The Research On The Secondary Metabolites From Xenorhabdus Nematophila SN313 And Their Inhibitive Activity Against Plant Pathogenic Fungi

Under natural circumstances,many organisms can cooperate with microorganisms and produce a group of chemicals which are capable of acting against insects,bacteria and viruses.These chemicals may precent their host from natural enemies such as parasites,parasitoids and pathogens.In the field of medical and agriculture,the metabolites produced by microbial symbionts are frequently used for the researches on antibiotics,antivirus drugs and microbial pesticides.The microbial symbionts in this paper are obtained from a certain symbiosis which is built by insects,the nematodes that invade into the insects and the microorganisms that live in the intestines of the nematodes.At the same time,the microbial symbionts are bacterial symbionts of entomopathogenic nematodes.After previously published papers were studied and pre-experiment was done,it was found that many metabolites showed inhibitive activity to the plant pathogenic fungi and these metabolites were produced by bacterial symbionts of entomopathogenic nematodes.To discover whether the metabolites that produced by these symbionts have beneficial effect on the prevention of agricultural plant pathogeny,we selected a dominant strain based on chemical and biological activities.Once the metabolites were obtained from the symbionts,they were isolated using column chromatography.Then high performance liquid chromatography(HPLC)was performed to purify the metabolites.After that,NMR was facilitated to determine the structures of the chemicals.Finally,96-well ELISA was performed and the growth rate of mycelium was measured to determine their inhibitive activity against bacteria.The main researches are followed:1.The culturing of bacterial symbionts of entomopathogenic nematodes and the selection of the dominant strain: the soil samples were mainly collected from Xingcheng,Tongliao,Zhaluteqi.The nematodes were induced into Galleria mellonella and they were cultured with NBTA.Fifty-two blue and green nascent strains were isolated.The crude extracts were obtained from these 52 strains using microbial fermentation.After that,the extracts were chemically selected by TLC and 11 strains showed obvious characters on the layer.The extracts from these 11 strains were chosen to be cultured with Botrytis cinerea and Phytophthora capsici.Therefore,the antimicrobial potential of these extracts could be monitored.Finally SN313 was selected to be the experiment strain.2.The identification of the target strain: Physiological and biochemical experiments were respectively performed to identify the target strain.Physiological and biochemical identification experiment was firstly performed on the representative bacteria and the reslt showed that this strain was gram-negative rod bacteria.In addition,it had active flagellum and showed negative potential on most bacteria in the physiological and biochemical experiments.Then 16 s r DNA method was performed to extract DNA from the target strain and the DNA sequence was analyzed.The result showed the length of the sequence was 1442 bp.After the sequence was uploaded to NCBI Gen Bank and the register number was MG385844.1.Once the phylogeny tree was built,it was found that the sequence was most closed to that from Xenorhabdus nematophila ATCC 19061T/FN667742.Based on the physiological and biochemical characters,the strain was finally identified as Xenorhabdus nematophila and named as SN313.3.The massive fermentation of the target strain and the activity selection of the crude extracts: 7.5 g crude extracts were obtained by microbial fermentation method,M media,XAD-16 macroporous resin and methanol.Then the growth rate of mycelium was measured to test the activity of the crude extracts on Botrytis cinerea,Phytophthora capsici,Thanatephorus cucumeris,Bipolaris sorokiniana,Sclerotinia sclerotiorum,Exserohilum turcicum,Pythium aphanidermatum and Fusarium graminearum.The result showed that the inhibitive activity of the crude extracts(100 μg/m L)against Phytophthora capsici(inhibitive rate = 88.70%)was higher than that against Thanatephorus cucumeris(inhibitive rate = 75.00%),Botrytis cinerea(inhibitive rate = 67.36%)and Bipolaris sorokiniana(inhibitive rate = 55.49%).Additionally,the inhibitive activity of the crude extracts against other four pathogens was not significant.Broth microdillution method was performed to measure the inhibitive activity of the crude extracts against Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Streptococcus pernyi.The result showed that the crude extracts only exhibited obvious inhibitive activity against Escherichia coli(MIC = 132.5 μg/m L)but exhibited insignificant activity against Staphylococcus aureus,Bacillus subtilis and Streptococcus pernyi(MIC > 250 μg/m L).4.Determining the active components in the ferment products of SN313 and the isolation and purification of the active components: the crude extracts were separated into 5 fractions(Fr.1-Fr.5)using silica gel column chromatography.Source tracking method was performed to compare different components’ activities against the representative of plant pathogenic fungi,Botrytis cinerea.The result showed that Fr.2 had the highest inhibitive activity against pathogens(inhibitive rate = 65.35%).So it was determined that Fr.2 was the active component.Column chromatography,TLC,HPLC and other chemical isolation techniques were performed to isolate and purify these active components.Finally 3 monomeric compounds were obtained.5.Identifying the structures of the active components in the ferment products of SN313: MS,NMR and previous researches helped to determine the structures of compounds 1,2 and 3.The three compounds were N-(2-hydroxyphenylacetyl)-tryptamine(1),phenazine-1-carboxylic acid(2)and cyclo(Pro-Trp)(3)respectively.Compound 1 was a newly discovered derivative of tryptamine.Compound 2 was a biopesticide that was already registered.6.Measuring the antimicrobial activity of the compounds: Based on the result of crude extract activity selection,Botrytis cinerea,Phytophthora capsici,Thanatephorus cucumeris and Bipolaris sorokiniana were chosen as the main targets in the antimicrobial activity experiment.Three compounds’ inhibitive activities against plant pathogenic fungi were monitored using 96-well ELISA.The result showed that compound 1 inhibited the growth of Phytophthora capsici and Botrytis cinerea(EC50 was 11.2 μg/m L and 28.94 μg/m L respectively).Compound 2 exhibited inhibitive potential against Botrytis cinerea,Phytophthora capsici and Bipolaris sorokiniana(EC50<40 μg/m L).Compound 3 showed inhibitive activity against Botrytis cinerea(EC50= 41.58 μg/m L).