Diversity Of Endophytes From The Halophyte Messerschmidia Sibirica And Study On The Secondary Metabolites Of One Endophytic Fungus Alternaria Sp. P8

It was well known that endophytes could improve the abilities of stress-resistance,diease-resistance,and promote the nutrition absorption of host plants.The secondary metabolites of endophytes play an important role in agricultural,industrial and medical applications.However,most studies of endophytes have focused on glycophytes or crops but few were for that in the extreme environment.Messerschmidia sibirica,as a pioneer species in costal salt marshes,is a salt-secreting halophyte in northern coastal areas of China.They play a crucial role in protecting ecological environment,and have important values in medicine,feeding,and ornamental gardening.Therefore,the aims of this study were to investigate endophytic bacteria community associated to M.sibirica,screen beneficial strains with bioactivity for agriculture,and identify the active component produced by endophytic strains.1.The community structure of endophyts associated to M.sibirica was analyzed with cultivation and free of cultivation,and strategies which halophytes have evolved to survive in coastal environment(such as high salinity and tidal changes)were discussed.A total of 198 endophytic bacteria and 82 fungi were isolated from 450 segments of healthy leaf,stem,and root tissues of M.sibirica.Phylogenetic analysis of the 16 S and ITS rRNA gene sequence revealed the diverse of endophytes of M.sibirica,with 41 different genera for bacteria and 31 genera for fungi.We collected leaf,stem,root,rhizosphere and bulk control soil samples,and sequenced the V5–V7 region of the bacterial 16 S rRNA gene using the Illumina HiSeq platform to identify bacterial communities originating from different plant habitats.We found that the species richness and diversity of rhizosphere bacteria exhibited larger amount than those of endophytic bacteria,but lower than those of the bulk control soil.In total,37 phyla and 438 genera were identified.Microbial-diversity analysis showed that Proteobacteria and Actinobacteria were the dominant phyla and that Pseudomonas,Bacillus,Sphingomonas,Streptomyces,Microbacterium,Rhizobium,and Nocardioides were the dominant genera.In a word,when the relative of endophytic bacteria is larger than 1%,the dominant bacteria isolated by both methods have great difference,and when is lower than 0.1%,these also show significant difference.These results illustrate that the species and structure of endophytic bacteria are associated with not only the dominant bacteria,but also lower abundance bacteria.2.Twenty-seven endophytic bacteria showed antimicrobial activity against one or more pathogens.Among these organisms,Bacillus toyonensis JM2 and B.subtilis BS13 exhibited a broad range of antimicrobial activities against all phytopathogens tested(such as,Phytophthora nicotianae,Pseudomonas syringae pv.lachrymans).According to morphology of endophytic fungi,30 strains were selected to be fermentated.Evaluation of the anti-phytopathogenic bacterial and fungal activities showed that 16 fungal extracts revealed significant antibacterial activity.Fusarium oxysporum P14 displayed the most potent antibacterial activity against Acidovorax avenae,Erwinia carotovora,P.syringae pv.lachrymans and Clavzbacter michiganensis,as strong as the positive control streptomycin.It was noteworthy that this was the first report on the application of endophytic strains of M.sibirica in antagonizing plant pathogens.3.According to chemical and biological screening,Alternaria sp.P8 was selected for further chemical investigation,and nine compounds(T1-T9)were obtained,including a new one(T1).These compounds belonged to three types,including benzofurans(T1-T5),perylenequinones(T6-T8),and steroid(T9).Evaluation of the anti-phytopathogenic bacterial and fungal activities,as well as phytotoxicity of these nine compounds revealed that most of them exhibited different levels of bioactivities,especially for alterperylenol.In addition,T6,T7,and T8 also showed significant inhibition aginst the germination of seeds of Amaranthus and Lettuce,which has the same efficacy as glyphosate.It was notable that the new compound also displayed potent phytotoxicity against the germination of seeds of Amaranthus and Lettuce.The diversity and community of endophytic microbes in M.sibirica were analyzed systematically for the first time in this paper.With pharmacological activity,we discussed the secondary metabolitic bioactivities and explored compound values in agricultural pathogens and weeding,which widened the application range of natural products.