Colletotrichum: complex species or species ... - CBS - KNAW

Damm et al.
stimulated by strawberry plant extracts, especially flower extracts
(Leandro et al. 2003). According to Buddie et al. (1999) secondary
conidia may be produced directly from germinating primary conidia,
and are smaller and more variable in shape, thus obscuring
differences between taxa. Additionally, C. acutatum forms simple
pigmented appressoria, but few or no setae (Simmonds 1965).
Guerber & Correll (1997, 2001) described Glomerella acutata,
the sexual morph of C. acutatum, as the product of mating
experiments, while some related species are homothallic, including
Ga. acutata var. fioriniae (Marcelino et al. 2008), later regarded as
a separate species (C. fioriniae, Shivas & Tan 2009) and an isolate
of a Glomerella species related to C. acutata from Acer platanoides
in the USA (LoBuglio & Pfister 2008). Talgø et al. (2007) observed
the sexual morph Ga. acutata on naturally infected fruits of
highbush blueberry in Norway. Numerous studies have shown
that C. acutatum is morphologically and phylogenetically diverse
(Sreenivasaprasad et al. 1994, Johnston & Jones 1997, Lardner
et al. 1999, Freeman et al. 2001a, Nirenberg et al. 2002, Talhinhas
et al. 2002, Guerber et al. 2003, Lubbe et al. 2004, Du et al. 2005,
Peres et al. 2005, Sreenivasaprasad & Talhinhas 2005, Talhinhas
et al. 2005, Johnston et al. 2008). Sreenivasaprasad et al. (1996)
were the first to recognise that C. acutatum was unusually diverse,
with strains showing divergence of 5.8 % in ITS-1 sequence
compared with levels of 2–4 % frequently found within other
fungal species, and they suggested splitting C. acutatum into two
species. Johnston & Jones (1997) recognised four morphological
groups, C. acutatum A–C and Glomerella miyabeana. Three of
these groups were supported by 28S nuclear ribosomal large
subunit rRNA (LSU) sequence data. Lardner et al. (1999), using a
combination of RAPDs and morphological/cultural data, identified
seven subordinate groups within C. acutatum. Sequences of a
200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and a 900-bp intron of the glutamine synthetase GS
distinguished seven main clades and several subclades within
strains that originated mainly from North America and New Zealand
(Guerber et al. 2003). The recognition of infraspecific groups
was more firmly established by Sreenivasaprasad & Talhinhas
(2005), who distinguished clades A1 to A8 based on rDNA ITS and
beta-tubulin DNA (TUB2) sequences. These clades were mostly
correlated with the groups that had been distinguished previously.
Whitelaw-Weckert et al. (2007) recognised an additional group A9.
At this point, it was widely presumed that C. acutatum was
a species complex containing a number of constituent taxa, but
there was substantial reluctance to recognise the clades involved
as independent species. This was due to the lack of differential
morphological and cultural characters. For example, C. lupini was
not recognised as formally separate from C. acutatum by Talhinhas
et al. (2002) or by Sreenivasaprasad & Talhinhas (2005). There
were some attempts to address these concerns via adoption of
formae speciales e.g. C. acutatum f. sp. pineum (Dingley & Gilmour
1972), C. acutatum f. sp. hakeae (Lubbe et al. 2004) and C.
acutatum f. sp. fioriniae (Marcelino et al. 2008), but this mechanism
for recognition of pathology-related taxa is now rarely used.
Gradually, separate species were recognised or accepted as
part of the C. acutatum species complex, e.g. C. lupini (Nirenberg
et al. 2002) and C. phormii (Farr et al. 2006). In a study using two
genes, ITS and TUB2, combined with morphological data, Shivas
& Tan (2009) recognised three distinct groups within C. acutatum
strains from Australia and accepted two new species, C. simmondsii
and C. fioriniae (formerly C. acutatum f. sp. fioriniae) for groups A2
and A3. Recently, a new species was described for group A4, C.
clavatum (Faedda et al. 2011).
Our research aims to present a comprehensive revision of
the C. acutatum species complex. We thoroughly survey the
constituent taxa and delineate additional species where needed.
We have examined a large number of C. acutatum s. lat. strains,
isolated from various hosts and in various geographic areas. Multilocus
molecular analysis is the basis of species recognition, but
morphological and cultural characters allowing alternative means
of species recognition are given where possible.
MATERIALS AND METHODS
Isolates
A total of 331 strains have been studied, mostly previously
identified as C. acutatum, as well as other related strains from the
CBS, IMI and other culture collections. Type material (holotypes
and epitypes) of the species studied are located in the Herbarium
of the Centraalbureau voor Schimmelcultures (CBS), Utrecht,
The Netherlands, in the IMI Fungarium, which is based in the
Royal Botanic Gardens, Kew (IMI and K(M)), UK, US National
Fungus Collections (BPI), Beltsville, Maryland, USA, the Botanic
Garden and Botanical Museum Berlin-Dahlem, Freie Universität
Berlin (B), Germany and in the dried collection of the Botanische
Staatssammlung München (M), Germany. All descriptions are based
on the ex-type, ex-epitype or ex-neotype culture as appropriate.
Features of other strains are added if deviant. Subcultures of the
types, epitypes and neotypes, respectively, as well as all other
isolates used for morphological and sequence analyses are
maintained in the culture collections of CBS and IMI (Table 1).
Morphological analysis
To enhance sporulation, autoclaved filter paper and doubleautoclaved
stems of Anthriscus sylvestris were placed onto the
surface of synthetic nutrient-poor agar medium (SNA; Nirenberg
1976). SNA and oatmeal agar (OA; Crous et al. 2009) cultures were
incubated at 20 °C under near UV light with 12 h photoperiod for
10 d. Measurements and photographs of characteristic structures
were made according to Damm et al. (2007). Appressoria on
hyphae were observed on the reverse side of SNA plates.
Microscopic preparations were made in clear lactic acid, with 30
measurements per structure and observed with a Nikon SMZ1000
dissecting microscope (DM) or with a Nikon Eclipse 80i microscope
using differential interference contrast (DIC) illumination. In the C.
acutatum species complex, conidia are usually formed in acervular
conidiomata and additionally in the aerial mycelium. Unless
mentioned otherwise, only conidia from conidiomata were used in
this study for morphological examination.
Colony characters and pigment production on SNA and
OA cultures incubated at 20 °C under near UV light with 12 h
photoperiod were noted after 10 d. Colony colours were rated
according to Rayner (1970). Growth rates were measured after 7
and 10 d.
Phylogenetic analysis
Genomic DNA of the isolates was extracted using the method of
Damm et al. (2008). The 5.8S nuclear ribosomal gene with the two
flanking internal transcribed spacers (ITS), a 200-bp intron of the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and partial
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