African Journal of Biotechnology Vol. 3 (3), pp. 195-198, March 2004
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2004 Academic Journals
Full Length Research paper
Colletotrichum circinans and Colletotrichum coccodes
can be distinguished by DGGE analysis of PCRamplified 18S rDNA fragments
Olajire Fagbola1+ and Mathew M. Abang2*
1
Federal Biological Research Centre for Agriculture (BBA), Messeweg 11/12, D-38104 Braunschweig, Germany.
German Collection of Microorganisms and Cell Cultures (DSMZ), Messeweg 11/12, D-38104 Braunschweig, Germany.
2
Accepted 29 December 2003
The rDNA 18S region of Colletotrichum circinans and C. coccodes was amplified by PCR to evaluate
this DNA region as a tool for species delineation. PCR amplification of the 18S of both species
produced 1.65 Kb long fragments that covered most of the entire 18S rDNA molecule. DGGE analysis of
the amplified fragments distinguished C. circinans from C. coccodes isolates. This result provides
molecular evidence that supports the current treatment of C. circinans as a species distinct from C.
coccodes, in spite of the failure of previous attempts at genetic differentiation of the two species based
on RFLP analysis of the rDNA ITS region.
Key words: DGGE, Colletotrichum circinans, Colletotrichum coccodes, molecular differentiation, species
delineation.
INTRODUCTION
The revision of Colletotrichum by von Arx (1957) was a
landmark in the taxonomy of plant pathogens, in which
750 “species” of Colletotrichum were reduced to 11 taxa
based on morphology rather than host-specificity. The
number of “accepted” species has now increased to 39,
based on more detailed studies of morphology, cultural
characteristics, and pathogenicity (Sutton, 1980, 1992).
Several taxa considered as host specialized forms by von
Arx (1957) were recognized as full species by Sutton
(1992). For instance, Sutton (1992) regarded C. circinans
as distinct from C. coccodes and other Colletotrichum
species, while von Arx (1957) regarded C. circinans as
an Allium-specific form of C. dematium (C. dematium
*Corresponding author. Present address: IRAD, P. O. Box 2123
Messa-Yaoundé, Cameroon, and Germplasm Program,
International Center for Agricultural Research in the Dry Areas
(ICARDA), P.O. Box 5466, Aleppo, Syria. E-mail:
m.abang@cgiar.org.
+
Present address: Dept of Agronomy, University of Ibadan,
Ibadan, Nigeria.
Abbreviation: ITS, internal transcribed
denaturing gradient gel electrophoresis.
spacer;
DGGE,
f.sp. circinans). The complete host range of most
Colletotrichum species is as yet unknown, and Sutton’s
treatment of the genus has been criticized for being
based on the uncritical assumption that Colletotrichum
species are host-specific (Cannon et al., 2000). Conidia
of C. coccodes are described as 16-24 µm long by 3-4
µm wide, straight, fusiform and abruptly tapered while
those of C. circinans are 19-21 x 3.5 µm, falcate, fusiform
and gradually tapered to each end (Sutton, 1992).
However, many Colletotrichum species produce
secondary conidia in culture that are generally smaller
and more varied in shape, especially when the culture is
old (Cannon et al., 2000). The common occurrence of
intermediate morphological features, and the recognition
that the currently “accepted” species may actually be
host-specific forms of species complexes or aggregates
has led to the use of biochemical and molecular markers
to verify species boundaries within Colletotrichum
(Cannon et al., 2000; Abang et al., 2002).
Analysis of the ITS regions, as well as the D1 and D2
domains of the nuclear rDNA, have been used
extensively to resolve taxonomic questions within
Colletotrichum (Sreenivasaprasad et al., 1996, Freeman
et al., 2000). Martín and García-Figuerez (1999) and
Abang et al. (2002) failed to distinguish C. coccodes from
196
Afr. J. Biotechnol.
C. circinans based on restriction analysis of their ITS
sequence. They hypothesized that the two species are
identical and could belong to the same taxon, in spite of
apparent morphological differences. DGGE analysis of
PCR-amplified rDNA has been used for species
delineation in fungal genera such as Pleomassaria and
Melampsoridium (Paavolainen et al. 2000), and DGGE
analysis of 18S rDNA has been successfully applied in
molecular differentiation of C. gloeosporoides and C.
acutatum (Fagbola et al., 2001). In this study, we applied
the technique to determine if molecular evidence
supports the treatment of C. circinans as a species
distinct from C. coccodes.
