Erland S, Henrion B, Martin F, Glover L A, Alexanders I J
Department of Microbial Ecology, University of Lund, Ecology Building, Helgonävagen 5, S-223 62 Lund, Sweden.
Equip de Microbiologie Forestière, Centre de Recherches Forestieres de Nancy, INRA, Champenoux 54280, France.
New Phytol. 1994 Mar;126(3):525-532. doi: 10.1111/j.1469-8137.1994.tb04251.x.
Sitka spruce mycorrhizas, macroscopically identified as being formed by Tylospora fibritiosa Donk, were sampled from a young and on old plantation and the mycobionts were isolated into pure culture. DNA was extracted and fragments of the ribosomal DNA (rDNA) were amplified using the polymerase chain reaction (PCR). The primers were directed to conserved regions of fungal rDXA and hybridize to a wide range of fungi. One amplified region includes the internal spacer (ITS) region which has a low degree of conservation. The JTS amplification products, which were approximately 600 base pairs (bp), were digested with a variety of restriction endonucleases in order to detect restriction fragment length polymorphisms (RFLPs). The RFLPs clearly separated T. fibrillosa from other ectomycorrhizal species but there were only minor differences between the T. fibrillosa isolates. PCR amplification of the ITS region, digestion with the endonudeasc HinfI and examination of the RFLPs produced proved to be a rapid method by which to distinguish T. fibriHosa from a large number of other basidiomyictes. The method was also applied to DNA extracted, from single mycotrhizal root tips. The imergenic spacer region (1GS) of the rDNA is more variable than the ITS region in several fungal species. The 5'end of the 25S and the intergenic region between the 25S and the 5S genes were amplified and analyzed as above. Polymorphisms between T. fibritiosa isolates within this region were limited and RFLPs were not useful m discriminating between isolates, suggesting a low genetic variability in this species.
从一片年轻和一片老龄人工林中采集了宏观上鉴定为由纤维状 tylospora donk 形成的西加云杉菌根，并将菌根共生体分离到纯培养物中。提取 DNA，并使用聚合酶链反应（PCR）扩增核糖体 DNA（rDNA）片段。引物针对真菌 rDXA 的保守区域，可与多种真菌杂交。一个扩增区域包括保守程度较低的内部间隔区（ITS）。对约 600 个碱基对（bp）的 ITS 扩增产物用多种限制性内切酶进行消化，以检测限制性片段长度多态性（RFLP）。RFLP 能清楚地将纤维状 tylospora 与其他外生菌根物种区分开来，但纤维状 tylospora 分离株之间只有微小差异。对 ITS 区域进行 PCR 扩增、用内切酶 HinfI 消化并检查产生的 RFLP，被证明是一种从大量其他担子菌中区分纤维状 tylospora 的快速方法。该方法也应用于从单个菌根根尖提取的 DNA。在几种真菌物种中，rDNA 的基因间隔区（IGS）比 ITS 区域更具变异性。如上述那样对 25S 的 5'端以及 25S 和 5S 基因之间的基因间隔区进行扩增和分析。该区域内纤维状 tylospora 分离株之间的多态性有限，RFLP 对区分分离株没有用处，这表明该物种的遗传变异性较低。
