2.1.1 Molecular Methods for Identification
of Sclerotia-Associated Fungi
Mycorrhizal fungi occur in highly diverse communities (Bruns 1995) with fine-scale
spatial partitioning (Dickie et al. 2002; Dickie and Reich 2005; Genney et al. 2006),
which can make them difficult to identify. Thus, researchers increasingly rely on
molecular methods to identify species based on belowground structures alone
(Horton and Bruns 2001), initially with restriction fragment length polymorphism
(RFLP) analysis, also known as amplified ribosomal DNA (rDNA) restriction
analysis. Denaturing gradient gel electrophoresis (DGGE) is also commonly used
(Kowalchuk et al. 2002; Opik et al. 2003; Bougoure and Cairney 2005; Landeweert
et al. 2005; Ma et al. 2005; Pennanen et al. 2005), as are clone libraries (Landeweert
et al. 2003; Renker et al. 2006) and terminal restriction fragment length polymorphism (T-RFLP) analysis. It has been suggested that T-RFLP is more sensitive than
DGGE for fungal identification (Brodie et al. 2003; Singh et al. 2006), although
obtaining sequences directly from samples may be easier with DGGE (Ma et al.
2005). T-RFLP also has significant cost advantages over construction of clone
libraries, although clone libraries are likely the most accurate method of identifying
species (Dickie and Fitz John 2007). Using clone libraries together with T-RFLP
may permit both techniques to be used to their full potential: using T-RFLP to
process large numbers of samples and constructing clone libraries for selected
samples to determine the identities of key species (Lindahl et al. 2006; Widmer
et al. 2006).
Generally, T-RFLP refers to the use of fluorescently labeled primers combined
with restriction digestion to visualize sequence variation in either single- or mixedspecies DNA samples (Dickie and Fitz John 2007). The T-RFLP technique was first
developed by Liu et al. (1997) as a tool for assessing bacterial diversity and
comparing bacterial community structure between environmental samples (Marsh
1999; Lukow et al. 2000; Kitts 2001). T-RFLP data are visualized as an electropherogram, with the size and relative fluorescence intensity of fragments containing the
labeled primer (terminal fragment lengths) observed as peaks. Variation in the
presence and location of restriction sites results in different species having terminal
fragments of different lengths. In T-RFLP, as used by Liu and colleagues, a single
fluorescent label and a single restriction digest are used. The number of peaks and the
similarity of peak profiles across samples are then analyzed (Dollhopf et al. 2001;
Edel-Hermann et al. 2004; Mummey et al. 2005).
Although Cg forming sclerotia are also known as ectomycorrhizal fungi, we
performed T-RFLP analysis (Liu et al. 1997), coupled with ITS region clone library
construction and sequencing (Schütte et al. 2008). Since this technique requires no
culturing, it may offer a rapid method for identifying sclerotia and their associated
fungal communities.
2 Fungal Communities of Sclerotia Grains from Forest Soils
19
of Sclerotia-Associated Fungi
Mycorrhizal fungi occur in highly diverse communities (Bruns 1995) with fine-scale
spatial partitioning (Dickie et al. 2002; Dickie and Reich 2005; Genney et al. 2006),
which can make them difficult to identify. Thus, researchers increasingly rely on
molecular methods to identify species based on belowground structures alone
(Horton and Bruns 2001), initially with restriction fragment length polymorphism
(RFLP) analysis, also known as amplified ribosomal DNA (rDNA) restriction
analysis. Denaturing gradient gel electrophoresis (DGGE) is also commonly used
(Kowalchuk et al. 2002; Opik et al. 2003; Bougoure and Cairney 2005; Landeweert
et al. 2005; Ma et al. 2005; Pennanen et al. 2005), as are clone libraries (Landeweert
et al. 2003; Renker et al. 2006) and terminal restriction fragment length polymorphism (T-RFLP) analysis. It has been suggested that T-RFLP is more sensitive than
DGGE for fungal identification (Brodie et al. 2003; Singh et al. 2006), although
obtaining sequences directly from samples may be easier with DGGE (Ma et al.
2005). T-RFLP also has significant cost advantages over construction of clone
libraries, although clone libraries are likely the most accurate method of identifying
species (Dickie and Fitz John 2007). Using clone libraries together with T-RFLP
may permit both techniques to be used to their full potential: using T-RFLP to
process large numbers of samples and constructing clone libraries for selected
samples to determine the identities of key species (Lindahl et al. 2006; Widmer
et al. 2006).
Generally, T-RFLP refers to the use of fluorescently labeled primers combined
with restriction digestion to visualize sequence variation in either single- or mixedspecies DNA samples (Dickie and Fitz John 2007). The T-RFLP technique was first
developed by Liu et al. (1997) as a tool for assessing bacterial diversity and
comparing bacterial community structure between environmental samples (Marsh
1999; Lukow et al. 2000; Kitts 2001). T-RFLP data are visualized as an electropherogram, with the size and relative fluorescence intensity of fragments containing the
labeled primer (terminal fragment lengths) observed as peaks. Variation in the
presence and location of restriction sites results in different species having terminal
fragments of different lengths. In T-RFLP, as used by Liu and colleagues, a single
fluorescent label and a single restriction digest are used. The number of peaks and the
similarity of peak profiles across samples are then analyzed (Dollhopf et al. 2001;
Edel-Hermann et al. 2004; Mummey et al. 2005).
Although Cg forming sclerotia are also known as ectomycorrhizal fungi, we
performed T-RFLP analysis (Liu et al. 1997), coupled with ITS region clone library
construction and sequencing (Schütte et al. 2008). Since this technique requires no
culturing, it may offer a rapid method for identifying sclerotia and their associated
fungal communities.
2 Fungal Communities of Sclerotia Grains from Forest Soils
19