| Phellodendron amurense Rupr. belongs to the broad-leaved trees of the Rutaceae. It is the associated tree species of broad-leaved Korean pine mixed forest, and one of the distinguished Three-Hardwood forests in the northeast of China, and also a rare medicinal plant and timber. It is on the verge of extinction due to the illegitimate collect chinese materia medica resources and the excessively cut for utilizing. Arbuscular mycorrhizal (AM) fungi are ubiquitous, obligate biotrophs, which live in symbiosis with the roots of most terrestrial plant species. AM fungi enhances the uptake by plants of certain nutrients, promotes plant growth, improves plant tolerance to drought and toxicity of pollutants, defends plants against pathogens, and so on. At present, there is no research on identification and the flora structural analysis of the AM fungi of Phellodendron amurense at home and abroad. In this research, molecular biological method was applied to the studies of identification and the flora structural analysis of the AM fungi of Phellodendron amurense. Therefore, it not only has the theoretical meaning to the enrichment and development of synecology significantly, but also laid the foundation for collection, protection, exploitation of AM fungi resources and study for functional flora in mycorrhizal of Phellodendron amurense.The root and rhizospheric soil samples of Phellodendron amurense were collected in the Logging Station of Northeast Forestry University. The root samples were investigated by means of acid fuchsin stain after they were lysised in alkaline solution. The spores of AM fungi were collected from Phellodendron amurense rhizosphere by wet sieving and decanting procedure and centrifugation using sucrose. They were identified morphologically and molecularly. To detect the AM fungi of Phellodendron amurense, the crude DNA that was extracted from the root samples of Phellodendron amurense was applied to Nested-PCR by the specific primers of AM fungi. Four species were obtained. They were Glomus intraradices, Glomus mosseae, Scutellospora calospora and Glomus versiforme. They were identified from morphologic characteristics and analysis for the large-subunit (25S) rDNA D1/D2 domain sequences. However, only G. intraradices, G. mosseae and Scu. calospora were detected from the root samples of Phellodendron amurense by Nested-PCR. It was indicated that the roots of Phellodendron amurense were not infected by G. versiforme.Meanwhile, Nested-PCR was conducted to specifically amplify NS31/Glol domain sequences of the small-subunit (18S) rDNA from the AM fungi in the root and rhizospheric soil samples of Phellodendron amurense, respectively. The conditions of DGGE were optimized by the Nested-PCR products. Then, the flora structure of the AM fungi of Phellodendron amurense was analyzed by sequencing, phylogenetic analysis and analytical techniques of DGGE profiles. The result indicated that the targeted product (230bp) successfully amplified from trace DNA by Nested-PCR. The optimum condition of DGGE was finally confirmed. The purified PCR products were used for DGGE analysis. Electrophoreses were run for 8 h at 130V on a 8.0% acrylamide/bis-acrylamide (37.5: 1) gel with a 30–60% denaturant gradient. All DGGE analysis was run at a constant temperature of 60℃. Sequencing, phylogenetic analysis and DGGE fingerprint analysis revealed that the above 2 samples generated diverse DGGE profiles. Distinct differences were detected in DGGE fingerprint at band numbers, densities and dominance. All sequences can be divided into three groups of microbial population, that is Glomus, Scutellospora and Hyponectria buxi. Glomus sp. (EF177624) and Glomus sp. (DQ085205) were the prevalent AM fungi in the root and rhizospheric soil of Phellodendron amurense, respectively. |
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