry. Asci clavate, 8-spored, bitunicate, 75–143(–210)
× 17–21 μm. Ascospores broadly ellipsoidal to fusoid, often with
www.studiesinmycology.org
an apiculus at each end, hyaline, smooth, aseptate, occasionally
becoming 1-septate, (14–)18–23(−26) × (7–)8−10(–11) μm (av.
of 73 ascospores = 20.8 × 9.2 μm), L/W = 2.2. Conidiomatal
aggregates morphologically indistinguishable from ascomatal
aggregates. Conidiomata globose and non-papillate to pyriform
with a short, acute papilla, entire locule lined with conidiogenous
cells. Conidiogenous cells holoblastic, hyaline, subcylindrical,
proliferating percurrently to form 1–2 annellations, or proliferating
at the same level to form periclinal thickenings. Conidia ellipsoidal
with apex round and base lat, unicellular, hyaline, old conidia
becoming 1–2-septate hyaline, or light brown with the middle cell
darker than the terminal cells, (12–)13.5–21(–24) × 4–6(–10) μm,
95 % conidence limits of 320 conidia = 16.9–17.3 × 5.4–5.6 μm (av.
± S.D. of 320 conidia = 17.1 ± 2.1 × 5.5 ± 0.8 μm), L/W ratio = 3.2 ±
0.6 with 95 % conidence limits of 3.1–3.2. Dichomera synasexual
morph: Conidia subglobose to obpyriform, brown, apex obtuse,
base truncate, 8–10.5(–12) × (6.5–)7–8(–9) μm, 1–3 transverse
septa, 1–2 longitudinal septa, and 1–2 oblique septa.
Type: New Zealand, Bay of Plenty, Te Puke, No 3 Road, Baldwin
Orchard, on small dead branch of Populus nigra, 17 Dec. 1981,
S.R. Pennycook, holotype PDD 45438.
Cultures: PDDCC 8003 = ATCC 58191 (ex-type) = ICMP 8003 =
CMW 9081.
Hosts: Plurivorous including Actinidia deliciosa (Pennycook &
Samuels 1985, Abdollahzadeh et al. 2013), Araucaria heterophylla
(Slippers et al. 2005b), Citrus sinensis (Cunnington et al. 2007),
Citrus sp. (Adesemoye et al. 2011), Cupressus funebris (Li et al.
2010), Diospyros kaki (Gadgil et al. 2005), Eriobotrya japonica
(Gadgil et al. 2005), Eucalyptus citriodora, Eucalyptus globulus,
Eucalyptus grandis, Eucalyptus saligna (Gezahgne et al. 2004),
Eucalyptus pellita (Barber et al. 2005), Eucalyptus urophylla
(Mohali et al. 2007), Ficus microcarpa (Mayorquin et al. 2012),
Grevillea robusta (Toljander et al. 2007), Heteropyxis natalensis
(Slippers et al. 2004a), Juglans regia (Inderbitzin et al. 2010,
Abdollahzadeh et al. 2013), Juniperus communis, Pinus pinea,
Thuja plicata, Thujopsis dolabrata (Alves et al. 2013), Kolkwitzia
amabilis (Cunnington et al. 2007), Leucadendron sp. (Marincowitz
et al. 2008), Leucospermum sp. (Marincowitz et al. 2008), Lilium
lancifolium (Woodward et al. 2006), Malus domestica (Pennycook
& Samuels 1985), Mangifera indica (Javier-Alva et al. 2009), Olea
africana (Cunnington et al. 2007), Olea europaea (Lazzizera
et al. 2008), Persea americana (Hartill 1991, Cunnington et al.
2007, Zea-Bonilla et al. 2007, McDonald & Eskalen 2011, MolinaGayosso et al. 2012), Pistacia vera (Cunnington et al. 2007,
Inderbitzin et al. 2010), Populus sp. (Gadgil et al. 2005), Protea
cynaroides (Marincowitz et al. 2008), Prunus armeniaca (Gramaje
et al. 2012), Prunus dulcis (Inderbitzin et al. 2010), Prunus persica
(Cunnington et al. 2007), Prunus avium (Abdollahzadeh et al.
2013), Pseudopanax laetus (Gadgil et al. 2005), Psidium guajava
(Mohali et al. 2007), Pyrus sp. (Abdollahzadeh et al. 2013), Pyrus
communis (Gadgil et al. 2005), Pyrus pyrifolia (Shen et al. 2010),
Pinus sp. (Abdollahzadeh et al. 2013), Quercus suber (Linaldeddu
et al. 2007), Rhododendron sp. (Varela et al. 2011), Ribes sp.
(Slippers et al. 2004a), Rubus fruticosus (Abdollahzadeh et al.
2013), Salix sp. (Abdollahzadeh et al. 2013), Sequoia gigantea
(Slippers et al. 2004a), Syzygium cordatum (Pavlic et al. 2007),
Syzygium paniculatum (Ploetz et al. 2008), Terminalia catappa
(Didier Begoude et al. 2010), Trachycarpus fortunei (Taylor &
Hyde 2003), unknown, palm (Taylor & Hyde 2003), Vaccinium
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PhilliPs et al.
Fig. 57. Neofusicoccum parvum. A. Vertical section through an aggregate ascoma. B, C. Asci. D, E. Details of ascus apex as seen by interference contrast (D) or phase contrast
(E). F. Ascospores. G, H. Conidiogenous cells. I. Hyaline, aseptate conidia. J. Coloured, 1- and 2-septate conidia. Scale bars: A = 50 μm, B, C, F, G = 10 μm, D, E, H–J = 5 μm.
corymbosum (Espinoza et al. 2009), Vitis vinifera (Cunnington et
al. 2007, Mohammadi et al. 2008, Phillips, et al. 2006, Úrbez-Torres
et al. 2006, Díaz et al. 2011, White et al. 2011).
Known distribution: Probably worldwide. Australia (Barber et al.
2005, Cunnington et al. 2007, Slippers et al. 2004a, Taylor & Hyde
2003), USA (California) (Úrbez-Torres et al. 2006b, Adesemoye
& Eskalen 2011, Inderbitzin et al. 2010, McDonald & Eskalen
2011, Mayorquin et al. 2012), Chile (Díaz et al. 2011, Espinoza
et al. 2009), China (Li et al. 2010, Taylor & Hyde 2003), Ethiopia
(Gezahgne et al. 2004), USA (Florida) (Ploetz et al. 2008), USA
(Georgia) (Woodward et al. 2006), Greece (Inderbitzin et al. 2010),
USA (Hawaii) (Marincowitz et al. 2008), Iran (Mohammadi et al.
144
2008, Abdollahzadeh et al. 2013), Italy (Lazzizera et al. 2008,
Linaldeddu et al. 2007), Mexico (Molina-Gayosso et al. 2012), New
Zealand (Gadgil et al. 2005, Hartill 1991, Pennycook & Samuels
1985, Slippers et al. 2005b), Peru (Javier-Alva et al. 2009),
Portugal (Phillips et al. 2006, Alves et al. 2013), South Africa (Didier
Begoude et al. 2010, Pavlic et al. 2007, Slippers et al. 2004a,
Slippers et al. 2004b, White et al. 2011), Spain (Úrbez-Torres et al.
2006a, Zea-Bonilla et al. 2007, Varela et al. 2011, Gramaje et al.
2012), Taiwan (Shen et al. 2010), Uganda (Toljander et al. 2007),
Venezuela (Mohali et al. 2007).
Notes: Phylogenetically, this species lies within a cluster of
morphologically highly similar species that can be distinguished
The Botryosphaeriaceae: genera and sPecies known from culTure
only on the basis of ITS and EF1-α sequence data. Neofusicoccum
parvum has, however, been distinguished by different researchers
from other species in this cluster based on the colour and septation
of conidia at the time of germination. Thus, the conidia become
2-septate and the central cells become pale brown in N. parvum,
while in the other species there is no colouration of the conidia at
the time of germination. But recently, in a phylogenetic study on
Neofusicoccum and Botryosphaeria species in Iran, Abdollahzadeh
et al. (2013) studied 34 N. parvum isolates and found that in all
of them the old conidia remained hyaline even after 10 wk.
Furthermore, the production of a yellow pigment on PDA was
reported in some isolates of an Iranian population of N. parvum,
which is a feature never seen previously. Neofusicoccum parvum
is emerging as a common and cosmopolitan species on a wide
variety of hosts. It is now recognised as an aggressive pathogen of
grapevines (e.g., Phillips 1998) as B. dothidea, van Niekerk et al.
2004), and possibly other woody hosts.
Neofusicoccum pennatisporum K. Taylor, Barber & T.I.
Burgess, Mycol. Res. 113: 346. 2009. MycoBank MB511826.
See Taylor et al. (2009) for illustrations.
Ascomata not reported. Conidiomata stromatic, supericial, darkbrown to black, cylindrical to triangular to irregular, mostly solitary,
rough with some mycelium, 300–1000 μm long and 100–500 μm
diam on pine needles but up to 2 mm long on agar. Conidiogenous
cells holoblastic, hyaline, cylindrical to lask shaped, 4–10(–12) ×
(1–)2–3(–4) μm. Conidia hyaline, usually aseptate, often with 1
septum but can have up to 5 septa with age, typically fusiform,
smooth-walled, apex obtuse, base frequently truncate but
sometimes rounded, (31–)40–50(–64) × 6–10 (–12) μm (av. of
100 conidia = 45.4 × 9.7 μm), L/W ratio = 4.6. Spermatia hyaline,
aseptate, fusiform, either rounded or truncate at both ends, (2–)3–
6(–7) × 1–2 μm (av. of 100 spermatia = 4.4 × 1.5 μm). Dichomera
synasexual morph not reported.
Culture characteristics: Colonies composed of appressed mycelial
mat with diffuse irregular edges, white centre, darkening slightly
with age, pycnidia produced profusely.
Type: Australia, Western Australia, Yalgorup National Park, from
healthy stem of Allocasuarina fraseriana, Jun. 2005, K.M. Taylor,
holotype PERTH 07693044.
Cultures: WAC 13153 = MUCC 510 (ex-type).
Host: Allocasuarina fraseriana (Taylor et al. 2009).
Known distribution: Western Australia (Taylor et al. 2009).
Notes: The conidia of N. pennatisporum are unusually long (40–
50 × 6–10 μm), when compared with other Neofusicoccum spp.,
including N. macroclavatum (25–35 × 6–8 μm), which is also found
in Western Australia, and N. protearum (25–30 × 7–8 μm), which
is the most closely related species to N. pennatisporum based on
ITS sequence data. In the phylogeny based on ITS and EF1-α
sequences, this species resides in a distinct clade as a sister
group to all other Neofusicoccum species. According to Taylor et al.
(2009), an isolate of N. pennatisporum produced the sexual morph
once on pine needles in culture. The ascospores have distinctive
www.studiesinmycology.org
protrusions at either end unlike ascospores of other Neofusicoccum
spp.
Neofusicoccum protearum (Denman & Crous) Crous,
Slippers & A.J.L. Phillips, Stud. Mycol. 55: 249. 2006. MycoBank
MB500880. See Denman et al. (2003) for illustrations.
Basionym: Fusicoccum protearum Denman & Crous, Mycologia
95: 301. 2003.
= Botryosphaeria protearum Denman & Crous, Mycologia 95: 301. 2003.
Ascomata pseudothecial, embedded in host tissue, up to 600 μm
diam, becoming erumpent, solitary or botryose, stromatic, dark
brown to black, with central, black ostioles; pseudothecial wall 6–15
layers thick, composed of brown textura angularis. Asci clavate to
subcylindrical, 8-spored, bitunicate, with a well-developed apical
chamber that becomes inconspicuous at maturity, 110–200 ×
15–21 μm. Pseudoparaphyses iliform, branched, septate, 3–5
μm wide. Ascospores irregularly biseriate, hyaline, nonseptate,
granular, becoming light brown with age, fusiform, widest in the
middle with obtuse ends, sometimes inequilateral, (25–)26–33(–
37) × (9–)10–12(–13) μm. Conidiomata stromatic, embedded in
host tissue, solitary or botryose, stromatic, globose, up to 500 μm
diam, wall 4–8 layers thick, composed of brown textura angularis,
becoming hyaline towards the inner region. Conidiophores
0–1-septate, hyaline, subcylindrical, rarely branched, 7–20(–30) ×
3–5 μm. Conidiogenous cells holoblastic, hyaline, subcylindrical,
rarely proliferating percurrently with 1−2 anellations, proliferating
predominantly at the same level with minute periclinal thickenings,
which become more prominent in older conidiogenous cells, 7−12
× 3−5 μm. Conidia hyaline, granular, ovoid to clavate when young,
becoming irregularly fusoid when mature, widest in the middle
with an obtuse apex and bluntly rounded or slightly lattened base,
(20–)25–30(–40) × 7–8(–10) μm in vivo. Spermatia produced
in same conidiomata as conidia, or in separate conidiomata.
