Isolation And Identification Of Secondary Metabolites From Xenorhabdus Bovienii SN52

The genera Photorhabdus and Xenorhabdus are entomopathogenic bacteria living in symbiotic mutualism with soil entomopathogenic nematodes (EPN) and are exclusively associated with the genera Heterorhabditis and Steinernema. Recent studies have shown that Xenorhabdus.spp as a source of secondary metabolites, it have developed low molecular weight toxins that are specifically active either against insects or against fungi or as a herbicide. It played a significant role in new sources of biopesticides. In resent years, the study of Xenorhabdus. spp mainly focus on the fermented liquid activity and insecticidal mechanism research rather than isolation identification and biological activity of the secondary metabolites study in our country. This article focus on the research of Xenorhabdus.spp. Mainly include:the isolation of entomopathogenic bacteria (ENB), the screening of biological EPB, the isolation identification and biological activity of the secondary metabolites. The main conclusions are as follows:1. Isolation and purification of EPB:collection 146 soils in Liaoning province, production methods for culturing EPB in insect hosts have been based on the White trap and NBTA medium. It got 43 EPB including 41 primary form and 2 secondary form.2. Screening of symbiotic bacteria with biological activity:The crude extractions of the 43 bacteria were got by using macroporous resin adsorption method and screened by using the HPLC detecting, which was subsequently obtained 9 different crude extract and named ET1, ET2, ET3, ET4, ET5, ET6, ET7, ET8, ET9. The bioactivity of the 9 crude extractions were determined by the plate confrontation method against Bipolaris sorokiniana, Botrytis cinerea, Magnaporthe oryzae, Setosphaeria turcica, Phytophthora capsici, Fusarium graminearum, Rhizoctonia solani. The 100 μg/mL concentration of inhibiting rate is used as a reference. The maximum inhibition rate for Botrytis cinerea is crude extract ET4, and it corresponds to SN52 of the strains.3. Identification of SN52 strain:The SN52 strain is identified by physiological and biochemical properties and 16S rRNA genes sequence analysis. The 16S rRNA genes of SN52 strain was amplified by PCR. The multi sequence homology was analyzed by clustal and Mega 6.0 software. The phylogenetic tree obtained by the neighbor-joining method showed that SN52 strain clustered together with Xenorhabdus bovienii, it can come up to 100%by comparing the 16S rRNA sequence of homology of the strain SN52. Together with the result of physiological and biochemical properties, SN52 strain should belong to the species of X. bovienii and named Xenorhabdus bovienii SN52 by this study.4. Isolation and purification of secondary metabolites from SN52:Bioassay-guided fractionation of secondary metabolites of Xenorhabdus bovienii SN52 resulted in the isolation of bioactive molecule. Six indole derivatives were isolated and purified from secondary metabolites of SN52 strain by silica gel column chromatography, Sephadex LH-20 chromatography and semi-preparative high-performance liquid chromatography. Their chemical structures were elucidated by spectroscopic technologies including nuclear magnetic resonance (NMR) and mass spectroscopy (MS), and were identified as xenocyloins A (M5), xenocyloins B (M6), xenocyloins C (M3), xenocyloins D (M4), xenocyloins E (M2) and xenocyloins F (M1), in which xenocyloins F (M1) was a new compound. 5. The bioactivity of the 6 indoles compound were determined by the plate confrontation method against Botrytis cinerea, chlorothalonil was positive control, in which the EC50 of these compound (from M1-M6) were 88.44 μg/mL100.94|μg/mL,43.90μg/mL,60.15 μg/mL, 21.78 μg/mL,34.43μg/mL, separately.