Septoria and Stagonospora Diseases of Cereals - CIMMYT ...

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Septoria passerinii Closely Related to the Wheat
Pathogen Mycosphaerella graminicola
S.B. Goodwin and V.L. Zismann
USDA-ARS, Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN, USA
Abstract
Septoria passerinii is known only from its anamorphic Septoria state; no teleomorph has been identified. In culture, S.
passerinii looks very similar to Mycosphaerella graminicola from wheat. Comparisons of the nucleotide sequences of the
internal transcribed spacer (ITS) regions of the ribosomal DNA of both species and of many other fungi in the Dothideales
revealed that the ITS regions of S. passerinii and M. graminicola differed by only 10 bases out of 571 total. Therefore, these
species are very closely related. Phylogenetic analysis showed that S. passerinii and M. graminicola grouped together within
a large cluster of Mycosphaerella species. Thus, S. passerinii almost certainly has a Mycosphaerella teleomorph.
Septoria passerinii causes
speckled leaf blotch on barley. The
colony and conidial morphologies
of S. passerinii and the wheat
pathogen Mycosphaerella graminicola
are very similar. Both are
pathogens of cereals, and they may
be closely related. However,
nothing is known about the
evolutionary relationships of S.
passerinii (Cunfer and Ueng, 1999);
no fruiting structures have been
found and its teleomorph remains
unknown.
Analyses of the internal
transcribed spacer (ITS) region of
ribosomal DNA have been very
useful for elucidating the
phylogenetic relationships of fungi.
This region contains the highly
variable ITS1 and ITS2, separated
by the conserved 5.8S ribosomal
RNA gene, and is bounded by the
highly conserved 18 and 28S
ribosomal RNA genes. This greatly
facilitates analysis, because
polymerase chain reaction (PCR)
primers within the conserved
regions of the 18 and 28S genes
amplify the intervening ITS region
of approximately 600 bp.
Furthermore, many fungal ITS
sequences are available in
GenBank, which expands the
number of species available for
comparison.
The objective of this research
was to assemble a database of ITS
sequences to test the hypothesis
that S. passerinii is a close relative of
M. graminicola. The alternative
hypothesis is that S. passerinii could
be more closely related to other
cereal pathogens, such as the
septoria nodorum pathogen of
wheat and barley, Phaeosphaeria
nodorum.
Materials and Methods
Isolates of species of
Mycosphaerella, Leptosphaeria, and
Phaeosphaeria were obtained from
various sources (Table 1). DNA was
extracted from lyophilized tissue
and the ITS regions were amplified
using primers ITS4 and ITS5.
Amplification was with the
following cycling parameters: 94 C
for 2 min, 30 cycles of 93 C for 30 s,
53 C for 2 min, 72 C for 2 min, and
a final extension of 10 min at 72 C.
Amplification products were
purified and cloned. Each clone
was sequenced in both directions
on an automated DNA sequencer
and several clones were sequenced
per isolate.
DNA sequence alignment,
genetic distance calculation,
bootstrap analysis (1000
replications), and a neighborjoining
tree was prepared using
Clustal X (http://www-igbmc.ustrasbg.fr/BioInfo/ClustalX/
Top.html). The final tree was
printed using NJplot.
Results
The ITS regions of all S.
passerinii isolates were 571 bases
long, including the primer regions,
and were essentially identical. A
BLAST search on GenBank
identified M. graminicola as the
closest match to S. passerinii.
Sequences of other species showing
good matches with S. passerinii in
the BLAST search were
downloaded from GenBank, along
with those of species of
Leptosphaeria, Phaeosphaeria, and
other fungi in the Dothideales
(Table 1). A multiple alignment
revealed that the ITS sequences of
S. passerinii and M. graminicola
differed by only 10 bases (data not