Spermatophores hyaline, smooth, branched, cylindrical,
0−2-septate, straight, unbranched or branched above, 12–17 × 2–3
μm. Spermatogenous cells discrete or integrated, hyaline, smooth,
cylindrical, proliferating via phialides with periclinal thickenings,
5–12 × 1.5–2.5 μm. Spermatia hyaline, smooth, aseptate, rodshaped with rounded ends, 3–6 × 1–1.5 μm. Dichomera synasexual
morph not reported.
Type: Of sexual morph: South Africa, Western Cape, Porterville,
Baanbreek Farm, on stems of Protea magniica, 27 Jul. 1997, S.
Denman, holotype PREM 57329; of asexual morph: South Africa,
Western Cape, Devon Valley, Protea Heights Farm, on stems of
Leucadendron salignum, 31 Oct. 1997, S. Denman & J. Taylor,
holotype PREM 57330.
Cultures: STE-U 4361 = CPC 4361 (ex-type culture of sexual
morph), STE-U 1775 = CBS 114176 (ex-type culture of asexual
morph).
Hosts: Protea and Leucadendron spp. (Denman et al. 2000, 2003,
Marincowitz et al. 2008), Santalum acuminatum (Taylor et al. 2009).
Known distribution: Australia, Portugal (continental and Madeira),
South Africa, Spain (Tenerife), USA (Hawaii) (Denman et al. 2000,
2003, Marincowitz et al. 2008, Taylor et al. 2009).
145
PhilliPs et al.
Notes: Neofusicoccum protearum was originally thought to be
restricted to Proteaceae, but it was recently isolated from Santalum
acuminatum (Taylor et al. 2009). See notes for N. pennatisporum.
Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.)
Crous, Slippers & A.J.L. Phillips, Stud. Mycol. 55: 249.
2006. MycoBank MB500881. See Slippers et al. (2004) for
illustrations.
Basionym: Fusicoccum ribis Slippers, Crous & M.J. Wingf.,
Mycologia 96: 96. 2004.
= Botryosphaeria ribis Grossenb. & Duggar, Tech. Bull. N.Y. Agric. Exp. St.
18: 128. 1911.
Ascostroma erumpent through the bark, pulvinate, 100–400 μm
diam. Ascomata pseudothecial, forming botryose aggregates of
up to 5–50, globose with central ostiole, papillate or not, brown to
black, 175–250 μm, pseudothecial wall comprising 5–15 layers of
textura angularis, outer region of dark brown or brown cells, inner
region 2−4 layers of hyaline cells lining the locule. Asci bitunicate,
clavate, 8-spored, 80–120 × 17–20 μm. Pseudoparaphyses
iliform, septate, rarely branched, 2–4 μm wide. Ascospores
fusoid to ellipsoid, often round at the ends then broadly ellipsoidal,
hyaline, unicellular, smooth with granular contents, biseriate in
the ascus, (14–)18–23(–27) × 6–8(–10) μm (av. of 80 ascospores
= 20.5 × 7.1 μm), L/W = 2.9. Conidiomata in same stromata as
ascomata and morphologically indistinguishable from them, or
solitary and embedded in young host shoots. Conidiogenous
cells holoblastic, hyaline, subcylindrical, proliferating percurrently
with 1–2 annellations, or proliferating at the same level to form
periclinal thickenings, 6–22 × 2–5 μm. Conidia fusiform, sometimes
irregularly fusiform, base subtruncate to blunt, hyaline, unicellular,
rarely septate with age, smooth with granular contents, (16–)19–
23(–24) × 5–6(–7) μm (av. of 90 = conidia 20.8 × 5.5 μm), L/W
= 3.8. Spermatia not reported. Dichomera synasexual morph:
Conidia subglobose, obpyriform or rarely obovoid to broadly
fusiform or fusiform, apex subobtuse to obtuse, base truncate to
bluntly rounded. Subglobose, obpyriform conidia (7–)8–13.5(–17)
× (6.5–)7–9.5(–10.5) μm, hyaline to pale brown when immature
with one transverse septum and 0–2 longitudinal septa, becoming
brown when mature with 1–4 transverse septa, 0–3 longitudinal
septa, and 0–4 oblique septa. Broadly fusiform to fusiform conidia
(12–)13.5–22.5(–24) × (5–)5.5–8 μm, brown with 2–7 transverse
septa, and 0–2 oblique septa.
Notes: For many years, B. ribis was was regarded as a synonym
of B. dothidea (e.g., Witcher & Clayton 1963, Barr 1972, English
et al. 1975, Maas & Uecker 1984, Pennycook & Samuels 1985,
Brown & Britton 1986, Smith et al. 1994), while others regarded
them as distinct species (e.g., Punithalingam & Holliday 1973,
Morgan-Jones & White 1987, Rayachhetry et al. 1996, Smith &
Stanosz 2001). The debate was inally settled when Slippers et
al. (2004) demonstrated that the two were phylogenetically and
morphologically distinct and Crous et al. (2006) showed that B.
dothidea and N. ribis reside in two distinct phylogenetic lineages.
Phylogenetically N. ribis resides in a cluster of cryptic species that
are dificult to separate based on morphology.
Neofusicoccum umdonicola Pavlic, Slippers & M.J. Wingf.,
Mycologia 101: 644. 2009. MycoBank MB512500. See Pavlic
et al. (2009) for illustrations.
Ascomata not reported. Neofusicoccum umdonicola is
morphologically similar to other related species in the N. parvum
/ N. ribis species complex. Conidia hyaline, unicellular, fusiform
to oval, apices tapered 15–23.5 × 4.5–6.5 μm (av. of 310 conidia
= 19.4 × 5.5 μm), L/W = 3.5). Neofusicoccum umdonicola differs
from other species in the N. parvum / N. ribis complex by uniquely
ixed nucleotides in four nuclear loci: ITS (EU821904) position 168
(C); EF1-α (EU821874) positions 62 (T); β-tubulin (EU821844)
position 40 (A); RNA polymerase II subunit (EU821934) position
280 (T).
Type: South Africa, Kwazulu-Natal Province, Kosi Bay from
symptomless branches and leaves, dying branches and pulp of ripe
fruits of Syzygium cordatum, Mar. 2002, D. Pavlic, a dry culture on
pine needles, holotype PREM 60068.
Cultures: CMW 14058 = CBS 123645 (ex-type), CMW 14060 =
CBS 123646.
Host: Syzygium cordatum (Pavlic et al. 2009).
Known distribution: South Africa (Pavlic et al. 2009).
Nots: See notes for N. cordaticola.
Type: Of asexual morph; uSA, New York, Ithaca, Ribes sp., 2000,
G. Hudler, holotype PREM 57368, lectotype of sexual morph; uSA,
New York, Geneva, on Ribes vulgare, 1911, J.G. Grossenbacher &
B.M. Duggar, holotype CUP-A (F.Col. 3408).
Neofusicoccum viticlavatum (Van Niekerk & Crous)
Crous, Slippers & A.J.L. Phillips, Stud. Mycol. 55: 249. 2006.
MycoBank MB500882. See van Niekerk et al. (2004) for
illustrations.
Cultures: CBS 115475 = CMW 7772 (ex-type), CMW 7054.
Basionym: Fusicoccum viticlavatum Van Niekerk & Crous,
Mycologia 96: 792. 2004.
Hosts: More than 250 hosts are listed for N. ribis (Farr et al. 2012).
However, many of the reports were published before the concept
of N. ribis (as Botryosphaeria ribis) was clariied by Slippers et al.
(2004) and thus the identiications are not reliable.
Known distribution: Although this species has been considered
to be distributed worldwide on numerous hosts this is based on
reports published prior to the establishment of a stable concept for
N. ribis (Slippers et al. 2004). Thus far it has been veriied only on
Ribis sp. in NY state, USA (Slippers et al. 2004).
146
Ascomata not reported. Conidiomata stromatic, embedded in host
tissue, solitary, stromatic, globose, up to 450 μm wide, wall 4–8 cell
layers thick, of brown textura angularis, becoming hyaline toward
inner region. Conidiophores 0–1-septate, hyaline, subcylindrical,
10–20 × 2.5–3.5 μm. Conidiogenous cells holoblastic, hyaline,
subcylindrical, proliferating percurrently with 1–3 proliferations,
or proliferating at same level (phialidic) with minute periclinal
thickening, 7–15 × 2.5–3.5 μm. Conidia hyaline, guttulate, ellipsoid
to clavate, widest in upper third, with an obtuse apex and lattened,
subtruncate base, aseptate, (15–)16–18(–20) × (6–)6.5–7.5(–8)
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 58. Neofusicoccum vitifusiforme. A. Conidiomata on pine needles in culture. B. Conidiogenous cells. C. Conidia. Scale bars: A = 1 mm, B, C = 10 μm.
μm, L/W ratio = 2.4. Spermatia not reported. Dichomera synasexual
morph not reported.
Culture characteristics: Colonies umbonate with undulating margins,
olivaceous on the surface, and dull green reverse, reaching a radius
of 26 mm after 3 d at 25 °C. Cardinal temperatures for growth: min
10 °C, max 35 °C, opt 30 °C.
Type: South Africa, Western Cape Province, Stellenbosch, on V.
vinifera, 2002, F. Halleen, holotype CBS H-7755.
Cultures: STE-U 5044 = CBS 112878 (ex-type), STE-U 5041 =
CBS 112977.
Host: Vitis vinifera (van Niekerk et al. 2004).
Known distribution: South Africa (Western Cape Province) (van
Niekerk et al. 2004).
Notes: Neofusicoccum viticlavatum is closely related to N.
mediterraneum and N. vitifusiforme. It can be differentiated from
N. vitifusiforme based on the characteristic clavate conidia of N.
viticlavatum and its smaller conidia. Conidia of this species are
much smaller (16–18 × 6.5–7.5 μm) than those of N. mediterraneum
(24 × 6 μm).
Neofusicoccum vitifusiforme (Van Niekerk & Crous)
Crous, Slippers & A.J.L. Phillips, Stud. Mycol. 55: 249. 2006.
MycoBank MB500883. Fig. 58.
Basionym: Fusicoccum vitifusiforme Van Niekerk & Crous,
Mycologia 96: 793. 2004.
Synasexual morph: Dichomera eucalypti (G. Winter) B. Sutton,
Mycol. Pap. 138: 182 1975.
Basionym: Camarosporium eucalypti G. Winter, Revue Mycol.,
Toulouse 8 (32): 212. 1886.
= Neofusicoccum corticosae Crous & Summerell, Fungal Divers. 23: 337.
2006.
Ascomata not reported. Conidiomata stromatic, solitary, globose to
obpyriform, up to 450 μm diam, conidioma wall 6–15 cell layers
thick, of brown textura angularis, becoming hyaline toward inner
region. Conidiophores 0–1-septate, hyaline, subcylindrical, 10–45 ×
www.studiesinmycology.org
2.5–5 μm. Conidiogenous cells holoblastic, hyaline, subcylindrical,
proliferating percurrently with numerous proliferations, or
proliferating at the same level (phialidic) with minute periclinal
thickening, 10–30 × 2.5–3.5 μm. Conidia hyaline, granular, fusoid
to ellipsoid, widest in the upper third with an obtuse apex and
lattened, subtruncate base, (18–)19–21(–22) × (4.5–)5.5–6.5(–8)
μm in vitro, L/W ratio = 3.3. Spermatia not reported. Dichomera
synasexual morph: Conidia subglobose, obpyriform or obovoid,
apex obtuse, base truncate to bluntly rounded, (9–)9.5–13(–14.5)
× (6.5–)8–10.5(–11) μm, hyaline to pale brown when immature
with 0–3 transverse septa, 0–2 longitudinal septa, and 0–2 oblique
septa, becoming brown when mature with 1–3 transverse septa,
0–3 longitudinal septa, and 0–2 oblique septa.
Culture characteristics: Colonies effuse with even, smooth margins,
white on the surface, and greenish olivaceous underneath, reaching
a radius of 31 mm after 3 d at 25 °C. Cardinal temperatures for
growth: min 10 °C, max 35 °C, opt 30 °C.
Type: South Africa, Western Cape Province, Stellenbosch, on V.
vinifera, 2002, J.M. van Niekerk, holotype CBS H-7756.
Cultures: STE-U 5252 = CBS 110887 (ex-type), STE-U 5050 =
CBS 110880.
Hosts: Eucalyptus corticosa (as N. corticosae) (Summerell et al.
2006), Eucalyptus sp., Eucalyptus camaldulensis, Eucalyptus
diversicolor, E. paucilora, Eucalyptus marginata, Eucalyptus
rubida, Eucalyptus viminalis (as D. eucalypti) (Barber et al. 2005,
Taylor et al. 2009, Sutton 1980), Olea europaea (Lazzizera et al.
