Restriction Analyses of PCR Amplified Partial SSU Ribosomal DNA to Distinguish Arbuscular Mycorrhizal Fungi from Other Fungi Colonizing Plant Roots

Roots of Glycine max and Miscanthus sinensis and soil samples were collected from various field sites at Goesan, Chungbuk in Korea. Microscopic observations of the roots indicated high colonization rates of both arbuscular mycorrhizal fungi (AMF) and other fungi. The partial small subunit of ribosomal DNA genes were amplified with the genomic DNA extracted from their roots by nested polymerase chain reaction (PCR) with universal primer NS1 and fungal specific primers AM1. Restriction fragment length polymorphism (RFLP) was analyzed using the combinations of three restriction enzymes, HinfI, AluI and AsuC21. Nucleotides sequence analysis revealed that ten sequences from Miscanthus sinensis and one sequence from Glycine max were close to those of arbuscular mycorrhizal fungi. Also, 33% of total clones amplified with NS31-AM1 primers from M. sinensis and 97% from G. max were close to Fusarium oxysporum or other pathogenic fungi, and they were successfully distinguished from AMF. Results suggested that these techniques could help to distinguish arbuscular mycorrhizal fungi from root pathogenic fungi in the plant roots. Especially, DNA amplified by these primers showed distinct polymorphisms between AMF and plant pathogenic species of Fusarium when digested with AsuC21.

Sequence Analyses of PCR Amplified Partial SSU of Ribosomal DNA for Identifying Arbuscular Mycorrhizal Fungi in Plant Roots

The genomic DNAs were extracted from roots of Glycine max and Sorghum bicolor, and compared with those from spores of two arbuscular mycorrhizal (AM) fungi, Glomus mosseae and Scutellospora heterogama. The partial small subunit (SSU) of ribosomal RNA genes were synthesized and amplified by polymerase chain reaction with the fungal specific primers, AM1 and NS31. By the recent molecular techniques, the presence of another AM fungal DNA were confirmed in the roots of two plants, and three sequences of rDNA fragments amplified were identified to be close to those of G. caledonium, G. fasiculatum and G. proliferum. The two AM fungi, both, were found to colonize at the cortical layers of plant roots collected in the fields, together.

The Observation of Arbuscular Mycorrhizal Roots in Horticultural Plants

To determine the degree of variability among the host plant species in their abilities to become colonized by arbuscular mycorrhizal fungi (AMF), the inoculum for AMF was collected from the various sites in Korea and was inoculated to the three horticultural plants; Tagetes patula, Torenia fournieri, and Salvia splendens. After 4-month growth under greenhouse, mycorrhizal root colonization rates and spore density were measured. The roots of T. patula showed higher colonization rate than both plants of T. fournieri and Salvia splendens. The mycorrhizal root colonization was influenced by both of the AM fungal inoculum and the host species or their interactions. The combination of the host and fungal species was suggested to be important for the application of AMF to horticultural crops.