Genetic Diversity Of Endophytic Fungi From Sophora Alopecuroides L. And Screening Its Strains Producing Quinolizidine Alkaloids

Endophytic fungi were widespread in the all kinds of terrestrial and aquatic plants which had currently been found. Its diversity was influenced by many factors. Different areas or site, climate conditions, and vegetation can affect the diversity of the endophytic fungi groups. Research showed that medicinal plants contain a large number of beneficial endophytic fungi which has close ecological relationship with the host. The secondary metabolites produced by the plant endophytic fungi had gotten more and more significant application in medicine, biology controls, and become the important direction in finding new active natural products. In this paper, we chose Sophora alopecuroides L. as the object, which is the important medicinal psammophyte in the Baijitan nature reserve of Lingwu, Ningxia. Endophytic fungi from S. alopecuroides L. were isolated, purification, morphological identification, ecological distribution analysis, molecular biology identification and genetic diversity analysis. The single alkali in total alkaloid of S.alopecuroides made optimization of detection system. Thus the strain of producing quinolizidine alkaloids was screened and its rDNA-ITS sequence was analyzed. The main conclusions were as follows:1. The213endophytic fungi were isolated from healthy S.alopecuroides in different soil and vegetation type of six sample regions in the Baijitan nature reserve of Lingwu, Ningxia. In the different organizations from the same plant, most number of endophytic fungi was in the roots, followed in the seed, in the leaf at least. The number of endophytic fungi from S.alopecuroides in light gray desert soil was more than in aeolian sandy soil. Morphological identification results show that the dominant genus of endophytic fungi were Fusarium, Alternaria and Phoma. Among of them, Fusarium had distribution in every region.2. The S.alopecuroides contained abundant species of endophytic fungi in the six sample regions of the Baijitan nature reserve of Lingwu, Ningxia. The213endophytic fungi were belonged to19genus and32species by analyzed of rDNA-ITS sequence. Among of them, the dominant species of endophytic fungi were Fusarium solani, Fusarium oxysporum, Alternaria alternata, Alternaria tenuissima and Phoma herbarum.3. To establish the system optimization of TLC and HPLC methods for simultaneous separation and determination of alkaloids of Sophora alopecuroides L. Thin layer chromatography (developing solvent was chloroform-methanol-ammomia=8:2:0.1). The chromatographic column of high performance liquid chromatography included Ultra IITM C18(5μm×250mm×4.6mm) and mobile phase consisting of a mixture of0.01mol/L phosphate buffer-methanol (45:55). The detection wavelength was216nm. The flow rate was1.0mL/min. The method has a good linearity and correlation coefficient: the sophoridine and matrine were200-900μg/mL. The mean recoveries (n=9) were99.47%and99.82%respectively.4. The one strain of producing quinolizidine alkaloids was XKYKDF40, which was preliminary screened by TLC and the mycelium extract of strains by HPLC in59representative strains of endophytic fungi of S.alopecuroides. The content of oxymatrine in the fermented liquid of XKYKDF40 was145.73μg/mL. By the morphology and molecular biology identification, strain XKYKDF40was one species of Exophiala genus. The highest similarity was to98%with endophytic fungi from one species of being called fungal ant.