2008, Úrbez-Torres et al. 2013), Prunus armeniaca, Prunus persica,
Prunus salicina (Damm et al. 2007), Vaccinium corymbosum (Kong
et al. 2010), Vitis vinifera (van Niekerk et al. 2004, Úrbez-Torres et
al. 2012).
Known distribution: Australia (Sutton 1980, Barber et al. 2005,
Summerell et al. 2006, Taylor et al. 2009), China (Kong et al. 2010),
Italy (Lazzizera et al. 2008), South Africa (van Niekerk et al. 2004,
Damm et al. 2007), USA (Úrbez-Torres et al. 2012, Úrbez-Torres
et al. 2013).
Notes: The fusiform conidia of N. vitifusiforme separate this species
from its closest relative N. viticlavatum, which has clavate conidia.
147
PhilliPs et al.
This species was originally thought to be restricted to Vitis species,
but it was later isolated from Olea europaea in Italy (Lazzizera
et al. 2008). The same authors showed that it is phylogenetically
indistinguishable from Dichomera eucalypti, which was conirmed
in the present study based on ITS and EF1-α. Thus, D. eucalypti
becomes a synonym and Eucalyptus can be regarded as an
additional host for the fungus. Furthermore, as mentioned earlier, in
the ITS phylogeny, N. corticosae grouped with N. vitifusiforme and
D. eucalypti and despite the lack of EF1-α sequence data for N.
corticosae it would appear that these three species are synonyms,
more information is needed to conirm this.
Neoscytalidium Crous & Slippers, Stud. Mycol. 55: 244.
2006. MycoBank MB500868.
Type species: Neoscytalidium hyalinum (C.K. Campb. & J.L.
Mulder) A.J.L. Phillips, Groenewald & Crous.
Coelomycetous synasexual morph: Hendersonula Speg., Anal.
Soc. Cient. Arg. 10: 160. 1880.
Ascomata not reported. Conidia occurring in arthric chains in
aerial mycelium, powdery to the touch, disarticulating, cylindricaltruncate, oblong-obtuse to doliiform, dark brown, thick-walled,
0–2-septate. Coelomycetous synasexual morph: Mycelium
immersed, branched, septate, hyaline. Conidiomata stromatic and
irregularly multilocular, or pycnidial and unilocular, blackish brown.
Conidiophores absent. Conidiogenous cells discrete, determinate
or indeterminate, hyaline, smooth, ampulliform, doliiform or
cylindrical, proliferating enteroblastically with conidia seceding at
the same level or at successively higher levels, periclinal thickening
distinct or not, with occasionally a single percurrent proliferation.
Conidia holoblastic, pale brown, smooth or verruculose, thin-walled,
1–3 (mostly 3)-euseptate, septa thick and prominent, cylindrical to
fusiform, apex obtuse, base truncate, eguttulate, occasionally with
a mucilagenous apical appendage.
Species descriptions
Neoscytalidium hyalinum (C.K. Campb. & J.L. Mulder)
A.J.L. Phillips, Groenewald & Crous, comb. nov. Fig. 59.
MycoBank MB805648.
Basionym: Scytalidium hyalinum C.K. Campb. & J.L. Mulder,
Sabouraudia, 15: 163, 1977.
= Torula dimidiata Penz., Michelia 2: 466. 1882.
≡ Scytalidium dimidiatum (Penz.) B. Sutton & Dyko, Mycol. Res. 93: 484.
1989.
≡ Fusicoccum dimidiatum (Penz.) D.F. Farr, Mycologia 97: 740. 2005.
≡ Neoscytalidium dimidiatum (Penz.) Crous & Slippers, Stud. Mycol. 55:
244. 2006.
= Hendersonula toruloidea Nattrass, Trans. Br. Mycol. Soc. 18: 197. 1933.
Ascomata not reported. Conidia occurring in arthric chains in
aerial mycelium, powdery to the touch, disarticulating, cylindricaltruncate, oblong-obtuse to doliiform, dark brown, thick-walled,
0–2-septate, 4–16.5 × 8.5 μm. Coelomycetous synasexual morph:
Conidiomata stromatic, immersed, eventually erumpent, dark
brown to black, unilocular to multilocular, globose, up to 2 mm
diam, wall of 7–12 cell layers, up to 20–43 μm thick, outer wall
of irregular, thick-walled, dark brown textura angularis, inner wall
of hyaline, thinner-walled textura angularis. Ostiole central to each
locule, circular, papillate. Conidiophores absent. Conidiogenous
cells lageniform to ampulliform, hyaline, discrete, collarette absent,
148
periclinal thickenings and cytoplasmic channel wide, arising from
the inner wall of the locules, 6.5–14 × 2.5–4 μm. Conidia holoblastic,
ellipsoid to nearly fusiform, hyaline, at irst aseptate, then becoming
1–2(–3)-euseptate, central cell dark brown, end cells hyaline to
pale brown, 10–16(–21) × 3.5–6.5 μm.
Lectotype: united Kingdom, sole of human foot, 20 Nov. 1973,
C.K. Campbell, CBS H-7745 (isotype of Scytalidium hyalinum).
Cultures: CBS 145.78 (ex-isotype).
Hosts: Human skin and nails (Campbell & Mulder 1977). According
to Sutton & Dyko (1989) it is plurivorous causing gummosis,
dieback, wilt and cankers on Acacia auriculiformis, Agathis
palmerstoni, Agave americana, Agave sisalana, Ananas comosus,
Ananas sativa, Citrus sinensis, Eucalyptus, Eucalyptus globulus,
Ficus carica, Fucraea sp., Ipomoea batatas, Juglans regia, Malus
pumila, Mangifera indica, Manihot utilissima, Melia azaderach,
Morus alba, Musa, Philidendron bipinnatiidum, Plumeria obtusa,
Populus alba, Prunus armeniaca, Sanseveria guineensis.
Known distribution: Tropical and sub-tropical regions of Europe,
Africa, Asia, North and South America.
Notes: Nattrass (1933) irst described this fungus under the name
Hendersonula toruloidea . Gentles and Evans (1971) reported the
same fungus from a dermatomycosis in patients from tropical areas
and a few years later, Campbell and Mulder (1977) introduced the
new species S. hyalinum as the cause of the same clinical lesions
as H. toruloidea. Since these irst descriptions, the production of
both arthroconidial and pycnidial synanamorphs has been shown
and led to several controversies in the nomenclature. Sutton and
Dyko (1989) transferred H. toruloidea to Nattrassia mangiferae
with the mycelial synanamorph named Scytalidium dimidiatum
based on Torula dimidiata. Farr et al. (2005) concluded from a
phylogenetic analysis that Nattrassia mangiferae and Scytalidium
dimidiatum belong in Fusicoccum and introduced the name
Fusicoccum dimidiatum to replace Scytalidium dimidiatum. Crous
et al. (2006) in a taxonomic revision of the Botryosphaeriaceae
concluded that Scytalidium is polyphyletic and proposed the
genus Neoscytalidium to accommodate S. dimidiatum as N.
dimidiatum. It has been suggested that S. dimidiatum and S.
hyalinum might be conspeciic and a new name (N. dimidiatum
var. hyalinum) has been suggested (Madrida et al. 2009).
Although Crous et al. (2006) included an isolate of S. hyalinum
in their study, they were not aware at the time that the isolate is
in fact linked to the isotype of S. hyalinum. Since S. hyalinum
is phylogenetically indistinguishable from N. dimidiatum and
is the older epithet we transfer S. hyalinum to Neoscytalidium
and reduce N. dimidatum to synonymy. Diseases reported to be
associated with this fungus tend to be more common in tropical
countries. It has been associated with freeze-damaged limbs of
Citrus spp. in California, and appears to be a wound pathogen of
this host. In Italy, it causes a shoot blight, canker and gummosis
disease of Citrus (Polizzi et al. 2009, 2011).
Neoscytalidium novaehollandiae Pavlic, T.I. Burgess, M.J.
Wingf., Mycologia 100: 862. 2008. MycoBank MB512103.
See Pavlic et al. (2008) for illustrations.
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 59. Neoscytalidium hyalinum. A. Conidiomata formed on pine needles in culture. B. Arthric chains of conidia. C. Conidiogenous cells of coelomycetous state. D. Conidia of
coelomycetous state. Scale bars: A = 500 μm, B–D = 10 μm.
Ascomata not reported. Conidiomata semi-immersed or supericial,
solitary or in multilocular stromata, black, with globose base, up to
300 μm diam and long neck, up to 600 μm long. Conidiogenous cells
holoblastic, cylindrical to subcylindrical, hyaline, the irst conidium
produced holoblastically and subsequent conidia enteroblastically,
(6–)7–10(–11) × (2–)2–3(–4) μm (av. = 8.6 × 2.5 μm). Conidia of
two types: (i) ellipsoidal to oval, apices rounded, initially hyaline,
unicellular, becoming cinnamon to sepia, and 0–1-septate or
2-septate with darker central cell, (8–)10.5–12.5(–14) × (3–)4–
5(–5) μm (av. = 11.5 × 4.4 μm, L/W = 2.6); (ii) variable in shape,
globose, subglobose to obpyriform with muriform septa, initially
hyaline becoming cinnamon to sepia, (8–)8.5–12.5(–15.5) × (5–)
5.5–7.5(–8) μm (av. = 10.6 × 6.9 μm, L/W = 1.5). Aerial mycelium
forms chains of arthroconidia, (5–)5.5–7.5(–9.5) × (3–)3.5–4.5(–5)
μm (av. = 6.5 × 4 μm, L/W = 1.6), unicellular, powdery to the touch,
disarticulating, cylindrical, oblong to obtuse to doliiform, thickwalled, initially hyaline becoming becoming cinnamon to sepia and
0–1-septate.
Culture characteristics: Colonies initially white to olivaceousbuff, becoming greenish olivaceous to citrine from the middle of
colonies within 7 d, and black (surface and beneath) with age, with
suppressed, moderately luffy mycelium, edges smooth. Optimum
growth at 35 ºC, covering the 90 mm diam Petri dish after 3 d in
the dark.
Type: Australia, Western Australia, Bell Gorge, on Crotalaria
medicaginea, Jul. 2006, T.I. Burgess, holotype PREM 60069.
Cultures: CMW 26170 = CBS 122071 (ex-type).
Hosts: Asymptomatic branches (sapwood) of Acacia synchronica,
Adansonia gibbosa, Crotalaria medicaginea and Grevillia agrifolia
(Pavlic et al. 2008). Pathogen of Mangifera indica and Ficus carica
(Ray et al. 2010).
Known distribution: northern Western Australia.
Notes: Although N. novaehollandiae is morphologically and
phylogenetically similar to N. dimidiatum (Punithalingam &
Waterston 1970, Crous et al. 2006), Pavlic et al. (2008) reported
muriform, dichomera-like conidia in the isolates that they studied
and for this reason they regarded it as a distinct species.
Phaeobotryon Theiss. & Syd., Ann. Mycol. 13: 664. 1915.
MycoBank MB3892.
Type species: Phaeobotryon cercidis (Cooke) Theiss. & Syd., Ann.
Mycol. 13: 664. 1915.
Ascomata black, immersed to erumpent, subglobose to ovoid,
multilocular, wall composed of layers of dark brown textura
angularis. Pseudoparaphyses hyphae-like, septate, constricted
at septa. Asci 8-spored, bitunicate, issitunicate, clavate to
cylindro-clavate, short-pedicellate, apically rounded with an
ocular chamber. Ascospores hyaline to brown, 2-septate, ellipsoid
to broad fusiform, with an apiculus at each end, immature asci
surrounded by a mucilagenous sheath. Conidiomata pycnidial,
stromatic, black, ostiolate, separate or aggregated, immersed
to erumpent, unilocular or multilocular, ostiolate. Ostiole circular,
central, papillate. Paraphyses hyaline, thin-walled, usually
aseptate, sometimes becoming 1–2-septate. Conidiogenous
cells holoblastic, hyaline, thin-walled, smooth, cylindrical to
doliiform. Conidia ellipsoidal to oblong or obovoid, ends rounded,
moderately thick-walled, initially hyaline, becoming brown, mostly
2-septate at maturity.
Notes: Phaeobotryon was introduced by Theissen & Sydow (1915)
to accommodate Dothidea cercidis. This taxon was considered a
distinct genus on account of its pale brown, 2-septate ascospores,
which were reported as hyaline in the original description. In their
broad concept of Botryosphaeria, von Arx & Müller (1954, 1975)
considered Phaeobotryon as a synonym of Botryosphaeria.
