结合态氮对满江红内生细菌群落组成和结构的影响

研究从超微结构和分子水平上探讨结合态氮对满江红(Azolla)叶腔中以共生藻占优势的微生物群落的影响。为排除外源污染并保留其内生菌的多样性,采用茎尖组织培养并添加结合态氮等方法,取得了表面无菌的含藻满江红(AmA)和无藻满江红(AmB)。电镜观察揭示, AmB较AmA的萍体表型有某种程度的修饰。AmA的微生物群落结构以共生藻、内生菌和宿主腺毛及其分泌物组成的生物被膜和藻囊为主要特征,而AmB的叶腔几乎中空。基于16S rRNA基因和nif H基因的高通量测序结果显示,生长于无氮培养液的AmA样品的微生物群落有相当高的多样性,共有17个可操作分类单元(OTU),分属4个细菌门,并有一个以Nostoc azollae为优势的固氮微生物亚群包括草螺菌、根瘤菌和Niveispirillum等。而生长于富含结合态氮培养液的AmB样品的群落多样性明显降低,仅有8个OTU,且以Nostoc azollae为优势的上述固氮微生物亚群全部消失。研究结果表明,通过调整氮素营养,可改变宿主植物的微生物群落组成与结构,进而改良植物的生长发育。

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.