RESULTS AND DISCUSSION
Total genomic DNA was extracted from isolates of C.
coccodes (DSM 62126, 66376, 70882, 70879, DSM
2492, 70880) and C. circinans (67846 and 71247) using
a modified CTAB procedure (Abang et al., 2002). The
oligonucleotide primers, FR1 (fungus-specific reverse
primer 1) and NS1 (forward primer), of Vainio and
Hantula (2000) were used for PCR amplification of the
small subunit rDNA of the isolates (Gomes et al., 2003).
The primer pair FR1 + NS1 produced 1.65 Kb long PCRfragments that covered most of the entire SSU rDNA
molecule. Good resolution of all of the products was
achieved using the denaturing gradients of 18-43 % and
no artefacts bands were observed (Figure 1).
The Colletotrichum strains were separated into two
distinct groups based on the migration rate of their
amplified 18S fragments following DGGE (Figure 2). The
amplified 18S bands of C. circinans migrated faster (band
“b”) and were clearly distinct from those of C. coccodes.
Colletotrichum coccodes isolates could be easily
distinguished by the slow migration rate of their amplified
18S bands in denaturing gradient gels (band “a”). The
migration of amplified 18S rDNA samples within each
Colletotrichum species was similar, suggesting that there
was little intraspecific variation. It is also likely that
intraspecific variation present was not properly resolved
by DGGE. However, this was not tested by sequencing.
DGGE is an electrophoretic technique that efficiently
separates DNA molecules according to their size, as well
as sequence differences (Myers et al., 1986, Lessa,
1992). In comparison to DNA sequencing, DGGE is a
simple and relatively cheap method that can be used in
genetic fingerprinting assays of large numbers of
samples. Application of this technique to a 1.65 Kb SSU
rDNA fragment was previously shown to be an efficient
method for differentiating taxa within the C.
gloeosporioides
group
species
associated
with
anthracnose disease of yam in Nigeria (Fagbola et al,
2001). Also, DGGE analysis of PCR-amplified SSU rDNA
clearly differentiated C. gloeosporioides from C.
acutatum. DGGE of PCR-amplified 18S fragments
bp
2036
1636
1018
506
Figure 1. Electrophoretic pattern of PCR-amplified 18S ribosomal
DNA on ethidium bromide-stained agarose gel, depicting a single
1.65 Kb long fragment for all the Colletotrichum strains analysed in
this study. M = molecular size marker (1 Kb DNA ladder, Gibco
BRL). Total genomic DNA was extracted from aerial
mycelium/conidia scraped off 7-day-old cultures using a modified
CTAB procedure (Abang et al., 2002). DNA quality was visually
assessed on 1% agarose gel following electrophoresis, and the
concentration was measured using a Dyna Quant 200 fluorometer
(Hoefer Pharmacia Biotech Inc., USA). The oligonucleotide primers,
FR1 (fungus-specific reverse primer 1) and NS1 (forward primer), of
Vainio and Hantula (2000) that target the 18S subunit of the fungal
genome were used for PCR amplification. PCR was performed with
a DNA thermal cycler 480 (Perkin-Elmer Cetus, Norwalk, CT, USA).
PCR amplification of DNA was carried out in 25 µl reaction mixtures
containing 2.5 µl of 10X reaction buffer, 3.5 µl of 25 mM MgCl2, 2.5
µl 2.0 mM dNTP mixtures, 0.5 µl DMSO (2% v/v), 0.5 µl each of the
primers at 10pmol concentration, 0.25 µl of Stoffel fragment, 1.0µl
template DNA, and 13.75µl water. Amplification condition was 8 min
at 94oC, followed by 25 cycles of 30s at 94oC, 45s at 48oC, 3 min at
72oC, and final extension for 10 min at 72oC. Products were first
analysed by electrophoresis in 1.5% (w/v) agarose gels and
ethidium bromide staining.
provided clearly interpretable results suggesting that this
method could be useful in further verification of species
delineation among the 39 “accepted” species currently
defined within Colletotrichum based on cultural
characteristics (Sutton, 1992). In previous studies,
restriction analysis did not reveal any difference in the
ITS sequence of C. coccodes and C. circinans (Martin
and Garcia-Figuerez, 1999; Abang, 2003). However, a
more intensive usage of different enzymes might have
differentiated the two species. It may seem surprising that
differences not evident in the variable ITS region were
revealed in the conserved 18S gene; however, Cannon et
al. (2000) have noted that the distinction between the ITS
and 18S as variable and conserved regions, respectively,
is not absolute. In fact, functional ribosomal sequences
have been used for species and intraspecific group
definition in Colletotrichum (Sheriff et al., 1994; Johnston
and Jones, 1997).
