T-RFLP Analysis For Diversity Of Endosymbionts Within Azolla Microphylla

The leaf cavities of the aquatic fern Azolla contain the symbiotic N2-fixing cyanobacterium Anabaena azollae Strasb. Utilization of the Azolla as a livestock feed and a bio-fertilizer for wetland paddy in China has a long history of centuries. Endophyte is a microorganism community with abundant biodiversity.Their existence and effect on the host plant have been ignored for they have been living in the healthy plant tissues without causing any diagnostic and co-evolvement in their hosts.The first description of symbiotic bacteria in Azolla by means of transmission electron microscopy (TEM) observations was make by Grilli(1964). Since then, the Azolla endophytes has become one of the interesting research topics.With the scientific development broadened and deepened, progress has been made on the study of the endophytic populations. Molecular methods have been developed rapidly over the last decade, and they have overcome many of the problems associated with conventional culture methods. Terminal restriction fragment length polymorphism (T-RFLP) analysis is a culturing-independent approach for analyzing microbial community in environments.In this study, T-RFLP fingerprinting method was validated for analysis of the diversity and community structure of the endobacteria, endophytic fungi and endophytic archaea in the cyanobateria-free Azolla microphylla. The combination of traditional methods and molecular tools were used to explore the phenotype polymorphism and genetic diversity of the endophytes within Azolla, so that to reveal potential role of the endophytes in phylogeny and synchronic evolution between the Azolla, cyanobacteria and other endophytes..Total DNA was directly extracted from host plant tissue containing the symbionts , and the endophyte population within Azolla was isolated through density graduation centrifugation.Using individual fluorescently labeling 16SrDNA primer pairs for bacteria, archaea and for fungi ITS primers, PCR was performed with total DNA as template separately.Followed by restriction digestion amplicons with HhaI and MspI.The T-RFLP profiles were produced using the GeneMarker? software and T-RFs were compared and analysed in RDP database.10 candidate bacterial identities and 97 candidate archaea identities were found in this investigation. The fingerprint of fungi T-RFs also proved its existence. Bacterial isolates obtained from the cyanobateria-free Azolla microphylla with different morphological characteristics showed the phenotype polymorphism of the endophytes on cellular level.The finding revealed the phenotype polymorphism and genetic diversity of endobacteria,endophytic fungi and endophytic archaea communities within Azolla microphylla at cellular and molecular levels.The results presented here confirm the existence of the endophytic archaea and fungi except the endobacteria firstly,which make a significant progress on the research of diversity of endosymbionts within Azolla microphylla.