Phillips et al. (2008) reinstated Phaeobotryon after they showed
that it is morphologically and phylogenetically distinct from all
other genera in the Botryosphaeriaceae. The 2-septate, brown
ascospores with an apiculus at each end are characteristic for
the genus. Only two species (P. mamane and P. cupressi) are
currently known in culture and they can be separated on the size
of their conidia.
Species descriptions
Phaeobotryon mamane Crous & A.J.L. Phillips, Persoonia
21: 45. 2008. MycoBank MB506581. See Phillips et al. (2008)
for illustrations.
Ascomata pseudothecial, dark brown to black, stromatic, globose,
aggregated in botryose clusters or separate, immersed, becoming
erumpent, ostiolate, up to 350 μm diam, wall consisting of 4–6 cell
www.studiesinmycology.org
149
PhilliPs et al.
Fig. 60. Phaeobotryon cupressi. A. Conidiomata formed on pine needles in culture. B, C. Conidia on conidiogenous cells D. Paraphyses and developing conidia. E, F.
Spermatogenous cells. G. Spermatia. H. Hyaline immature conidia. I. Mature and germinated, hyaline and septate or aseptate conidia. J, K. Mature, brown septate or aseptate
conidia in two different focal planes to show verruculose inner surface of the wall. L. Brown chlamydospores. Scale bars: A = 500 μm, B, D, H–J, L = 10 μm, C, G, K = 5 μm, E,
F = 2.5 μm.
layers of dark brown textura angularis. Pseudoparaphyses hyaline,
smooth, multiseptate, with septa 10–23 μm apart, constricted
at septa, 3–4 μm wide. Asci bitunicate, 8-spored, stipitate,
thick-walled with thick endotunica and well-developed apical
chamber, 120–150(–200) × 25–30 μm, with biseriate ascospores.
Ascospores ellipsoid to ovate, (30–)37–40(–45) × (11–)13–15(–16)
μm, 2-septate, with three cells of equal length, not constricted at
septa, inely verruculose, widest in middle with conical apiculus
at one or both ends. Spermatogonia morphologically similar to
conidiomata, also formed in culture. Spermatia hyaline, rod-shaped
with rounded ends, 3–5 × 2 μm. Conidiomata pycnidial, stromatic,
ostiolate, separate or aggregated, globose, black, immersed to
erumpent, unilocular, up to 350 μm diam, wall consisting of 4–6
layers of brown textura angularis. Conidiogenous cells cylindrical
to doliiform, hyaline, smooth, proliferating percurrently near apex,
10–14 × 4–8 μm. Conidia ellipsoid to oblong or subcylindrical or
obovoid, brown, smooth to inely verruculose, moderately thickwalled, granular, guttulate, ends rounded, 1(–2)-septate, base with
inconspicuous scar, slightly lattened, (30–)35–38(–43) × (12–)14–
15(–16) μm.
Type: uSA, Hawaii, Manna Koa Park, Saddle Road, on stems
of Sophora chrysophylla, Jul. 2005, W. Gams, holotype CBS
H-20109.
150
Cultures: CPC 12440 = CBS 122980 (ex-type).
Host: Sophora chrysophylla (Phillips et al. 2008).
Known distribution: USA (Hawaii) (Phillips et al. 2008).
Note: Asexual morph dothiorella/spencermartinsia-like, but with up
to two transverse septa and apiculi at either end of the ascospores.
Phaeobotryon cupressi Abdollahz., Zare & A.J.L. Phillips,
Persoonia 23: 6. 2009. MycoBank MB513236. Fig. 60.
Ascomata not reported. Conidiomata pycnidial, stromatic,
supericial, dark-brown to black, mostly unilocular on pine
needles and up to 650 μm diam, mostly multilocular on Populus
twigs, individual or aggregated, thick-walled, ostiolate. Ostiole
central, circular, non-papillate. Paraphyses hyaline, thin-walled,
arising from the conidiogenous layer, extending above the level
of developing conidia, up to 42 μm long, 4.8 μm wide, usually
aseptate, sometimes becoming up to 2-septate, tip rounded,
occasionally branched. Conidiophores absent. Conidiogenous
cells hyaline, smooth, thin-walled, cylindrical, holoblastic, phialidic,
proliferating internally with visible periclinal thickening, 7–14 × 2–5
The Botryosphaeriaceae: genera and sPecies known from culTure
μm. Conidia thick-walled, initially hyaline, oval, both ends broadly
rounded, aseptate, forming a single septum at germination, rarely
becoming brown and 1-septate, internally verruculose when aged,
(19.5–)21–28(–30) × (10–)11–15(–17) μm, 95 % conidence
limits = 24–25 × 12–12.5 μm (av. ± S.D. = 24.8 ± 1.9 × 12.4 ±
1.3 μm), L/W ratio = 2. Spermatogonia globose, dark-brown to
black, supericial, occasionally immersed in pine needle or Populus
tissue. Spermatophores cylindrical, hyaline, aseptate becoming
1–2-septate, branched, 7–13 × 1.5–2.5 μm. Spermatogenous cells
hyaline, thin-walled, phialidic, proliferating internally, giving rise to
periclinal thickening, 6–10 × 1–2 μm. Spermatia oval, thin-walled,
hyaline, aseptate 2–4 × 1–2 μm. Chlamydospores intercalary,
brown, smooth, thick-walled, formed within the agar medium.
Culture characteristics: Colonies on PDA with abundant aerial
mycelium towards periphery, appressed in the centre, becoming
grey-olivaceous to olivaceous-grey at the surface, and greyolivaceous in reverse after 2 wk in the dark at 25 °C, reaching 46–
53 mm diam after 4 d in the dark at 25 °C. Cardinal temperatures
for growth: min 5 °C, max > 35 °C, opt 25 °C.
Type: Iran, Golestan Province, Gorgan, City Park, on twigs of
Cupressus sempervirens, 15 Aug. 2006, M.A. Aghajani, holotype
IRAN 13940F.
Cultures: IRAN 1455C = CBS 124700 (ex-type).
Host: Cupressus sempervirens (Abdollahzadeh et al. 2009),
Juniperus scopulorum (Alves et al. 2013).
Known distribution: Iran (Abdollahzadeh et al. 2009), USA (Alves
et al. 2013).
Notes: This species differs from P. quercicola and P. mamane in
its smaller conidia, and has been collected only from Cupressus
species. The hyaline, aseptate conidia of P. cupressi are supericially
similar to those of other Diplodia species with hyaline conidia.
Furthermore, conidial dimensions of P. cupressi are similar to those
of Diplodia cupressi (21.5–30.5 × 12–16 μm) as reported by Alves
et al. (2006). It is thus possible that P. cupressi has been mistaken
for D. cupressi in the past. Pycnidial paraphyses in Phaeobotryon
clearly distinguish this genus from Diplodia.
Pseudofusicoccum Mohali, Slippers & M.J. Wingf., Stud.
Mycol. 55: 249. 2006. MycoBank MB500884.
Type species: Pseudofusicoccum stromaticum (Mohali, Slippers &
M.J. Wingf.) Mohali, Slippers & M.J. Wingf., Stud. Mycol. 55: 249.
2006.
Resembling species of Fusicoccum, but distinct in having conidia
encased in a persistent mucous sheath. Conidia are also more
cylindrical than in Fusicoccum species.
Notes: Pseudofusicoccum was introduced by Crous et al. (2006)
for species that are morphologically similar to Fusicoccum and
Neofusicoccum but phylogenetically distinct from both of these
genera. While it was originally based on Ps. stromaticum a further
ive species have subsequently been added to the genus. Species
are distinguished primarily on the dimensions of their conidia and
on pigment production in culture. Thus far no sexual morphs have
been found. The species appear to be restricted to tropical or
sub-tropical regions and occur mainly as endophytes. There is no
evidence of host-speciicity.
Key to Pseudofusicoccum spp.
1.
1.
Forms a violet pigment in culture ............................................................................................................................................................. 2
No violet pigment in cultures ................................................................................................................................................................... 3
2.
2.
Conidia on average greater than 30 μm long ..................................................................................................................... Ps. violaceum
Conidia on average smaller than 25 μm long ................................................................................................................... Ps. adansoniae
3.
3.
Conidia on average smaller than 30 μm long .......................................................................................................................................... 4
Conidia on average = 30 μm or more long .................................................................................................................. Ps. kimberleyense
4.
4.
Conidia on average 7 μm or more wide .................................................................................................................................................. 5
Conidia on average smaller than 7 μm wide ................................................................................................................... Ps. stromaticum
5.
5.
Conidia 20–26 × 6.5×7.5 μm .............................................................................................................................................. Ps. olivaceum
Conidia 21–29 × 7–8 μm .................................................................................................................................................. Ps. ardesiacum
dNA phylogeny
Species descriptions
Six species can be distinguished in the ITS phylogeny (Fig. 61).
Support for Ps. ardesiacum and Ps. kimberleyensis is very low
and the branch lengths for these two species are very short.
Morphologically they are also very similar, although conidia of
Ps. kimberleyensis are, on average, longer than those of Ps.
ardesiacum.
Pseudofusicoccum adansoniae Pavlic, T.I. Burgess, M.J.
Wingf., Mycologia 100: 855. 2008. MycoBank MB512048.
See Pavlic et al. (2008) for illustrations.
www.studiesinmycology.org
Ascomata not seen. Conidiomata semi-immersed, solitary,
globose, papillate, chestnut, covered by hyphal hairs, up to 500
μm diam. Conidiogenous cells holoblastic, smooth, cylindrical,
hyaline, the irst conidium produced holoblastically and
subsequent conidia enteroblastically, (9–)10–15(–16) × (1.5–)2–
151
PhilliPs et al.
100
P. stroma*cum CBS 117448
P. stroma*cum CBS 117449
P. olivaceum CBS 124939
98
54
P. olivaceum CBS 124940
100
P. adansoniae WAC 12689
P. adansoniae CBS 122055
P. violaceum CBS 124936
97
57
100
P. violaceum CBS 124937
P. ardesiacum WAC 13294
P. ardesiacum CBS 122062
P. kimberleyense CBS 122058
P. kimberleyense CBS 122059
Neofusicoccum parvum CMW 9081
Botryosphaeria dothidea CBS 115476
10 changes
Fig. 61. One of six equally most parsimonious trees obtained from combined ITS
and EF1-α sequence data for species in Pseudofusicoccum. Bootstrap values from
1000 replicates are given at the nodes.
3(–3.5) μm (av. 12.7 × 2.4 μm). Conidia ellipsoid, occasionally
slightly bent or irregularly shaped, apices rounded, smooth
with ine granular content, hyaline, thin-walled, covered with a
persistent mucus layer, unicellular, forming 1 or 2 septa before
germination, (19–)21–24(–26) × (3.5–) 4.5–6(–6.5) μm (av. size
of conidia = 22.5 × 5.2 μm), L/W = 4.3.
Culture characteristics: Colonies initially white with moderately
dense, appressed mycelial mat, submerged mycelium turning greyolivaceous to olivaceous-black from the middle of colony after 3–5
d and becoming dark slate-blue with age, aerial mycelium slightly
luffy, becoming dense, cottony with age, sometimes remaining
white to smoke-grey, usually turning pale olivaceous-grey within 7
d and becoming olivaceous-grey to iron grey with age; conidiomata
readily formed from the middle of colony within 7–10 d, covering the
entire surface of the colony and immersed in the medium. Optimum
growth at 30 °C, covering the 90 mm diam Petri dish after 4 d in
the dark.
Type: Australia, Western Australia, Derby, on Adansonia gibbosa,
Jul. 2006, T.I. Burgess, holotype PREM 59841 (a dry culture on
pine needles).
Cultures: CBS 122055 = CMW 26147 (ex-type).
Hosts: Adansonia gibbosa, Acacia synchronica, Eucalyptus sp.,
Ficus opposita (Pavlic et al. 2008), Adansonia gregorii, Grevillea
agrifolia (Sakalidis et al. 2011).
Known distribution: Australia (Pavlic et al. 2008, Sakalidis et al.
2011).
152
Notes: This species appears to be a non-specialised endophyte
since it has been found on asymptomatic hosts residing in ive
widely separate genera. It has been found only in Australia.
Pseudofusicoccum ardesiacum Pavlic, T.I. Burgess, M.J.
Wingf., Mycologia 100: 858. 2008. MycoBank MB512051.
See Pavlic et al. (2008) for illustrations.
Ascomata not seen. Conidiomata semi-immersed, solitary,
globose, papillate, chestnut, covered by hyphal hairs, up to 510 μm
diam. Conidiogenous cells holoblastic, smooth, cylindrical, hyaline,
the irst conidium produced holoblastically and subsequent conidia
enteroblastically, (6–)7.5–10(–11) × (2.7–)3–4(–4.5) μm (av. = 8.6
× 3.5 μm). Conidia ellipsoid to rod-shape, straight or slightly bent,
apices rounded, smooth with ine granular content hyaline, thinwalled, covered with a persistent mucus layer, unicellular, forming
1–3 septa before germination, (17.5–)21–29(–32) × (6.5–)7–8(–9)
μm (av. = 25 × 7.5 μm), L/W = 3.3.
