Metabolite Components And Functional Study Of Exudate From Sclerotinia Ginseng

Ginseng(Panax ginseng C.A.Meyer)is a precious perennial plant,which has a long history of cultivation in northeast China and possesses important economic and medicinal value.Sclerotinia ginseng Wang et Chen is a kind of necrotrophic fungi that can infect the root of ginseng,causing root rot disease and irreparable economic losses.Exudation is a common feature in the early stage of sclerotial development.Exudate is an important metabolite produced by nuclear-producing fungi in the development of sclerotia,and is closely related to the development and pathogenesis of sclerotia.By using plant pathology and molecular biology methods,the biological characteristics,metabolic components,proteomics,pathogenicity and effect of soil microbial community structure of droplets were systematically studied and the results were as follows:1.Systematic discussion on the pathogenic mechanism of the exudate of S.ginseng.It was proved for the first time that pectinase(PG)and carboxymethyl cellulose(Cx)were contained in the exudate of S.ginseng,and the PG activity in the exudate was significantly higher than that in the hyphae.Inoculation of ginseng roots with exudate can produce disease symptoms similar to S.ginseng and the rise of various defense enzyme systems(PAL,POD,CAT,SOD,PPO)of ginseng root can be induced.It was found that comparing with mycelium infection the exudate had the characteristics of rapid infection,strong pathogenicity,short duration of disease course and slow spread of disease spot.2.The biological characteristics of the exudate of S.ginseng were systematically studied.By studying the biological characteristics of the exudate,the following results had been obtained.Experimental results showed that the optimal medium was PDA.The optimal carbon sources were sucrose and glucose on mycelial growth and exudate production,respectively.Three kinds of organic nitrogen including peptone,beef extract and yeast extract powder could promote the growth of mycelium.The most optimal nitrogen source for sclerotia formation was yeast extract powder.Mycelial growth rate was the relative high on concentration of 60 g/L of glucose,sucrose and soluble starch,but glucose occupied the highest.30 g/L glucose was the optimal concentration of carbon source for the exudate of S.ginseng.High concentration of yeast extract powder(30~60 g/L)contributed to the formation of sclerotium,but it could inhibit the production of exudate.3 g/L yeast extract powder with 30 g/L sucrose not only conducive to the growth of mycelium,but also facilitated the production of exudate.The optimal pH of mycelium growth,sclerotium formation and exudate droplets was 5.3.It was the first time to prove that the exudate had allelopathic effect.The exudate promoted the mycelium growth of S.ginseng,and the mycelial growth rate is significantly higher at the concentration of 4%.The effects of different treatments on the microbial community structure of ginseng rhizosphere soil were compared.UPGMA taxonomic tree analysis of bacteria showed that exudate group and mycelium suspension group could make the bacterial community structure more similar in soil.By UPGMA tree analysis of fungi,it was found that exudate group and exudate-mycelium suspension group could produce similar soil fungal community structure.It was also observed from the UPGMA taxonomy tree that the fungi abundance of ascomycetes was higher in the fungal communities treated with exudate group,mycelium suspension group and exudate-mycelium suspension group,indicating that exudate and pathogen had similar effects on the composition and richness of soil fungal communities.4.Identification of the components of the exudate of S.ginseng.Metabolic composition was analyzed by integrating flame atomic absorption spectrometry(FAAS),nano-reagent spectrophotometry,gas chromatography-MS(GC/MS)and high performance liquid chromatography(HPLC).It was first determined that K,Na and Mg ions,free ammonia,oxalic acid,caffeic acid,ferulic acid,gallic acid,tannic acid,vanillic acid,inositol,glucose and trehalose were contained in the exudates.It could be further speculated that exudates play a certain role in pathogenicity and effect of soil microbial community structure by identifying the components of exudates.5.De novo sequencing is an important molecular technique for studying non-model organisms.14,904 unigenes were assembled from S.ginseng,of which 12,424 unigenes were annotated into at least one database.Through GO functional analysis,it was found that the subcategories with the most enrichment in the three GO terms were cell processes,metabolic processes,cells,cell part,catalytic activity and transport activity.Comparing S.sclerotiorum with S.ginseng,we found 17,638 homologous genes,which proved that the two pathogens have high affinity.The four pg genes(pg1,pg3,pg5 and pg6)were clearly divided into four different taxa,which proved that each gene was relatively independent in evolution.Two pac1 homologous genes identified from S.ginseng and other pathogens revealed that they were all had a relative closely relationship with the fungi of Sclerotiniaceae.6.Analysis of protein components of exudate of S.ginseng by proteomics technique.By using proteomics technology,59 proteins were identified and classified into six categories: carbohydrate metabolism(39%),oxidation-reduction process(12%),transport and catabolism(5%),amino acid metabolism(3%),other functions(18%)and unknown protein(23%).In the carbohydrate metabolism,many proteins are involved in the remodeling and modification of the fungal cell wall,and some proteins are involved in the pathogenicity of the pathogen.The pathogenicity and virulence of the identified proteins are also discussed in this paper.A class of protein(Ssp1)in the unknown protein group is specifically expressed during sclerotia development.Many proteins in the redox process were related to the formation of fungal melanin.