Phytopathogenic Dothideomycetes - CBS - KNAW

Species concepts in Cercospora
cercosporin is not produced by all species (Assante et al. 1977,
examples cited by Goodwin et al. 2001, see also review by Weiland
et al. 2010). Nutritional and environmental conditions influence
the production of cercosporin, making it useless for application
in Cercospora taxonomy (Jenns et al. 1989). Genomic studies in
recent years attempt to understand the metabolic pathway used
to produce cercosporin and C. nicotianae has become the model
organism for these studies (e.g. Chung et al. 2003, Choquer et al.
2005, Chen et al. 2007, Amnuaykanjanasin & Daub 2009).
In an attempt to address some of the shortcomings highlighted
in the previous paragraph, we have obtained diseased plant
material and/or cultures from as many hosts and countries
as possible over several years. We sequenced the ITS locus
(including ITS1, 5.8S nrRNA gene and ITS2), as well as parts of
four genomic protein coding genes, namely translation elongationfactor
1-alpha, actin, calmodulin and histone H3 for each culture.
Our primary objective was to re-evaluate the species concept of
known Cercospora species by consolidating the results of multilocus
phylogenetic analyses with morphological characteristics
produced on host plants and different media. A secondary
objective was to test whether Cercospora species, in general,
were host-specific.
MATERIALS AND METHODS
Specimens and isolates
Dried specimens and cultures used in this study are maintained
in herbaria and culture collections of Genebank, National Institute
of Agrobiological Sciences, Japan, (MAFF), the Mycological
Herbarium and Culture Collection, laboratory of Plant Pathology,
Mie University, Japan (MUMH or MUCC) and the Centraalbureau
voor Schimmelcultures (CBS-KNAW Fungal Biodiversity Centre,
Utrecht, The Netherlands), or the working collection of P.W. Crous
(CPC), housed at CBS (Table 1). A global set of isolates (Table 1)
was either obtained from personal culture collections, the culture
collection of the CBS or recollected on diseased plant material, and
grown in axenic culture. Symptomatic leaves with leaf spots were
chosen for isolations of Cercospora spp. as explained in Crous
(1998). To obtain ascospore isolates, excised lesions were placed
in distilled water for approximately 2 h, after which they were placed
on the bottom of Petri dish lids, over which the plate containing 2
% malt extract agar (MEA) (Crous et al. 1991, 2009c) was inverted.
Germinating ascospores were examined after 24 h, and singleascospore
cultures established on MEA as explained by Crous
(1998). Colonies were sub-cultured onto oatmeal agar (OA), V8-
juice agar (V8), 2 % potato-dextrose agar (PDA) or MEA (Crous et
al. 2009c) and incubated at 25 °C under continuous near-ultraviolet
light, to promote sporulation.
DNA extraction, amplification and phylogeny
Genomic DNA was isolated from fungal mycelium grown on the
agar plates following the protocol of Lee & Taylor (1990) or the
UltraClean Microbial DNA Isolation Kit (Mo Bio Laboratories,
Inc., Solana Beach, CA, USA). All isolates were sequenced with
five genomic loci. The primers ITS5 or ITS1 and ITS4 (White et
al. 1990) were used to amplify the internal transcribed spacers
areas as well as the 5.8S rRNA gene (ITS) of the nrDNA operon.
Part of the actin gene (ACT) was amplified using the primer set
ACT-512F and ACT-783R (Carbone & Kohn 1999) and part of the
translation elongation factor 1-a gene (EF) using the primer set
EF1-728F and EF1-986R (Carbone & Kohn 1999). The primer set
CAL-228F and CAL-737R (Carbone & Kohn 1999) was used to
amplify part of the calmodulin gene (CAL) whereas the primer set
CylH3F and CylH3R (Crous et al. 2004c) was used to amplify part
of the histone H3 gene (HIS). Additional degenerate primers were
developed from sequences obtained from GenBank as alternative
forward and reverse primers for some of the loci during the course
of the study (Table 2); however, these were rarely used but based
on their degenerate design could be of use to the broader scientific
community. The protocols and conditions outlined by Groenewald et
al. (2005) were followed for standard amplification and subsequent
sequencing of the loci.
Sequences of Septoria provencialis (isolate CPC 12226)
were used as outgroup based on availability and phylogenetic
relationship with Cercospora (Crous et al. 2004b, 2006b). The
Cercospora sequences were assembled and added to the outgroup
sequences using Sequence Alignment Editor v. 2.0a11 (Rambaut
2002), and manual adjustments for improvement were made by
eye where necessary. Gaps present in the ingroup taxa and longer
than 10 characters were coded as a single event for all analyses
(see TreeBASE).
Neighbour-joining analyses using the HKY85 substitution
model were applied to each data partition individually to check the
stability and robustness of each species clade under each data set
using PAUP v. 4.0b10 (Swofford 2003) (data not shown, discussed
under the species notes where applicable). Alignment gaps were
treated as missing data and all characters were unordered and of
equal weight. Any ties were broken randomly when encountered.
The robustness of the trees obtained was evaluated by 1 000
bootstrap replications (Hillis & Bull 1993).
MrModeltest v. 2.2 (Nylander 2004) was used to determine the
best nucleotide substitution model settings for each data partition.
Based on the results of the MrModeltest, a model-optimised
phylogenetic re-construction was performed for the aligned
combined data set to determine species relationships using MrBayes
v. 3.2.0 (Ronquist & Huelsenbeck 2003). The heating parameter
was set at 0.3 and the Markov Chain Monte Carlo (MCMC) analysis
of four chains was started in parallel from a random tree topology
and lasted until the average standard deviation of split frequencies
came below 0.05. Trees were saved each 1 000 generations and
the resulting phylogenetic tree was printed with Geneious v. 5.5.4
(Drummond et al. 2011). New sequences generated in this study
were deposited in NCBI’s GenBank nucleotide database (www.
ncbi.nlm.nih.gov; Table 1) and the alignment and phylogenetic tree
in TreeBASE (www.treebase.org).
Isolates of Cercospora sp. Q were screened with five more loci
to test whether additional loci could distinguish cryptic taxa within
this species. This species was selected based on the intraspecific
variation present in Fig. 2 (part 5) and also the range of host species
and countries represented. The primer set GDF1 and GDR1
(Guerber et al. 2003) was used to amplify part of the glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) gene, primer set NMS1 and
NMS2 (Li et al. 1994) for part of the mitochondrial small subunit rRNA
gene and part of the chitin synthase (CHS) gene was amplified using
the primers CHS-79F and CHS-354R (Carbone & Kohn 1999). Part
of the gene encoding for a mini-chromosome maintenance protein
(MCM7) was amplified using primers Mcm7-709for, Mcm7-1348rev,
Mcm7-1447rev (Schmitt et al. 2009) and part of the beta-tubulin
gene using mainly the primers T1, Bt2b and TUB3Rd (see Table 2
for references).
www.studiesinmycology.org
131