Culture characteristics: Colonies initially white with sparse to
moderately dense appressed mycelial mat; submerged mycelium
dark violet to dark blue (middle of the colony) and smoke grey to
grey-olivaceous toward edges within 3–5 d, becoming violaceous
grey to slate blue with age, aerial mycelium slightly luffy, becoming
dense, cottony with age, turning smoke grey to pale purplish grey
in the middle of colony and smoke grey to grey-olivaceous toward
edges after 5–7 d, becoming lavender grey with age; occasional
columns of aerial mycelium in the middle of colony, reaching the lid,
colonies slightly irregular with sinuate edges, conidiomata readily
formed in culture and immersed in aerial mycelia on the entire
colony surface within 7–10 d. Optimum growth at 30 ºC, covering
the 90 mm diam Petri dish after 4 d in the dark.
Type: Australia, Western Australia, Mount Hardman, Great
Northern Highway, on Adansonia gibbosa, Jul. 2006, T.I. Burgess,
holotype PREM 59843 (a dry culture on pine needles).
Cultures: CMW 26159 = CBS 122062 (ex-type).
Hosts: Adansonia gibbosa, Eucalyptus sp. (Pavlic et al. 2008).
Known distribution: Western Australia (Pavlic et al. 2008).
Notes: This species is probably an endophyte not restricted to any
host since it has been found on dying branches of Adansonia and
in asymptomatic branches of Eucalyptus sp. (Pavlic et al. 2008). It
is known only from Australia.
Pseudofusicoccum kimberleyense Pavlic, T.I. Burgess,
M.J. Wingf., Mycologia 100: 857. 2008. MycoBank
MB512049. See Pavlic et al. (2008) for illustrations.
Ascomata not seen. Conidiomata semi-immersed, solitary,
globose, papillate, chestnut brown, covered by hyphal hairs,
up to 500 μm diam. Conidiogenous cells holoblastic, smooth,
cylindrical to subcylindrical, hyaline, the irst conidium produced
holoblastically and subsequent conidia enteroblastically, (7–)
8.5–11(–14) × (2.5–) 3–3.5(–4) μm (av. = 9.8 × 3.3 μm). Conidia
ellipsoid, straight or slightly curved, apices rounded, smooth
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 62. Pseudofusicoccum stromaticum. A. Conidiomata developing on pine needle in culture. B. Conidiogenous cells. C. Conidia. The mucilagenous sheath is visible on the
conidium in the insert. Scale bars: A = 1 mm, B, C = 10 μm.
with ine granular content, hyaline, thin-walled, covered with a
persistent mucus layer, unicellular, forming 1–4 septa before
germination, (24–)28–33(–34) × (6.5–)7–8(–8.5) μm (av. = 30.7
× 7.4 μm), L/W = 4.1.
occasionally granular, guttulate, surrounded by a persistent mucoid
sheath, apex and base blunt to broadly rounded, bacilliform, (18–)
20–25.5(–30.5) × (6–)6.5–7.5(–9) μm (av. = 22.8 × 7.0 μm).
Culture characteristics: Colonies slightly irregular with sinuate
edges, initially white, forming a moderately dense, appressed
mycelial mat, submerged mycelium citrine to grey-olivaceous
from the middle of colony after 3–5 d, becoming olivaceous-black
to black with age, aerial mycelium slightly luffy, becoming dense,
cottony with age, smoke-grey to pale olivaceous-grey. Optimum
growth at 30 ºC, covering a 90 mm diam Petri dish after 4 d in the
dark.
Type: South Africa, Mpumalanga Province, Kruger National
Park, Pretoriuskop, on an asymptomatic branch of Pterocarpus
angolensis, Sep. 2005, J. Roux, holotype PREM 60328.
Type: Australia, Western Australia, Tunnel Creek National Park,
on Acacia synchronica, Jul. 2006, T.I. Burgess, holotype PREM
59842 (a dry culture on pine needles).
Cultures: CMW 26156 = CBS 122058 (ex-type).
Hosts: Adansonia gibbosa, Acacia synchronica, Eucalyptus sp.
and Ficus opposita (Pavlic et al. 2008).
Culture characteristics: Cultures luffy, initially white to amber at the
centre, olivaceous at the edges, becoming white to olivaceous with
age. Optimum temperature for growth 25 °C.
Cultures: CMW 20881 = CBS 124939 (ex-type), CMW 22637 =
CBS 124940, CMW 22643 = CBS 124941 (ex-paratype).
Host: Pterocarpus angolensis (Mehl et al. 2011).
Known distribution: South Africa (Mehl et al. 2011).
Notes: In addition to the host on which it was described, this
species has also been found on Terminalia sericea (Mehl et al.
(2011), suggesting it is a common endophyte on other tree species.
Known distribution: Western Australia (Pavlic et al. 2008).
Note: The wide range of hosts and absence of symptoms on the
hosts suggest that this species is a non-specialised endophyte
known only in Australia.
Pseudofusicoccum olivaceum Mehl & Slippers, Mycologia
103: 537. 2011. MycoBank MB513501. See Mehl et al. (2011)
for illustrations.
Ascomata not seen. Conidiomata on host and on pine needles on
water agar pycnidial, stromatic, subcuticular, unilocular, dark brown,
mostly solitary, applanate, covered with hyphae/mycelium, wall
composed of three layers: an outer layer of thick-walled dark to light
brown textura angularis; a middle layer of thin-walled light brown
cells; an inner layer of thin-walled hyaline cells, (480–)530–650(–
690) μm diam. Ostiole central, circular, papillate. Conidiogenous
cells hyaline, holoblastic, smooth, cylindrical, guttulate, proliferating
percurrently to form one or two indistinct annellations, or
proliferating at the same level giving rise to periclinal thickenings.
Paraphyses (3–)4.5–8.5(–12.5) × (1.5–)3–4.5(–6.5) μm (av. =
6.6 × 3.7 μm). Conidia hyaline, thin-walled, unicellular, aseptate,
www.studiesinmycology.org
Pseudofusicoccum stromaticum (Mohali, Slippers & M.J.
Wingf.) Mohali, Slippers & M.J. Wingf., Stud. Mycol. 55: 249.
2006. MycoBank MB500885. Fig. 62.
Basionym: Fusicoccum stromaticum Mohali, Slippers & M.J.
Wingf., Mycol. Res. 110: 408. 2006.
Ascomata not seen. Conidiomata large, supericial, multilocular,
locule totally embedded without ostioles when formed on on
MEA, smaller, uniloculate, ostiolate on pine needles; eustromatic,
covered with hyphae, locule walls consisting of a dark brown
textura angularis, becoming thinner and hyaline towards the
conidiogenous region. Conidiogenous cells hyaline, holoblastic,
smooth, cylindrical, producing a single apical conidium, the
irst conidium produced holoblastically and subsequent conidia
produced enteroblastically, proliferating at the same level forming
periclinal thickenings, (10–)11–15(–17) × (1.5–)2–3 μm (av. = 13
× 2.5 μm, L/W = 5.3). Conidia hyaline, thin to slightly thick-walled,
aseptate, granular, cylindrical, straight to slightly curved, apex and
base blunt to bluntly rounded, surrounded by a persistent mucous
sheath, (19–)20–23(–24) × (4–)5–6 μm (av. = 21.5 × 5.5 μm), L/W
= 4.
153
PhilliPs et al.
Culture characteristics: Colonies luffy, greenish olivaceous with
reverse olivaceous after 15 d on MEA at 25 °C, reaching 70–75 mm
diam on MEA after 4 d in the dark at 25 °C. Cardinal temperatures
for growth: min 15 °C (little or no growth), max < 40 °C (no growth
at 40 °C), opt 30–35 °C.
Type: Venezuela, Portuguesa State, Acarigua, Smurit Company,
on branches of Eucalyptus urophylla, Feb. 2003, S. Mohali,
holotype PREM 58237.
Cultures: CMW 13366 (ex-holotype), CMW 13434 = CBS 117448,
CMW 13435 = CBS 117449.
Hosts: Eucalyptus spp. (Mohali et al. 2006), Acacia mangium
(Mohali et al. 2006), Mangifera indica (Marques et al. 2012).
Known distribution: Brazil (Marques et al. 2012), Venezuela (Mohali
et al. 2006).
Notes: Pseudofusicoccum stromaticum was originally isolated from
asymptomatic as well as dead and dying branches and stems of
Eucalyptus and Acacia mangium trees in Venezuela. The presence
of the fungus in asymptomatic branches of two different host genera
suggests that it is a generalist endophyte. However, it has been
reported to cause die-back of Mangifera indica in Brazil (Marques
et al. 2013).
Pseudofusicoccum violaceum Mehl & Slippers, Mycologia
103: 542. 2011. MycoBank 513500. See Mehl et al. (2011)
for illustrations.
Ascomata not seen. Conidiomata on the host and on pine needles
on water agar pycnidial, stromatic, supericial, unilocular, dark
brown, mostly solitary, more or less globose/circular, covered with
hyphae/mycelium, wall composed of three layers: an outer layer
of thick-walled, dark to light brown textura angularis, a middle
layer of thin-walled light brown cells, and an inner layer of thinwalled hyaline cells, (470–)500–615(–660) μm diam. Ostiole
central, circular, papillate. Conidiogenous cells hyaline, holoblastic,
smooth, cylindrical, proliferating percurrently to form one or two
distinct annellations, or proliferating at the same level giving rise
to periclinal thickenings, (5.5–)6–11(–17) × (2.5–)3.5–5(–6.5) μm
(av. = 8.6 × 4.3 μm). Paraphyses not seen. Conidia hyaline, thinwalled, unicellular, aseptate, granular, guttulate, surrounded by a
persistent mucoid sheath, apex and base blunt to broadly rounded,
cylindrical, (26.5–)29.5–36(–39.5) × (8–)8.5–10.5(–11.5) μm (av. =
33.0 × 9.5 μm).
Culture characteristics: Cultures with luffy mycelium, initially white
to amber in the center and violet on the edges, turning olivaceous to
greenish black in the centre and becoming olivaceous to greenish
black with age. Optimum temperature for growth 30 °C.
Type: South Africa, Mpumalanga Province, Mawewe Nature
Reserve, on an asymptomatic branch of Pterocarpus angolensis,
Dec. 2005, J.W.M. Mehl & J. Roux, holotype PREM 60333.
Cultures: CMW 22679 = CBS 124936 (ex-type), CMW 22671 =
CBS 124938 (ex-paratype).
154
Host: Pterocarpus angolensis (Mehl et al. 2011).
Known distribution: South Africa (Mehl et al. 2011).
Notes: The violet pigment formed in cultures of this species was
considered to be distinctive for Ps. violaceum (Mehl et al. 2011).
However, a similar pigment is also found in Ps. ardesiacum (Pavlic
et al. 2008). Nevertheless, the two species can be distinguished
based on conidial dimensions and are clearly differentiated in ITS
and EF1-α phylogenies. The wide host range suggests that this is
a non-specialised endophyte.
Spencermartinsia A.J.L. Phillips, A. Alves & Crous,
Persoonia 21: 51. 2008. MycoBank MB511762.
Type species: Spencermartinsia viticola (A.J.L. Phillips & J. Luque)
A.J.L. Phillips, A. Alves & Crous, Persoonia 21: 51. 2008.
Ascomata pseudothecial, ostiolate. Pseudoparaphyses thin-walled,
hyaline, septate, constricted at septa. Asci bitunicate, 8-spored,
clavate, stipitate, developing amongst thin-walled, septate
pseudoparaphyses, with biseriate ascospores. Ascospores hyaline
when young, brown when mature, uniseptate with an apiculus
at each end. Conidiomata pycnidial, stromatic. Conidiophores
absent. Conidiogenous cells lining inner surface of conidiomata,
holoblastic, proliferating internally producing periclinal thickenings,
or proliferating percurrently to form annellations. Conidia initially
hyaline, becoming dark brown and 1-euseptate within the pycnidial
cavity often while still attached to the conidiogenous cell, thickwalled, externally smooth, internally verruculose, broadly rounded
at the apex, base truncate.
