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

本研究探讨了一种分子标记诊断方法作为鉴别丛枝菌根真菌（amam）种类的有效方法。在此基础上，研究了日本草地AMF群落结构的地理变异及其影响因素。设计了一种新的PCR引物，特异扩增了丛枝菌根真菌18S rDNA的一部分。用该引物构建的PCR-DGGE能够区分包括细端古孢子菌在内的5种AMF，这些AMF的18S rDNA未被其他AMF特异性引物扩增。因此，PCR-DGGE分析方法可作为分析农田AMF群落结构的有效方法。在日本9个草原上采集了芒草和结缕草。采样点选择不同土壤环境草地：异氧土、非异氧土和黄壤。采用PCR-DGGE方法，构建系统发育树，确定这些突出的DGGE条带的碱基序列。样本植物DGGE条带主要属于Glomus-Ab、Ac和Ad组，较少属于glomus - b组和Acaulosporaceae。就土壤类型而言，Glomus-Ab组在所有草地的两种土壤类型中均有分布，而Glomus-Ac、Ad组在不同土壤类型和寄主植物中表现出不同的趋势。根际孢子群落与菌根中的真菌种类不同，以红球囊菌（Glomus rubiformme）、高斯芽孢菌（Acaulospora koskei）等真菌群落占优势。以播种的白三叶草为研究对象，对影响AMF群落结构的因素进行了研究。在成熟草地群落中，主要定植有Glomus-Ab、Ad、B类群和Acaulosporaceae，而在新耕草地中，主要定植有Glomus-B类群和Acaulosporaceae。此外，土壤中高水平的有效磷对定植AMF物种的多样化造成了抑制。

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.