Study On Bacteriostatic And Insect Repellant Activities Of Ginsenosides From Ginseng Stem And Leaf Against Several Major Plant Diseases And Insect Pests In Vegetables

As the rapid development of vegetable industry,high density planting induces the increased occurrence of plant diseases and insect pests at both pathogenic species and amount,leading to an elevated trend in economic losses.For a long time,the overuse of chemical pesticide and bactericide,on the one hand,results in resistance of pest and pathogen even developing into cross and multi resistance,which increases the difficulty of disease control.On the other hand,this destroys ecological balance to some extent,as well as enhances pesticide residue and reduces the safety of vegetable products.Thus,it is extremely urgent to adopt biological control.Some natural products show inhibitory effects on pathogen growth as well as antifeedant,repellant and poison effects on pests.Developing ginsenoside into a botanical pesticide could achieve both pest and disease controlling goals as well as environment protecting,ecological balance maintaining and agricultural product safety guaranteeing goals.The aboveground part of ginseng will fall off every season,and these resources containing ginsenoside are discarded.Therefore,make use of these discarded resources for ginsenoside extraction will tremendously lower the production cost.Ginsenosides have a long degradation cycle and are harmless to plants.Additionally,to develop ginsenoside into a plant protection product not only has the insect disease prevention roles,but also has healthcare function for human beings.This advantage could not be found in any other botanical pesticide,bactericide or fungicide.In this study ginsenosides were extracted from ginseng stem and leaf,Central composite design–response surface methodology?CCD-RSM?was employed to optimize extraction process.Pathogens of common vegetable diseases were isolated from infected leaves,and inhibitory effects of ginsenosides on growth rate of these pathogenic bacteria and funguses were tested.Then antimicrobial spectrum was obtained and structure-function relationship was also confirmed.Botrytis cinereal,which was sensitive to ginsenosides,was selected for further study.The influences of ginsenosides on spore germination,mycelial morphology,ultrastructure and transcription were tested to uncover the underlying mechanism.Meanwhile,the antifeedant activity of ginsenosides against plutella xylostella was evaluated as well.The main results are as follows:1.according to CCD-RSM results,the optimum parameters for ginsenoside extraction are 65.0%alcohol?v/v?,liquid-to-solid ratio of 29.5:1 and ultrasonic treatment for 60 min.HPLC result showed that the sum amount of nine ginsenosides in the extract were 66.19 mg·g^-1.2.Nine pathogens caused vegetable diseases were isolated,among which a fungus was found in tomato for the first time.Morphological identification indicated that the newly found fungal was Alternaria tenuissima,and the sequences of ITS?MF796663?and HIS?MF688839?region was 100%identity with A.tenuissima.The pathogenicity identification obtained the same morphological feature as original infected plant grown on field condition.The inoculum density has a positive relationship with disease index,while has a negative relationship with plant height and biomass.This is the first report concerning A.tenuissima infect tomato in China.3.The antibacterial spectrum of ginsenoside was screened.Ginsenosides had significant inhibitory effects on both bacteria and funguses.The inhibitory effects showed positive relationship to applied concentration,but it differed between pathogens.The inhibitory effects of ginsenosides on five bacterial was showed in a descendent order:Erwinia carotovora subsp.carotovora,Ralstonia solanacearum,Clavibacter michiganensis subsp.michiganense,Clavibacter michiganensis subsp.michiganense and Pseudomonas syringae pv.lachrymans.The EC50 of ginsenosides for Alternaria tenuissima,Botrytis cinereal,Alternaria brassicae var.phaseoli and Rizoctonia solani were 1.31 mg·mL^-1,0.04 mg·mL^-1,0.28 mg·mL^-1 and 109.08 mg·mL^-11 respectively.4.Ginsenosides showed significant inhibitory effect on B.cinereal,with a suppression ratio of 97.55%when applied concentration was 1 mg·mL^-1.Structure-function relationship indicated that ginsenoside type,sugar type and sugar number have obvious influence on inhibitory effect against pathogen.On one hand,panoxadiol ginsenosides have higher inhibitory effect on pathogen.On the other hand,glucose number in ginsenoside has positive relationship to inhibitory effect,while other types of sugar number in ginsenoside has negative relationship with inhibitory effect.In addition,synergistic interaction was observed within single ginsenosides.The EC₅₀0 of ginsenosides is 4.84 mg·mL^-11 for B.cinereal spore germination.Further,ginsenosides make B.cinereal hypha morphology changed obviously,including uneven thickness,growth point malformation and hypha collapse.Similarly,ultrastructure was also destroyed by ginsenosides,such as vacuole in cytoplasm,cytoplasm external flow and mitochondria degradation.5.Differently expressed genes in B.cinereal at three stages?hypha formation,logarithmic phase and sclerotium formation?,which are response to ginsenosides treatment,are screened by RNA-seq technique.These differently expressed genes are mainly distribute to general function prediction only,garbohydrate transport and metabolism and amino acid transport and metabolism functional categories.There are seventeen genes differently expressed at all three stages,and most of them related to sterol biosynthesis,carbohydrate metabolism and fatty acid metabolism pathways.Ginsenosides may restrain B.Cinerea growth by lowering its membrane stability,correspondingly B.Cinerea may revolt to ginsenosides stress by elevating ergosterol synthesis and ABC transporter protein level.6.Ginsenosides showed significant antifeedant activity on P.xylostella larvae.The AFC50 of total ginsenosides for the second instar larval of P.xylostella were 4.98 and5.03 mg/mL at 24 h and 48 h respectively in non-choice assay,and they were 2.74 and4.14 mg/mL respectively at 24 h and 48 h in choice assay.The residue rate of ginsenosides in the pest became gradually higher as applied concentration increased.Feeding with ginsenosides generally resulted in decrease of GST,AChE and CarE activities,but increase of MFO activity in P.xylostella.This research highlighted that ginsenoside showed varying inhibitory activity on different pathogens,with the strongest activity on B.cinereal.This is the first time to investigate the structure-function relationship of ginsenoside and inhibitory activity,indicating that inhibitory activity of ginsenoside is related to skeletal structure type,monosaccharide category and monosaccharide number.Also,this is the report that A.tenuissima infected tomato in China.Further,this work uncovered the physiology mechanism that ginsenoside restrained A.tenuissima growth by destroying cell structure as well as lowering stability of cytomembrane and organelle membrane.In response to ginsenoside stress,A.tenuissima increased synthesis of ergosterol and expression of ABC transporter to maintain membrane stability.On the other hand,the research revealed that the antifeedant activity of ginsenoside on P.xylostella may correlate to the suppression of GST,AChE and CarE activities by ginsenoside.This research will help further understanding the role of ginsenosides on plant evolution,and provides new insight to perceive the role of secondary metabolite in plant defense.