Notes: Spencermartinsia was introduced by Phillips et al. (2008)
for species similar to Dothiorella but that differ in having 2-celled
ascospores with an apiculus at either end of the ascospores. This
minor difference was considered to be taxonomically meaningful
since the presence or lack of apiculi on ascospores also separates
other genera in this family, such as Barriopsis (no apiculus)
from Sphaeropsis (apiculus present), and this was supported
phylogenetically. Nevertheless, this is a tenuous and dificult
morphological character to apply, especially since a sexual morph
has been reported only for S. viticola and it is not clear whether
this is a consistent character for the genus. Furthermore, with the
addition of further species in Dothiorella, the phylogenetic distinction
between the two genera is becoming less obvious. However, we
continue to recognise Spencermartinsia as a separate genus
pending further phylogenetic and morphological studies including
additional species. Spencermartinsia is presently monotypic based
on S. viticola. Based on phylogenetic analyses, two recently
described species, S. uruguayensis and S. pretoriensis have been
re-combined in Dothiorella (see above).
dNA phylogeny
Based on ITS and EF1-α sequence data, Spencermartinsia is
clearly separated from Dothiorella. In the phylogenetic analyses two
main clades are recognised in Spencermartinsia (Figs 32, 33). The
irst clade constitutes S. viticola while the other includes three subclades including four isolates CBS 500.72 (Diplodia medicaginis),
CBS 117006, ICMP 16827 and ICMP 16828, representatives of
three distinct species. Isolate CBS 117006 identiied by Luque
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 63. Spencermartinsia viticola. A. Ascomata erumpent through the host bark. B. Ascoma cut through horizontally revealing the white contents with dark spots corresponding
to asci and ascospores. C. Vertical section through an ascoma. D. Septate paraphyses. E. Clavate ascus containing eight biseriate, dark brown, 1-septate ascospores. F.
Ascospores. G. Ascospores with small, rounded apiculi (arrows). H. Conidiomata partially erumpent through the host bark. I, J. Conidiogenous cells. K. Conidia. Scale bars: A,
H = 500 μm, B = 250 μm, C = 50 μm, D–G, I–K = 10 μm.
et al. (2005) as B. viticola, exhibited some differences in culture
morphology and sequence data from the ex-type strain and
other strains as discussed by Phillips et al. (2008), reside in a
www.studiesinmycology.org
distinct clade. The two isolates ICMP 16827 and ICMP 16828 on
Citrus sinensis from New Zealand constitute a distinct clade as
representatives of a new species. Furthermore, isolate CBS 500.72
155
PhilliPs et al.
previously characterised as Diplodia medicaginis formed another
distinct clade and is clearly a missidentiication. These species are
not described here due to their uncertain taxonomic status.
Species descriptions
Spencermartinsia viticola (A.J.L. Phillips & J. Luque) A.J.L.
Phillips, A. Alves & Crous, Persoonia 21: 51. 2008. MycoBank
MB511763. Fig. 63.
Basionym: Dothiorella viticola A.J.L. Phillips & J. Luque, Mycologia
97: 1116. 2005.
= Botryosphaeria viticola A.J.L. Phillips & J. Luque, Mycologia 97: 1116. 2005.
Ascomata dark brown to black, stromatic, pyriform,
pseudothecial, isolated or in botryose clusters up to 2 mm diam,
initially immersed in host, partially erumpent at maturity, up to
240 μm diam, ostiole circular, central, papillate, wall up to 60
μm thick, of dark brown thick-walled textura angularis, and lined
with thin-walled, hyaline cells. Pseudoparaphyses thin-walled,
hyaline, frequently septate, slightly constricted at septum, 3.5–
4.5(–5) μm wide. Asci arising from base of ascoma, stipitate,
clavate, thick-walled, bitunicate with a thick endotunica and a
well-developed apical chamber, 8-spored, irregularly biseriate,
100–110 × 25–30 μm. Ascospores oblong, ovate to sub-clavate,
mostly 1-septate, slightly constricted at septum, dark brown,
moderately thick-walled, finely verruculose on inner surface,
often inequilateral, widest in lower 1/3 to middle of apical cell,
often with a small rounded projection at tip and base of spore,
basal cell tapering towards obtuse base, (19–) 22.5–23.5(–27)
× (8.5–)10.5–11(–14.5) μm (av. ± S.D. = 23.1 ± 0.2 × 10.9
± 0.1 μm). Conidiomata pycnidial, stromatic, separate or
aggregated into botryose clusters up to 2 mm diam, individual
conidiomata spherical to globose, black, immersed, partially
erumpent when mature, unilocular, 200–360 μm diam, thickwalled, wall consisting of three layers: an outer layer of dark
brown, thick-walled textura angularis, a median layer of dark
brown thin-walled cells textura angularis, and an inner layer
of thin-walled, hyaline cells. Ostiole single, central, circular,
papillate. Conidiophores absent. Conidiogenous cells discrete
or integrated, cylindrical to broad lageniform, (5–)8.5–10(–14) ×
(3–)4.5–5(–7) μm, hyaline, smooth, holoblastic, indeterminate,
proliferating at same level to form periclinal thickenings or rarely
proliferating percurrently giving rise to 1–2 annellations. Conidia
brown, oblong to subcylindrical, septate, occasionally slightly
constricted at septum, moderately thick-walled, externally
smooth, internally finely verruculose, ends rounded, often with
a truncate base, (16–)20–20.5(–26) × (7–)9–9.5(–12) μm (av. ±
S.D. = 20.4 ± 0.1 × 9.3 ± 0.1 μm), L/W ratio = 2.2.
Culture characteristics: Colonies on PDA reaching 40 mm in radius
after 3 d at 25 °C. Aerial mycelium present, colonies cottony, dark
olive to greyish, darkening from the center of the colony after 3
d, colony fully darkened after 6–10 d. Conidiomata produced after
20–30 d in culture at 23 °C under near UV black light (12/12 h
photoperiod). Cardinal temperatures for growth: min 5 °C, max
< 35 °C, opt 20–25 °C.
Type: Spain, Catalonia, Vim-bodí, near the Monastery of Poblet,
on pruned canes of Vitis vinifera cv. Garnatxa Negra, Aug. 2004, J.
Luque & S. Martos, holotype LISE 95177.
156
Cultures: CBS 117009 (ex-type), CBS 302.75.
Hosts: Citrus sp. (Adesemoye & Eskalen 2011, Inderbitzin et al.
2010), Populus cathayana (Zhang et al. 2009), Poniciana gilliesii
(Phillips et al. 2008), Prunus persica and P. salicina (Damm et al.
2007), Vitis vinifera (de Wet et al. 2009, Luque et al. 2005, Qiu et
al. 2011, Úrbez-Torres et al. 2007).
Known distribution: Australia (Qiu et al. 2011), China (Zhang et
al. 2009), France (Phillips et al. 2008), South Africa (Damm et
al. 2007, de Wet et al. 2009), Spain (Luque et al. 2005) and USA
(Adesemoye & Eskalen 2011, Inderbitzin et al. 2010, Úrbez-Torres
et al. 2007).
Notes: The sexual morph is extremely rare compared to the
abundant asexual morph. The ex-type isolate of Spencermartinsia
viticola (CBS 117009) clustered with an isolate previously identiied
as Diplodia spegazziniana (CBS 302.75), which is clearly a
misidentiication.
Sphaeropsis Sacc., Michelia 2: 105. 1880. MycoBank
MB9992.
= Phaeobotryosphaeria Speg., Ann. Inst. Rech. Agron. 17, 10: 120. 1908.
Type species: Sphaeropsis visci (Alb. & Schwein.) Sacc., Michelia
2: 105. 1880.
Ascomata pseudothecial, brown to black, unilocular, thick-walled.
Pseudoparaphyses hyaline, septate. Asci bitunicate, 8-spored,
thick-walled with thick endotunica and well-developed apical
chamber. Ascospores brown, aseptate with small apiculus at
either end. Conidiomata pycnidial, stromatic, immersed to
erumpent, thick-walled, wall composed of several layers of
dark-brown textura angularis. Ostiole single, central, papillate.
Paraphyses hyaline, aseptate, thin-walled. Conidiogenous
cells hyaline, discrete, proliferating internally to form periclinal
thickenings. Conidia oval, oblong or clavate, straight, aseptate,
moderately thick-walled.
Notes: Sphaeropsis was introduced by Saccardo (1880) for
species of Diplodia with brown, aseptate conidia with S. visci as
the type species. Since then more than 600 species have been
described (MycoBank accessed 10 Jul. 2013) mostly on the basis
of host association. However, few of these names are currently
in use and cultures are not available for the species that deine
them. The well-known pine pathogen that has been known as
Sphaeropsis sapinea is clearly not a species of Sphaeropsis and
is retained in Diplodia.
Phillips et al. (2008) established the connection between the
asexual and the sexual morph in S. visci. A bitunicate ascomycete,
with characters corresponding to Phaeobotryosphaeria, occurring
on Viscum album produced in culture a coelomycete with large,
brown, aseptate conidia typical of Sphaeropsis and corresponding
to the current concept of S. visci. Phillips et al. (2008) applied the
one fungus-one name concept and chose Phaeobotryosphaeria
in favour of Sphaeropsis. However, following the ammendments
to the ICBN ratiied at the 18th Botanical Congress in Melbourne,
it is now clear that priority of names will no longer be based on
the life stage of the fungus. Thus, the older name Sphaeropsis
(1880) takes priority over Phaeobotryosphaeria (1908). To correct
this, new combinations are introduced here together with the
descriptions of the species considered by Phillips et al. (2008).
Pycnidial paraphyses distinguish Sphaeropsis morphologically
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 64. Sphaeropsis citrigena. A–C. Asci with brown ascospores. D. Pseudoparaphyses. E–G. Brown, aseptate ascospores with apiculi (arrows). H. Conidium developing on a
conidiogenous cell with periclinal thickenings. I. Hyaline, aseptate conidia. J. Conidiomatal paraphyses. Scale bars A = 50 μm, B–D = 20 μm, E–J = 10 μm.
from Diplodia while the striate conidia of Lasiodiplodia differentiate
it from Sphaeropsis, which has smooth-walled conidia. Although
more than 600 names exist in Sphaeropsis, only four species are
currently known in culture. The distinctly pitted conidial walls of S.
porosum distinguish it from the other two species. The paraphyses
with swollen tips and conidia that soon become pigmented
distinguish S. visci from S. citrigena in which conidia remain hyaline
www.studiesinmycology.org
for long periods, rarely become pigmented and paraphyses tips
are not swollen. The only known cultures of S. eucalypti have not
sporulated and thus could not be included in the key, which relies
on characters of the asexual morph.
157
PhilliPs et al.
Fig. 65. Sphaeropsis eucalypti. A, B. Asci with ascospores. C. Ascospores. Scale bars = 10 μm. Scale bar in A applies to B.
Key to Sphaeropsis spp.
1.
1.
Conidial wall distinctly pitted ..................................................................................................................................................... S. porosa
Conidial wall not pitted ............................................................................................................................................................................ 2
2.
2.
Conidiomatal paraphyses with swollen tips ................................................................................................................................... S. visci
Conidiomatal paraphyses not swollen at tip ............................................................................................................................ S. citrigena
Sphaeropsis citrigena (A.J.L. Phillips, P.R. Johnst. &
Pennycook) A.J.L. Phillips & A. Alves, comb. nov. MycoBank
MB805463. Fig. 64.
Basionym: Phaeobotryosphaeria citrigena A.J.L. Phillips, P.R.
Johnst. & Pennycook, Persoonia 21: 50. 2008.
Ascomata pseudothecial, brown to black, separate or aggregated,
immersed, becoming erumpent, ostiolate, wall composed of several
layers of dark brown textura angularis. Pseudoparaphyses hyaline,
smooth, 4–6 μm wide, multiseptate, with septa 11–26 μm apart,
constricted at septa. Asci bitunicate, 8-spored, stipitate, thick-walled
with thick endotunica and well-developed apical chamber, 180–230
× 35–43(–50) μm, with biseriate ascospores. Ascospores reddishbrown when mature, ellipsoid to ovoid with both ends rounded, with
an apiculus at either end, aseptate, externally smooth, internally inely
verruculose, widest in middle to upper third, (27.5–)29–37.5(–38.5) ×
(14.5–)15.5–18(–19.5) μm. Conidiomata immersed to erumpent and
supericial, unilocular, up to 500 μm wide, wall composed of several
layers of dark brown textura angularis. Paraphyses hyaline, aseptate,
up to 25 μm long and 3–3.5 μm wide, apex not swollen. Conidiogenous
cells hyaline, discrete, proliferating internally to form periclinal
thickenings, 8–11 × 4–6.5 μm. Conidia oval, apex obtuse, base obtuse
or truncate, moderately thick-walled, initially hyaline, becoming brown,
externally smooth, internally inely verruculose, aseptate, (27–)28–33(–
34) × (14.5–)15–18.5(–19) μm.
Type: New Zealand, Northland, Kerikeri, Davies Orchard (#2),
Inlet Road, on recently dead bark-covered twigs of Citrus sinensis,
6 Sep. 2006, S.R. Pennycook, P.R. Johnston & B.C. Paulus,
holotype PDD 89238.
Culture: ICMP 16812 (ex-type).
