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

在这项研究中，我们研究了分子标记诊断作为有效的方法来区分丛枝菌根真菌（AMY）的物种。在此基础上，研究了日本草地丛枝菌根真菌群落结构的地理变异及土壤因子对其的影响。设计了一对新的PCR引物，对日本草地丛枝菌根真菌18 SrDNA的一部分进行了特异性扩增。利用该引物进行的PCR-DGGE分析，可区分包括细毛孢在内的5个AMF种，而其它AMF特异性引物均未扩增出其18 SrDNA。因此，PCR-DGGE分析方法作为一种新的有效的方法来分析AMF的群落结构在田间。采样点选在不同土壤环境的草地上：他化的红壤、非他化的红壤和黄壤。采用PCR-DGGE技术对这些差异条带进行系统发育树分析，确定其碱基序列。DGGE分析表明，Glomus-Ab、Ac和Ad类群的DNA条带较明显，Glomus-B类群和无梗孢菌科的DNA条带较少。就土壤类型而言，Glomus-Ab类群在两种土壤类型中都有分布，而Glomus-Ac、Ad类群在不同土壤类型和不同寄主植物中的分布趋势不同。根际土壤中的孢子群落以红色球囊霉、考氏无梗囊霉等为主，而菌根中的真菌种类较少。以人工白色三叶草草地为研究对象，研究了影响丛枝菌根真菌群落结构的因子。在成熟草地群落中，Glomus-Ab、Ad、B类群和无梗囊霉科主要定植，而在新垦草地群落中，Glomus-B类群和无梗囊霉科主要定植。此外，土壤中高水平的有效磷也限制了AMF的多样性。

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.