Agriculture Reference
In-Depth Information
nucleic acid sequences to be used to identify the organism. A method for identifying
the presence of the target sequences in the sample also needs to be concentrated to
achieve successful diagnosis of the pathogen. Identification of DNA sequences can
either be achieved by developing a method that can exploit the variations present in
the unknown conserved genes or by screening random parts of the fungal genome
that exhibit the required specificity (Ward
et al.,
2004).
The
ribosomal DNA (rDNA)
operon is mainly targeted for development of
diagnostic markers because of their presence in all organisms at high copy number
that allow very sensitive detection. The ribosomal DNA operon comprises three
functionally and evolutionary conserved genes, the large subunit gene, the small
subunit gene and the 5.8S gene interspaced with a variable spacer region called the
internal transcribed spacer (ITS) in a unit, which is repeated many times in the
nuclear genome (Schmidt, 1994). Some of the rDNA regions are well conserved
throughout the evolutionary process and other parts are variable even within a
species. Based on these variations, multiple strategies have been adopted to
characterize pathogens (Louws
et al.,
1999). The conserved regions allow probes or
primers (often referred to as universal primers) from one species to be used to detect
and amplify ribosomal gene fragments from a broad range of phylogenetically
diverse genera, families or even kingdoms (McCartney
et al.,
2003). However, the
equivalent rDNA gene fragments detected in different organisms have sequence
variations that can be exploited for the subsequent selection of pathogen-specific
primer sequences allowing the identification of the pathogen in question. rDNA
sequences of different organisms available in publicly-available databases are far
greater than any other region of the genome (Ward
et al.,
2004).
The
internal transcribed spacer (ITS) region
that separates repeat units within
tandem arrays of the nuclear DNA (nrDNA) genes are ubiquitous in nature
and found in all eukaryotes. The ITS region is a part of the transcriptional unit of
nrDNA and in part it has been demonstrated to reflect some functional value
(Morales
et al.,
1993). The ITS region has been used extensively in fungal taxonomy
(Seifert
et al.,
1995) and is known to show variation between and within species
(Nazar
et al.,
1991; O'Donnell, 1992). Species-specific diversity within the ITS
region has been demonstrated across a variety of fungi. Mugnier (1998) has used the
ITS and 5.8S sequences to infer the relationships between the classes within the
division
Euascomycotina
. Studies of ITS regions from powdery mildews (Mori
et al.,
2000),
Aspergillus
(Henry
et al.,
2000),
Alternaria
(Pryor and Gilbertson,
2000);
Pythium
(Paul, 2000);
Verticillium
(Zare, 2000);
Tilletia tritici
(Josefsen and
Christiansen, 2002);
Fusarium culmorum
(Mishra
et al.,
2002);
Phytophthora
(Blomquist and Kubisiak, 2003) and
Phellinus
(Fischer
and Binder, 2004) have
revealed significant sequence divergence at the interspecific level indicating the
potential of using the ITS region for the identification of pathogens at species level.
Other genes are also becoming more widely used as targets for diagnostic
development and pathogen characterization. The beta-tubulin genes are widely used
as second common targets for diagnostic development in fungi (McCartney
et al.,
2003) and have been extensively studied by Hirsch
et al.
(2000, 2002), Schroeder
et al.
(2002) and Drogemuller
et al.
(2004). Apart from the ITS and beta-tubulin
region, regions pertaining to the endochitinase gene (Lieckfeldt
et al
., 1999, 2000),
