Isolation,Structure Identification,and Nematicidal Mechanism Of Active Compounds From Paenibacillus Polymyxa KM2501-1

Meloidogyne incongnita is one kind of soil transmission plant parasitic nematode that caused very serious losses to agricultural production worldwide.Because of the characteristics of easy culture,short life span and clear genetic background,Caenorhabditis elegans is often used as a model organism in molecular mechanism research.In the previous study,we found that Paenibacillus polymyxa KM2501-1 has very good contact nematicidal and fumigant activity against M.incognita in vitro,and shows pretty good control effect on M.incognita in greenhouse experiment.In present study,we purified and structural identified the nematicidal compounds from P.polymyxa KM2501-1 against M.incongnita by the method of silica-gel column chromatography,high performance liquid chromatography,Liquid chromatography-mass spectrometry,nuclear magnetic resonance,solid-phase micro-extraction,and gas chromatography-mass spectrometry;besides,researching on the contact nematicidal and fumigant activity against M.incongnita,and the activity influence the chemotaxis of M.incongnita of volatile organic compounds from P.polymyxa KM2501-1;what’s more,researching on the mechanism of nematicidal volatile organic compounds furfural acetone produced by P.polymyxa KM2501-1 attracts and kills C.elegans by the methods of target prediction,comparative transcriptomics,biochemical staining,and nematode mutants activity detection.The primary results are listed as follows:（1）Purification,structural identification and nematicidal activity verification of non-volatile nematicidal small molecule substance from P.polymyxa KM2501-1Fermented supernatant fluid of P.polymyxa KM2501-1 was extracted by methonal and obtaining the crude active compounds,the pure namticidal compounds was obtained by 3 times of positive silica-gel column chromatography and 2 times of semi-prepative high performance liquid chromatography.Structural identification of the pure nematicidal compound by liquid chromatography-mass spectrometry and nuclear magnetic resonance.The molecular weight of the active compound is 244 Da,the molecular formula is C₁₄H₁₆N₂O₂,and the chemical name is cyclo（Pro-Phe）.Then Verification of the nematicidal activity of cyclo（Pro-Phe）and proving its nematicidal activity against M.incongnita.（2）Structural identification and activity exploration of the volatile organic compounds from P.polymyxa KM2501-1Eleven volatile organic compounds were identified from P.polymyxa KM2501-1 by the method of solid-phase micro-extraction and gas chromatography-mass spectrometry.Of which 8 including furfural acetone,2-undecanol,4-acetylbenzoic acid,2-decanol,2-nonanol,2-undecanone,2-decanone,and 2-nonanone had contact nematicidal activity;6 including furfural acetone,2-undecanol,2-decanol,2-nonanol,2-undecanone,and2-decanone had fumigant activity;acetone,2-decanol,and furfural acetone acted as attractants to M.incognita;whereas 2-undecanone acted as a repellent;4-acetylbenzoic acid showed as an attractant at low concentration（1-10 mg/L）and as a repellent at high concentration（100-10000 mg/L）towards to M.incognita.According to the activity of the volatile organic compounds towards to M.incognita,some comprehensive strategies including（?）honey-trap’against M.incognita were proposed.P.polymyxa KM2501-1 and the volatile organic compounds produced by it disrupted the intestine and pharynx of M.incognita.（3）The mechanism of furfural acetone attracts and kills C.elegansFurfural acetone is a volatile organic compound produced by P.polymyxa KM2501-1,having both contact nematicidal activity and fumigant activity against M.incognita,and also function as a stable attractant.We found that furfural acetone can attract nematodes to nearby and then kill it by fumigant or contact in a 90 mm plate;the mode of“honey-trap”that furfural acetone against M.incognita was proposed.Through the method of C.elegans mutants activity detection,the olfactory signal transduction pathway of C.elegans recognise fufural acetone was described,the process of C.elegans recognise fufural acetone is mediated by both of the olfactory neurons AWA and AWC.Prediction of furfural acetone kill C.elegans by apoptosis and necrosis pathway by methods of id Target,pharm Mapper and comparative transcriptomics.And verification of furfural acetone kill C.elegans by apoptosis and necrosis pathway by propidium iodide staining,acridine orange staining,and C.elegans mutants activity detection.Determining the signal transduction pathway of necrosis and apoptosis that furfural acetone kill C.elegans.Illustrating the signal transduction pathway of furfural acetone attract and kill C.elegans by the method of C.elegans mutants activity detection.C.elegans recognise fufural acetone and induced attractant activity by both of the olfactory neurons AWA and AWC.And furfural acetone kill C.elegans by apoptosis and necrosis pathway.