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Molecular and cultural assessment of chytrid and Spizellomyces populations in grassland soils

     Department of Microbiology, Colorado State University, Fort Collins, Colorado, 80523-1677 USA

We developed a molecular method for the detection and quantification of members of the genus Spizellomyces in the environment and used this technique, together with traditional cultural techniques, to measure the effects of cultivation and nitrogen availability on Spizellomyces populations in grassland soils. Primer sets specific for Spizellomyces acuminatus and S. kniepii were developed by sequencing internal transcribed spacer 2 (ITS2) of the gene encoding ribosomal RNA for 9 isolates within the genus Spizellomyces, 5 representatives of different genera within the order Spizellomycetales and one member of the order Chytridiales. These primers were used with fungal-specific primers in a nested PCR approach to generate a specific molecular signal for S. acuminatus and S. kneipii in a soil from which S. acuminatus had previously been recovered. Using MPN-PCR (a quantitative molecular technique) and traditional cultural techniques, we found that chytridiomycetous fungi, including members of the genus Spizellomyces, are abundant in the grassland ecosystems studied. No significant differences in occurrence were observed between native and disturbed control soils but it appeared in 2 separate MPN assays and one MPN-PCR assay that chytrid populations increased in response to disturbance. No significant differences in chytrid or Spizellomyces populations were observed with variations in nitrogen availability. The primer sets and protocols developed in this study worked well to complement traditional cultural data to better assess Spizellomyces populations in the environment. These molecular approaches should provide a foundation for further work with these interesting and oft neglected fungi.

Key words: Chytridiomycota, disturbance, ITS, MPN-PCR, quantification, soil

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