Highlights of the Didymellaceae - Studies in Mycology

AveSkAMp et al.
thresholds than can be obtained with single-copy loci. Despite the
general practicality of using ITS in barcoding, the locus is relatively
conservative and may oversimplify species delimitations or blur
generic boundaries in some groups (Nilsson et al. 2008). In the
present study, a combination of four loci is therefore applied. These
include two loci that are renowned for their capacity to resolve
phylogenies above family level, namely parts of the LSU (Large
Subunit – 28S) and SSU (Small Subunit – 18S) nrDNA. Additinally
two loci were applied that mainly provide resolution at species level
– or even below. In addition to the abovementioned ITS regions,
also part of the β-tubulin gene was utilised, which was successfully
applied in a preliminary study on Phoma species of the section
Peyronellaea (Aveskamp et al. 2009a).
For the present study, four objectives were defined. The
main objective of this study was to reach consensus on the
circumscription of the genus Phoma. A modified definition of the
genus is not only helpful in taxonomy, but will also be of interest
to plant quarantine officers (Aveskamp et al. 2008). Teleomorph
associations of Phoma are still uncertain, and here we attempt to
shed light on the sexual state of Phoma s. str. Species representing
all Phoma sections were included and DNA sequences were
compared with those of other species in the Pleosporales.
Secondly, we aimed to integrate morphological and cultural
features with DNA sequence data to resolve the generic limits of taxa
currently placed in the Didymellaceae. The number of genera in this
family is still unclear. Although De Gruyter et al. (2009) found a series
of genera that, according to their reconstructed phylogeny, clustered
in this family, many were not clearly defined or were morphologically
distant from each other, although all anamorph taxa found are
accommodated in the coelomycetes (Sutton 1980). Examples
of these taxa were included in this study, although the number of
Ascochyta, Coniothyrium and Microsphaeropsis species is too high
to take all infrageneric taxa of these adjacent genera into account.
Further, we aimed to validate the Phoma sections, which are
widely applied in Phoma species recognition. Are the sections
representing evolutionary units, and what is the taxonomical value
of the characters used to define the sections? To judge the value
of the Boeremaean taxonomic system, representative species of
all sections were studied, including the sectional type species.
The main focus was, however, to resolve the sections associated
with Didymellaceae. A single generic name, based on priority but
regardless of whether it is an “anamorph” or “teleomorph” genus,
is used for all unambiguous monophyletic phylogenetic lineages
(Crous et al. 2006, 2009a, b). Finally, we aimed to assess the
molecular variation within species that have historically been
placed in Phoma. Genes were tested for their potential reliability as
standard barcoding genes for Phoma species.
For this study, a sequence data set was generated and
morphological data assembled for the more than 300 wellvouchered
strains available in the culture collections of CBS (CBS-
KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands) and
PD (Plantenziektenkundige Dienst, Dutch Plant Protection Service,
Wageningen, the Netherlands). In addition, five species recognised
in a recent study in the section Peyronellaea (Aveskamp et al.
2009a) have also been included, as well as several strains that
could not be associated with any of the species that were accepted
in Phoma by Boerema et al. (2004), and that were maintained
as unnamed Phoma species in the culture collections mentioned
above. These strains were recognised as taxonomic novelties
and are described at species or variety level in the present paper.
Furthermore, several species were relocated to more appropriate
genera based on the results obtained.
4
MATERIALS AND METHODS
Strain selection
A total of 324 strains, belonging to 206 species were selected for
the present study. The majority of these species (159) belonged to
the genus Phoma or its associated teleomorphs, the remainder to
genera that are regularly confused with this genus and that belong
to the Pleosporales according to the studies published by De
Gruyter et al. (2009). Besides the anamorphous species that were
included, representatives of the teleomorph genera Didymella,
Leptosphaeria, Leptosphaerulina, Macroventuria and Pleospora
were also included. The recently described genus Atradidymella
(Davey & Currah 2009) was not available for study and therefore
excluded.
Strains were obtained from CBS and PD culture collections in
lyophilised form or from the liquid nitrogen collection. Freeze-dried
strains were revived overnight in 2 mL malt/peptone (50 % / 50 %)
liquid medium. Subsequently, the cultures were transferred and
maintained on oatmeal agar (OA, Crous et al. 2009c). The strains
that were stored at -196 °C were directly plated on the same agar
medium.
DNA extraction, amplification and sequence
analysis
Genomic DNA extraction was performed using the Ultraclean
Microbial DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA,
U.S.A.), according to the instructions of the manufacturer. All DNA
extracts were diluted 10 × in milliQ water and stored at 4 ºC before
their use as PCR templates.
For nucleotide sequence comparisons fragments of four loci
were analysed: LSU, SSU, ITS, and TUB. Amplification of LSU and
SSU was conducted utilising the primer combination LR0R (Rehner
& Samuels 1994) and LR7 (Vilgalys & Hester 1990) for LSU
sequencing and the primer pair NS1 and NS4 (White et al. 1990)
for SSU. The PCRs were performed in a 2720 Thermal Cycler
(Applied Biosystems, Foster City, California) in a total volume of
12.5 μL. The PCR mixture contained 0.5 μL 10 × diluted genomic
DNA, 0.2 μM of each primer, 0.5 Unit Taq polymerase E (Genaxxon
Bioscience, Germany), 0.04 mM (SSU) or 0.06 mM (LSU) of
each of the dNTP, 2 mM MgCl 2 and 1 × PCR buffer E incomplete
(Genaxxon Bioscience). Conditions for amplification for both
regions were an initial denaturation step of 5 min at 94 °C, followed
by 35 cycles of denaturation, annealing and elongation and a final
elongation step of 7 min at 72 °C. For the SSU amplification, the
35 cycles consisted of 30 s at 94 °C, 50 s at 48 °C and 90 s at
72 °C; for the LSU 45 s at 94 °C, 45 s at 48 °C and 2 min at 72 °C.
The loci ITS and TUB were amplified as described by Aveskamp
et al. (2009a), using the primer pairs V9G (De Hoog & Gerrits van
den Ende 1998) and ITS4 (White et al. 1990) for ITS sequencing
and the BT2Fw and BT4Rd primer pair (Aveskamp et al. 2009a)
for sequencing of the TUB locus. PCR products were analysed
by electrophoresis in a 1.0 % (w/v) agarose gel containing 0.1 ug/
mL ethidium bromide in 1 × TAE buffer (0.4 M Tris, 0.05 M glacial
acetetic acid 0.01 M ethylenediamine tetraacetic acid [EDTA], pH
7.85). The amplicons were visualised under UV light. Hyperladder
I (Bioline, Luckenwalde, Germany) was applied as size standard.
The obtained amplicons were sequenced in both directions
using the same primer combinations, except for LSU, where an
additional primer, LR5 (White et al. 1990) was further required