The Study Of Endophyte Diversity In The Gentiana Straminea Maxim.

Gentiana straminea Maxim.is a traditional Chinese medicine,belonging to Genus Gentiana,Family Gentianaceae.its main active substance is gentiopicroside,a iridoid glycoside,which has many medical functions.At present,the wild G.straminea Maxim.resources are scarce,so it is of great significance to seek its substitute resources.Endophytes can produce the same or similar active metabolites as the host through long-term coevolution with their host plants.Studying on the community structure of endophyte provides a theoretical basis for the protection and utilization of G.straminea Maxim.resources and the development of the endophytic resources.In this paper,the roots,stems,leaves and flowers were chose as experimental materials.The diversity of the cultured endophytic bacterium and fungi in the different tissue parts of G.straminea Maxim.were studied by traditional culture method.Meanwhile,the metabolites gentiopicroside from those endophytes were detected by HPLC.The abundance and community structure of the non-culturable endophytic bacterium and fungi were studied by q PCR and PCR-DGGE methods separately.Besides,The contents of gentiopicroside in different tissue parts were determined by HPLC,and the correlation between the diverisity of endophytes and gentiopicroside contents was analyzed.The results are as follows:(1)the gentiopicroside content of G.straminea Maxim.in different parts was roots>flowers>stems>leaves,the content in roots reaches(7.11±1.44)%and was significantly higher than those in other tissue parts.(2)The number of the cultured endophytic bacterium and fungi obtained from the root of G.straminea Maxim.was the highest,which were(8.77±0.71)×10~5 cfu/g and(2.00±1.05)×10~4 cfu/g respectively.Moreover,the number of the cultured endophytic bacterium and fungi in some G.straminea Maxim.were significantly positively correlated with the content of gentiopicroside.13 endophyties were isolated from G.straminea Maxim,belonging to 3 phyla,6 classes,8 orders,9 families,10 genera,including 10 strains of bacteria and 3 strains of fungi.Among them,the strains of bacteria QJ2(Duganella sp.),QJ6(Rahnella sp.),QJ10(Pseudomonas sp.)and strain of fungi QJ11(Aspergillus sp.)and QJ13(Aspergillus sp.)had very significant positive correlation with the gentiopicrin content(P<0.01),and the dominant strains included QJ2(Duganella sp.)、QJ3(Rahnella sp.)、QJ5(Pseudomonas sp.).Beisides,strain QJ3 and QJ9 may produce gentiopicroside,and they belonged to Xanthomonas and Pantoea respectively.(3)The abundance range of non-culturable endophytic bacterium and fungi in different tissue parts of G.straminea Maxim.was 1.82×10~5-8.55×10~5 copies/g dry weight and 5.57×10~4-1.63×10~5 copies/g dry weight respectively.The abundance of the non-culturable endophytic bacterium was highest in roots and lowest in flowers.The similarity of the non-culturable endophyte communities in G.straminea Maxim.was analyzed by UPGMA method.Non-culturable endophytic bacterium in different parts of G.straminea Maxim.had certain differences,while the difference between leaves and other parts was the largest.The difference of the non-culturable endophytic fungi in flowers was the largest.(4)The species number(S)and diversity indices including Shannon-Wiener index(H’),Simpson’s index(C)and Piolou’s evenness index(J)were analyzed.The species number and the Shannon-Wiener index of the non-culturable endophytic bacterium in roots and leaves of G.straminea Maxim.were higher than those in stem and flowers,and the differences among the Simpson’s indices or the evenness indices in roots,stems,leaves and flowers were not significant.Both of the species number and the Shannon-Wiener index of the non-culturable endophytic fungi were highest in the roots of G.straminea Maxim,but the differences among the Shannon-Wiener index or the species number of the non-culturable endophytic fungi in the roots,stems,leaves and flowers of G.straminea Maxim.were not obvious.Besides,the differences among the Simpson’s indices or the evenness indices in roots,stems,leaves and flowers were also not obvious.To sum up,the Shannon-Wiener index of the non-culturable endophyte was highest in the roots of G.straminea Maxim.,the non-culturable endophytes were evenly distributed in the different tissues parts of G.straminea Maxim.and the dominant groups of endophytes were not obvious.(5)The abundance of the non-culturable endophytic bacterium and fungi were positively correlated with gentiopicroside contents.The Shannon-Wiener index and the species richess of the non-culturable endophytic bacterium were positively correlated with gentiopicroside contents,and the Shannon-Wiener index and the species number of the non-culturable endophytic fungi were also positively correlated with gentiopicroside contents.This indicated that the diversity of endophyties in G.straminea Maxim.may be related to gentiopicroside.Conclusively,the genetic diversity of endophytes from G.straminea Maxim.were rich.The culturable study showed that endophytic bacterium and endophytic fungi in different tissue parts were different in the contents and the dominant groups.In this study,strains that may produce gentiopicroside were isolated,which were QJ3(Xanthomonas)and QJ9(Pantoea).The q PCR assay showed that the roots were richer in the non-culturable endophytes,especially the non-culturable endophytic bacterium.The results of PCR-DGGE showed that the Shannon-Wiener diversity index of the non-culturable endophytic bacterium was the largest,and the endophytic distribution among different tissues was relatively uniform and the dominant species were not prominent.Correlation analysis showed that the quantity of the culturable endophytes and the Shannon-Wiener diversity indices and the species number of the non-culturable endophytes in G.straminea Maxim.had a positive correlation with the contents of gentiopicroside.