158
Notes: Conidia of P. citrigena remain hyaline for long periods
and dark conidia are rarely encountered. Conidial dimensions
of this species are similar to those of S. visci, but its ascospores
are reddish-brown in contrast to the pale brown ones of S. visci.
Furthermore, S. visci appears to be speciic to Viscum species
while S. citrigena has been found only on Citrus species.
Sphaeropsis eucalypticola (Doilom, J.K. Liu, & K.D. Hyde)
A.J.L. Phillips, comb. nov. MycoBank MB805464. Fig. 65.
Basionym: Phaeobotryosphaeria eucalypti Doilom, J.K. Liu & K.D.
Hyde, Fungal Divers. 57: 190. 2012.
Ascomata black, dark brown, aggregated, initially immersed in
tissue becoming erumpent through cracks in bark, solitary, or
gregarious, multiloculate, globose to subglobose, wall composed
of several layers of dark brown cells of textura angularis.
Pseudoparaphyses 3–4 μm wide, septate, constricted at septa.
Asci 8–spored, bitunicate, issitunicate, cylindro-clavate or clavate,
with a short pedicel, apically rounded with an ocular chamber,
(90–)97−110(−125) × 28–30 μm (av. = 106 × 29 μm). Ascospores
overlapping biseriate, hyaline when young, becoming dark brown
when mature, aseptate, ellipsoid to ovoid, ends rounded, with a
minute apiculus at each end, smooth, widest in the middle, 27–35 ×
11–14 μm (av. = 30 × 12 μm). Asexual state not seen.
Type: Thailand, Chiang Rai Province, Muang District, on dead twig
of Eucalyptus sp., 8 Aug. 2011, M. Doilom, holotype MFLU 120753.
Cultures: MFLUCC 11-0579 = CBS 133993.
The Botryosphaeriaceae: genera and sPecies known from culTure
Fig. 66. Sphaeropsis porosa. A. Pycnidium with elongated neck. B. Conidium developing between paraphyses. C. Paraphyses. D. Conidia and conidiogenous cells. E, F.
Immature conidium at two different levels of focus to show the pores in the conidium wall. G, H. Mature conidium at two different levels of focus to show verruculose inner surface
of the wall. Scale bars: A = 500 μm, B–H = 10 μm.
Hosts: Eucalyptus sp. (Liu et al. 2012).
Known distribution: Thailand (Liu et al. 2012).
Notes: Liu et al. (2012) could not induce asexual sporulation of S.
eucalypti in culture and our attempts with the ex-type culture were
also unsuccessful.
Sphaeropsis porosa (Van Niekerk & Crous) A.J.L. Phillips &
A. Alves, comb. nov. MycoBank MB805465. Fig. 66.
Basionym: Diplodia porosum Van Niekerk & Crous, Mycologia 96:
790. 2004.
= Phaeobotryosphaeria porosa (Van Niekerk & Crous) Crous & A.J.L. Phillips,
Persoonia 21: 51. 2008.
Ascomata not reported. Conidioma solitary, unilocular, ostiolate,
globose to obpyriform, up to 400 μm wide, conidioma wall 4–8 cell
layers thick, of dark brown textura angularis, becoming hyaline
toward inner region. Conidiophores reduced to conidiogenous
cells. Conidiogenous cells lining cavity, holoblastic, hyaline,
subcylindrical to ampulliform, 6–10 × 5–7 μm, rarely proliferating
percurrently. Conidia hyaline, guttulate, ovoid to broadly ellipsoid
with a bluntly rounded apex, and lattened base, wall 2 μm thick,
with pores 1 μm wide, becoming medium brown with age, (38–)42–
45(–47) × (20–)22–25(–30) μm in vitro, L/W = 1.9.
Culture characteristics: Colonies lat with undulating margins, dark
green on the surface and dull green underneath, reaching a radius
www.studiesinmycology.org
of 32 mm after 3 d at 25 °C. Cardinal temperatures for growth: min
10 °C, max 30 °C, opt 25 °C.
Type: South Africa, Western Cape Province, Stellenbosch, on Vitis
vinifera, 2002, J.M. van Niekerk, holotype CBS H-12039.
Cultures: STE-U 5132 = CBS 110496 (ex-type).
Host: Vitis vinifera (van Niekerk et al. 2004).
Known distribution: South Africa (Western Cape Province) (van
Niekerk et al. 2004).
Notes: Van Niekerk et al. (2004) did not mention pycnidial paraphyses
in Diplodia porosum, but they were clearly seen when their isolates
were re-examined (Fig. 3). This species is unique within the
Botryosphaeriaceae because of its large, thick-walled conidia with
large pores (1 μm wide) that are easily seen by light microscopy.
However, the pitted walls, although unique and distinctive, should be
regarded as informative at the species level in the same way that this
character was regarded in the original description.
Sphaeropsis visci (Alb. & Schwein.) Sacc., Michelia 2: 105.
1880. MycoBank MB281898. Fig. 67.
Basionym: Sphaeria atrovirens var. visci Alb. & Schwein., Consp.
fung. (Leipzig): 48. 1805.
≡ Ceuthospora visci (Alb. & Schwein.) Sollm., Hedwigia 2: 189. 1863.
≡ Sphaeropsis visci (Alb. & Schwein.) Sacc., Michelia 2(no. 6): 105. 1880.
≡ Sphaeropsis visci (Alb. & Schwein.) Sacc. f. visci, Michelia 2(no. 6):
159
PhilliPs et al.
Fig. 67. Sphaeropsis visci. A. Immature asci. B. Mature ascus with brown, aseptate ascospores. C–F. Brown, aseptate ascospores with apiculi (arrows). G. Conidioma formed in
culture on a pine needle. H, I. Conidia forming on conidiogenous cells between paraphyses (arrows). J. Developing conidia. K. Paraphyses. L. Brown, aseptate mature conidia.
Scale bars: A, B = 20 μm, C–F, H–L = 10 μm, G = 50 μm.
160
The Botryosphaeriaceae: genera and sPecies known from culTure
105. 1880.
≡ Botryosphaerostroma visci (Alb. & Schwein.) Petr., Beih. Rep. spec.
nov. regn. veg. 42: 127. 1926.
= Sphaeria visci DC., in de Candolle & Lamarck, Fl. franç., Edn 3 (Paris) 6:
146. 1815.
≡ Diplodia visci (DC.) Fr., Summa veg. Scand., Section Post. (Stockholm):
417. 1849.
≡ Microdiplodia visci (DC.) Potebnia, Ann. Mycol. 8(1): 63. 1910.
≡ Ascochytella visci (DC.) Petr., Ann. Mycol. 23(1/2): 111. 1925.
≡ Botryosphaerostroma visci (DC.) Petr., Ann. Mycol. 23(1/2): 111. 1925.
≡ Pseudodiplodia visci (DC.) Petr., Sydowia 7(5–6): 304. 1953.
≡ Metadiplodia visci (DC.) Zambett., Bull. trimest. Soc. mycol. Fr. 70(3):
295. 1955.
= Dothidea visci Kalchbr., Hedwigia 8: 117. 1869.
≡ Anthostomella visci (Kalchbr.) Sacc., Syll. fung. (Abellini) 1: 293. 1882.
≡ Anthostoma visci (Kalchbr.) Sacc., Nuovo G. bot. ital. 23(2): 224. 1916.
≡ Phaeobotryon visci (Kalchbr.) Höhn., Sber. Akad. Wiss. Wien, Math.naturw. Kl., Abt. 1 128: 591. 1919.
≡ Botryosphaeria visci (Kalchbr.) Arx & E. Müll., Beitr. Kryptl. Schweiz
11(no. 1): 41. 1954.
≡ Phaeobotryosphaeria visci (Kalchbr.) A.J.L. Phillips & Crous, Persoonia
21: 47. 2008.
= Macrophoma visci Aderh., Arb. biol. Anst. Land-u. Forstw. 4: 462. 1905.
Ascomata pseudothecial, brown to black, uni- or multiloculate,
separate, immersed, ostiolate, up to 500 μm diam, wall composed of
several layers of dark brown textura angularis. Pseudoparaphyses
hyaline, smooth, 4–6 μm wide, multiseptate, with septa 11–19(–
26) μm apart, constricted at septa. Asci bitunicate, 8-spored,
ascospores biseriate in the ascus, stipitate, thick-walled with
thick endotunica and well-developed apical chamber, 180–230 ×
35–50 μm. Ascospores pale-brown when mature, ovoid, aseptate,
externally smooth, internally inely verruculose, widest in middle
with an apiculus at either end, (27.5–)31–37.5(–38.5) × (14.5–)15–
19(–19.5) μm. Conidiomata immersed to erumpent and supericial,
unilocular, up to 300 μm wide, wall composed of dark brown textura
angularis. Paraphyses hyaline, aseptate, up to 40 μm long and 4
μm wide with a bulbous tip 5 μm diam. Conidiogenous cells hyaline,
discrete proliferating internally to form periclinal thickenings, (4–)
8.5–11 × 4–6.5 μm. Conidia oval, apex obtuse, base obtuse or
truncate, moderately thick-walled, initially hyaline, becoming brown,
externally smooth, internally inely verruculose, (27–)29–33(–50) ×
(14.5–)15.5–19(–22) μm.
Holotype: Germany, on Viscum album, Albertini & Schweinitz,
holotype not found and presumably lost. ukraine, National Nature
Park ‘Svjatie Gory’, on branches of Viscum album, 10 Mar. 2007,
Á. Akulov, neotype here designated CWU (MYC) AS 2271
(MBT176099).
Cultures: CBS 122526, CBS 122527 (ex-neotype).
Host: Viscum album (Sutton 1980, Phillips et al. 2008).
Known distribution: Austria, Czechoslovakia, Egypt, Romania
(Sutton 1980), Ukraine (Phillips et al. 2008).
Notes: Sphaeropsis was introduced by Saccardo (1880) for
Diplodia species with brown, aseptate conidia. He designated S.
visci, based on Sphaeria atrovirens var. visci, as the type species.
The connection between the asexual and sexual morphs was
established by Phillips et al. (2008). Single ascospore isolations
from a botryosphaeria-like ascomycete on CWU (MYC) AS 2271
resulted in cultures of a coelomycete indistinguishable from
S. visci, thus proving the connection between the two states.
This specimen is herein designated as neotype. Features that
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distinguish the sexual morph from others with brown ascospores
in the Botryosphaeriaceae are the aseptate ascospores with an
apiculus at either end.
Tiarosporella Höhn, Ber. Deutsch. Bot. Ges. 37: 159. 1919.
MycoBank MB10233.
Type species: Tiarosporella paludosa (Sacc. & Fiori ex P. Syd.)
Höhn., In: Weese, Mitt. bot. Inst. tech. Hochsch. Wien 1(3): 83.
1924.
Characterised by having conidia formed from smooth, hyaline
conidiogenous cells that lack periclinal thickenings and percurrent
proliferations. The hyaline, subcylindrical to fusiform conidia have
irregular mucoid appendages.
Tiarosporella graminis var. karoo B. Sutton & Marasas,
Trans. Brit. Mycol. Soc. 67 (1): 73. 1976. MycoBank
MB353200. For illustrations see Sutton & Marasas (1976).
Aerial mycelium composed of hyaline to light brown, septate,
branched, smooth, encrusted, thin-walled hyphae and strands of
coarse, thick-walled, dark brown, smooth or verrucose hyphae, 6–12
μm wide and consisting of cylindrical cells, 12–45 μm long which
sometimes round off to form chains of globose, 1-celled, thick-walled,
dark brown, chlamydospore-like cells. Pycnidia begin to develop
after 7 d, embedded in the surface of the agar, single or in small
groups, dark brown to black, rostrate and the elongate necks are
covered with grey-olivaceous to brown, simple, septate, smooth or
verruculose, straight or lexuous pycnidial hairs with obtuse ends.
Conidiogenous cells formed from the cells lining the inner walls of
the pycnidia, holoblastic, determinate, simple, cylindrical and slightly
tapered towards the apex, hyaline, 12–18 × 1.5–2.5 μm. Conidia
acrogenous, solitary, hyaline, smooth, thin-walled, straight, fusiform
with truncate base and obtuse apex, 21–28 × 5–8 μm. During
development, conidia are enclosed in a gelatinous sheath that may
remain as an apical, hyaline, cone-like appendage.
Type: South Africa, Cape Province, Colesberg, on Eriocephali sp.,
16 Feb. 1971, W.F.O. Marasas, holotype IMI 186782.
Cultures: IMI 186783 = CBS 118718.
Hosts: Eriocephalus sp., Nestlera sp., Tribulus terrestris (Sutton &
Marasas 1976)
Known distribution: South Africa (Sutton & Marasas 1976)
Notes: Conidia of T. graminis var. graminis resemble those of T.
graminis var. karroo in shape, though they are somewhat larger
(20–29.5 × 7–9 μm), than those of T. graminis var. karroo (21–28 ×
5–8 μm) (Sutton & Marasas 1976).
