Study On The Diversity And Bioactivity Of Endophytic Fungi In Baishouwu

Objectives: To analyze the diversity and distribution of population structure of endophytic fungi of Baishouwu,and screen the active endophytic fungal strains with same cytotoxic activity as their host or promoting the growth of the plant,it is hoped to explore the interaction between plants and endophytes and its cultivation and application,as well as to provide a theoretical basis to search for new antitumor natural products in endophytes.Methods:To characterize the diversity and population structure among different types of plant organs(roots,stems,leaves),species(C.bungei,C.auriculatum,C.wilfordii)and localities(Jinan,Linyi,Taian)of endophytic fungal of Baishouwu by a High-throughput18 S r DNA gene Illumina Mi Seq sequencing technique;The endophytic fungi were isolated and identified from the roots,stems and leaves of C.bungei by traditional culture method with molecular identification and biodiversity analysis.The cytotoxic activity of the endophytic fungi of C.bungei was detected by MTT method in vitro.The endophytic fungi of C.bungei were co-cultured with A.thaliana to screen the endophytic fungi which could promote the growth of A.thaliana.Results:(1)The results performed that the main factor influencing the distribution and communities of endophytes was a type of plant organs,rather than the species and localities by using a High-throughput 18 S r DNA gene Illumina Mi Seq sequencing approach.This indicates that the colonization of endophytic fungi in plants is tissue-specific.Moreover,Rank abundance curves demonstrated that the richness and evenness of the roots of endophytes of C.bungei from Jinan(JTG)were the highest,while the evenness of the leaves of endophytes of C.bungei from Jinan(JTY)was the lowest,the richness of the stems of endophytes of C.bungei from Jinan(JTJ)was the lowest.Furthermore,three localities samples had relatively similar fungal communities while the endophytic fungal communities of three different species and three different tissues were significantly different,as indicated by PCA and UPGMA analysis.The Fusarium,Exophiala,Plectosphaerella,Botryosphaeria,and Filobasidium were the predominant fungal genera in the seven samples.(2)It was indicated that 90 strains of endophytic fungi were isolated and identified from roots,stems and leaves of C.bungei by traditional isolation and culture method,and the results showed that Fusarium was the dominant genus in the root and Alternaria was the dominant genus in the stems and leaves.Differences between traditional isolation culture method and high-throughput sequencing:at the level of species,the endophytic fungi isolated and identified by traditional analytical culture method are less than 1/30 of the number of OTU detected by high-throughput sequencing;At the level of genera,18 genera were isolated and identified by traditional isolation culture,but only 4 genera were common among the first 50 genera obtained by high-throughput sequencing analysis,namely Fusarium,Colletotrichum,Botryosphaeria and Aspergillus.(3)The anti-tumor activity of endophytic fungi was detected by MTT method.Further studies showed that 13 endophytic fungi of C.bungei had good anti-tumor activity in vitro,accounting for 14.4% of the total.Among them,A.tenuissima LTJ2 and A.alternata LTJ6 of stems had significant anti-tumor activity.(4)It was found that F.nematophilum LTG5 and Ascomycota sp.JG12 could significantly promote the growth and lateral root development of A.thaliana.Conclusions: The study first reported that the main factor influenced the distribution and communities of endophytes was a type of plant organs,rather than the species and localities by using a High-throughput sequencing approach.This indicates that the colonization of endophytic fungi in plants is tissue-specific;The endophytic fungal strains of 90 kinds of morphology were isolated by traditional isolation culture method;Thirteen strains of endophytic fungi of C.bungei with anti-tumor activity were screened in vitro,accounting for 14.4% of the total;Among them,A.tenuissima LTJ2 and A.alternata LTJ6 of stems of Alternaria genus have the most significant anti-tumor activity and are worth further study;It was shown that F.nematophilum LTG5 and Ascomycota sp.JG12 could significantly promote the growth of A.thaliana.The results showed that endophytic fungi of C.bungei could promote the growth of plants.It was hopeful that the above studies could provide a theoretical basis for better understanding the diversity of endophytic fungi of Baishouwu and improving the development and utilization of the medicinal materials and their endophytes.