Diversity Of Endophytic Bacteria Within Azolla Microphylla

Azolla is a heterosporous water fern, which lives in a symbiotic relationship with filamentous nitrogen-fixing cyanobacteria. In China, Azolla has been used as green manure for rice cultivation and animal feed for centuries. Recent use of Azolla as wastewater bio-filter and in biologically based life support systems incorporated in bio-regenerative space device is now in progress in several laboratories and has attracted the researchers worldwide. Since 1970s, eubacteria have been found in the leaf cavities of Azolla and considered as the third partner of the mutualistic symbiosis and as a model for the study of plant-mirobe interactions.An attempt has been made using tissue culture technique to gain a cyanobateria-free Azolla microphylla in our lab so that possible external bacterial contamination could be excluded, followed by using a combination of 16S rDNA-PCR-DGGE(denatured gradient gel electrophoreisis), electron microscopy and traditional plating methods in order to explore the phenotype polymorphism and genetic diversity of the endophytic bacteria within Azolla and to reveal its potential role of the endophytic bacteria in phylogeny and synchronic evolution between the Azolla, cyanobacteria and bacteria.Total DNA including bacterial DNA was directly extracted from host plant tissue containing the symbionts, and the bacterial population within Azolla was isolated through density graduation centrifugation. 16S rDNA-PCR was performed by using both total DNA and the bacterial population as templates. DGGE profile of the purified PCR products showed 22 bands due to different base pairs of the individual fragments among which 11 bands were sequenced and 5 sequences were shown to belong to 5 uncultivated bacteria via Blastn and using software DNAMAN.20 bacterial isolates, which were obtained from A. microphylla with and without the symbiotic cyanobacteria, with different morphological and biochemistry reaction characteristics were found to grow on 10 different bacterial media, including LB, NA, TRN, Ashby, Maconkey etc. At least two kinds of nitrogen-fixing bacteria were showed to occur in root tissue of the host. Based on scanning and transmission electron microscopy observations (SEM and TEM), the endophytic bacteria could be divided into 20 types according to their ultrastructural characteristics.The information from DGGE analysis combined with the results gained from traditional plating methods and EM observation, it is suggested that there is a complex bacterial community with Bacillus cereus and its variants, Pseudomonas, Agrobacterium, Alcaligenes and Azospirillum as dominant species within Azolla micwphylla. The finding revealed the phenotype polymorphism and genetic diversity of endophytic bacteria within Azolla micwphylla at cellular, subcellular and molecular levels.