Reconstitution Of The Cycad-Cyanobacteria Association

Cyanobacteria with the capacity to nitrogen-fixation are one of the larger groups of photosynthetic prokaryotes, forming symbiosis with a wide variety of plants. An attention has recently been given to their symbiotic relationship with Cycads which are unique among the gymnosperms. It will favor to create artificially the nitrogen-fixing cyanobacteria-crop symbioses by revealing the mechanism of cyanobacteria-cycads symbioses.The symbionts within the coralloid roots of Cycads revoluta and the free-living cyanobacteria in the soil surrounding the roots were isolated and cultured. The PCR fingerprintings of the cyanobacterial isolates were examined with primers STRRmod, STRRspecial and Hip TG. The results showed that the PCR fingerprintings of the cyanobacterial isolates from the coralloid root tissues could be distinguished from those of cyanobacterial isolates from the surrounding soils. The diversity of the isolates from different branches of the root and the surrounding soils were demonstrated as well.The cyanobacyerial isolates were purified by heat incubation and by using antibiotics. A successful reconstitution between the sterile Cycad plantlets and the purified cyanobacterial isolates, which were used as inoculants, was made. An isolate from the coralloid root of the reconstituted Cycad plantlet was showed to have an identical STRR-PCR fingerprinting of the original inoculant, RZHA4. It is also confirmed that cyanobacterium strain RZHA4 was the only symbiont of the reconstituted cycad plantlet due to its 100% homological 16SrRNA sequence. These results has led to a suggestion that the strain RZHA4 is probably belong to Nostoc based on the alignment between RZHA4 and 100 cyanobacterial strains by NCBI blast.