Identify Of Microbial Community And Detection Neurotoxin From Cycas Fairylakea Coralloid Root

In this study, coralloid root samples from Cycas fairlakea in Shenzhen Fairy Lake Botanical Garden were collected and the symbiotic cyanobacteria were isolated and identified by morphological and molecular biological metheds as Nostoc sp. Isolates collected from the green rings in the transverse sections of coralloid roots of Cycas fairylakea were cultured with normal and nitrogen-free BG-11 medium. Bacterial community composition grown in Nostoc cultures were investigated with metagenomic sequencing method using Illumina MiSeq 16 S rRNA gene amplicons. The results showed similar dominant bacterial sequences in cultures with and wihout nitrogen, while a couple of differences existed. For example, high portions of Rhizobium and Bradyrhizobium were detected in nitrogen-free cultures, which indicated that besides cyanobacteria, other nitrogen-fixing bacteria may also provide nitrogen for cycads. This is the first metagenomic study of bacterial community associated with symbiotic cyanobacteria within cycad coralloid roots.In this study, liquid chromatography tandem mass spectrometry(LC-MS/MS) method was applied to detect the possible BMAA and its structure isomer DAB in the Nostoc sp. cultures isolated from Cycas fairlakea. The retention time of BMAA?DAB were relatively stable within standards and spiked samples, with BMAA eluted earlier than DAB. BMAA and DAB also showed similar ion mass spectra, while BMAA has m/z 118.8 fragment ions that DAB does not have. More fragment ions were found between m/z 90 to m/z 125 in the standards of BMAA than in DAB. For all the tested culture samples, free and protein-bound BMAA were detected in samples from nitrogen-free cultures, while in samples from normal cultures, free BMAA and protein-bound DAB were detected.In this study, a symbiotic cyanobacterium in Cycas fairylakea coralloid root were isolated and identified as Nostoc sp. For the first time, the bacterial community compositon associated with this symbiotic cyanobacterium was investigated through metagenomic methods. Moreover, neurotoxic amino acids BMAA and DAB were detected in extracts of this Nostoc sp. culture. As Nostoc sp. might also occur in freshwater ecosystems, it is necessary to develop standardized testing method and environment safety thresholds of these two toxins. Study of the chronic toxicity of BMAA and DAB, their toxic mechanism and their environmental behavior are also needed.