Molecular Characterization of Fungal Species From Pure Cultures and Environmental Samples




Timothy Tarbell

Introduction

Traditionally, the species composition of fungal communities has been determined through a combination of media culturing and identification based on macro or microscopic features. However, these techniques have several drawbacks, including the over or underestimation of fungal species within a community based on the presence or absence of fruiting bodies, the inability of certain species to be grown in culture, and the difficulty in identifying fungi to the species level based on microscopic features.

Quick and simple methods for determining the species composition of fungal communities based on sequencing particular regions of the fungal genome have proven a reliable alternative to traditional methods.
Ribosomal genes and spacers regions within the fungal genome are good candidates for amplification via the polymerase chain reaction (PCR) because they are comprised of highly conserved tracts with heterogeneous regions in between. The conserved tracts are ideal for universal primer design that can allow for the amplification and sequencing of heterogeneous regions. Most molecular fungal species identification relies on the amplification and sequencing of the internal transcribed spacer (ITS) region of the fungal genome, which is highly variable among species or even populations of the same species. This region lies between the small subunit (SSU) and the large subunit (LSU) ribosomal RNA (rRNA) genes and contains two noncoding spacer regions (ITS-A and ITS-B) separated by the 5.8S rRNA gene. In fungi, the ITS region is typically 650900 bp in size, including the 5.8S gene and is usually amplified by the universal primer pair ITS 1 and ITS 4 designed by White et al. (1990).

The primary objective of this study was to determine the ability of PCR to amplify the ITS regions of fungal samples for the purposes of sequencing and species identification. The method was tested using DNA extracted from nine unidentified fungal cultures, as well as from three soil samples. Because the ITS regions of different fungal species varies in both size and nucleotide sequence, the second objective of this study was to test the ability of single stranded conformational polymorphism (SSCP) to generate a unique banding pattern for each fungal species present in mixed sample.

Figures


Figure 1-(A) The ITS primer pair used in this study. (B) rRNA genes showing the location of primers ITS-1 and ITS-4.


Figure 2-PCR products generated from culture samples using the ITS primer pair. Numbers below each lane indicate the culture number from which tissue samples were taken. Black labels above each lane indicate the identity of each fungus based on the alignment of their sequenced ITS regions with published sequences in the NCBI database. Samples were run on a 2% agarose gel.


Figure 3-PCR products generated from three soil samples using the ITS primer pair. The species identity of each fungus was determined based on the alignment of their sequenced ITS regions with published sequences in the NCBI database. Samples were run on a 2% agarose gel.


Figure 4-SSCP products generated through amplification of the ITS region of P. expansum, C. uredinicola, and M. hiemalis. Lanes 1-3 show SSCP products generated by the three species individually. Lane 4 shows SSCP products generated by a reaction containing the DNA of all three species.


This study had two major objectives. The first was to determine the effectiveness of using PCR to amplify the ITS region of fungal DNA for sequencing and eventual species identification. The second was to determine a suitable method for adequately separating PCR products generated in reactions containing multiple species.

Amplification and sequencing of the ITS region of unidentified fungi in culture led to the successful identification of all nine to the species level. This technique also allowed for the identification of three fungal species from three different soil samples.

Fractionation of mixed samples on 2% agaros gels did not yield enough resolution to separate PCR products of similar sizes. SSCP analysis showed that individual fungal species produced a distinct banding pattern that was not based on PCR product size. Separation of bands generated by a mixed reaction adequately showed the presence of multiple species. More intensive screening of fungal species using this technique could lead to the compilation of a fungal species barcode.

Full Paper

Acknowledgments

I would like to thank Dr. Rubin, Bo Liu, and Leleesha Samaraweera for all their guidance and encouragement during this project. I would also like to thank Dr. Amy Tuininga and Pam Greengarten for providing me with the fungal cultures that made these experiments possible.


This document was last modified 05/13/2008.
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