THE EFFECTS OF COMBINED NITROGEN ON THE MICROBIAL COMMUNITY COMPOSITION AND STRUCTURES WITHIN AZOLLA MICROPHYLLA

The aquatic fern Azolla is symbiotically associated with N₂-fixing cyanobacterium, Nostoc azollae, which resides in the leaf cavity of its host and supply total N requirement of the association by fixing atmospheric N₂. It has been demonstrated that addition of combined nitrogen significantly inhibits the N₂-fixation activity of the cyanobiont to negatively affect the growth of Azolla. Since the 1980’s, more and more bacteria species have been found in the leaf cavity and are thought to be the third partner, bactbiont, of the symbiotic association. We explored the effect of combined nitrogen on the microbial community dominated by cyanobiont within Azolla at molecular and ultrastructural levels. To exclude the exotic contamination and retain the native endophyte, both the sterilized Azolla containing microbial community dominated by Nostoc azollae (AmA) and the sterilized Nostoc azollae-free Azolla (AmB) were obtained by using stem apex culture and adding combined N in the medium. The SEM showed that compared with AmB, the phenotype of AmA fronds was modified to some extend by morphology, and the structure of the microbial community within the leaf cavity of AmA was characterized by both the formation of biofilm and the algal pocket which were composed of cyanobiont, bactbiont, the host hairs and their secretions. In contrast, no biofilm and algal pocket were found in case of AmB. High throughout sequencing based on 16S rRNA and nifH gene indicated a remarkable diversity of microbial community within AmA cultured in N-free medium with totally 17 operational taxonomic unit (OTU) sequences, belonging to 4 phyla. A diazotroph endosymbiont consortium including Herbaspirillu, Niveispirillum, Rhizobium dominated by Nostoc azollae was found in AmA samples. Compared with AmA, the diversity of the bactbiont community in AmB samples was dramatically reduced with only 8 OTUs and the diaozotrophic consortium, particularly Nostoc azollae, were completely disappeared. These findings showed for the first time that it is possible to improve the growth and development of host plant by altering its endophyte community composition and structure through adjusting N nutrient supply.