| The endophytes of medicinal plants participate in biochemical pathways and produce analogous or novel bioactive compounds and become an important source of new medicines. The purpose of this study was to investigate the diversity and biological activity (antimicrobial activity, antitumor activity, antioxidant activity) of endophytes colonized in Lycium barbarum L. planted in Ningxia, aim to screening for pharmaceutically active endophytes. The study will lay the foundation for exploring the synthesis mechanism of active substance of endophytes, and help to reveal its coevolution relationship with host plants, the correlation between endophytes and traditional Chinese medicinal plants. The main research results are as follow:(1) 81 endophytes were isolated from different organs and tissues of Lycium barbarum by tablet coating method, of thoses include 39 bacteria,31 fungi, and 11 actinomycetes. The 16S rRNA and rDNA-ITS gene were sequenced to clarify the diversity of endophytes. The result showed that endophytic bacteria could be phylogenetically classified into 7 families and 11 genera, Bacillus was a predominant genus and was isolated in all tested tissues; endophytic fungi could be classified into 1 familiy and 3 genera, Aspergillus spp. was a predominant genus; endophytic actinomycetes could be classified into 3 families and 4 genera, Streptomyces spp. was a predominant genus. The quantity of endophytic bacteria in the different tissues were roots>leaves> flowers> fruits; the diversity of endophtes were flowers> roots> leaves> fruits.(2) Escherichia coli, Staphylococcus aureus, Colletotrichum nigrum, Exserohilum turcica and Fusarium graminearum as indicator pathogens, the agar diffusion were used to determine the antimicrobial activity of 46 representative endophytes of Lycium barbarum. The result showed that 29 strains had inhibition activity against at least one of the five tested pathogens, among which Bacillus strains EBR2, EBR7, EBL3 and Brevundimonas EBR3 displayed the strongest inhibition activity against the Colletotrichum nigrum and Exserohilum turcica. The majority of isolates showed weak inhibition effect against E. coli and S.aureus. All of endophytic fungi isolates had no antimicrobial activity. Mycobacterium strain EAF8 showed stronger inhibition activity against S. aureus.(3) The hydroxyl radical scavenging activities and the total antioxidant capacities of endophytes were determined by Fenton reaction and test kits respectively. The result showed that there are 26 endophytes have antioxidant activity and showed distinct difference. The antioxidant activity of endophytic fungi was stronger than endophytic bacteria and actinomycetes. Aspergillus strains EFL1(IC50 was 0.42 mg·L-1), EFL9(113.63±1.021)U·mL-1 and EFL6((188.5±0.549) U·mL-1,and IC50 is 0.3 mg·L-1) displayed the strongest antioxidant activity.(4) The metabolite of 36 representative endophytes were used to determine the antitumor activity in vitro with the methods of MTT (Methyl Thiazolyl tetrazolium) and CCK-8 (Cell Counting Kit-8). The results indicated that 32 strains could inhibit one of the tested tumor cells growth, as well as 17 strains could inhibit the growth of A549 and HeLa. All of endophytic fungi have significantly antitumor activities. Aspergillus strains EFL1 had strongest toxicity to tumor cells and the activities IC50 were 0.22×102 mg/L and 0.28×102 mg/L to different tumor cells.Endophytes of Lycium barbarum are genetically diverse and most of them have a strong antimicrobial, antioxidant and antitumor activity. |
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