Community Structure analysis of arbuscular mycorrhizalfungi in grassland ecosystem using DNA analysis

在本研究中，我们探讨了分子标记诊断作为丛枝菌根真菌(AMY)种类鉴别的有效方法。并在此基础上，研究了日本草原丛枝菌根真菌群落结构的地理变异及土壤因素对其的影响。针对丛枝菌根真菌18S rDNA部分片段的特异扩增，设计了新的聚合酶链式反应引物。用该引物建立的聚合酶链式反应-凝胶电泳法可以鉴别出5种AMF菌株，其中18S rDNA的18S rDNA未被其他报道的AMF特异引物扩增。因此，发展了一种新的分析AMF群落结构的新方法--聚合酶链式反应-凝胶电泳法。采样点取自不同土壤环境的草地：化感红壤、非化感红壤和黄壤。进行了PCR-DGGE分析，构建了系统发育树，确定了这些显著DGGE条带的碱基序列。DNA的DGGE谱带主要集中在球孢菌科、Ac和Ac组，少数属于球菌科和无菌球菌科。就土壤类型而言，各草地土壤类型中均有球状抗体类群的分布，但Ac、Ad类群在不同土壤类型和寄主植物中表现出不同的趋势。根中的孢子群落以红色球孢菌、无梗霉等为主，与菌根中的真菌种类不同。利用人工种植的白三叶草地，对影响丛枝菌根真菌群落结构的因素进行了研究。在成熟草地群落中，球囊菌属-Ab、Ad、B组和无球菌科占优势，而在新开垦草地、球囊菌属-B组和无球菌科中主要定植。此外，土壤中高水平的有效磷对定植的AMF物种的多样性造成了抑制。

In this Study, we examined a molecular marker diagnosis as effective method to discriminate of Arbuscular Mycorrhizal Fungi (AMY) species. And on the basis of the method, geographic variation of AMF community structures in Japanese grassland and soil factors affecting on those are studied.New PCR primer was designed for specific amplification of a part of 18S rDNA of arbuscular mycorrhizal fungi. The PCR-DGGE with this primer enabled to discriminate 5 AMF species including Archaeospora leptoticha, whose 18S rDNA had not be amplified with other AMF specific primers reported previously. Thus, the new PCR-DGGE analysis method as an effective way to analyze AMF community structure in fields were developed.Miscanthus sinensis and Zoysia japonica were collected from 9 grasslands in Japan. Sampling sites were chosen from different soil environment grasslands : allophenic andosoil, non-allophenic andosoil and yellow soil. PCR-DGGE was performed and the phylogenetic tree was made to decide the base sequence of these prominent DGGE bands. Prominent DGGE bands of DNAs in sampled plants were belonged to Glomus-Ab, Ac, and Ad group and little to Glomus-B-group and Acaulosporaceae. Concerning soil types, Glomus-Ab group was distributed over both soil types in all grassland, but Glomus-Ac, Ad group were shown different tendency among soil types and host plants. Spore communities in root rhizoshere were dominant with Glomus rubiforme, Acaulospora koskei and so on unlike fungal species in mycorrhiza. Using sown white clover grassland, factors effecting AMF community structures were examined. In mature community of grassland, Glomus-Ab, Ad, B group and Acaulosporaceae were mainly colonized, but in newly plowed grassland, Glomus-B group and Acaulosporaceae. Additionally, high levelof available phosphate in soil caused a restraint to diversification of colonized AMF species.