Tiarosporella tritici B. Sutton & Marasas, Trans. Brit. Mycol.
Soc., 67 (1): 74. 1976. MycoBank MB324614.
Aerial mycelium composed of hyaline to light brown, septate,
branched, smooth or encrusted thin-walled hyphae and strands of
161
PhilliPs et al.
very coarse, thick-walled, dark brown to black, verrucose hyphae
7.5–16.5 μm wide and consisting of cylindrical cells, 12–40 μm long
that sometimes round off to form chains of intercalary, globose,
thick-walled, smooth or verruculose chlamydospore-like cells, 8–14
μm diam. Immersed mycelium dark brown to black. Pycnidia begin
to develop after 7 d and numerous mature pycnidia are present
throughout the Petri dish after 14 d, particularly on PDA, semiimmersed in the surface of the agar, single or 2–15 aggregated
in large, pulvinate, botryose, stromatoid groups up to 3 mm diam,
dark brown to black, globose, rostrate, unilocular or multilocular,
up to 200 μm diam, walls thick, composed of large, thick-walled,
dark brown pseudoparenchymatous cells that become paler and
thin-walled towards the inner conidiogenous region, ostiole circular,
up to 65 μm diam, formed at the apex of an apical beak that is up
to 400 μm long and covered with hyaline to light brown, simple,
septate, straight or lexuous, smooth or verruculose pycnidial
hairs with obtuse ends. Conidiogenous cells formed from the
cells lining the inner wall of the pycnidia, holoblastic, determinate,
simple, hyaline, cylindrical, 9–14 × 4–5 μm. Conidia acrogenous,
solitary, hyaline, smooth, thin-walled, eguttulate, straight, oval to
fusiform, apex obtuse, base truncate, 29–38 × 12–17 μm. During
development some conidia are enclosed in a gelatinous sheath
that later becomes everted into an apical, irregularly infundibuliform
appendage up to 23 μm long and 29 μm wide.
Type: South Africa, Orange Free State, Heilbron, dried culture
isolated from dead stems of Triticum aestivum, 18 Jan. 1973,
W.F.O. Marasas, holotype PREM 44966.
Cultures: IMI 186786 = CBS 118719 (ex-type).
Host: Triticum aestivum (Sutton & Marasas 1976)
Known distribution: Free State Province, South Africa (Sutton &
Marasas 1976).
Notes: Conidia of T. tritici are much larger than those of all other
known species of Tiarosporella and the shape of the appendage
is also different. Of the 14 species of Tiarosporella that have
been named to date, DNA sequence data are only available for T.
graminis, T. madreeya, T. tritici and T. urbis-rosarum (Crous et al.
2006, Jami et al. 2012).
Tiarosporella urbis-rosarum Jami, Gryzenh., Slippers
& M.J. Wingf., Cryptogam. Mycol. 33: 256. 2012. For
illustrations see Jami et al. (2012).
Conidiomata (on sterile twigs of Acacia karroo) pycnidial, dark
black, up to 200 μm diam, immersed, unilocular, with long necks
(4–9 mm); wall of 5–7 layers of dark brown textura angularis,
becoming thin-walled towards inner region. Conidiogenous cells
holoblastic, hyaline, cylindrical, (5–)5.5–9.5(–11) × (3–)3.5–4(–5)
μm. Conidia ovoid, smooth, granular, thin-walled, aseptate, apices
rounded, (21–)23.5–29.5(–34) × (8–)9–10(–11) μm (from Jami et
al. 2012).
Culture characteristics: Colonies on MEA appressed, centres dirty
white, becoming dark grey at the edges; reverse dark grey to black.
Growth at 5–35 °C, with optimal growth rate of 14.4 mm / d at
25 °C.
162
Type: South Africa, Free State Province, Bloemfontein, on healthy
wood of Acacia karroo, Jun. 2008, M. Gryzenhout, holotype PREM
60698.
Cultures: CMW 36477 = CBS 130405 (ex-type).
Host: Acacia karroo.
Known distribution: Free State and Gauteng Provinces of South
Africa.
Note: Tiarosporella urbis-rosarum is morphologically similar to T.
tritici (conidia 29–38 × 12–17 μm), but has smaller conidia (23.5–
29.5 × 9–10 μm).
dIScuSSIoN
In this paper we considered only those genera and species of the
Botryosphaeriaceae that are known to exist in culture, and thus
accept 17 genera in the family. These genera are characterised
based on 17 lineages in a multi-locus phylogeny. In a recent
phylogenetic study of the Botryosphaeriales, Liu et al. (2012)
included Auerswaldia in the Botryosphaeriaceae based on fresh
collections of A. lignicola and A. dothiorella. However, they did
not include ITS sequence data in their analyses because they
claimed that it was not suitable to segregate taxa at the generic
and species level. In our analyses, A. lignicola clustered within
Lasiodiplodia and A. dothiorella in Dothiorella. For this reason,
we argue that there is no evidence to suggest that Auerswaldia
should be regarded as a distinct genus in the Botryosphaeriaceae.
Indeed, Liu et al. (2012) state that depending on the method used
to generate the phylogeny, A. lignicola clustered in the Diplodia /
Lasiodiplodia clade in the RAxML analysis, but in Dothiorella when
Maximum Parsimony was used. Furthermore, in the combined
EF1-α and β-tubulin analysis, this species always clustered in
Dothiorella irrespective of the phylogenetic method used. In the
present paper we found that a combination of SSU, ITS, LSU,
EF1-α and β-tubulin gave a clear separation of the genera and this
was consistent between the different phylogenetic methods (MP,
ML). This is also consistent with a previous multi-locus phylogeny
(Phillips et al. 2008) of a smaller sub-set of the family.
Most of the genera revealed by the multi-locus phylogeny in
this study can be distinguished based on their morphology. This is
especially true for characteristics of the conidia and to a lesser extent on
the presence or absence of paraphyses in the conidiomata. However,
some genera cannot be separated using morphological characters.
For example, conidia of Botryosphaeria are indistinguishable from
those of Neofusicoccum when the range of variation for each genus
is taken into consideration. Although there is some evidence that
pycnidial paraphyses are found only in Botryosphaeria, this has not
been conirmed for all the species. Nevertheless, paraphyses have
never been reported in any Neofusicoccum species.
Another dificult pair of genera to distinguish is Spencermartinsia
and Dothiorella. The conidial characters of species in both of
these genera are identical, being pigmented and 1-septate. In
both genera, the conidia become pigmented and septate even
while they are attached to conidiogenous cells, and this character
distinguishes them from Diplodia. Phillips et al. (2008) introduced
Spencermartinsia for species similar to Dothiorella but differed in
the presence of an apiculus on the ascospores, which is absent
The Botryosphaeriaceae: genera and sPecies known from culTure
from Spencermartinsia species. Although this is a small difference,
it is supported by phylogenetic data and is also a useful character
to separate Barriopsis (no apiculus) from Phaeobotryosphaeria
(apiculus present). However, the status of these two genera needs
to be re-evaluated in the light of the multi-locus analysis presented
here and by Slippers et al. (2013, this volume), in which the
phylogenetic distinction is unclear.
Although ITS alone was usually suficient to separate species
within each genus of the Botryosphaeriaceae, inclusion of EF1-α
resulted in a more robust separation, and was considered essential
in some genera such as Diplodia, Lasiodiplodia and Neofusicoccum.
We therefore recommend at least these two loci for species
separation within the Botryosphaeriaceae. With the increase in
the number of species recognised in phylogenetic studies, the use
of morphological data for species identiication is becoming less
useful. Although we have provided keys for species identiication
in each genus, the resulting identiication should be interpreted
with caution. For example, in Neofusicoccum the range of variation
within a species is becoming more apparent as additional isolates
are studied and often the variation overlaps considerably with
other species. Furthermore, phylogenetic inference is revealing
cryptic species complexes that cannot be distinguished based on
morphology alone (see for example Pavlic et al. 2009a, b, Sakalidis
et al. 2011). In this regard, in addition to ITS and EF1-α sequence
data, data from the β-tubulin, RPB2 and other loci have been useful,
and were at times necessary to provide convincing evidence of
multigene phylogenetic concordance to separate cryptic species
(see also Sakalidis et al. 2012).
Recognising the isolate identiied by Liu et al. (2012) as A.
lignicola is in fact a species in Lasiodiplodia has helped to resolve
a long-standing problem regarding the connection between the
asexual and the sexual morphs in Lasiodiplodia. As explained in
the notes for L. theobromae, the connection between the asexual
and sexual morphs of L. theobromae has not been deinitively
conirmed, and thus the characteristics of the sexual morph are
also not clear. Liu et al. (2012) clearly demonstrate the asexual /
sexual morph connection for L. lignicola and conirmed that mature
ascospores are dark brown. This has also recently been observed
for other species of Lasiodiplodia (Crous, unpubl. data). For this
reason, we have amended the description of Lasiodiplodia to
include brown ascospores. In recent studies, several new species
have been introduced in Lasiodiplodia, and frequently these species
are recognised based on minor differences in ITS sequences with
great emphasis placed on EF1-α sequence data (Abdollahzadeh et
al. 2010). It would seem that this genus should be the subject of a
more detailed analysis based on additional gene loci to provide a
robust phylogenetic basis for species deinitions.
In each genus of the Botryosphaericaceae the species share
a common general morphology, which complies to a great extent
with the deinition of a genus (Singer 1975, Booth 1978, Crous et
al. 2009). However, in Diplodia, several different morphologies are
seen and these lie within separate phylogenetic lineages. The typical
morphology, as seen in D. mutila and related species, consists of
hyaline, aseptate, thick-walled conidia that become dark brown and
1-septate. Another major group, which includes D. seriata, D. pinea
and their relatives, has conidia that turn brown at an early stage
of development and remain aseptate. These two morphological
groups cluster in two well-supported clades. This would give the
impression that Diplodia consists of two separate genera. However,
D. corticola and D. quercivora have the characteristics typical of
the D. mutila group, but form a clade near the root of the Diplodia
phylogenetic tree. Furthermore, D. cupressi and D. tsugae with
www.studiesinmycology.org
conidia indistinguishable from D. mutila, cluster with D. bulgarica
(pale brown, aseptate conidia) in another clade that lies between
the D. mutila and D. seriata clades. Thus, for the present, we have
chosen to consider Diplodia as a genus with two morphologies
rather than to provide separate genera or sections for them.
Following the recent changes to the nomenclature of
pleomorphic fungi, and in particular the abolition of dual
nomenclature for a single fungus, we have introduced some new
combinations. With regard to Botryosphaeria / Fusicoccum, the
oldest genus is Fusicoccum Corda (1829), not Botryosphaeria
Ces. & De Not. (1863). However, Botryosphaeria is the type
genus of Botryosphaericeae and Botryosphaeriales, and is well
entrenched in the user community. For these reasons we have
retained Botryosphaeria and have made several recombinations of
Fusicoccum species.
Phillips et al. (2008) reinstated Phaeobotryosphaeria for species
with dark brown, aseptate ascospores that have a hyaline apiculus
at either end, and asexual morphs in Sphaeropsis. In the present
paper we decided to revert to using the generic name Sphaeropsis
for these species. Sphaeropsis Sacc. (1880) is an older name than
Phaeobotryosphaeria Speg. (1908), and is also better established
with the plant pathological community. Although Sphaeropsis has
been applied incorrectly in the past, we believe that the confusion
has now been resolved and the genus is clearly circumscribed.
Ever since Crous et al. (2006) sub-divided Botryosphaeria the
position of B. mamane has been uncertain, apparently residing
outside of Botryosphaeria. Furthermore, conidia of B. mamane
are considerably larger than those of any other species in
Botryosphaeria. In our ITS phylogenies the ex-type cultures of B.
mamane formed a clade within Cophinforma conirming that this is
a suitable genus for it.
The present study provides the irst phylogenetic overview and
morphological synthesis of the species of Botryosphaeriaceae that
are presently known from culture. We trust that this will provide
a stable platform to accommodate the numerous undescribed
species that still await description, or recollection and epitypiication
to ensure a stable genetic application of names in the family.
AcKNowledGeMeNTS
We would like to thank the curators of the numerous fungaria and Biological
Resource Centres cited in this paper, for making specimens and cultures available
for examination over the past 15 yr, without which this study would not have been
possible. Part of this work was supported by Fundação para a Ciência e a Tecnologia
(Portugal) through grant PEst-OE/BIA/UI0457/2011. Artur Alves and Alan Phillips
were supported by the programme Ciência 2008, co-funded by the Human Potential
Operational Programme (National Strategic Reference Framework 2007–2013) and
the European Social Fund (EU).
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