Diversity Of Endophytes From Spiranthes Sinensis And The Antifungal Activity Of One Endophytic Bacterium

Spiranthes sinensis（Pers.）Ames is an important Chinese medicine and an ecological indicator plant,which has high medicinal and ecological values.However,the product of Spiranthes sinensis has declined sharply in recent years due to human overexploitation and environmental pollution.Plant endophytes are potential resources because of their metabolites,which are widely used in medical treatment,agriculture and industry.This study focused on the diversity of endophytes from Spiranthes sinensis（Pers.）Ames,the antifungal activity of endophytes and the extraction of antifungal substances from a candidate strain to apply it in agriculture.The main results are as follows:A total of 263 endophytes were obtained from the tissues of root,stem,leave,and inflorescence of Spiranthes sinensis.Based on the morphological and phylogenetic approaches,endophytic fungi and endophytic bacteria were identified based on internal transcribed spacer r DNA regions（ITS）and 16S ribosomal RNA（16S r RNA）gene analyses,respectively.The results indicated that the endophytes were assigned to 25genera of fungi and 11 genera of bacteria.The dominant endophytic fungi were Fusarium（20%）,followed by Alternaria（8%）and Penicillium（8%）.The dominant endophytic bacteria were Burkholderia（19%）,Bacillus（18%）and Pseudomonas（17%）.The endophytic strains were evaluated against three major plant pathogens（Fusarium oxysporum,Botrytis cineria,Alternaria solani）by the plate confrontation tests.Among them,9 strains of fungi and 8 bacterial isolates showed broad spectrum antifungal activity and one bacterial strain YZU 173017 exhibited the best inhibitory activity against those three pathogens.The 16S rRNA gene and the whole genome of the strain YZU 173017 were sequenced and analyzed.Based on phylogenetic analyses of both sequences,the strain was identified as Bacillus velezensis.The whole genome size is 3940997 bp containing30 scaffolds and 46.414%GC nucleotides,coding 2512 protein sequences around 61.9%of whole genome and predicted 85 t RNA and 10 r RNA.The genes enriched in secondary metabolites synthesis,transport and metabolism according to GO,COG and KEEG databases.In the analysis of anti SMASH,19 gene clusters of secondary metabolites were obtained,which relevant metabolites including fengycin,bacillus aene,macrolactin,butirosin,bacilysin,bacillibactin,difficidin,surfactin,plipastatin,plantazolicin,kijanimicn,and so on.Among them,most gene clusters were related to antagonistic activity.The biological characteristics of the strain YZU 173017 indicated that it was a gram-positive bacterium producing protease,amylase,but no cellulose.It could form biofilm but not resistant to high salt,strong acid and base.In addition,the antifungal activity of fermentation broth showed stronger ability than the cell extracts.The fermentation broth exhibited resistance to high temperature and protease K,which can well inhibited the growth of nine agricultural pathogenic fungi resulting in the damage of mycelia expansion and deformation and the preventing of spore germination.Both lipopeptide and protein extracts of the fermentation broth showed antimicrobial activities,while protein extract indicated better action.The protein antagonistic substance was obtained by 80%（w/v）（NH₄）₂SO₄ saturation precipitation and purified by DEAE-Sephrose TM anion exchange.A relatively clear band with molecular mass of42k Da was observed after SDS-PAGE,which was predicted to be important antimicrobial proteins produced by the strain YZU 173017.