The Effects Of Ginsenosides On The Cylindrocarpon Destructans Pathosystem And Its Mechanism

Plant-pathogen co-evolution has made the interaction between plants and fungi very complicated.Pathogens infect the plant to obtain nutrients and produce harmful substances,and plants defend themselves by producing large amounts of low-molecular-mass natural products(i.e.secondary metabolites).Ginsenosides are a class of secondary metabolites produced by Panax,including P.ginseng C.A.Meyer,and provide an effective defense against phytopathogenic fungi.Ginseng rusty rot is the most widespread of ginseng diseases.Therefore,we choose ginsenosides and ginseng root rusty rot pathogens as object to study the interaction and mechanism between them.Four pathogens coming from different sources was identified,the results confirmed that all of them were Cylindrocarpon,but not the same species.To detect the transformation ability of four different species,ginsenoside Rb1 was used as the substrate.It was found that all of them could convert Rb1,but the transformed products were different.The pathway of sp.1 and sp.3 was Rb1?G-XVII?F2,and the pathway of sp.2 and sp.4 was Rb1?G-XVII/Rd?F2.Sp.1 had the only intermediate G-XVII,and the conversion rate was relatively high,this strain was screened for the further experiment.And sp.1 was identified as Cylindrocarpon destructans.Through a lot of experimental exploration of isolatied enzyme from the fermentation of C.destructans,we found that the glycosidase from C.destructans which transformed Rb1 into G-XVII required the presence of coenzyme to carry out catalysis effectively.To investigate the role that ginsenosides(and some of their metabolites)play in interactions between plants and C.destructans,we systematically determined the anti-fungal activities of six major ginsenosides(Rb1,Rb2,Rc,Rd,Re and Rg1),along with the metabolites of ginsenoside Rb1(G-XVII and F2),against the ginseng root pathogen C.destructans and non-ginseng pathogens F.graminearum,E.turcicum(Pass.)Leonard et Suggs,P.megasperma and P.oryzae.Our results showed that the growth of both ginseng pathogens and non-pathogens could be inhibited by using the proto-panaxatriol(PPT)ginsenosides Re and Rg1.In addition,the growth of the non-pathogens could also be inhibited by using proto-panaxadiol(PPD)ginsenosides Rb1,Rb2,Rc and Rd,whereas the growth of ginseng pathogen C.destructans was enhanced by ginsenosides Rb1 and Rb2.In contrast,G-XVII and F2 strongly inhibited the hyphal growth of both C.destructans and the non-pathogens tested.Furthermore,addition of sucrose to the media increased the growth of C.destructans,whereas glucose did not affect the growth.Moreover,C.destructans and all four non-pathogens were able to deglycosylate PPD ginsenosides using a similar transformation pathway,albeit with different sensitivities.All results suggest that the pathogenicity of C.destructans against ginseng root is independent of its ability to deglycosylate ginsenosides.Then,components of C.destructans cytomembrane were analyzed by GC-MS,and four main sterols were contained on the cell membrane,among which the highest is ergosterol.This showed that C.destructans did not inhibited by ginsenosides was not for lack of sterols.The interaction of ginsenosides and sterols from C.destructans cytomembrane was studied by constructing ergosterol-phospholipid liposome,artificial cell membrane and protoplast of C.destructans.Interestingly,ginsenoside Rb1 could damage cell membrane by binding sterols on that,but the growth of hypha was promoted.While,Re showed inhibitory effect though it was not able to combine with sterols.Therefore,we infer that there are other unknown substances in C.destructans which could identifie and interact with ginsenosides.The whole genome of C.destructans was sequenced.Results showed that the genome size was 60.4 M and GC-content was 51.8%.And 2095 transmembrane proteins,455 typical secretory proteins,3403 non-classical secretory proteins and 170 effect factors were selected as possible ginsenosides identification factors,and lay the foundation for follow-up study of the interaction mechanism of ginseng and C.destructans.