Isolation And Identification Of Compounds Active On Nematodes From Pseudomonas Putida MCCC 1A00316 And The Nematicidal Mechanism Of Its Metabolites 2-undecanone Against Caenorhabditis Elegans

In recent years,Meloidogyne incognita has become the second largest agricultural hazard.Although the use of chemical control methods has obvious effects,it has brought many negative effects,such as environmental pollution,endangering human and animal safety,and developing the drug-resistant root-knot nematodes,etc,so there is an urgent need for environmentally friendly control methods.Among them,biological control methods have received more and more attention,and the research of development of biological control agents has become a research hotspot.In this study,we isolated and identified a variety of nematode-killing compounds from the secondary metabolites of Pseudomonas putida,MCCC 1A00316,and studied the target proteins and signaling pathways of nematicidal volatile 2-undecanone acting on the Caenorhabditis elegans.The main findings are as follows:(1)A new functional nematicidal activity compound was discovered through isolation and identificationFurther research on nematocidal secondary metabolites in the fermentation broth of P.putida 1A00316,and a new functional natural product with nematicidal activity was obtained.After identification the molecular composition and structure analysis,the compound was determined to be cyclodipeptide cyclo(L-Pro-L-Leu).And the cyclic dipeptide has strong nematicidal activity and a certain degree of egg hatching inhibition against Meloidogyne incognita.(2)Study on multiple modes of action of volatile compounds produced by bacterial fermentation brothIt was confirmed by three-compartment petri plate experiment that Pseudomonas putida 1A00316 can produce volatile nematicidal compounds.Eight volatile substances were identified,and the nematicidal activity,fumigation activity and chemotaxis activity of seven compounds(including dimethyl-disulfide,2-nonanone,2-undecanone,2-octanone,(Z)-Hexen-1-ol acetate,1-(ethenyloxy)-octadecane,and 1-undecene)against M.incognita were studied.We found that 5 volatile compounds other than 1-undecene and 1-(ethenyloxy)-octadecane have strong nematicidal activity against J2 larvae and inhibition activity against egg hatching in the direct contact way,and only 2-undecanone had fumigation activity against the J2 larvae of M.incognita.In addition,except for the volatile compound 2-octanone,the other 6 volatile substances show an inhibitory activityon egg hatching.The results of chemotaxis experiments show that these 7 volatile substances are effective against M.incognita and all of them repelled M.incognita.Based on the analysis of the above data,it can be seen that the 2-undecanone exhibits a variety of action modes of strong contact killing,fumigating activity and avoiding activity.Then the nematicidal mechanism of action was further studied.(3)Identification of target protein of 2-undecanone acting nematodesWe used the drug affinity responsive target stability method to look for the target proteins of 2-undecanone acting on C.elegans.And two potential target proteins of2-undecanone acting on C.elegans,Hsp 70 A and V-ATPase A,were identified.2-Undecanone had strong binding activity to Hsp 70 A and V-ATPase A with dissociation constants of 117.33 ± 16.26 n M and 301.67 ± 43.92 n M by using micro Scale thermophoresis method,respectively.By constructing a plasmid targeting RNA interference of the target proteins,it was found that the mortality of nematodes after interference of genes hsp-1,vha-13 and dual genes hsp-1 / vha-13 were significantly lower than those of the control group,and the lysosome did not rupture in a wide range,so it also showed that Hsp 70 A and V-ATPase A were target proteins of 2-undecanone acting on C.elegans in vivo.(4)Elucidates the signal pathway of 2-undecanone acting on C.elegansTranscriptome analysis has preliminarily determined that 2-undecanone causes cell necrosis by inducing lysosomal rupture in C.elegans.2-Undecanone enters the cell membrane of C.elegans as a small molecule.Then it activates calmodulin ITR-1 or CLP-1 to significantly increase the intracellular calcium ion concentration,and high concentration of calcium ion activates calpain TRA-3,leading to lysosomal rupture and cathepsins release.We ask what is the ruptured mechanism of lysosomal membrane? On the one hand,2-undecanone binds to the target protein Hsp 70 A in the lysosome,which significantly reduces the acid sphingomyelinase 1 and 2 activity,accumulated the sphingomyelin and decreased the ceramide level,and that makes lysosomal membrane unstable,and the lysosomal permeability gradually increases and even membrane rupture occours.On the other hand,2-undecanone influences the V-ATPase A activity and interferes with the normal function by binding to protein V-ATPase A in the lysosome,which weakens its acidification ability and aggregats a large number of broken tissues and proteins,and that makes lysosomal function abnormal.The combined effect of these twoaspects ruptured the lysosome of C.elegans,and released internal cathepsins ASP-3,ASP-4 and other hydrolases,causing cell necrosis and eventually nematode death.In this study,based on the preliminary work of MCCC 1A00316,a number of active compounds with multi-action modes against M.incognita were further discovered,such as cyclo(L-Pro-L-Leu)and 7 volatile substances.A systematic study was conducted on the nematicidal molecular mechanism of one of the prominent volatile compounds,2-undecanone,and two target proteins were found and verified: Hsp 70 A and V-ATPase A,revealing a new necrosis signaling pathway for C.elegans by targeting Hsp 70 A and V-ATPase A proteins.