Isolation And Identification Of Secondary Metabolites From Photorhabdus Temperata SN35 And Their Antifungal Activities Against Phytopathogenic Fungi

Photohabdus temperata is a symbiotic bacterium parasitic in the intestine of Heterorhabditis.Photohabdus temperata are carried into insects by nematodes and multiply in large numbers and kill their hosts.At present,stilbene compounds isolated from secondary metabolites of Photohabdus temperata have many biological activities.In order to promote the development and application value of isopropylstilbene in the field of agricultural antibiotics,nine compounds were isolated and purified from the crude extract of the fermentation broth of Photohabdus temperata SN35 by solid phase extraction,column chromatography and high performance liquid chromatography.The structures of these nine active compounds were determined by NMR and MS spectroscopy and related literature research,and their biological activities were preliminarily studied.Finally,Plackett-Burman test and response surface methodology were used to optimize the shake flask fermentation conditions of Photohabdus temperata SN35.The main results are as follows:1.Separation,purification and identification of secondary metabolites of SN35 strain: A large amount of liquid fermentation was carried out on Photohabdus temperata SN35 to obtain 48 L fermentation liquid.A total of 12.90 g crude extract was obtained by solid-phase adsorption of macroporous resin and extraction of dichloromethane.Nine compounds were isolated and purified from the crude extract of the fermentation broth of Photohabdus temperata SN35 by solid phase extraction,column chromatography and high performance liquid chromatography.The structures of nine active compounds were determined by NMR and MS spectroscopy and related literature.They were temperatacinol A(1),temperatacinol B(2),temperatacinol C(3),temperatacinol D(4),3-hydroxy-2-isopropyl-5-Phenethylphenyl carbamate(5),2-(1-hydroxy propan-2-yl)-5-[2-phenylethyl]benzene-1,3-diol(6),2-(1-hydroxypropan-2-yl)-5-[(E)-2-phenylethenyl]benzene-1,3-diol(7),2-ethyl-5-[(E)-2-phenylethenyl]benzene-1,3-diol(8),and 2-isopropyl-5-[(E)-2-phenylethenyl]benzene-1,3-diol(9),named compounds 1-9.Compound 1 is a new compound.Compounds 2 and 3 are isolated and purified for the first time,which have been elucidated from metabolites of Photohabdus temperata by HPLC/MS analysis.Compounds 4-9 are known compounds.2.Antifungal activity of compounds 1-9: The biological activity of compounds 1-9 was determined by mycelial growth rate method and micro broth dilution method.The results showed that compound 9(isopropylstilbene)had a good inhibitory effect on the mycelial growth of E.turcicum,Botrytis cinerea,R.solani Kühn,with EC50 values of compound 9 were 10.5 ?g/m L,9.2 ?g/m L ? 9.8 ?g/m L,respectively.The inhibitory effect on P.grisea was slightly worse,and the EC50 value was 29.4 ?g/m L.Compounds 5,6 and 7 had a general inhibitory effects on E.turcicum,Botrytis cinerea,R.solani Kühn,but poor inhibitory effects on P.grisea.Compounds 1,2,3 and 4 had weak inhibitory effects on four pathogenic fungi,and their EC50 values were all greater than 50 ?g/m L.By comparing the chemical structures and corresponding biological activities of nine compounds,it can be inferred that linking styrene(phenylethyl)on C-5 is more helpful than linking alkyl chain to improve the antimicrobial activity of the compounds.3.The in vitro and in vivo antifungal activities against Botrytis cinerea of isopropylstilbene: Based on the results of screening in vitro biological activity of compounds,isopropylstilbene,a compound with better biological activity,was further studied.Firstly,the inhibition of isopropylstilbene on spore germination of Botrytis cinerea was determined by concave slide method.The results showed that the inhibition of isopropylstilbene on spore germination of Botrytis cinerea was 63.01%(100% chlorothalonil)at 10 ?g/m L.Then the control effect of isopropylstilbene on gray mould was tested by pot experiment of tomato.The results showed that the control effect of 5% isopropylstilbene on gray mould of tomato reached 72.12% at the concentration of 200 ?g/m L,which slightly lower than that of chlorothalonil(81.61%).Finally,isopropylstilbene carried out a simple indoor pesticide damage test.The results showed that 5% isopropylstilbene had no toxicity to cucumber and tomato seedlings at the concentration of 500 ?g/m L.4.Establishment of quantitative method for isopropylstilbene: A rapid method for the determination of isopropylstilbene in Photohabdus temperata fermentation broth by high performance liquid chromatography was established.The chromatographic conditions were as follows: chromatographic column: Agilent ZORBAX Eclipse XDB,4.60 × 250 mm,5 ?m;mobile phase: 68% methanol water 0.1% formic acid solution;injection volume: 10 ?L;flow rate: 1.0 m L/min,column temperature: 30-39?;detection wavelength: 247 nm.The precision of the method is 0.56-2.62%,the recovery is 93.71-109.62%,the linear equation is Y = 10069 X + 3.7629(R2 = 0.9974),the linear range is 0.002-5 g/L,the minimum detection limit is 2 mg/L calculated by 3 times SNR,and the quantitative limit is 10 mg/L calculated by 10 times SNR.5.Selection of dominant strains and optimization of fermentation conditions: The dominant strains were screened from 18 species of Photohabdus temperata in the laboratory stock by thin layer chromatography and high performance liquid chromatography.The results showed that the content of isopropylstilbene in fermentation broth of Photohabdus temperata SN35 was the highest.The shaking flask fermentation conditions of Photohabdus temperata SN35 were optimized by single factor test,Plackett-Burman test and response surface methodology.The results of single factor experiment showed that the yield of isopropylstilbene was greatly increased by adding leucine in M medium,and the yield of isopropylstilbene was increased when adding 3.01 g/L sodium chloride in M medium.The optimized M medium composition was 6.13 g/L of glucose,33.23 g/L of peptone,1.50 g/L of magnesium sulfate,2.46 g/L of ammonium sulfate,0.86 g/L of potassium dihydrogen phosphate,1.11 g/L of potassium dihydrogen phosphate,1.72 g/L of sodium sulfate,3.00 g/L of leucine,3.01 g/L of sodium chloride,1 L of distilled water and 7.2-7.6 of p H value.The yield of isopropylstilbene was 2.22 times higher than that before optimization.