Department of Microbiology, Colorado State University, Fort Collins, Colorado, 80523-1677 USA.
Mycologia. 2002 May-Jun;94(3):411-20.
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.
我们开发了一种用于检测和定量环境中 Spizellomyces 属成员的分子方法，并使用该技术以及传统的文化技术来测量培养和氮可用性对草原土壤中 Spizellomyces 种群的影响。通过对属内 9 个分离物的核糖体 RNA 编码基因的内部转录间隔区 2（ITS2）进行测序，开发了针对 Spizellomyces acuminatus 和 S. kniepii 的引物对，这 9 个分离物属于 Spizellomyces 属，5 个代表不同属的 Spizellomycetales 目和一个 Chytridiales 目成员。这些引物与真菌特异性引物一起用于巢式 PCR 方法，以在先前已从土壤中回收出 Spizellomyces acuminatus 的土壤中产生 Spizellomyces acuminatus 和 S. kneipii 的特定分子信号。使用 MPN-PCR（一种定量分子技术）和传统的文化技术，我们发现包括 Spizellomyces 属成员在内的壶菌在研究的草原生态系统中非常丰富。在原生和干扰对照土壤之间未观察到发生的显着差异，但在 2 个单独的 MPN 测定和 1 个 MPN-PCR 测定中，壶菌种群似乎因干扰而增加。氮可用性的变化并未观察到壶菌或 Spizellomyces 种群的显着差异。在本研究中开发的引物对和方案很好地补充了传统的文化数据，以更好地评估环境中的 Spizellomyces 种群。这些分子方法应为进一步研究这些有趣且经常被忽视的真菌提供基础。