This is the second report of the application of DGGE of
PCR-amplified 18S rDNA for genetic differentiation of
Colletotrichum species, after the differentiation of C.
Fagbola and Abang
b
a
Figure 2. Denaturing gradient gel electrophoresis profiles depicting
differential migration of PCR-amplified 18S ribosomal DNA
fragments of C. coccodes isolates DSM 62126, 66376, 70882,
70879, DSM 2492, 70880 (arrow points to slow-migrating bands
“a”), and C. circinans isolates 67846 and 71247 (arrow points to
fast-migrating bands “b”). The following fungal species, from top to
bottom, were used as standards (Lanes M): Colletotrichum sp.,
Sclerotium tuliparum, Trichoderma harzianum, Myrothecium
cinctum, Ustilago nuda, Myrothecium leucotrichum, and Penicillium
simplicissimum. DSM isolates were kindly supplied by Dr Peter
Hoffmann from the German Collection of Microorganisms and Cell
Cultures (DSMZ, Braunschweig, Germany), while Dr Helgard
Nirenberg provided the other Colletotrichum strains from the fungal
collection of the Federal Biological Research Centre for Agriculture
(BBA, Berlin, Germany). PCR products from all isolates were
analysed using DGGE (D GENE System; Bio-Rad, Inc., Hercules,
CA) (Gomes et al., 2003). The DGGE solution was made of 7.5%
(w/v) acrylamide/bisacrylamide (37.5:1) gels. The denaturant
gradient was 18-43% which were produced with 100% denaturing
solution containing 40% deionized formamide and 7 M urea. The
gels were run in 1X TAE-buffer at a constant temperature of 58oC
and 180V for 18 h. The gels were silver stained to visualise the
electrophoretic mobility pattern of the isolates. Silver staining
solutions used were 10% (v/v) ethanol plus 0.5% acetic acid for
fixation, freshly made 0.1% (w/v) silver nitrate for staining, freshly
prepared developing solution containing 0.01% (w/v) sodium
borohydride, 0.15% formaldehyde, 1.5% (w/v) NaOH and 0.75%
(w/v) sodium carbonate solution to stop the development. Gels
were mummified and dried for documentation and analysis.
gloeosporioides and C. acutatum (Fagbola et al., 2001).
The technique may be applied in any of the many
instances where multiple Colletotrichum species cause
anthracnose disease on the same host (e.g. Martín and
García-Figueres, 1999).
Because
electrophoretic
197
behaviour directly reflects differences in nucleotide
composition of the fragments screened, the assay
provides clear evidence of polymorphism among the
taxonomic entities investigated. This study was
handicapped by our inability to find more C. circinans and
C. coccodes isolates for analysis. It would be necessary
to analyse more strains within each species, and to
obtain DNA sequence data from multiple loci (including
the SSU rDNA gene) to establish the taxonomic
boundaries around C. circinans and C. coccodes.
Results obtained in the present study support the
treatment of C. circinans as a species distinct from C.
coccodes, in contrast to previous studies (Martín and
García-Figuerez, 1999; Abang et al., 2002) that failed to
find molecular evidence in support of Sutton’s (1992)
treatment of the two taxa as distinct species. Clarification
of species boundaries in Colletotrichum is a matter of
urgency and great practical importance, especially for
plant pathologists. Not only does our ability to accurately
identify disease causal agents depend on it but precise
data on host ranges and geographic distributions of
species cannot be assembled until these problems are
resolved.
ACKNOWLEDGEMENTS
The first author was supported by AvH Stiftung,
Germany. The authors would like to thank Drs Kornelia
Smalla (BBA, Braunschweig) and Stephan Winter
(DSMZ, Braunschweig) for their support of this work, and
Drs Peter Hoffmann (DSMZ, Braunschweig, Germany)
and Helgard Nirenberg (BBA, Berlin, Germany) for kindly
providing C. coccodes and C. circinans reference
isolates.
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