The Study Of Diversity Analysis And Bioactive Constituents From Ceriops Tagal And It's Endophytic Fungi

The habitats of mangrove plants are often referred as a special group from marine life and composed of a diverse salt-tolerant plants.Mangrove endophytic fungi are a relatively underappreciated reservoir of bioresources,which comprise the second largest ecological group of marine fungi.The specialized ecological niche renders them capable of developing unique biosynthetic pathways and producing impressive numbers of metabolites for the purposes of ecological adaptability.The plant Ceriops tagal is a typical true mangrove species,historically used as folk medicinal plant for the treatment of various ailments,which were demonstrated to be an important and well-known traditional herbal.However,C.tagal endophytic fungi are rarely studied.In order to find new biologically active secondary metabolites that were indispensable for pharmaceutical or agrochemical applications,we investigated endophytic fungi diversity of C.tagal collected from three localities and five different tissues,chemistry and bioactivity of metabolites of biological active strain Cytospora sp.A total number of 408 isolates were recovered from 975 tissues of C.tagal,which 164 taxa were identified as 45 species belonging to 23 genera of 10 orders by combining morphology with molecular biology.Of which,97.8%are in phylum Ascomycota,2.2%are in Basidiomycota.The colonization and isolation frequencies of the fungal endophytes from different tissues showed significant differences,in the following decreasing order:flowers>hypocotyls>twigs>roots>leaves(location A),flower>twigs>roots(location B)and fruits>twigs>roots>leaves(location C).The Shannon-Wiener diversity index(H')and Shannon evenness(E)indicated that the endophytic fungi diversity of Ceriops tagal are closely related to host habitats,which were in the order of location A(salinity=19‰)>location C(salinity=16‰)>location B(salinity=11‰),which indicated that the fungal diversity might probably in proportion to salinity.The endophyte assemblages were influenced by location and tissues.In addition,the distributions of dominant fungi at different taxonomic levels including orders,genera and species levels were greatly influenced by location and tissue.Xylariales is demonstrated to be the dominant orders in location A,Hypocreales is the dominant orders in location B and C,Pestalotiopsis is dominant genus in location A,Fusarium is dominant genus in location B and C,Neopestalotiopsis chrysea is dominant species in location A,Fusarium proliferatum and Fusarium equiseti is dominant species in location B and C respectively.Xylariales is the dominant order in twigs and roots,Botryosphaeriales,Hypocreales and Capnodiales is the dominant orders in leaves,flower,hypocotyls and fruits respectively;Neofusicoccum,Colletotrichum,Nodulisporium,Fusarium and Cladosporium is the dominant genus in roots,tiwgs,leaves,hypocotyls and fruits,flower,respectively.Fusarium proliferatum,Pestalotiopsis microspore,Colletotrichum gloeosporioides,Aspergillus sydowii are revovered from location A,B and C in common.Most of the predominant fungi showed spatial heterogeneity and tissue specificity or preference.Some rarely occured endophytes exhibited high tissue specificity,i.e.,Pestalotiopsis neglecta is only recovered from tiwgs,Fusarium oxysporum only recovered from hypocotyls and fruits related to location C,Phellinus noxius only recovered from leaves related to location B,Cytospora sp.recovered from twigs related to location A.Isolation and identification of compounds from mangrove plant Ceriops tagal and its endophytic Cytospora sp..The EtOH extracts from the hypocotyls and fruits of C.tagal were chromatographed on Silica gel,SephadexLH-20 and HPLC respectively to yield 23 compounds.14 compounds were elucidated by means of spectrospcopicm methods including H-NMR?13C-NMR?DEPT-135?1H-1HCOSY?HSQC?HMBC?ESI-MS?EI-MS as well as chemical method,which contains eleven triterpenoids(compounds 1-3,5-11),one sesquiterpenes(compounds 4)and two steroids(compounds 12 and 13).Sesquiterpenes were isolated from Ceriops tagal for the first time.Screening endophytic fungi with biological activities and optimizating fermentation conditions by antimicrobial activities models methods.And selected the target strains Cytospora sp.(MG948459)combined chemical screening.The EtOAc extracts of Cytospora sp.were chromatographed over Silica gel,SephadexLH-20 and HPLC respectively to yield 28 compounds.18 compounds were elucidated by including ESI-MS,HR-ESI-MS,1D-NMR 1H NMR,13C NMR,DEPT)and 2D-NMR(,H-1H COSY,HSQC,HMBC)as well as chemical method,which includes three sesquiterpenes(C1?C13?C14),five phenols(C6?C10?C11?C12?C15),six steroids(C5?C7?C8?C9?C16?C17),one pyrone(C3),one benzofuranone(C4),two isocoumarin(C2?C18).Compound C1 is a new sesquiterpene,compound C2 is a new pyrone,compound C3 is a new sesquiterpenes.C13 and C14 is a reverse cis isomers.All compounds from Ceriops tagal and its endophytic fungi Cytospora sp.were evaluated for their antimicrobial and cytotoxic activities.Antimicrobial assay were conducted using E.coli,C.Albicans,MRS A,P.aeruginosa,C.gloeosporioides,M.oryzae,and B.subtilis,HepG2 cells was selected for cytotoxicity evaluation.The results indicated 50%of the isolated compounds form Ceriops tagal(13,3,6,7,11)and 73%compounds from Cytospora sp.(C6,C7,C9,C1,C2,C4,C12,C13,C14,C15,C16)showed strong antimicrobial activities.Compounds Cll showed strong antiotumor activities,the IC50 for HepG2 was 3.50±0.68 ?g/mL.