Research On Key Metabolites During Parasitism Of Sclerotinia Sclerotiorum By Coniothrium Minitans Based On Metabonomics

Disease caused by Sclerotinia sclerotiorum is a worldwide distributed important crop disease,and Coniothyrium minitans is a mycoparasiticfungus on S.sclerotiorum.Up till now,most of the studies about the interactions between C.sclerotiorum and S.sclerotiorum are obtained from the gene and protein level,and few studies have been conducted from the metabolic level.In order to study the key metabolites in the parasitic process,LC-MS-based metabolomic analysis was used in this study to figure out the metabolic effects of NOX related genes on C.minitans,to conduct the pathway enrichment analysis of those metabolites which change during the parasitic process,and to screen out the key metabolites closely related to the parasitism,their biological functions are verified as well.The main results obtained are as follows:1 Metabolic characteristics of non-parasitic C.minitans.Through the metabolites comparison of the mutant strain Qnoxl-6 with the wild strain zs-1,it was found that after knocking out the genes associated with NADPH oxidase,a large number of metabolites of C.minitans were significantly up-regulated.By collecting samples at different culture times and comparing the metabolites which have changed significantly,14 overlapped metabolites were obtained.Mostly are acids and glucosides.Notably,7 of 14 metabolites only occured in the mutant strains,including coniferin(antioxidant),3 carboxylic acids,1 phenol,1 steroidal saponin and 1 other substance.These results suggest that knocking out NOX-related genes,in addition to triggering the metabolism of antioxidant compounds in the mutant,will also lead to the metabolism of other new secondary metabolites.These metabolites are considered to be associated with cell differentiation and conidia formation of C.minitans.2 Screening of the key metabolites during the parasitic process.Compared the co-culture of C.minitans and S.sclerotiorum with the mono-culture of the two strains separately,the results of metabolic total ion chromatograms showed that C.minitans had significant metabolic disturbances after exposure to S.sclerotiorum.52 overlapped matabolites in the two groups were screened,most of which were acids and glycosides.Targeted metabolic pathway analysis results showed up to 10 different metabolic pathways were enriched,including:a significant enrichment of the amino acid-related pathways,glycerol phospholipid metabolism and glutathione pathways.It indicates an avigorous life activities of fungi during the process of parasitism,and the parasitic processes involve the production of metabolites related to many complex life activities;Besides,the parasitism involves cell apoptosis in addition to the conduction of complex signaling molecules and the metabolise of glycerophospholipids were used to the mutual recognition of each other,moreover,phospholipids may take participate in the defense of parasitism.A non-parasitic experiment(mutant strain Qnoxl-6 co-culture with S.sclerotiorum)was conducted to further narrow the range of metabolites closely related to the parasitic behavior.Through compared screening of multi-component,and perturbation analysis of the screened metabolites,biological function analysis as well,it was preliminarily determined that 2-Phenylacetamide and Sinapic acid are key substances of parastic.3 Research on the biological function of parasitic key metabolites.Additive experiments of sinapic acid(S.A.)as an external source showed:S.A.has little effect on the growth of C.minitans;however,S.A.significantly inhibits the growth of S.sclerotiorum and resulted in an abnormal growth.The metabolites pathway enrichment analysis shows that the addition of low concentrations of S.A.results in the abnormal oxidation and energy metabolism of S.sclerotiorum,which leads to a significant up-regulation of NAD salvage pathways.High concentration of S.A.inhibits the phospholipid anabolic pathway of Sclerotinia sclerotiorum.It is concluded that S.A.affects the biological function of S.sclerotiorum's cell membrane,leading to its slow growth and abnormal growth.The experiment of S.A.addition on the co-culture process showed that the addition of S.A.caused a significant up-regulate in the number of acids,glucosides,phenols and amides compared to the non-addition group.It indicates that the addition of S.A,has an enhanced effect on the changed metabolites caused by co-culture.In addition,the results of pathway enrichment analysis indicate that the addition of S.A.sinapinic acid will affect the energy metabolism,cell recognition and the conduction of signal molecules of C.minitans when exposed to S.sclerotiorum.It further proved that S.A.is a key metabolite in the process of C.minitans exposure to S.sclerotiorum.